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Adaptation to mitochondrial stress requires CHOP-directed tuning of ISR – Science Advances

By daniellenierenberg

INTRODUCTION

Mitochondrial diseases are a heterogeneous group of devastating disorders characterized by respiratory chain dysfunction (1). Although mitochondrial disorders have distinct tissue and organ presentation, they seem to activate common stress responses evolved to mitigate the negative impact of respiratory deficiency on cellular and organismal metabolism (1). It appears that mitochondrial stress responses precede respiratory chain deficiency, thereby suggesting that they constitute an early event in pathogenesis of mitochondria-related diseases (2). This suggests that monitoring the activation and/or alteration of mitochondrial stress responses may provide early diagnostic markers in these conditions. Moreover, manipulation of mitochondrial stress responses may be beneficial for patients with mitochondrial disease and thus therapeutically exploited (3, 4).

Initially, the mitochondrial unfolded protein response (UPRmt) was postulated to be a common stress response to respiratory chain dysfunction (5). UPRmt constitutes a transcriptional program that up-regulates mitochondrial chaperones and proteases aimed to restore the loss of organelle proteostasis. Notwithstanding that UPRmt was first described to be triggered by the accumulation of misfolded proteins within the mitochondrial matrix in mammalian cells (5), most of the subsequent mechanistic studies were performed in Caenorhabditis elegans (6). In contrast, many aspects of the mammalian UPRmt signaling are less well understood. In mammalian cells, it is thought that mitochondrial proteotoxic stress leads to CHOP [CCAAT/enhancer binding protein (C/EBP) homology protein] up-regulation resulting in up-regulated transcription of UPRmt-responsive genes (5, 7). The CHOP-binding sites in the UPRmt gene promoters are presumably flanked by two conserved regions named the mitochondrial UPR elements 1 and 2 (MURE1 and MURE2) (7, 8). The role of CHOP in governing transcription of UPRmt genes is however controversial as the transcription factors that bind to MURE1 and MURE2 elements have not been identified (7, 9). Nevertheless, multiple studies confirmed up-regulation of the CHOP mRNA in cells derived from patients with various mitochondrial disorders, as well as mitochondrial disease models (2, 1012). This illustrates that although CHOP plays a pivotal role in mammalian mitochondrial stress responses, the underpinning mechanisms of its actions in the context of mitochondrial dysfunction are still obscure.

Recently, it became clear that unlike in C. elegans, mammalian UPRmt may not be the primary response to mitochondrial dysfunction but rather function as a part of more complex mitochondrial stress response (1114). Mammalian cells treated with mitochondrial toxins exhibit transcriptional reprogramming mimicking the integrated stress response (ISR) arm of the UPR, which is centered on the activating transcription factor 4 (ATF4) (13, 14). Consistent with this, studies carried out in models with defects in different steps of mitochondrial DNA (mtDNA) expression and protein synthesis revealed activation of ISR transcriptional signatures (11, 12). ISR hallmarks are increased eIF2 phosphorylation, reduction in ternary eIF2:tRNAiMet:guanosine 5-triphosphate (GTP) complex levels, and subsequent inhibition of global protein synthesis that is paralleled by selectively induced translation of a subset of inhibitory upstream open reading frame (uORF) containing stress-responsive mRNAs, including ATF4, CHOP, and GADD34 (15). CHOP induction during ISR is thought to lead to cell death via induction of Growth Arrest and DNA Damage-Inducible Protein 34 (GADD34)mediated eIF2a dephosphorylation and activation of Endoplasmic Reticulum Oxidoreductase 1 Alpha (ERO1A) endoplasmic reticulum (ER) oxidase (16).

CHOP is a multifunctional transcription factor that dimerizes with members of the C/EBP and ATF/cyclic adenosine 3,5-monophosphate response element binding protein families (17). Although up-regulated in response to a wide variety of stresses such as growth arrest and DNA damage, amino acid and glucose deprivation, hypoxia, and ER stress, the role of CHOP in cellular physiology is incompletely understood. CHOP is considered to induce apoptosis, but its transcriptional targets largely overlap with those of ATF4, including genes promoting cell survival and growth (16, 18). These findings highlight the intricate interaction partnerdependent roles of CHOP under different stresses and in various tissues. They also point out the importance of understanding the context-dependent role of CHOP under different physiological conditions. In the context of mitochondrial respiratory chain dysfunction, the role of CHOP is particularly important as CHOP was proposed to be the main transcription factor that conveys specificity of the mitochondrial stress response (5).

Here, we aimed to decipher the role of CHOP in the regulation of the mitochondrial stress response. As a model for the most common cause of mitochondrial diseases, namely, loss of mitochondrial translation, we used mice deficient in the mitochondrial aspartyl transfer RNA (tRNA) synthase DARS2 specifically in heart and skeletal muscle (DARS2 KO) (2). We demonstrate a beneficial role of CHOP in mitochondrial mutants as its loss leads to a marked shortening of life span in DARS2/CHOP double knockout (DKO) as compared to DARS2 KO animals. The beneficial effects of CHOP appear to be independent of UPRmt activation but rather mediated by attenuation of harmful overactivation of the ISR and a consequent metabolic imbalance. We also provide mechanistic evidence that these effects stem from the interplay between CHOP, ATF4, and C/EBP in regulation of mitochondrial ISR targets.

To determine the in vivo function of CHOP in the context of mammalian mitochondrial dysfunction, we intercrossed whole-body Chop/ mice (CHOP KO) with heart and skeletal muscle-specific DARS2-deficient mice (Dars2fl/fl; Ckmm-Cre+/tg; DARS2 KO) (fig. S1, A and B) (2). The resulting animals deficient in both CHOP (whole body) and DARS2 (heart and skeletal muscle) (Dars2fl/fl; Ckmm-Cre+/tg; Chop/ and DKO) were born in Mendelian ratios (fig. S1C). We previously showed that DARS2 depletion mediated by Ckmm-Cre expression induces dilated cardiomyopathy preceding any pathological phenotypes in skeletal muscle (2). Hence, we monitored the effects of CHOP loss on pathologies caused by DARS2 abrogation in the heart.

Approximately from 2 weeks of age, a large number of DKO mice became increasingly susceptible to sudden death during a routine ear-clipping handling for genotyping. This procedure was tolerated well up to postnatal day 13 (P13) by mice of all four genotypes; hence P13 (1) was defined as the early stage of heart dysfunction in DKO animals (DKOE). It appeared that the deterioration of the health status of DKO mice characterized by lower spontaneous cage activity, piloerection, unsteady gait, and overall droopiness is a very rapid process as the interval from the first apparent symptoms to death of the mice at around P17 (2) was between 24 and 48 hours. This interval was defined as the late/terminal stage in DKO mice (DKOL). Consequently, the life expectancy of DKOs was severely reduced (>60%) compared to DARS2 KOs, signifying the essential role for CHOP in adaptation to impaired mitochondrial protein synthesis in heart (Fig. 1A). CHOP deficiency in the absence of DARS2 resulted in dilated cardiomyopathy (Fig. 1B and fig. S1, D to F) characterized by increased expression of mRNAs encoding cardiac hypertrophy markers Nppa and Nppb (Fig. 1C). Although no gross morphological changes were observed upon hematoxylin and eosin (H&E) staining, ultrastructural analyses suggested a disrupted myocardial organization, characterized by severely disorganized sarcomeric structures, expected to cause disturbances in contractile function of DKOL hearts (Fig. 1, D and E). Therefore, DKOL animals display very similar pathological changes, as compared to the terminal stage DARS2 KO mice (2), whereby the onset of these pathologies is markedly accelerated upon CHOP loss.

(A) Kaplan-Meyer survival curves for wild-type (WT; n = 36), CHOP KO (n = 35), DARS2 KO (n = 47), and DKO animals (n = 60). The life span of DKO in comparison to DARS2 KO mice is significantly decreased (P < 0.0001; log-rank test and Gehan-Breslow-Wilcoxon test). The viability of CHOP KO mice was WT-like in a 12-month follow-up. (B) Heart gross morphology. (C) Fold changes of the cardiac hypertrophy markers Nppa and Nppb obtained from the RNA sequencing dataset at P17 (2) (n = 4). Bars represent means SD [multivariate analysis of variance (MANOVA) followed by one-way ANOVA and Tukeys multiple comparisons test, **P < 0.05, ***P < 0.001, and ****P < 0.0001]. (D) H&E staining; (n = 3) at P17 (2). Scale bars, 50 m. (E) Transmission electron microscopybased analyses of cardiac tissue biopsies; (n = 1) at P17 (2). Scale bars, 2 m.

We next sought to identify pathways that are affected by the CHOP deficiency in the context of DARS2 KO. To this end, we compared global changes in mRNA levels to corresponding changes in the proteome in CHOP KO, DARS2 KO, and DKOL versus control hearts using the anota2seq algorithm (19). Scatter plots comparing mRNA and protein changes in DARS2 KO hearts revealed alterations in protein levels that were mainly independent of the mRNA levels, thus arguing for a prevalent impact of translational and/or protein stability changes on the proteome (Fig. 2A, fig. S2A, and table S1). In contrast, DKOL animals primarily showed congruent changes in mRNA and protein levels, which accounted for ~75% of detected alterations in protein levels (Fig. 2A, fig. S2A, and table S1).

(A) Scatter plots of total mRNA and protein fold changes (FC) comparing CHOP KO, DARS2 KO, or DKOL to WT. The numbers of significantly regulated genes are indicated for translation/protein stability (red), and mRNA abundance (green). RNA sequencing and quantitative proteomics were performed on hearts of animals at P17 (2) (n = 4). (B) A GO network of overrepresented terms among genes regulated via changes in translation/protein stability (up-regulated, light red; down-regulated, dark red) and mRNA abundance (up-regulated, light green; down-regulated, dark green) in DKO versus WT. Nodes represent identified GO terms, while the pie chart within each node indicates the proportion of genes regulated. (C and D) Heatmap of protein expression (P) and total mRNA (T) log2 fold changes of (C) the OXPHOS subunits grouped in respective complexes and (D) OXPHOS assembly factors (n = 4). (E) In organello translation assay (left) of cardiac mitochondria at P17 (2). De novo protein synthesis was determined after 1 hour of 35S-methionine pulse labeling; protein turnover was assessed after 3 hours of the cold chase. Coomassie brilliant bluestained gel was used as a loading control. Relative protein synthesis and turnover rates (right) (n = 3). (F) Oxygen consumption of intact cardiac mitochondria at P17 (2). State 3: adenosine 5-diphosphate (ADP)stimulated respiration using CI or CI + CII substrates. State 4: Respiration upon addition of oligomycin. ETS, maximum respiration upon mitochondrial uncoupling (CI) and after addition of rotenone (CII) (n = 3 to 4). Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Gene Ontology (GO) analysis performed using ClueGO (20) and annotation from the GO consortium (21) on genes whose expression was reduced indicated that oxidative phosphorylation, electron transport, complex I assembly, adenosine 5-triphosphate (ATP) biosynthesis, fatty acid oxidation, and heart contraction are predominantly disrupted in DKO hearts (Fig. 2B). This is consistent with the impairment of mitochondrial energy production and heart failure in DKO animals and similar to other models of mitochondrial cardiomyopathy (11). In contrast, translation, tRNA metabolism, mitochondrial RNA, and glutathione metabolism were primarily up-regulated pathways (Fig. 2B). We observed further perturbations in apoptotic pathways, amino acid catabolism, and purine nucleotide metabolism that contained a combination of up- and down-regulated gene expression changes (Fig. 2B).

A general down-regulation of steady-state levels of individual oxidative phosphorylation (OXPHOS) subunits detected in DARS2 KO hearts was further decreased in DKOL animals (Fig. 2C and fig. S2B). Intriguingly, while in DARS2 KO animals, most of the changes in the levels of OXPHOS subunits were not accompanied by alterations in mRNA abundance, numerous OXPHOS subunit-encoding genes exhibited congruent changes in mRNA and protein levels in DKOL animals (Fig. 2C). These include three of four subunits of succinate dehydrogenase (SDH; complex II), a complex fully encoded by nuclear DNA, usually up-regulated upon mitochondrial translation defects. This was further confirmed using an enzyme-histochemical assay, showing that substantial cyclooxygenase (COX) deficiency observed in DKOL animals is not accompanied by a compensatory SDH up-regulation (fig. S2C), as observed in DARS2 and other mitochondrial mutants (2, 22). Furthermore, while we detected a general compensatory up-regulation of OXPHOS assembly factors in DARS2 KO hearts, many were either unaltered or down-regulated in DKOL samples (Fig. 2D).

Although Dars2 deletion primarily interferes with mitochondrial protein synthesis, at P17, only a moderately dysbalanced mitochondrial translation was observed in DARS2 KO (Fig. 2E). In contrast, mitochondrial de novo protein synthesis in DKOL mice was significantly decreased and severely dysregulated, whereas the protein turnover remained unaffected (Fig. 2E). The exaggerated translation defect observed in DKOL animals was not caused by a decrease in mtDNA or mtRNA levels (fig. S2, D and E). Some mtRNAs were up-regulated (e.g., mt-COX3 and mt-ND1) in both DARS2 KO and DKO hearts, possibly as a compensatory response to defective protein synthesis (Fig. 2E and fig. S2E).

Severe dysregulation of mitochondrial translation in DKOL was accompanied with a strong decrease in the respiration capacity of all inducible states in mitochondria isolated from DKOL hearts (Fig. 2F). In contrast, no major defects in DARS2 KO heart mitochondria respiration were observed, thus suggesting compensation for the mitochondrial protein synthesis defect (Fig. 2F).

Unexpectedly, a comparable defect at the level of assembled respiratory chain complexes and supercomplexes was detected in DARS2 KO and DKOL mice despite higher levels of individual OXPHOS subunits in DARS2 KO (Fig. 2C and fig. S2F). These data suggest that, at early stages of DARS2 deficiency, nascent nuclear-encoded OXPHOS subunits are not efficiently incorporated in respiratory chain complexes in DARS2 KO hearts and are likely turned over at higher rates. Although DKOL and DARS2 KO mitochondria have comparable levels of respiratory chain supercomplexes (fig. S2F), DKOL mitochondria fail to sustain normal respiration (Fig. 2F). This suggests that the OXPHOS activity is further indirectly affected by CHOP deficiency that might lead to disruption of mitochondrial integrity or supply of critical metabolites.

CHOP deficiency in the context of mitochondrial dysfunction is expected to blunt the mitochondrial stress response (5). Therefore, by analyzing changes in the transcriptome, we compared pathways that are affected in DARS2-deficient hearts before and after CHOP depletion (table S2).

In DARS2 KO heart, relatively few mRNAs changed their expression, and most were up-regulated. Notably, using Cytoscape plug-in iRegulon, we demonstrated that two-thirds of these transcripts overlapped with an ISR signature activated by ATF4, which was also identified as the most prominent regulator of gene expression in DARS2 KO hearts (Fig. 3A and tables S2 and S3) (18, 23, 24). The most up-regulated transcripts in DARS2 KO hearts encoded enzymes involved in one-carbon metabolism, serine biosynthesis, and trans-sulfuration, as well as Gdf15 and Fgf21 (Fig. 3, A and B, and table S2), the two cytokines shown to be excreted from tissues upon OXPHOS deficiency (25, 26). Similar changes (Fig. 3A) were previously described in other cellular and in vivo models for mitochondrial OXPHOS defects, confirming that DARS2 deficiency causes a stress response relevant for many mitochondrial disease states (1114).

(A) Heatmap of total mRNA fold changes (log2) of significantly changed ATF4 target genes [as predicted by Cytoscape plug-in iRegulon (23, 24)], in DARS2 KO animals compared to WT controls. Black boxes above DKO and below CHOP KO rows indicate their respective significantly changed transcripts as compared to WT controls (n = 4). (B) Fgf21 log2 raw expression counts (sequenced reads, +0.5) as this gene was not detected in multiple WT and CHOP KO samples and hence was excluded during data filtering. Of note, these samples will obtain negative log2 values (n = 4). (C) Western blot analysis (left) and quantification of ISR markers (right). HSC70 was used as a loading control. Antibodies used were raised against proteins indicated in panels. Experiments were performed on cardiac lysates of mice at P17 (2) (n = 3). (D) p-eIF2/eIF2 ratio quantified from (C). (B to D) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (E) Western blot analysis and quantification of UPRmt markers in WT, CHOP KO, DARS2 KO, and DKO animals at P17 (2). Antibodies used were raised against proteins indicated in panels. HSC70 was used as a loading control. Bars represent means SD; no significant differences were detected (MANOVA: Wilks test, P = 0.176; Hotelling-Lawleys test, P = 0.183; Pollais test, P = 0.232) (n = 3). (F) Heatmap of total mRNA fold changes (log2) for the selected alleged CHOP target genes involved in apoptosis (n = 4).

The ISR activation in DARS2 KO hearts was confirmed by increased eIF2 phosphorylation, accompanied by up-regulation of ATF4 (Fig. 3, C and D). These effects were further potentiated by CHOP loss, whereby induction of both eIF2 phosphorylation and ATF4 was more pronounced in DKOL relative to DARS2 KO hearts (Fig. 3, C and D). Transcript and protein levels of almost all ATF4 targets were highly up-regulated in DKOL as compared to DARS2 KO animals (Fig. 3, A to C). Consistently, further analysis of binding motifs in genes up-regulated in DKOL hearts established ATF4 as the most prominent signature (table S3) (23, 24). The most up-regulated transcripts in DARS2 KO and DKOL showed a notable overlap. To this end, of the top 11 most up-regulated transcripts, 8 overlapped, despite the 40-fold difference in the number of overall changes between the two models (table S2). The only difference was that these transcripts were, on average, more than 10-fold more up-regulated in DKOL than in DARS2 KO hearts (table S2). In contrast, UPRmt markers were not significantly changed in DARS2 KO or DKOL animals, adding evidence that UPRmt is neither an early nor prominent stress response in mammalian cells and tissues upon mitochondrial OXPHOS dysfunction (Fig. 3E). Instead, our data suggest a central role for ISR and ATF4-dependent regulation in the context of mitochondrial dysfunction in vivo and point to an unexpected role of CHOP in the suppression of the transcriptional overactivation of ATF4 targets.

CHOP is proposed to be involved in the regulation of apoptosis upon ER stress, although the exact mechanism remains controversial, as exogenously expressed CHOP has also been reported to positively regulate genes involved in protein synthesis and not apoptosis (16, 18). Henceforth, we analyzed changes in the expression levels of various apoptotic genes reported to be CHOP targets (27). Notably, proapoptotic members of the B-cell lymphoma 2 (BCL-2) family (Puma/Bbc3, Bid, Bax, and Bim/Bcl2l11) and genes encoding proteins involved in the activation or execution of apoptosis (Dr5/Tnfrsf10b, Casp3, and Ero1l) were not suppressed but often further up-regulated upon loss of CHOP in DARS2-deficient animal (Fig. 3F). Similarly, the steady-state level of proapoptotic protein BCL2-associated X protein (BAX) was up-regulated, and we observed a higher cleavage of caspase 3 in DKOL hearts as compared to control animals (fig. S3A). These results suggest that, unexpectedly, apoptosis may be up-regulated in DARS2-deficient hearts upon CHOP depletion and thus contribute to the detrimental phenotype observed in DKOL mice.

As we observed major changes in the abundance of proteins involved in amino acid metabolism, we next measured amino acid levels by liquid chromatographytandem mass spectrometry. While only minor perturbations in amino acid levels were observed in DARS2 KO hearts, most amino acids were significantly up-regulated in DKO mice (fig. S3B). Of note, serine, glutamine, glutamate, and aspartate levels were not significantly changed in either DARS2 KO or DKOL relative to control hearts (fig. S3B). The unaltered serine levels, despite the increased levels of enzymes involved in serine synthesis [Phosphoglycerate dehydrogenase (PHGDH), Phosphoserine Aminotransferase 1 (PSAT1) , and Phosphoserine Phosphatase (PSPH)], suggest an increased flux of serine-derived one-carbon units for further methylation reactions into the one-carbon cycle. Similarly, glutamine and glutamate are likely used to replenish tricarboxylic acid cycle intermediates and aspartate production that is essential for nucleotide synthesis and cell proliferation (28, 29). Increased levels of citrate and isocitrate in DKOL, but not DARS2 KO, hearts suggest that glutamine primarily undergoes reductive metabolism (fig. S3C), as seen in the patient-derived cell lines harboring mtDNA mutations (30). Increased citrate levels can propagate intracellular acidosis, leading to hypocalcemia caused by reduced availability of Ca2+, further contributing to reduced contractility of the heart through a vicious circle of the excitation-contraction-metabolism impairment (31). Additional effects of elevated citrate levels on the regulation of metabolic enzyme and/or chromatin dynamics by acetylation may further contribute to accelerated pathological phenotypes observed in DKOL hearts.

Next, we tested whether mitochondrial stressinduced ISR has a beneficial or detrimental role in conditions of mitochondrial dysfunction. For these analyses, we took advantage of two cell models for mitochondrial respiratory chain dysfunction: (i) mouse skin fibroblasts with severe mitochondrial dysfunction caused by the loss of COX10 (COX10 KO), an early assembly factor of the respiratory cytochrome c oxidase (32); and (ii) mouse embryonic fibroblasts (MEFs) treated with actinonin, an inhibitor of mitochondrial peptide deformylase causing impairment in mitochondrial translation (33).

In the COX10 KO cells, a robust activation of the ISR was detected as evidenced by increased levels of phosphorylated eIF2, ATF4, and ATF4 targets (Fig. 4A and fig. S4A). To test whether increased ATF4 levels are a direct result of ISR activation, we incubated COX10 KO cells with the ISR inhibitor (ISRIB) (34). This treatment abrogated ATF4 induction and attenuated up-regulation of its downstream targets at both transcript and protein levels (Fig. 4A and fig. S4A). The phosphorylation of eIF2 remained unchanged (Fig. 4A), which was expected as ISRIB bolsters guanine-nucleoside exchange activity of eIF2B without affecting on phospho-eIF2 levels (34). Similarly, increased ATF4 levels induced by actinonin treatment were suppressed by ISRIB (Fig. 4B). Mirroring the results from DKOL mice, loss of CHOP combined with mitochondrial dysfunction induced by actinonin treatment greatly increased ATF4 protein and transcript levels, and expression of ATF4 targets Shmt2, Pycr1, and Mthfd2 (Fig. 4, B and C).

(A) Western blot analysis (left) and relative protein levels (right) of ISR markers and ATF4 downstream targets in immortalized COX10 KO and WT fibroblasts upon 48-hour treatment with DMSO () or ISRIB (+). (B) Western blot analysis of WT and CHOP KO MEFs treated for 48 hours with DMSO () or actinonin (+) in the presence (+) or absence () of ISRIB during the last 4 hours before protein isolation. (C) Relative transcript levels in WT and CHOP KO MEFs treated for 48 hours with DMSO (control) or actinonin. Tbp expression was used for normalization (n = 3). (D) Growth curves of respective exponential growth phases of WT and CHOP KO MEFs treated with DMSO (control), actinonin, and +/ISRIB, respectively. Curves were determined using linear regression (n = 3). Bars represent means SD. (E) Western blot analysis of heart lysates from 4-week-old WT and DARS2 KO animals treated with control (DMSO) and ISRIB, according to the experimental setup presented in the schematic illustration (top). Animals are treated with daily injections of saline (control) or ISRIB solution for 7 days (blue boxes), starting at P19, and euthanized at P27 (red line) (n = 3). (F) Quantification of ISR markers (top), OXPHOS subunits (bottom), and p-eIF2/eIF2 ratio (right) from the Western blot analysis at (E). (A, B, and F) Antibodies used were raised against proteins indicated in panels. HSC70 was used as a loading control. (A, C, and F) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Prevention of ISR overactivation in CHOP KO MEFs by ISRIB treatment resulted in a partial rescue of the proliferation defect induced by actinonin (Fig. 4D). In turn, wild-type (WT) cells treated with actinonin and CHOP KO cells grown under control conditions showed minor growth defects, which were not further affected by ISRIB (Fig. 4D). Therefore, CHOP deficiency, only in conditions of mitochondrial dysfunction, results in a detrimental ISR activation, which can be partially rescued by ISRIB treatment.

To assess the effect of ISRIB treatment in vivo, DKOL mice and respective controls were injected with ISRIB (5 g/g) for up to 7 or 12 days, starting from 1 week of age (fig. S4B). Unfortunately, neither protocol resulted in the suppression of ATF4 levels or downstream targets in either DKO or DARS2 KO animals nor did it affect steady-state levels of OXPHOS subunits (fig. S4, C and D). However, this is not unexpected given the fact that ISRIB inhibits low-level ISR activity but does not affect strong ISR signaling (35), as observed in DKO mice. In contrast, a 7-day treatment of DARS2 KO animals with ISRIB, starting from P20, resulted in an apparent reduction of ISR markers (Fig. 4, E and F). Nevertheless, ISRIB-mediated suppression of ISR in DARS2 KO animals up to 4 weeks of age was not beneficial as it also prevented compensatory complex II (CII) up-regulation.

One of the hallmarks of the acute ISR is suppression of global protein synthesis, accompanied by translational activation of some uORF-containing mRNAs (15). To further understand the consequences of ISR activation in our model, we measured the global protein synthesis rate at P6, P13, and P17 in vivo in DKO and control hearts (36). At P6, cytoplasmic translation of all four genotypes was similar, in agreement with no phenotypes observed at this time point (fig. S5A). Coinciding with increased eIF2 phosphorylation, a 70% decrease in general protein synthesis was detected in mice at P13 (DKOE; Fig. 5A and fig. S5B). Within a few days, this effect seems to be reversed as we detected fully recovered protein synthesis rates in DKOL hearts at P17 (Fig. 5B and fig. S5C). This was despite unaltered eIF2 phosphorylation levels and activation of ATF4 and its targets that were comparable between DKOE and DKOL hearts (fig. S5D). These findings suggested a transition from acute to prolonged ISR, characterized by recovery of global protein synthesis and ongoing translation of ISR-sensitive mRNAs (37). These distinctions in global protein synthesis levels reflected different phenotypes of DKOE and DKOL mice. In the acute ISR, when global translation is strongly down-regulated, DKOE (P13 1) animals cope better with the mitochondrial translation defect when compared to DARS2 KO animals (Fig. 5C). This is illustrated by the unaffected levels of OXPHOS complexes and supercomplexes in DKOE animals (Fig. 5D and fig. S5E). However, these effects are reversed when DKO animals reach the prolonged ISR stage, which is characterized by partial recovery of global mRNA translation and sustained ATF4-mediated transcriptional reprograming (fig. S5D). This reactivation of normal translation is likely to result in ER stress, and further energy crisis as protein synthesis is highly energy demanding (38). Consistently, we detected increased levels of the ER-chaperone binding immunoglobulin protein (BIP) in P17 DKOL hearts, which mirrored findings in DARS2-deficient hearts at the terminal state of 6 weeks of age (Fig. 5E). Levels of several ER Ca2+ transporter proteins were also profoundly disturbed [Ryanodine receptors (RyR), Sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), and The inositol 1,4,5-trisphosphate receptor type 2 (IP3RII)], which may explain defects in the conductive system of the heart (Fig. 5F). Perturbed Ca2+ homeostasis due to the dysregulation of the ER Ca2+ transporters and increased Ca2+ release by ERO1-stimulated IP3R activation may also contribute to ER stress leading to the development of fatal cardiomyopathy (Fig. 5, E and F). Therefore, although strong activation of ISR, as seen in DKOE animals, brings brief protection from the mitochondrial dysfunction, it cannot be sustained over prolonged period of time and results in a detrimental switch to a prolonged ISR program leading to additional ER stress, loss of Ca2+ homeostasis, and premature death.

(A and B) The relative protein synthesis rate of animals injected with puromycin at (A) P13 and (B) P17. Bars represent means SD (one-way ANOVA and Tukeys multiple comparisons test, **P < 0.01 and ***P < 0.001) (n = 4). (C) De novo synthesis in mitochondria isolated from WT, CHOP KO, DARS2 KO, and DKOE and DKOL animals after 1 hour of 35S-methionine pulse labeling followed by SDS-PAGE. Coomassie bluestained gel was used as a loading control. (D) Blue native polyacrylamide gel electrophoresis (BN-PAGE) and subsequent Western blot analysis of OXPHOS complexes and supercomplexes in mitochondria isolated from WT, CHOP DO, DARS2 KO, and early (DKOE) and late-stage (DKOL) DKO animals. Subunit-specific antibodies (left) were used to detect respective complexes and supercomplexes (right) (n = 3). (E) Western blot analysis of BIP levels in WT, CHOP KO, DARS2 KO, and DKO at P17 (2) (top) and WT and DARS2 KO at 6 weeks (bottom) (n = 3). (F) Western blot analysis proteins involved in the Ca2+ metabolism in WT, CHOP KO, DARS2 KO, and DKOL at P17 (2) (n = 3). (E and F) HSC70 was used as a loading control (n = 3).

The prolonged activation of ISR in DKOL hearts may have adverse effects on cellular and organismal fate. GADD34, a regulatory subunit of the enzyme dephosphorylating eIF2, is thought to function as ISR rheostat acting to restore protein synthesis and block excessive ATF4 activation (15). Unexpectedly, although CHOP was proposed to be a primary Gadd34 transcriptional activator (16), DKOL animals at P17 showed a significant up-regulation of Gadd34 transcripts to similar levels as those observed in terminal, 6-week-old DARS2 KO animals (Fig. 6A). This result suggests that CHOP may play a GADD34-independent role in the suppression of the overactivation of ATF4 induction and ATF4-mediated transcriptional reprogramming.

(A) Relative Gadd34 transcript levels at P17 (2) WT, CHOP KO, DARS2 KO, and DKO animals, as well as in 6-week-old WT and DARS2 KO mice. Bars represent means SD, samples were normalized to WT mice of the respective age (P17: one-way ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001; 6 weeks: unpaired Students t test) (n = 4). (B) Coimmunoprecipitation (co-IP) of CHOP from WT, CHOP KO, DARS2 KO, and DKOL hearts. The CHOP and C/EBP interaction was monitored with Western blotting using an antibody against C/EBP. One percent of the input fractions was used as loading controls. Asterisks indicate the immunoglobulin G heavy and light chains. (C) Western blot analysis (left) and quantification (right) of the three CEBP isoforms LAP1, LAP2, and LIP in CHOP KO MEFs treated for 48 hours with actinonin along with the respective control (n = 3). (D) Western blot analysis (left) and quantification (right) of steady-state levels of ISR markers in actinonin-treated (48 hours) CHOP KO MEFs expressing the CEBP LIPL120T mutant variant along with the respective controls (n = 3). (E) Western blot analysis of the ATF4 and three CEBP isoforms in actinonin-treated (48 hours) CHOP KO MEFs expressing the CEBP LIP WT and CEBP LIPL120T mutant variant along with the WT cells and respective controls (n = 4). (C to E) Antibodies used were raised against proteins indicated in the panels. HSC70 was used as a loading control. (A, C, and D) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) (n = 3).

As a prerequisite for DNA binding, CHOP needs to heterodimerize with other transcription factors (17). To this end, to identify CHOP-interacting partners that may play a role in mitochondrial stress responses, we immunoprecipitated CHOP from DARS2 KO heart extracts, followed by mass spectrometry (table S4). Notably, besides CHOP, only six proteins were identified. Among those, the most enriched protein and the only transcription factor was C/EBP (table S4). These results were confirmed by Western blot analysis following coimmunoprecipitation (co-IP) against CHOP (Fig. 6B). Notably, CHOP and C/EBP appear to interact only upon mitochondrial dysfunction (i.e., DARS2 KO), despite similar levels of C/EBP in WT and DARS2 KO hearts (Fig. 6B and fig. S6A). The mass spectrometry analysis of C/EBP immunoprecipitates corroborated these results (table S5). In DKO hearts, C/EBP instead interacted with ATF4 and ATF3 (table S5). Previously, the induction of Atf3 was detected in the terminal stages mitochondrial stress responses along with UPRmt (12).

Further interplay of the three proteins is illustrated by the fact that mitochondrial dysfunction in C/EBP-deficient cells exacerbated the ISR stress and led to ATF4 activation similar to CHOP KO (fig. S6B). Interaction of CHOP with C/EBP was previously proposed in the context of mitochondrial dysfunction, wherein CHOP/C/EBP dimers are thought to bind and activate the promoters of UPRmt-responsive genes (5). Consistent with these results, we propose that C/EBP-CHOP heterodimers might act as suppressors of ATF4 overactivation upon mitochondrial dysfunction.

C/EBP is primarily regulated at the translational level and exists in three different isoforms, two activating [Liver-enriched activator protein (LAP1) and LAP2], and one inhibitory [Liver-enriched inhibitor protein (LIP)] (39). The C/EBP target genes are presumably positively regulated by LAP1/2 proteins, whereas LIP binding is thought to repress the transcription of respective promoter (39), although recently more complex functions have been proposed for C/EBP LIP in vivo (40). To further dissect the interplay between CHOP and C/EBP, we assessed the levels of all three C/EBP isoforms in different models of mitochondrial dysfunction. COX10 KO cells with strong chronic mitochondrial dysfunction presented an increase of all C/EBP isoforms (fig. S6C). Acute mitochondrial dysfunction caused by actinonin treatment in MEFs or DARS2 deficiency in heart had a milder effect on the levels of LAP isoforms (Fig. 6C and fig. S6D). Still, C/EBP LIP levels were strongly increased by actinonin treatment in WT cells (Fig. 6C). Notably, this effect was strongly blunted in CHOP-deficient cells and DKOL mice, indicating that an increase in C/EBP LIP levels is dependent on the CHOP presence (Fig. 6C and fig. S6D). In general, the CHOP presence seems to have a positive effect on the C/EBP levels in MEFs, indicating a regulation opposite to that of ATF4.

Under ER stress, CHOP and C/EBP LIP are shown to act in concert to exert their respective functions in the nucleus (41). According to the proposed model, CHOP depends on the interaction with C/EBP LIP to enter the nucleus, while the interaction with CHOP is thought to mask the nuclear export signal (NES) of C/EBP LIP, thereby reducing its exclusion from the nucleus and subsequent proteasomal degradation (41). To test whether C/EBP LIP plays a role in the direct regulation of the mitochondrial dysfunctioninduced ISR, we expressed mutant LIPL120T, carrying a leucine-to-threonine substitution predicted to disrupt NES (42), in CHOP KO cells treated with actinonin (Fig. 6D). The expression of LIPL120T in CHOP KO cells resulted in intense ablation of basal and actinonin-induced ATF4 mRNA and protein levels and a marked decrease in the mRNA and protein levels of its downstream targets, even in the absence of mitochondrial insult (Fig. 6D and fig. S6E). Moreover, expression of LIPL120T mutant resulted in decreased expression of the endogenous C/ebp gene (fig. S6E). Intriguingly, moderate overexpression of WT C/EBP LIP in CHOP KO cells resulted in a mild further increase of ATF4 levels upon mitochondrial dysfunction (Fig. 6E). In contrast, C/EBP LIPL120T mutant suppresses ATF4 while also decreasing endogenous C/EBP levels (Fig. 6E). These results also suggest that mutant C/EBP LIPL120T does not require CHOP for its action.

It has been shown that ER stress leads to interdependent translocation and retention of C/EBP and CHOP inside the nucleus (41). Therefore, we next investigated the effects of mitochondrial stress on subcellular localization of C/EBP, CHOP, and ATF4. In both WT and CHOP KO cells, C/EBP and ATF4 were detected mainly in the nucleus (fig. S6F). The expression of either WT or mutant C/EBP LIP did not affect the subcellular localization of ATF4 in CHOP KO cells (Fig. 7A). Therefore, the ATF4 levels in CHOP-deficient cells appear not to be regulated through alterations in subcellular localization of LIP. Alternatively, in the absence of CHOP, C/EBP LIPL120T may bind ATF4 and prevent its translocation to the nucleus, thus promoting its degradation. To test this hypothesis, we incubated WT and CHOP KO cells in the presence or absence of the proteasome inhibitor MG132. In control conditions, both ATF4 and C/EBP were rapidly degraded, and only a modest fraction was retained and transported to the nucleus (Fig. 7B and fig. S6G). The rate of turnover, however, appeared not to be affected by mitochondrial function or CHOP deficiency (Fig. 7B and fig. S6G). In turn, mitochondrial dysfunction, induced by actinonin treatment, induced translocation of ATF4 to the nucleus and promoted activation of ISR. Of note, the expression of LIPL120T mutant resulted in lower levels of ATF4 in all fractions (Fig. 7B and fig. S6G). Overall, these results suggest that fine-tuning of mitochondrial stress responses is dependent on CHOP:C/EBP LIP interaction but not their subcellular localization nor their potential effects on the nuclear translocation of ATF4.

(A) Cell fractionation followed by the Western blot analysis of the ATF4 and three C/EBP isoforms in actinonin-treated (48 hours) WT or CHOP KO MEFs expressing WT C/EBP LIP or C/EBP LIPL120T mutant. (B) Cell fractionation followed by the Western blot analysis of the ATF4 and three C/EBP isoforms in actinonin-treated (48 hours) CHOP KO MEFs expressing WT C/EBP LIP and C/EBP LIPL120T mutant along with the WT cells and respective controls. The MG132 (15 M) was applied in the last 6 hours of the actinonin treatment. Elevated protein ubiquitination reflects proteasome inhibition. (A and B) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and H3K4me3 were used as loading controls and to determine quality of fractionation (n = 3). (C) CHOP levels increase early upon mitochondrial dysfunction leading to its association with C/EBP. The interaction with C/EBP likely promotes translocation of CHOP to the nucleus where it negatively regulates Atf4 levels and transcription of downstream ISR targets. Abrogation of CHOP results in increased ATF4:C/EBP association and transcription of ISR-regulated genes, created with BioRender.com.

Understanding of the mitochondrial stress response in mammals remains incomplete. In the present study, we uncovered an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBP, and ATF4. Contrary to its previously proposed role as a transcriptional activator of UPRmt, we present strong evidence that CHOP, through its interaction with C/EBP, attenuates prolonged ISR and mitochondrial cardiomyopathy through regulation of ATF4 levels (Fig. 7). Our results argue that upon mitochondrial dysfunction, the interaction of CHOP with C/EBP is needed for the adjustment of an ATF4-regulated transcriptional program. Very early upon DARS2 depletion, Chop is increasingly expressed (2) and forms a complex with C/EBP, which might facilitate the translocation of CHOP:C/EBP heterodimers to the nucleus. Regulation of ATF4 levels by C/EBP isoform LIP inhibition was proposed during ultraviolet (UV) stress, but CHOP was shown not to play a role in this context (43).

Similar to CHOP, C/EBP is a pleiotropic transcription factor that contributes to the regulation of homeostasis in several tissues, including bone, skin, and fat (40). We showed that in the context of mitochondrial dysfunction, the C/EBP accumulates in the cell (in particular, LIP isoform) and dimerizes with CHOP to presumably prevent overactivation of an ATF4-mediated response. In the absence of CHOP, C/EBP dimerizes with ATF4, which correlates with further induction of ISR. Our data suggest that C/EBP also dimerizes with ATF3 when CHOP is absent in DKO animals. ATF3 is shown to be activated during the second stage of ISR (12, 44). Once expressed, ATF3 binds promoters of ISR-responsive genes, leading to a subsequent suppression of transcription back toward the basal level (44). It is possible that also in the DKO animals, ATF3:C/EBP interaction is part of the feedback loop intended to suppress the ATF4 overactivation. In contrast, the interaction of ATF4 with C/EBP positively activates targeted genes under different conditions (45), which might have a deleterious outcome leading to, e.g., skeletal muscle atrophy (46). In contrast, we show that a dominant-negative C/EBP LIPL120T fully suppresses Atf4 and C/ebp overexpression upon mitochondrial dysfunction and down-regulates even basal levels of these transcription factors. Our findings thus suggest that C/EBP acts as a promiscuous transcription factor in the context of mitochondrial dysfunction, whereby differential transcriptional activity and associated functional outcomes are determined via interactions with CHOP and ATF4 (Fig. 7C). Further work is however required to dissect precise mechanisms of the observed interplay between CHOP, ATF4, and C/EBP.

CHOP is a transcription factor that is ubiquitously expressed at very low levels but quickly activated by a variety of insults such as ER stress, amino acid deprivation, glucose starvation, and UV irradiation (47). To date, CHOP was mostly studied in the context of ER stress, where it was proposed to regulate many pro- and anti-apoptotic genes in the late phase of ISR (47, 48). While numerous functions related to cell proliferation, differentiation, and development have been described for this transcription factor, in unstressed conditions, CHOP-deficient mice do not present any conspicuous phenotype (48, 49). Nevertheless, these mice seem to be protected from transient renal insufficiency caused by acute tubular necrosis (49). CHOP depletion seems to be beneficial in various other conditions, e.g., by delaying the onset of metabolic disease in several diabetic models (50), protecting livers from diet-induced hepatosteatosis (51), or delaying the onset of brain ischemia-induced neuronal cell death (52). Collectively, these studies suggest that loss of CHOP often leads to beneficial effects by delaying apoptosis in vivo. Unexpectedly, in mitochondrial mutants, CHOP depletion does not seem to decrease levels of proteins involved in the activation of apoptosis, as even the proposed bona fide CHOP targets BH3 interacting-domain death agonist (BID), Bcl-2-like protein 11 (BIM), ERO1A, and Tribbles homolog 3 (TRIB3) further increase their levels in DKO mutants.

We also provide evidence that CHOP loss is detrimental in mitochondrial mutants as it leads to early-onset fatal mitochondrial cardiomyopathy. This is, at least in part, mediated by the overactivation of ISR that is paralleled by inhibition of global protein synthesis and appears to be beneficial for a short time as DKOE animals maintain higher levels of OXPHOS complexes and balanced mitochondrial translation. However, loss of CHOP mitigates sustained suppression of protein synthesis in vivo that results in rapid loss of OXPHOS complexes and mitochondrial respiration. This is likely to affect mitochondrial import capacity leading to vicious cycle of damaging events. Simultaneously, mRNA translation rates are restored in DKOL around P17, coinciding with a detrimental phenotype. This is partly reminiscent of a transition from the acute to prolonged ISR in the cellular model of ER stress (37). During the acute ISR phase, global translation is reduced, and only a subset of stress-responsive mRNAs are translated, whereas the prolonged ISR is characterized by recovery of global translation while still allowing execution of acute ISR translational programs (37). While the prolonged ISR appears to have a beneficial effect in vitro by preventing cell death under conditions of ER stress (37), we show that in vivo, mitochondrial dysfunction in the heart impedes a sustained chronic ISR program. To this end, recovery of protein synthesis escalates ER stress possibly by increasing ER load. Recovery of global translation is also expected to significantly increase the energy demand and thereby result in energy depletion caused by massively reduced respiratory capacity due to DARS2 loss. According to the energy starvation hypothesis, suboptimal ATP supply predisposes for the contractile dysfunction observed during heart failure (53). It was shown that even very few cardiomyocytes with severe mitochondrial dysfunction are sufficient to promote ventricular arrhythmias, which lead to heart failure (54). Considering the severe impairment of electron transport chain (ETC) function in DKO mice, the occurrence of cardiac arrhythmias in those animals, contributing to the pathology, seems likely.

The pathology observed in DKOL animals is not a DARS2-specific phenomenon but a prevalent cardiac phenotype in mutants affecting mitochondrial gene expression and translation, as shown by a comparative study of five different models (11). At the molecular level, we demonstrated markedly increased serine synthesis and remodeling of the one-carbon cycle in hearts of DARS2 KO, DKOL mice, and cell culture models, attributable to OXPHOS deficiency and not to the loss of DARS2 in particular. Moreover, similar changes are described in other models and different tissues (11, 13, 14, 55). The vast majority of these alterations have been attributed to ATF4, which has been identified as a major regulator of amino acid metabolism feeding into the folate cycle during ISR induced by different stress signals including mitochondrial dysfunction (13, 14, 56). Although ATF4 may be activated by several different pathways, such as nuclear respiratory factor 2 (NRF2) stabilization or mechanistic (previously mammalian) target of rapamycin (mTOR) signaling (57, 58), we showed that ATF4 up-regulation caused by mitochondrial OXPHOS deficiency could be successfully prevented by suppression of the ISR.

In conclusion, we found a regulatory mechanism that fine-tunes the activation of the ISR upon mitochondrial dysfunction. We showed that CHOP is needed to prevent excessive activation of the ATF4-mediated stress response that results in cardiotoxic effects. This is mediated by CHOP interaction with C/EBP, which likely promotes CHOP:C/EBP heterodimer translocation to the nucleus. Our results also highlight an unforeseen opportunity of exploring a therapeutic intervention targeting ATF4 activity in various mitochondrial diseases.

DARS2 KO (Dars2fl/fl; Ckmm-Cre+/tg) mice were generated as previously described (2). WT control animals (Dars2fl/fl; Ckmm-Cre+/+ and Dars2+/fl; Ckmm-Cre+/+) were also obtained from this breeding. CHOP KO [B6.129S(Cg)-Ddit3tm2.1Dron/J] mice were obtained from the Jackson laboratory. Those mice are characterized by a Chop::LacZ KO allele, resulting in the whole-body KO of Chop (Chop/) (49).

Conditional Dars2-floxed mice (Dars2fl/fl) were crossed to CHOP KO mice (Chop/) to obtain CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/). Triple transgenic mice were generated by intercrossing of CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/), with transgenic mice harboring one copy of the Cre recombinase under control of the striated muscle creatine kinase (Ckmm) promoter (Ckmm-Cre+/tg). Resulting heterozygous triple transgenic mice (Dars2+/fl; Ckmm-Cre+/tg; Chop+/) and CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/) were used to lastly generate CHOP KO (Dars2+/fl; Ckmm-Cre+/+; Chop/ and Dars2fl/fl; Ckmm-Cre+/+; Chop/) and DKO (Dars2fl/fl; Ckmm-Cre+/tg; Chop/) mice. Genotyping for the Dars2 allele was performed as previously described (2). Genotyping for the Ckmm-Cre and Chop alleles was performed following the instructions of the Jackson laboratory using the protocol 22415 along with the primers oIMR3884, oIMR3885, and oIMR3886 for the Chop allele and the protocol Tg(Ckmm-Cre)5Khn along with the primers oIMR1085, oIMR6754, oIMR8744, and oIMR8745 for the Ckmm-Cre allele, respectively (www.jax.org). One- to 6-week-old animals were used in experiments approved and authorized by the Animal Ethics Committee of North-Rhein Westphalia (Landesamt fr Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen) following the German and European Union regulations. Animal work was performed in conformity with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations.

Immortalized MEFs and fibroblasts were cultured in standard conditions, at 37C and 5% CO2. The cell culture medium was composed of Dulbeccos modified Eagles medium [glucose (4.5 g/liter), GlutaMAX, and sodium pyruvate; Gibco Life Technologies] supplemented with 10% Fetal Bovine Serum Premium, South American Origin (Biowest) and penicillin-streptomycin (Pen-Strep) (Gibco Life Technologies). In conditions of mitochondrial dysfunction (induced either genetically or by treatment), the medium was additionally supplemented with uridine (50 g/ml). At 90% confluency, cells were split cell typedependently in ratios ranging from 1:4 to 1:20.

Generation of immortalized MEF lines. Embryos from embryonic day 13.5 of intercrossed CHOP KO (Chop/) mice were used to isolate primary MEFs (59). Immortalization was achieved by transformation with the SV40 T antigen.

Drug treatments. For induction of mitochondrial dysfunction by actinonin treatment, 80% confluent cells were treated for 48 hours with 100 M actinonin (Sigma-Aldrich). Proteasome was inhibited with 15 M MG132 for the last 6 to 8 hours of treatment as indicated. Inhibition of the ISR was achieved by 4- or 48-hour 1 M ISRIB (Sigma-Aldrich) treatments of 90% confluent cells. All compounds were solubilized in dimethyl sulfoxide (DMSO). Untreated cells were supplemented with corresponding amounts of the solvent. Treatments were renewed on a daily basis.

Transfection. Transfection of plasmids conferring hygromycin resistance (pTK-Hyg LIP, pTK-Hyg LIPwestern, pTK-Hyg LAP, and pTK-Hyg C/EBP) was performed with Lipofectamine 2000 or Lipofectamine LTX (Invitrogen) according to the manufacturers instructions using the forward transfection procedure. Seventy-two hours after transfection, the culture medium was replaced by hygromycin-supplemented (100 g/ml) medium for negative selection of untransfected cells. Transfected cells were maintained in hygromycin-supplemented (100 g/ml) medium.

Cell growth estimation. To estimate differences in cell growth caused by CHOP deficiency and/or mitochondrial dysfunction, an equal number of cells were seeded and treated as indicated. The numbers of cells were determined at the indicated time points using the Countess Automatic Cell Counter (Invitrogen) combined with trypan blue staining.

Freshly collected hearts were immediately transferred into 10 ml of prechilled mito-isolation buffer (MIB) [100 mM sucrose, 50 mM KCl, 1 mM EDTA, 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and 0.2% bovine serum albumin (BSA) free from fatty acids (pH adjusted to 7.2)] supplemented with 1 g of subtilisin (Sigma-Aldrich) per mg of tissue. Approximately 20 long strokes of a Potter S (Sartorius) homogenizer at 1000 rpm were required for homogenization. After centrifugation (800g, 5 min, 4C), the mitochondria-containing supernatant was transferred into a fresh tube. Pelleted mitochondria (8500g, 5 min, 4C) were resuspended in 30 ml of MIB and subjected to a third centrifugation step (700g, 5 min, 4C). Last, mitochondria were pelleted (8500g, 5 min, 4C) and resuspended in 100 l of macrophage inflammatory protein without BSA. Protein concentration of mitochondria was determined using Bradford reagent (Sigma-Aldrich) according to the manufacturers instructions. Mitochondria were either immediately used (respirometry or in organello translation) or snap-frozen and stored at 80C.

High-resolution respirometry using an Oxygraph-2k (OROBOROS Instruments) and a carbohydrate substrate-uncoupler-inhibitor titration protocol was conducted to determine mitochondrial oxygen consumption rates. First, the respiration medium (120 mM sucrose, 50 mM KCl, 20 mM tris-HCl, 1 mM EGTA, 4 mM KH2PO4, 2 mM MgCl2, and 0.1% BSA) was added to the Oxygraph chamber, and air equilibration was performed. Next, 25 g of freshly isolated cardiac mitochondria was added. The respiration medium was supplemented with 2 mM pyruvate, 0.8 mM malate, 2 mM glutamate, and 2 mM adenosine 5-diphosphate (ADP) to assess CI-dependent respiration. By providing additional 4 mM succinate, convergent CI- and CII-dependent respiration was determined. Inhibition of ATP-synthase-complex V (CV) by addition of oligomycin (1.5 g/ml) allowed evaluating the coupling efficiency. The maximal capacity of the electron transfer system (ETS) was assessed by titration of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (0.5 M increments). Maximal capacity of the ETS of CII solely could be determined by inhibition of CI through addition of 0.5 M rotenone. Last, inhibition of CIII by supplementation of 2.5 M antimycin A allowed the determination of the residual oxygen consumption.

De novo mitochondrial translation was assessed by incubation (1 hour, 37C, on rotating wheel) of 1.5 mg of freshly isolated mitochondria in 1 ml of 35S-translation buffer [100 mM mannitol, 10 mM Na-succinate, 80 mM KCl, 5 mM MgCl2, 1 mM KH2PO4, 25 mM Hepes (pH 7.4), 5 mM ATP, 200 M GTP, 6 mM creatine phosphate, creatine kinase (60 g/ml), cysteine (60 g/ml), tyrosine (60 g/ml), amino acids (60 g/ml) (Ala, Arg, Asp, Asn, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, and Val), 35S-methionine (7 l/ml)]. Subsequently, mitochondria were pelleted (12,000g, 2 min) and resuspended in 1 ml of nonradioactive translation buffer containing methionine instead of 35S-methionine. Half of the sample (pulse fraction) was pelleted again, resuspended in 100 l of SDSpolyacrylamide gel electrophoresis (PAGE) loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% -mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue], and lysed (30 min, room temperature) before transfer at 20C. For the cold chase allowing to estimate the protein turnover, the remaining 500 l of resuspended mitochondria was incubated for 3 hours at 37C on a rotating wheel. Subsequently, the chase fraction was pelleted, resuspended in 100 l of SDS-PAGE loading buffer, and lysed as the pulse sample before.

Separation of mitochondrial proteins was achieved by SDS-PAGE. Ten microliters per sample was loaded on a 15-cm-long, 15% polyacrylamide gel and run in a SE600X Chroma Deluxe Dual Cooled Vertical Protein Electrophoresis Unit (Hoefer) overnight at 80 V continuously. After fixing (50% methanol and 10% acetic acid) for 30 min, staining in Coomassie solution, and destaining (20% methanol and 10% acetic acid) of the polyacrylamide gel, the latter one was placed on Whatman paper (GE Healthcare) and dried (2 hours, 80C) in a gel dryer. For detection of radioactive signals of de novo synthetized proteins, Amersham Hyperfilm MP (GE Healthcare) was exposed to the dried polyacrylamide gel.

Cellular protein lysates. Washed cell pellets were resuspended in cold radioimmunoprecipitation assay buffer [150 mM NaCl, 1% Triton X-100 (v/v), 0.5% Na-deoxycholate (w/v), 0.1% SDS (w/v), 50 mM tris-HCl (pH 7.4), 50 mM NaF, and 2 mM EDTA] supplemented with 1 protease inhibitor cocktail (Sigma-Aldrich) and 1 PhosSTOP phosphatase inhibitor cocktail (Roche). Next, cells were incubated 30 min on ice with brief vortexing every 10 min. Following 2 45-s sonication, the lysates were cleared (10 min, 20,000g, 4C) and transferred into fresh tubes.

Cardiac tissue protein lysates. Homogenization of 25 mg of cardiac tissue samples in 400 l of cold organ lysis buffer [50 mM Hepes (pH 7.4), 50 mM NaCl, 1% Triton X-100 (v/v), 0.1 M NaF, 10 mM EDTA, 0.1% SDS (w/v), 10 mM Na-orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 1 protease inhibitor cocktail (Sigma-Aldrich), and 1 PhosSTOP phosphatase inhibitor cocktail (Roche)] was performed with the Precellys CK 14 (Bertin Technologies) (5000 rpm, 30 s). Cleared protein lysates (45 min, 20,000g, 4C) were transferred into fresh tubes. Determination of protein concentration was performed with Bradford reagent (Sigma-Aldrich) according to the manufacturers instructions. Protein lysates were stored at 80C.

SDSpolyacrylamide gel electrophoresis. Protein samples were dissolved in SDS-PAGE loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% -mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue] before denaturation. Depending on the required range of protein sizes, the proteins were separated on 8 to 15% acrylamide gels [stacking gel: 5% acrylamide-bisacrylamide (37.5:1), 12.5 mM tris-HCl, 0.1% SDS (w/v), 0.25% Ammonium persulfate (APS), and 0.25% Tetramethylethylenediamine (TEMED) (pH 6.8); separating gel: 8 to 15% acrylamide-bisacrylamide (37.5:1), 37.5 mM tris-HCl, 0.1% SDS (w/v), 0.1% APS, and 0.1% TEMED (pH 8.8)] in running buffer [25 mM tris-HCl, 250 mM glycine, and 0.1% SDS (w/v) (pH 8.3)].

Western blot. Transfer of proteins on a nitrocellulose membrane by Western blot was conducted in transfer buffer (30 mM tris-HCl, 240 mM glycine, 0.037% SDS, and 20% methanol) at 400 mA for 2 hours at 4C. For a first evaluation of the transfer, shortly washed membranes (dH2O) were stained with Ponceau S solution (Sigma-Aldrich). Depending on the antibody requirements, destaining and blocking of membranes were performed for 1 hour either in 5% milk-PBST (Phosphate-Buffered Saline/Tween) or 3% BSA-TBST (Tris-Buffered Saline/Tween) on a gently shaking platform before subsequent immunodecoration with the indicated antibodies according to the manufacturers instructions. Secondary horseradish peroxidasecoupled antibodies (1:5000) were incubated for 1 hour before detection by Pierce ECL Western blotting substrate (Thermo Fisher Scientific). Densitometry-based quantification of Western blots was performed with ImageJ and Image Studio Lite Software.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed on the basis of the NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to the manufacturers instructions. For analysis of mitochondrial supercomplexes, 10 g of mitochondria was lysed with 4% of digitonin. Analysis of individual mitochondrial complexes was conducted after lysis of 10 g of mitochondria in 1% n-dodecyl--D-maltoside (DDM). After completion of lysis (15 min on ice), lysates were cleared (30 min, 20,000g, 4C), and the resulting supernatant was loaded on a 4 to 16% bis-tris gradient gel. Subsequently, proteins were transferred to an Amersham Hybond polyvinylidene difluoride membrane (GE Healthcare) by Western blot and subsequently immunodecorated with indicated antibodies.

Independently normalized label-free proteomics and RNA sequencing data were scaled before analysis using the anota2seq algorithm (version 1.4.2) (19). Furthermore, datasets were reduced to genes identified on both platforms resulting in a total of 2556 mRNAs for analysis. Analysis of changes in protein levels and total mRNA was performed using the anota2seqAnalyze function to identify differences between CHOP KO, DARS2 KO, and DKO compared to WT. Changes were considered significant when passing the following parameters within the anota2seqSelSigGenes function: maxPAdj = 0.15, minSlopeTranslation = 1, maxSlopeTranslation = 2, selDeltaPT = log2(1.2), selDeltaP = 0, and selDeltaT = 0. Changes in translation or protein stability, as well as changes in mRNA abundance, were characterized using the anota2seqRegModes() function. GO analysis (60) was performed in Cytoscape (v 3.8.0) (23) using the ClueGO (v 2.5.7) app (20). Within ClueGO, four gene lists were provided corresponding to the identified modes for regulation of gene expression using anota2seq (i.e., translation/protein stability and mRNA abundance) divided into up- and down-regulated mRNAs. GO term inclusion parameter was set to a 5 gene overlap and <4% of total genes present in the GO term. For the resulting network, GO term grouping and fusion parameters were enabled, and only GO terms with a false discovery rate of <5% were displayed. Furthermore, anota2seq was applied on the full RNA sequencing dataset (14,174 protein coding transcripts) following the same approach as above. Master regulators among significantly up-regulated total mRNAs in the DARS2 KO versus WT comparison were detected using iRegulon (v1.3) with default settings (24).

The Q5 Site-Directed Mutagenesis Kit (New England Biolabs) was used to introduce a point mutation (L120T) in the pTK-Hyg LIP plasmid (41). For primer design, the New England Biolabs (NEB) online design software NEBaseChanger was used. All three steps described in the protocol [exponential amplification, Kinase, Ligase & DpnI treatment (KLD) reaction, and transformation] were performed as indicated in the manual.

Protein synthesis was determined using the nonradioactive technique called surface sensing of translation described in (61). This assay is based on the incorporation of the structural analogue of tyrosyl-tRNA puromycin in nascent polypeptide chains and subsequent detection of puromycylated proteins using an anti-puromycinspecific antibody.

Briefly, mice were injected at the indicated time points intraperitoneally with 0.04 mol of puromycin dissolved in phosphate-buffered saline (PBS) per gram of body weight. Thirty minutes after injection, the animals were euthanized, and collected tissues were snap-frozen in liquid nitrogen. Subsequently, protein lysates of the collected tissues were prepared and processed by SDS-PAGE and Western blot. The relative signal intensity of the anti-puromycinspecific antibody is proportional to the relative protein synthesis rates at the time point of puromycin injection.

Briefly, mice were injected intraperitoneally with 5 g of ISRIB (stock solution: 5 mg/ml in DMSO, dissolved in PBS up to the weight-dependent injection volume of 30 to 50 l) per gram of body weight or the corresponding amount of PBS-dissolved solvent (DMSO) on a daily basis for the indicated time periods. One day after the last injection, the animals were euthanized, and collected tissues were snap-frozen in liquid nitrogen. Subsequently, protein lysates of the collected tissues were prepared and processed by SDS-PAGE and Western blot.

Numerical data are expressed as means SD. Statistical analysis was performed using the indicated statistical tests. If not indicated differently, statistical significance was considered for P < 0.05. With exception of multivariate analysis of variance (MANOVA) and omics analyses, all statistical tests were performed, and graphs were plotted using GraphPad Prism 8.0 software. MANOVA was performed with XLSTAT version 2020.3 software.

Acknowledgments: We wish to thank the CECAD Imaging and Proteomics Core Facilities for excellent support. Funding: The work was supported by Aleksandra Trifunovics grants of the Deutsche Forschungsgemeinschaft [DFG; German Research Foundation (SFB 1218)Projektnummer 269925409 and TR 1018/8-1] and the Center for Molecular Medicine Cologne, University of Cologne. S.K. received scholarship from the Cologne Graduate School of Ageing Research (CGA). I.T. acknowledges Senior Scholar Award from Le Fonds de recherche du QubecSant (FRQS) and support from Canadian Institutes for Health Research (MOP-363027) and Joint Canada-Israel Health Research Program (JCIHRP) (108589-001) to I.T. and O.L. O.L.s lab was supported by grants from the Swedish Research Council (2016-02891), the Swedish Cancer Society (19 0314), and the Wallenberg Academy Fellows program (2013.0181). M.H.s laboratory is supported by NIH R01 DK060596 grant. Author contributions: Conceptualization: A.T., S.K., C.O., K.Sz., O.L., I.T., and M.H. Data curation: S.K., C.O., A.T., S.B., O.L., and K.Sz. Formal analysis: S.K., C.O., A.T., S.B., O.L., and K.Sz. Funding acquisition: A.T., S.K., O.L., I.T., and M.H. Investigation: S.K., C.O., A.K., K.Se., K.Sz., C.L., S.B., and O.L. Visualization: S.K., C.O., A.T., O.L., I.T., and M.H. Writing: A.T., S.K., C.O., O.L., and I.T. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Further information and requests for resources and reagents should be addressed to and will be fulfilled by A.T. Mouse and cell lines requests include signing of material transfer agreement.

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Clinical Application of Cytokines in Cancer Immunotherapy | DDDT – Dove Medical Press

By daniellenierenberg

Introduction

Cancer is a disease characterized by the abnormalities in the regulation of cell proliferation and differentiation. Many factors contribute to cancer development including genetics,1 lifestyle, and environmental carcinogens, among others.2 Cancer is the second leading cause of death worldwide after cardiovascular disease, accounting for 9.6 million deaths in 2018 according to data from the International Agency for Research on Cancer. Lung cancer is the leading cause of cancer death (18.4%), followed by breast cancer (11.6%), and prostate cancer (7.1%).3 Clinical manifestations include pain, bleeding, lumps and ulcers at the site of disease, along with systemic symptoms such as weight loss and fatigue leading to cachexia. Traditional treatment modalities including surgery, radiotherapy, and chemotherapy have various disadvantages and cause side effects that are in some cases severe. Immunotherapies such as blockade of programmed death (PD)-1 and programmed death ligand 1 (PD-L1) immune checkpoints; chimeric antigen receptor T cell immunotherapy (CAR-T); using the monoclonal antibody against cancer antigen; and cytokine therapy offer a promising alternative to the conventional treatment approaches for cancer.4 In particular, cytokine therapy has shown encouraging results in both basic and clinical research settings.5

Cytokines are small proteins produced by various cells (immunocytes and non-immunocytes) as molecular messengers to communicate with each other or with other cells. Cytokines have versatile roles in several steps of the cancer immunity cycle including cancer antigen presentation, T cell priming and activation, effector T cell infiltration in cancer site, and cancer cell death, as shown in Figure 1. More importantly, cytokine-mediated signaling pathways control the direction of nave CD4+ T cell differentiation and thus determine the effects of anticancer immunity (Figure 2 and Table 1). Briefly, transforming growth factor (TGF-) signaling in nave CD4+ T cells is required for the differentiation of regulatory T cells (Tregs) and T helper type 17 (Th17) cells, both of which promote tumor progression. Additionally, Th17 cell differentiation and clonal expansion require a cocktail of cytokines (IL-6, IL-21, IL-23, IL-1, and TGF-).610 IL-17 secreted by Th17 cells guides macrophages and neutrophils to cancer sites and induces cancer-promoting inflammation. Th17 cells themselves also exert antitumor effects in the melanoma microenvironment by potentiating the functions of CD8+ T cells and T helper type 1 cells (Th1 cells).11 IL-10, IL-11, IL-4, and IL-13 are critical for the differentiation and development of T helper type 2 cells (Th2 cells),1217 whereas IL-12, IL-18, IL-1, and interferon (IFN)- promote Th1 cell development and activity.1823 Th1 cells modulate tumor-suppressing pathways by stimulating IFN- secretion and enhancing the cytotoxicity of natural killer (NK) cells and CD8+ T cells, while Th2 cells inhibit the anticancer immune responses by blocking Th1 cell differentiation and the release of IFN-. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)- promote cancer progression by directly facilitating angiogenesis, although recombinant TNF- has been shown to enhance the effect of combined chemotherapy regimens by increasing the permeability of tumor blood vessels.2427

Figure 1 Cytokines in the cancer immunity cycle.6 1) Antigens from dead cancer cells are captured by APCs, mainly by DCs. 23) DCs present cancer antigens to T cells to prime the adaptive immune response. 45) Activated effector T cells infiltrate cancer cells and then 6) kill cancer cells. Dead cancer cells release cancer antigens to continue the immune cycle. Cytokines that have been shown to promote or inhibit the anticancer immune responses are highlighted.

Abbreviations: IFN, interferon; IL, interleukin; TGF-, transforming growth factor ; TNF, tumor necrosis factor.

Figure 2 Varied roles of cytokines involved in anticancer immunity. Different cytokines determine nave CD4+ T cell fate to Tregs, Th17, Th1 or Th2, and further regulate anticancer immunity. IL-12, IL-18, IL-1, IL-10 and IL-11 secreted by dendritic cells (DCs) drive Th1 or Th2 cell differentiation. TGF-, IL-11, IL-6, and IL-21 are important signals for Treg and Th17 cell differentiation. IFN-, IL-2, IL-15, and IL-7 secreted by Th1 cells enhance the anticancer effects of cytotoxic T lymphocytes, NK cells, B cells, and macrophages, which can be suppressed by IL-4, IL-13, and IL-10 secreted by Th2 and Treg cells. IL-17 secreted by Th17 cells play a role in the induction of cancer-promoting anticancer inflammation by MDSCs. VEGF and TNF- promote cancer progression by facilitating angiogenesis. Cytokines functions are shown in text boxes; those that promote anticancer immunity are in red while those that inhibit anticancer immunity are in black.

Because the roles of cytokines are diverse and precise applications of cytokines are greatly needed, it is urgent to update the progresses of cancer immunotherapy with cytokines. Here, we review total 2339 clinical trials using or by targeting cytokines for precise treatment of cancers registered with ClinicalTrials.gov; summarize the therapeutic efficacy of typical cytokines based on clinical data; and highlight progress in the development and application of nanomaterials for cytokine-based therapy.

In order to review clinical application of cytokines in cancer therapy, we have searched all the known cytokines in ClinicalTrials.gov. In the advanced search page of ClinicalTrials.gov, we entered cytokine name, such as IL-2, in other term section and chose completed in the recruitment status section. Then, we download the search results and screen the trials item by item to make sure that the intervention of the trial includes cytokine-based drugs and the condition of the trial is cancer. As a result, we got 25 cytokines with clinical trials that had completed recruitment in ClinicalTrials.gov, and we also checked 2 cytokines (IL-10 and IL-17) without published clinical studies for cancers because of their crucial roles in anticancer immunity. Finally, we screen out 2630 clinical trials using cytokines as either therapeutic agents or targets in treating cancers registered with ClinicalTrials.gov that had completed recruitment up to January 2021.

It is interesting that G-CSF, GM-CSF, VEGF, IL-2 and IFN- are the five most studied cytokines (Figure 3A and Supplementary Table 1), which could be explained by the fact that they have been discovered and clinically studied very early (Figure 4A and B) and they play very important roles in cancer treatment. VEGF is the most studied target for the treatment of most types of cancer because the role of VEGF in angiogenesis induction, cell proliferation and promoting vascular permeability is extremely important for cancer growth, migration and infiltration. CSF can promote proliferation and differentiation of multiple immune cells such as macrophage, granulocytes, and mononuclear phagocytes, and thus is widely used as medication to stimulate the production of white blood cells following chemotherapy. Similarly, IL-2 is used to stimulate T cell production for enhancing anti-cancer immunity. IFN- can directly inhibit tumor cell proliferation and augment anti-tumor immunity by promoting MHC expression, antigen presentation, and the function of tumor-infiltrating Th1 cells, CTLs and macrophages. The clinical trials of cytokines cover nearly all cancer types (Figure 3B) but most of the clinical trials are done on melanoma and hematological malignancies because the two cancer types have better responses and outcomes than other cancers in the immune therapy.28,29

Figure 3 Clinical research status of cytokines. Number of cancer clinical trials using cytokine-based drugs treating all cancer types (A) or each cancer type (B) registered with ClinicalTrials.gov as of January 2021.

Figure 4 Historical timelines of cytokine research. (A) Timeline of cytokine discovery. The time point is the year in which the cytokines, EPO,69 IFNs,70,71 EGF,72 G-CSF,73,74 FGF,75 IL-1,76 IL-2,77 IGF,78 TNF,79 GM-CSF,80 TGF-,81 IL-3,82 IL-4,83 IL-6,84 IL-7,85 IL-10,86 IL-12,87 IL-13,88 VEGF,89 IL-11,90 IL-15,91 IL-17,92 IL-18,93 IL-21,94,95 and CCL21,96,97 were first described. (B) Timeline of the first clinical trials of cytokines for cancer treatment. The time point is the year that the trial was first registered with ClinicalTrials.gov. Clinical trial registry (NCT) numbers are shown.

The year of discovery of each cytokine and the year of the first clinical trial with the cytokine for cancer treatment are shown, respectively, in Figure 4A and B, which gives a visualized understanding of research progresses of cytokines in certain years. The main cytokines were discovered in the last 3 decades of last century and the clinical trials were carried out intensively between 1998 and 2008. The interval time from the discovery to the first clinical trial of certain cytokine is varied with the maximum of 95 years (EPO) and minimum of 7 years (IL-21). The cytokine-based drugs could be grouped into two types: cytokine drugs and drugs targeting cytokines.

IL-2, type I IFN, IL-12, chemokine (C-C motif) ligand (CCL) 21, and colony-stimulating factors (CSF) family cytokines are known to promote anticancer immunity. Although IFN-, TNF-, and IL-1 families play a dual role in the cancer immunity cycle, they are widely studied for their anticancer activity. In this section, we present the efficacy of these cytokine-based drugs in cancer treatment.

There are 268 trials registered with ClinicalTrials.gov using IL-2 for cancer treatment. Of the 52 trials for which results are available, 7 treated cancer with IL-2 alone, including 3 trials using IL-2 and 4 using IL-2 derivatives (hu14.18-IL12, denileukin diftitox [ONTAK], and ALT-801) for the treatment of melanoma, breast cancer, metastatic renal cell carcinoma (mRCC), and neuroblastoma. There were 45 trials investigating the effects of IL-2 combined with other therapies. In general, melanoma and leukemia responded better than other types of cancer to IL-2 treatment and IL-2 performed more outstanding when combined with other therapies in cancer treatment. The objectives of clinical studies using IL-2 in cancer treatment are summarized in Figure 5.

Figure 5 Application of IL-2 in 52 clinical trials for cancer therapy. IL-2 has been used in combination with lymphocytes, NK cells, genetically engineered cells, monoclonal antibodies, and tumor antigens as well as with radiotherapy, chemotherapy, and chemoradiotherapy. Dose finding, selected studies, and effects of recombinant IL-2 are shown. Numbers in parentheses are the number of clinical trials.

In 1992, high-dose aldesleukin became the first cytokine approved by the US Food and Drug Administration (FDA) for the treatment of mRCC based on an objective response rate (ORR) of 14% in 255 patients.30 In 2006, a new trial using high-dose aldesleukin for the treatment of mRCC was conducted by the Cytokine Working Group to evaluate the clinical utility of PD-L1, B7 homolog 3 protein, carbonic anhydrase 9, plasma VEGF, and fibronectin levels as biomarkers for therapeutic response monitoring. PD-L1 and B7 homolog 3 protein were identified as candidate markers but require independent validation.31 The IL-2 derivative hu14.18-IL-2, which consists of 2 molecules of IL-2 covalently linked via the Fc region, has demonstrated long-term tumor control in animal models.32 In Phase I and II trials, hu14.18-IL-2 prolonged the tumor-free survival period in some patients with recurrent stage III or stage IV melanoma following resection.33

The anticancer efficacy of IL-2 may be enhanced when it is used in combination with other immunotherapies and chemotherapy agents. In one trial, 6 of 11 patients with non-Hodgkin lymphoma treated with IL-2 plus rituximab achieved complete or at least partial remission (NCT00994643). A Phase III trial reported that IL-2 combined with other immunotherapeutic reagents, including dinutuximab and granulocyte/macrophage (GM)-CSF, enhanced the efficacy of isotretinoin in the treatment of neuroblastoma after stem cell transplantation; the 3-year event-free survival rates for isotretinoin with and without immunotherapy is 62.9% against 48.1%, respectively (NCT00026312). Results from 3 other trials supported the survival benefits of combination treatment (NCT01334515, NCT01592045, and NCT01041638). In addition to immunotherapy, data from 27 trials suggest that chemotherapy drugs such as ONTAK, etoposide, cyclophosphamide can increase the antitumor activity of IL-2.

Given the therapeutic effects of IL-2, other members of the IL-2 family including IL-7, IL-15, and IL-21, that are known to act independently or synergistically with IL-2 in the anticancer immune response have been investigated for the treatment of breast cancer, renal cell cancer, melanoma, and leukemia. However, in a trial of IL-7 in patients with metastatic castration-resistant prostate cancer (NCT01881867), the number of T cells per 300,000 peripheral blood mononuclear cell was not higher than in the comparator group. In trials investigating the efficacy of intravenous (NCT01385423 [phase I]) or subcutaneous (NCT02395822 [phase II]) recombinant human IL-15 in enhancing the effects of NK cell therapy in patients with acute myelogenous leukemia, 32% of patients in the phase I trial and 40% of those in the Phase II trial achieved complete remission.34 In a phase II trial evaluating the efficacy and safety of IL-21 in the treatment of malignant melanoma (NCT01152788), IL-21 did not demonstrate a clinical benefit over dacarbazine, with a progression-free survival (PFS) of 1.87 vs 2.04 years, although IL-21 was associated with fewer adverse events.

Type I IFNs including IFN- and IFN- play an essential role in the presentation of cancer antigens by mediating the maturation and activation of dendritic cells (DCs) and inducing the expression of major histocompatibility complex I molecules on tumor cells.35,36 Since 1996, there have been 248 trials investigating the therapeutic potential of IFN- in the treatment of cancers including melanoma and leukemia, with results for 76 available on ClinicalTrials.gov. Although there is in vitro evidence that IFN- more potently inhibits tumor cell proliferation than IFN-, there have been no clinical trials demonstrating its efficacy in cancer therapy.

A study conducted from 1988 to 2010 evaluating the efficacy of high-dose IFN--2b in 1150 patients who had undergone resection for stage II or III melanoma (NCT00003641) found no improvements in 5-year relapse-free survival and overall survival (OS) rates. In addition to treating melanoma, IFN- has been used as first-line treatment for mRCC, but was found to be less effective than the tyrosine kinase inhibitor su011248 in a phase III trial (NCT00083889). Various forms of IFN- including pegylated (PEG)-IFN- and recombinant adenovirus (rAd)-IFN (encoding IFN-2b) have been evaluated in clinical studies. Two trials compared the efficacy of PEG-IFN- and IFN- in different types of cancer; in patients with melanoma, the median OS was 25.63 months with PEG-IFN- vs 20.67 months with IFN- (NCT03552549), whereas in chronic myelogenous leukemia, the 12-month survival rate was slightly higher in the IFN- group than in the PEG-IFN- group (91.3% [158/173] vs 90.1% [154/171]) (NCT03547154). In both trials, more severe adverse effects were reported in patients receiving PEG-IFN- treatment. In another phase II study (NCT01687244), rAd-IFN showed promising results in patients with Bacillus Calmette-Gurin-refractory or relapsed bladder cancer.

The antitumor activity of IFN- can be dramatically enhanced by including other types of immunotherapy in the treatment regimen. In a phase III trial initiated in 2004 (NCT00738530), 649 patients with mRCC received IFN- alone or with bevacizumab; PFS was 5.5 and 10.2 months, respectively, and ORR was 12.5% and 32.4%, respectively. When the chemotherapy drug vinblastine was added to the regimen, the PFS was increased to 274 days (NCT00520403). Results from 5 other trials supported the effects of IFN- in combination with bevacizumab. A trial assessing the efficacy of pembrolizumab (anti-PD-1) plus sylatron (PEGIFN--2b) for the treatment of advanced cholangiocarcinoma was initiated in 2017, but no patients completed the study due to adverse effects (NCT02982720).

As the sole type II IFN, IFN- is a typical pro-inflammatory cytokine that exerts antitumor effects by suppressing proliferation and promoting apoptosis in tumor cells and inducing necrotic death and inhibiting angiogenesis in tumors. However, IFN- was shown to upregulate PD-L1 expression on tumor cells, which suppressed anticancer immunity through the binding of PD-L1 to its receptor PD-1 on lymphocytes.23 Despite these conflicting roles in cancer, the therapeutic potential of recombinant or adenovirus-delivered IFN- is being investigated in 22 trials, although only 2 have posted results. In a phase II trial (NCT00501644), 59 patients with ovarian or fallopian tube cancer or primary peritoneal cancer were treated with subcutaneous GM-CSF and IFN- before and after intravenous carboplatin; the ORR was 56% and median time to progression was 6 months. However, there was no control group in this trial. Another phase II trial assessed the efficacy of IFN- combined with 5-fluorouracil (FU), leucovorin, and bevacizumab in patients with metastatic colorectal cancer (CRC) (NCT00786643), but the specific contribution of IFN- to the treatment effect was not investigated. In summary, the efficacy of IFN- in cancer therapy has yet to be established.

IL-12, which is mainly produced by antigen-presenting cells, plays an important role in regulating innate and adaptive immune responses. There are 47 registered Phase IIII trials evaluating the efficacy and safety of intratumoral IL-12 administration either alone (22 trials) or with other immunotherapies (eg, DCs, T cells, and vaccines; 17 trials) for the treatment of melanoma, Merkel cell carcinoma, ovarian carcinoma, head and neck squamous cell carcinoma, and other cancers. In most cases a plasmid encoding IL-12 was used. A phase III trial that enrolled 51 patients with melanoma optimized the therapeutic strategy (NCT01502293): patients underwent 5 treatment cycles at 3-month intervals consisting of 3 intratumoral injections of IL-12 plasmid immediately followed by in vivo electroporation, which resulted in an ORR of 32.1% higher than the other two groups (underwent 9 cycles [25.0%] and 2 cycles [25.0%] at 6-week intervals, respectively). On the other hand, tumor-infiltrating CD8+T cells expressing IL-12 showed unsatisfactory results for the treatment of metastatic melanoma in a phase I/II trial (NCT01236573).

TNF was initially recognized as an antitumor cytokine. However, endogenous TNF induces the expression of multiple cytokines that act on M2 macrophages to stimulate the extracellular matrix remodeling as well as the differentiation of myeloid endothelial progenitor cells, which promotes tumor angiogenesis.27 These findings suggest that TNF can serve as either therapeutic target or agent. The first clinical trial of TNF for cancer treatment was initiated in February 1992; to date, there have been 20 trials involving at least 1152 participants in which TNF or related biological agents were used to treat 3 main tumor typesnamely, melanoma, CRC, and head and neck cancer. Only one study has published results. Etanercept, a TNF inhibitor, was investigated for the treatment of idiopathic pneumonia in patients with leukemia and lymphoma after stem cell transplantation (NCT00309907), but the results did not reflect the effect of TNF inhibitor.

The anticancer efficacy of CSF family cytokines including GM-CSF, granulocyte (G)-CSF, erythropoietin (EPO), and IL-3, has been widely studied in clinical settings. To date, 1311 clinical trials enrolling over 200,000 cancer patients treated with GM-CSF and G-CSF alone or in combination have been registered at ClinicalTrials.gov; of these, 96% and 94% studied the effects of GM-CSF and G-CSF in combination therapy, respectively (Figure 6A).

Figure 6 The number and ratio of clinical trials of cytokine combined with other agents in cancer treatment. (A) Relative ratio of clinical trials using GM-CSF, G-CSF and VEGF receptor inhibitors alone or in combination. (B and C) Number of clinical trials using cytokine-based drugs alone or in combination.

EPO exhibits pro-proliferative and anti-apoptotic activities in multiple nonhematopoietic cell types including tumor cells.37 EPO has been used to alleviate cancer- and chemotherapy-related anemia. The first clinical trial of EPO for cancer treatment was initiated in 2003 and to date, 15 trials without results have been published at ClinicalTrials.gov.

IL-3, also known as multi-CSF and hematopoietic cell growth factor, has been the focus of 7 clinical trials. A single-arm trial study evaluating the efficacy of DT388IL3 fusion protein for the treatment of patients with acute myeloid leukemia or myelodysplastic syndromes reported an overall response rate of 81.8% (NCT00397579).

IL-1 and IL-18 are members of the IL-1 family; IL-1 is an important regulator in innate immunity,38 and both cytokines stimulate IFN- production by T cells and NK cells. IL-1 has dual roles in anticancer immune response. Clinically, patients with high IL-1 concentrations in tumors have poor prognoses.39 Anakinra is an IL-1 receptor antagonist that is commonly used to treat rheumatoid arthritis; its antitumor efficacy has been assessed in 8 trials. In a phase II trial, anakinra combined with dexamethasone was used to treat multiple myeloma and plasma cell neoplasm (NCT00635154); the 6-month progression-free rate was 90.7%. 6 clinical trials are investigating a recombinant human IL-18, namely SB-485232, for the treatment of patients with melanoma, lymphoma, and ovarian neoplasms, but no results have been published.

Chemokines and their receptors mediate immunocytes trafficking into the cancer microenvironment, playing roles in promoting or inhibiting cancers. CCL21, together with CCL19, regulates the migration of DCs and T cells to secondary lymphoid organs when binding to their receptor CCR7, thus plays an important role in adaptive immunity and immune tolerance.40 Intratumoral injection of CCL21 enhances the infiltration of T cells and DCs in tumor.41 To date, 3 cancer clinical trials using CCL21 have been registered at ClinicalTrials.gov. In a phase II trial (NCT01433172), CCL21 combined with GM.CD40L vaccine (tumor antigen expressing GM-CSF and CD40L) was used to treat lung adenocarcinoma; the 6-month progression-free survival rate was higher in the combination group than in the GM.CD40L group (15.2% [5/33] vs 9.4% [3/32]).42 For chemokine (C-X-C motif) ligand (CXCL)12, CXCL8, CCL2, CCL3 and CCL5 which are involved in cancer progression and metastasis, few clinical trials studied drugs targeting their receptors, CXCR4, CXCR 1/2 and CCR2, and their effects for cancers have not been verified.43

TGF-, VEGF, epidermal growth factor (EGF), insulin-like growth factor (IGF) and broblast growth factor (FGF), IL-4, IL-13, IL-10, IL-6, IL-11, and IL-17 are known to inhibit anticancer immune response. In this section, we present efficacy of cancer therapy by targeting these cytokines.

TGF- is an oncogenic factor that facilitates evasion of systemic immune surveillance.44 The clinical efficacy of various inhibitors of TGF- signaling including GC1008 (fresolimumab, anti-TGF- monoclonal antibody), TEW-7197 (TGF- receptor activin-like kinase [ALK]4/ALK5), and AP 12009 (TGF-2 antisense oligodeoxynucleotide) has been investigated in metastatic breast cancer, RCC, recurrent or refractory high-grade glioma, and advanced melanoma. In a phase II trial examining the efficacy and safety of combined fresolimumab (1 or 10 mg/kg) and local radiotherapy in the treatment of metastatic breast cancer (NCT01401062), overall response rates were 100% with both low and high drug doses and the rate of serious adverse events was 27% and 25%, respectively. The results of 7 other trials of TGF- inhibitors in cancer treatment have yet to be reported.

Angiogenesis is a vital step in tumor progression and metastasis. Sustained expression of VEGF during tumor development induces the formation of tumor vasculature.45 Various VEGF receptor (VEGFR) inhibitors either alone or in combination with other drugs have been investigated for cancer treatment. These inhibitors include antibodies against VEGFR (eg, bevacizumab, ramucirumab, and ranibizumab); inhibitors of receptor protein kinases (eg, axitinib and vandetanib); soluble decoy receptors containing VEGFR domains (eg, aflibercept); and small molecules that interfere with the binding sites of VEGFR (eg, vatalanib). There are 301 trials registered at ClinicalTrials.gov for the treatment of various cancers (CRC, breast cancer, ovarian cancer, non-small-cell-lung cancer, lymphoma, etc) using VEGFR inhibitors, of which 35% have examined the effects of VEGFR inhibitor monotherapy (Figure 6A).

A large phase III clinical trial that enrolled 1690 participants investigated the efficacy of docetaxel alone or with vandetanib in non-small-cell-lung cancer (NSCLC) (NCT00312377). Median PFS was longer with the combination therapy than with docetaxel alone (17.3 vs 14 weeks), although median OS was comparable between the 2 groups (10.6 vs 10 months). In another phase III trial of 913 patients with NSCLC (NCT00532155), aflibercept increased the median OS of docetaxel from 10.05 to 10.41 months and prolonged median PFS from 4.11 to 5.19 months. In a phase III trial examining the efficacy of aflibercept vs a placebo in 1226 patients with metastatic CRC who had failed to respond to the FOLFIRI regimen (irinotecan, 5-FU, and leucovorin) (NCT00561470), median OS was increased from 12.6 to 13.5 months while median PFS was increased from 4.67 to 6.90 months. Besides combination with chemotherapy, the efficacy of VEGF inhibitors combined with other immunotherapies has been evaluated in 40 clinical trials. As described in the paragraph of IFN-, bevacizumab in conjunction with IFN- showed clinical benefits in mRCC and melanoma patients. Thus, VEGFR inhibition is an effective therapeutic strategy for the treatment of multiple cancers.

In addition to VEGF, growth factors such as EGF, IGF and FGF, have been shown to be crucial for the development and progression of certain cancers. Clinically, human epidermal growth factor receptor 1 and 2 (HER1 and HER2), IGF-1 receptor (IGF-1R) and FGF receptor (FGFR) have been found to be overexpressed in various cancers, particularly in breast and lung cancers.4648 There are 206, 71 and 14 trials for blocking EGFR, IGF-1R and FGFR, respectively, with small molecule inhibitors or monoclonal antibodies in treating cancers registered with ClinicalTrials.gov that had completed recruitment. Unsurprisingly, most of these trials are for lung and breast cancers: 71/206 trials of HER1/2 inhibitors and 16/71 of IGF-1R inhibitors are for treating lung cancers; and 44/206 trials of HER 1/2 inhibitors and 9/71 trials are for treating breast cancers. Their effects in combination of chemotherapeutics have been generally studied. A phase II trial (NCT00986674) demonstrated that carboplatin and paclitaxel are more effective when given with cixutumumab (anti-EGFR antibody) and cetuximab (anti-IGF-1R antibody) than with cetuximab alone in treating advanced non-small cell lung cancer, with overall response rates of 22%, 21.7% and 11%, respectively. However, a phase II trial (NCT00684983) showed that cixutumumab did not enhance the effects of capecitabine and lapatinib ditosylate (EGFR and HER2 inhibitors) in treating HER2-positive stage IIIB-IV breast cancers. Five trials with published results showed limited effects of FGFR inhibitors. A phase II trial studied the effects of dovitinib (a multitargeted inhibitor of FGFR and VEGFR) for patients with advanced lung cancer or CRC who have progressed on anti-VEGF treatment, and the overall response rate was 14.3%. Overall, blockade of growth factor receptors brings considerable therapeutic effects when combining with chemotherapy in treating certain cancers.

IL-4 and IL-13 function as immunosuppressive cytokines that inhibit antitumor immunity by enhancing the Th2 cell response and blocking Th1 cell differentiation.16 Mutated forms of IL-4 and IL-13 receptors highly expressed in multiple human tumor cell lines.49,50 Based on these observations, targeted drugs were developed by linking Pseudomonas exotoxin to IL-4 or IL-13 (IL4-PE38KDEL and IL13-PE38QQR, respectively).

Since 2001, there have been 7 cancer trials of IL-4 registered at ClinicalTrial.gov. It was shown in vitro that IL-4 can inhibit the growth of Kaposi sarcoma cells,51 and one trial assessed the efficacy of IL-4 in the treatment of 48 patients with Kaposi sarcoma (NCT00000769) although no findings have been published. IL-4 was also administered as an adjuvant to enhance the effect of a DC vaccine in the treatment of Wilms tumor (NCT00001564) and Ewing sarcoma (NCT00923910),52 but the outcome of these trials is unknown.

The first clinical trial using IL13-PE38QQR (for the treatment of malignant gliomas) was initiated in 2000. Since then, there have been 10 clinical trials involving over 500 participants with malignant gliomas who were treated with IL-13-PE38QQR. It is difficult to conclude these trials as the results have not been published. In one phase III trial of 300 patients with recurrent malignant gliomas (NCT00064779), IL13-PE38QQR was directly infused into the tumor tissue for 96 hours. After 15 days, patients underwent surgery to excise the recurrent tumors and received another infusion. However, no results have been posted for this or any other trial investigating IL13-PE38QQR.

IL-10 functions as an immune suppressor that inhibits the cancer immunity cycle.53 To date, there have been no reports from ClinicalTrial.gov evaluating the efficacy of IL-10 for cancer treatment, although many trials have examined the use of IL-10 for the treatment of autoimmune disease such as rheumatic arthritis.

The IL-6 cytokine family, which includes IL-6 and IL-11, participates in the activation of oncogenic signal transducer and activator of transcription (STAT)3.54 Twenty trials of IL-6 for cancer treatment (multiple myeloma, lymphoma, mRCC, and prostate cancer) have been registered at ClinicalTrials.gov, mostly involving siltuximab, an IL-6 antagonist approved by the FDA for the treatment of multicentric Castleman disease. In a phase II trial of 88 patients with myeloma (NCT00911859), siltuximab combined with VELCADE (a prescription medication for myeloma) resulted in a higher complete response rate (26.5% vs 22.4%) and overall response rate (87.8% vs 79.6%) than VELCADE alone; in the second part of this trial (286 patients; NCT00401843), PFS of the 2 groups was 245 and 232 days, respectively. Given its role in hematopoiesis, IL-11 has been investigated for its potential to increase platelet counts in patients with chronic myelogenous leukemia in 2 trials.

IL-17 is a pro-oncogenic cytokine that is mainly produced by Th17 cells and induces the production of IL-6 by tumor cells to activate the IL-6/STAT3 signaling pathway.55 Elevated IL-17 expression is related to poor prognosis in patients with invasive ductal carcinoma. But no cancer clinical trial using or targeting IL-17 has been published in ClinicalTrial.gov.

According to review of hundreds of clinical trials, we know that efficacy of cytokines as therapeutic drugs on clinical outcomes are limited. One possible reason for this is that because of the short half-life of cytokines in the blood, frequent high doses are required to achieve lasting therapeutic effects. For example, the effective dosage of IL-2 is 600,000 IU/kg administered every 8 hours for 5 days; moreover, 3 treatment cycles are needed for its activity. Because of this, adverse events generally occur in patients receiving cytokine therapy, include fatigue, chills, fever, chest pain, and musculoskeletal pain.56 More serious adverse events are gastrointestinal disorders (eg, stomachache, diarrhea, and gastritis), cardiac abnormalities (eg, myocardial infarction, nodal tachycardia), and disorders of the immune system (eg, anaphylaxis) and blood and lymphatic systems (eg, anemia and febrile neutropenia).

Nanomaterials used as carriers to deliver cytokines to target tissues can improve the stability of cytokines in blood and reduce their toxicity. At the same time, the unique features of nanomaterials have advantages for the therapeutic application of cytokines including aqueous solubility, prolonged circulation time, and preferential accumulation at tumor sites.57 Recent studies on nanomaterials used for cytokine loading are summarized in Table 2.

Table 2 Nanomaterials for Therapeutic Delivery of Cytokines

Nanomaterials can improve the stability and bioactivity of cytokines. For example, sustained released over a period of 1 month was achieved for IFN- encapsulated in poloxamer-blend microspheres.58 Chitosan coated with pJME/GM-CSF (plasmid DNA) was more effective than naked pJME/GM-CSF in promoting DC recruitment.59 Nanoscale liposomal polymeric gels loaded with TGF- inhibitor and IL-2 delayed tumor growth and increased NK cell activity and the number of tumor-infiltrating T cells.60 Nanomaterials can also reduce the toxic effects of cytokine therapy; for instance, encapsulation in PEG liposomes abrogated the diarrhea induced by TNF in rats with subcutaneous BN175 sarcomas.61 Gold nanoparticles were found to enhance the accumulation of TNF around blood vessels in a mouse model of epithelial carcinoma, leading to a significant decrease in tumor volume.62 Magnetic nanoparticles carrying human IFN-2b were enriched in the liver upon application of a magnetic field and compared to the control group, the volume of human liver cancer cell-derived tumors in nude mice was reduced by about 30%.63 Nanomaterials are good auxiliaries for cytokine gene therapy. Chitosan coated with plasmids encoding the cytokines IL-15 and IL-21 suppressed tumor growth and prolonged survival in mice.64,65 PEGpoly (lactic-co-glycolic acid)PEG nanoparticles were shown to be effective carriers for IL18 gene delivery.66 Magnetic nanoparticles carrying a plasmid encoding a small interfering RNA targeting gene encoding epidermal growth factor receptor reduced endogenous epidermal growth factor receptor expression in U251 glioma cells, resulting in tumor regression in vivo.67 Two clinical trials involving 168 patients have investigated the efficacy of colloidal gold-bound TNF for the treatment of primary or metastatic cancer (NCT00356980 and NCT00436410, respectively), but the results have yet to be reported.

Besides, artificial oncolytic viruses are well-established carriers for cytokine gene therapy. There are 17 cytokines including CCL2, CCL5, CCL19, CXCL11, FGF2, FLT3L, GM-CSF, IFN-/, IFN-, IL-2, IL-7, IL-12, IL-15, IL-18, IL-23, IL-24, and TNF- that are delivered by artificial oncolytic viruses derived from adenovirus, herpesvirus, paramyxovirus, poxvirus, or rhabdovirus. More details could be found in the recent review wrote by Pol et al.68

Although animal studies showed that properties and efficacy of cytokine-based drugs can be improved by nanomaterials, sufficient clinical studies are required to support the conclusion. Two ongoing clinical trials involving 168 patients aim to investigate the efficacy of colloidal gold-bound TNF for the treatment of primary or metastatic cancer (NCT00356980 and NCT00436410, respectively), but the results have yet to be reported.

As important immune regulators, cytokine-based drugs offer many possibilities for cancer treatment. Large amounts of cytokines can be readily produced using eukaryotic or prokaryotic expression systems as the cDNA sequences of most cytokines are available, which makes cytokines attractive to new drug development. However, our statistical results (Supplementary Table 1) indicate that a large number of clinical trials of cytokine-based drugs ended up without published results mainly because of the low efficacy, serious adverse effects, and antagonistic roles in immunoregulation. These problems are partly overcome by delivering with nanomaterials or oncolytic viruses in animal experiments, or combining with immunotherapies or chemotherapeutic agents or both (Figure 6B and C) in clinic. Such strategies would be used and improved in the future clinical trials. Moreover, clarifying the immune-regulatory mechanisms of cytokines can improve their efficacy and safety in cancer therapy.

APC, antigen-presenting cell; B7-H3, B7 homolog 3 protein; CAR-T, chimeric antigen receptor T cell; CCL, C-C motif chemokine ligand; CRC, colorectal cancer; CSF, colony-stimulating factor; CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T lymphocyte-associated protein 4; CXCL, chemokine (C-X-C motif) ligand; DC, dendritic cell; EGF, epidermal growth factor; EMT, epithelial-to-mesenchymal transition; EPO, erythropoietin; FDA, US Food and Drug Administration; FGF, broblast growth factor; Fu, fluorouracil; G, granulocyte; GM, granulocyte/macrophage; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; ILC, innate lymphoid cell; i.v., intravenous injection; IU, international unit; JAK, Janus kinase; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; mRCC, metastatic renal cell carcinoma; NK, natural killer; NKT, natural killer T; NSCLC, non-small-cell-lung cancer; ORR, objective response rate; OS, overall survival; PD-1, programmed death 1; PD-L1, programmed death ligand 1; PEG, pegylated; PFS, progression-free survival; PGE2, prostaglandin E2; PLGA, poly (lactic-co-glycolic acid); rAd, recombinant adenovirus; STAT, signal transducer and activator of transcription; s.c., subcutaneous injection. TAA, tumor-associated antigen; TAM, tumor-associated macrophage; TGF-, transforming growth factor ; Th, T helper cell; TNF, tumor necrosis factor; Treg, regulatory T cell; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

This work is supported by the National Natural Science Foundation of China (Grant No. 31800006) to YQ; Natural Science Foundation of Guangdong Province (Grant No. 18zxxt26) to YQ; Guangzhou Basic and Applied Basic Research Foundation (Grant No. 202002030127) to JS; Guangdong Basic and Applied Basic Research Foundation (Grant No. 2021A1515012324) to JS; the Fundamental Research Funds for the Central Universities (Grant No. 20ykzd08) to JS; Natural Science Foundation of Guangdong Province (Grant No. 2018A030313563) to JS; Program for Guangdong Introducing Innovative and Entrepreneurial Teams (Grant No. 2016ZT06S252) to JS; Guangdong Financial Fund for High-Caliber Hospital Construction to JS.

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

The authors declare they have no competing financial interests and other onflicts of interest in this work.

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Clinical Application of Cytokines in Cancer Immunotherapy | DDDT - Dove Medical Press

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Stem Cell Therapy Market Report | Know the Cutting-Edge Innovations and Future Trends of Market – BioSpace

By daniellenierenberg

Of late, there has been an increasing awareness regarding the therapeutic potential of stem cells for management of diseases which is boosting the growth of the stem cell therapy market. The development of advanced genome based cell analysis techniques, identification of new stem cell lines, increasing investments in research and development as well as infrastructure development for the processing and banking of stem cell are encouraging the growth of the global stem cell therapy market.

One of the key factors boosting the growth of this market is the limitations of traditional organ transplantation such as the risk of infection, rejection, and immunosuppression risk. Another drawback of conventional organ transplantation is that doctors have to depend on organ donors completely. All these issues can be eliminated, by the application of stem cell therapy. Another factor which is helping the growth in this market is the growing pipeline and development of drugs for emerging applications. Increased research studies aiming to widen the scope of stem cell will also fuel the growth of the market. Scientists are constantly engaged in trying to find out novel methods for creating human stem cells in response to the growing demand for stem cell production to be used for disease management.

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It is estimated that the dermatology application will contribute significantly the growth of the global stem cell therapy market. This is because stem cell therapy can help decrease the after effects of general treatments for burns such as infections, scars, and adhesion. The increasing number of patients suffering from diabetes and growing cases of trauma surgery will fuel the adoption of stem cell therapy in the dermatology segment.

Global Stem Cell Therapy Market: Overview

Also called regenerative medicine, stem cell therapy encourages the reparative response of damaged, diseased, or dysfunctional tissue via the use of stem cells and their derivatives. Replacing the practice of organ transplantations, stem cell therapies have eliminated the dependence on availability of donors. Bone marrow transplant is perhaps the most commonly employed stem cell therapy.

Osteoarthritis, cerebral palsy, heart failure, multiple sclerosis and even hearing loss could be treated using stem cell therapies. Doctors have successfully performed stem cell transplants that significantly aid patients fight cancers such as leukemia and other blood-related diseases.

Global Stem Cell Therapy Market: Key Trends

The key factors influencing the growth of the global stem cell therapy market are increasing funds in the development of new stem lines, the advent of advanced genomic procedures used in stem cell analysis, and greater emphasis on human embryonic stem cells. As the traditional organ transplantations are associated with limitations such as infection, rejection, and immunosuppression along with high reliance on organ donors, the demand for stem cell therapy is likely to soar. The growing deployment of stem cells in the treatment of wounds and damaged skin, scarring, and grafts is another prominent catalyst of the market.

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On the contrary, inadequate infrastructural facilities coupled with ethical issues related to embryonic stem cells might impede the growth of the market. However, the ongoing research for the manipulation of stem cells from cord blood cells, bone marrow, and skin for the treatment of ailments including cardiovascular and diabetes will open up new doors for the advancement of the market.

Global Stem Cell Therapy Market: Market Potential

A number of new studies, research projects, and development of novel therapies have come forth in the global market for stem cell therapy. Several of these treatments are in the pipeline, while many others have received approvals by regulatory bodies.

In March 2017, Belgian biotech company TiGenix announced that its cardiac stem cell therapy, AlloCSC-01 has successfully reached its phase I/II with positive results. Subsequently, it has been approved by the U.S. FDA. If this therapy is well- received by the market, nearly 1.9 million AMI patients could be treated through this stem cell therapy.

Another significant development is the granting of a patent to Israel-based Kadimastem Ltd. for its novel stem-cell based technology to be used in the treatment of multiple sclerosis (MS) and other similar conditions of the nervous system. The companys technology used for producing supporting cells in the central nervous system, taken from human stem cells such as myelin-producing cells is also covered in the patent.

Global Stem Cell Therapy Market: Regional Outlook

The global market for stem cell therapy can be segmented into Asia Pacific, North America, Latin America, Europe, and the Middle East and Africa. North America emerged as the leading regional market, triggered by the rising incidence of chronic health conditions and government support. Europe also displays significant growth potential, as the benefits of this therapy are increasingly acknowledged.

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Asia Pacific is slated for maximum growth, thanks to the massive patient pool, bulk of investments in stem cell therapy projects, and the increasing recognition of growth opportunities in countries such as China, Japan, and India by the leading market players.

Global Stem Cell Therapy Market: Competitive Analysis

Several firms are adopting strategies such as mergers and acquisitions, collaborations, and partnerships, apart from product development with a view to attain a strong foothold in the global market for stem cell therapy.

Some of the major companies operating in the global market for stem cell therapy are RTI Surgical, Inc., MEDIPOST Co., Ltd., Osiris Therapeutics, Inc., NuVasive, Inc., Pharmicell Co., Ltd., Anterogen Co., Ltd., JCR Pharmaceuticals Co., Ltd., and Holostem Terapie Avanzate S.r.l.

This study provides a particularized anatomy according to the L.E.A.P mechanism

The regional analysis offers market assays across:

The study, prepared through the L.E.A.P mechanism adds a dimension of infallibility and assures precise information on all the growth dynamics.

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https://www.tmrresearch.com/stem-cell-banking-market

https://www.tmrresearch.com/animal-stem-cell-therapy-market

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Heart attack recovery aided by injecting heart muscle cells that overexpress cyclin D2 – The Mix

By daniellenierenberg

Researchers used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has high clinical relevance to human disease.

Researchers used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has high clinical relevance to human disease.In a large-animal study, researchers have shown that heart attack recovery is aided by injection of heart muscle cells derived from human induced pluripotent stem cell line, or hiPSCs, that overexpress cyclin D2. This research, published in the journal Circulation, used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has higher clinical relevance to human disease, compared to studies in mice.

An enduring challenge for bioengineering researchers is the failure of the heart to regenerate muscle tissue after a heart attack has killed part of its muscle wall. That dead tissue can strain the surrounding muscle, leading to a lethal heart enlargement.

Heart experts thus have sought to create new tissue applying a patch of heart muscle cells or injecting heart cells to replace damaged muscle. Similarly, they have tried to stimulate division of existing heart muscle cells near the damaged area. This current study, led by researchers at the University of Alabama at Birmingham, shows progress in both goals.

After the experimental heart attack, heart tissue around the infarction site was injected with about 30 million bioengineered human cardiomyocytes that were differentiated from hiPSCs. These cells also overexpress cyclin D2, part of a family of proteins involved in cell division.

Compared to control human cardiomyocytes, the cyclin D2-cardiomyocytes showed enhanced potency to repair the heart. They proliferated after injection, and by four weeks, the hearts had less pathogenic enlargement, reduced size of dead muscle tissue and improved heart function.

Intriguingly, the cyclin D2-cardiomyocytes stimulated not only their own proliferation, but also proliferation of existing heart muscle cells around the infarction site of the pig heart, as well as showing angiogenesis, the development of new blood vessels.

These results suggest that the cyclin D2-cardiomyocyte transplantation may be a potential therapeutic strategy for the repair of infarcted hearts, said study leader Jianyi Jay Zhang, M.D., Ph.D., the chair of Biomedical Engineering, a joint department of the UAB School of Medicine and the UAB School of Engineering.

This ability of the graft cyclin D2-cardiomyocytes to stimulate the proliferation of nearby existing heart cells suggested paracrine signaling, a type of cellular communication where a cell produces a signal that induces changes in nearby cells.

Exosomes small blebs or tiny vesicles that are released by human or animal cells and contain proteins and RNA from the cells that release them are one common form of paracrine signaling.

Zhang and colleagues found that exosomes that they purified from the cyclin D2-cardiomyocyte growth media indeed promoted proliferation of cultured cardiomyocytes. In addition, the treated cardiomyocytes were more resistant to programmed cell death, called apoptosis, induced by low oxygen levels. The exosomes also induced proliferation of various other cell types, including human umbilical vein endothelial cells, human vascular smooth muscle cells and 7-day-old rat cardiomyocytes that have almost undetectable proliferation.

Part of the cargo that exosomes carry are microRNAs, or miRNAs. These short pieces of RNA have the ability to interact with messenger RNA in target cells, and they are robust players of gene regulation in cells. Humans have more than 2,000 miRNAs with different RNA sequences, and these are thought to regulate a third of the genes in the genome.

So, the researchers documented which microRNAs were present in exosomes from the cyclin D2-overexpressing cardiomyocytes and in exosomes from non-overexpressing cardiomyocytes. As expected, they found differences.

Jianyi Jay Zhang, M.D., Ph.D.Together, the exosomes from both types of cells contained 1,072 different miRNAs, and 651 were common to the two exosome groups. However, 332 miRNAs were found only in the cyclin D2-overexpressing cardiomyocytes, and 89 miRNAs were specific for the non-overexpressing cardiomyocytes. In preliminary work of characterizing the effects of specific miRNAs, one particular miRNA from the cyclin D2-overexpressing exosomes was shown to stimulate proliferation when delivered into rat cardiomyocytes.

Thus, as the therapeutic potential of exosomes for improving cardiac function becomes more evident, combining an exosome-mediated delivery of proliferative miRNAs with transplantation of cyclin D2-overexpressing cardiomyocytes, or cell products, could become a new promising strategy for upregulating proliferation of the recipient cardiomyocytes and reducing cardiac fibrosis, Zhang said. Altogether, our data suggest that cardiac cell therapy, involving cardiomyocytes with enhanced proliferation capacity, may become an efficacious future strategy for myocardial repair and prevention of congestive heart failure in patients with acute myocardial infarctions.

UAB Department of Biomedical Engineering co-authors with Zhang, in the study Cyclin D2 overexpression enhances the efficacy of human induced pluripotent stem cell-derived cardiomyocytes for myocardial repair in a swine model of myocardial infarction, are Meng Zhao, Yuji Nakada, Yuhua Wei, Weihua Bian, Anton V. Borovjagin, Yang Zhou and Gregory P. Walcott.

Additional co-authors are Yuxin Chu and Min Xie, Division of Cardiovascular Disease, UAB Department of Medicine; Wuqiang Zhu, Department of Cardiovascular Diseases, Physiology and Biomedical Engineering, Mayo Clinic Arizona, Scottsdale; Thanh Nguyen, UAB Informatics Institute; and Vahid Serpooshan, Emory University and Georgia Institute of Technology, Atlanta.

Support came from National Institutes of Health grants HL114120, HL131017, HL149137 and HL134764.

At UAB, Zhang holds the T. Michael and Gillian Goodrich Endowed Chair of Engineering Leadership.

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Heart attack recovery aided by injecting heart muscle cells that overexpress cyclin D2 - The Mix

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Allogeneic Mesenchymal Stem Cell Segment Is Expected To Lead In the Global Rheumatoid Arthritis Stem Cell Therapy Market over the Forecast Period,…

By daniellenierenberg

The report on the Rheumatoid Arthritis Stem Cell Therapy market provides a birds eye view of the current proceedings and the impact on the COVID-19 pandemic. Further, the report ponders over the various factors that are likely to impact the overall dynamics of the market once the COVID-19 pandemic subsides. The current trends, growth opportunities, restraining factors, and drivers are discussed in the report in detail.

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The growing prevalence and recurrence of rheumatoid arthritis is expected to be the major factor driving the growth of the rheumatoid arthritis stem cell therapy market over the forecast period. Although doctors do not know the exact cause of rheumatoid arthritis, but certain risk factors are observed to be associated with it.

These risk factors include age (most common between the age of 40 and 60), family history, gender, environment (a toxic chemical in the environment can up the odds), obesity and smoking. Changes in lifestyle and eating habits are contributing to the growing prevalence of rheumatoid arthritis.

Rheumatoid Arthritis Stem Cell Therapy Market: Segmentation

Tentatively, the global rheumatoid arthritis stem cell therapy market can be segmented on the basis of treatment type, application, end user and geography.

Based on treatment type, the global rheumatoid arthritis stem cell therapy market can be segmented into:

Based on application, the global rheumatoid arthritis stem cell therapy market can be segmented into:

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Based on distribution channel, the global rheumatoid arthritis stem cell therapy market can be segmented into:

Based on geography, the global rheumatoid arthritis stem cell therapy market can be segmented into:

Rheumatoid Arthritis Stem Cell Therapy Market: Regional Outlook

Geographically, the global rheumatoid arthritis stem cell therapy market can be segmented into viz. North America, Latin America, Europe, Asia-Pacific excluding Japan (APEJ), Japan and the Middle East and Africa (MEA). North America is expected to be the dominant region in the global rheumatoid arthritis stem cell therapy market, owing to the presence of various key players.

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The rheumatoid arthritis stem cell therapy market in Asia Pacific excluding Japan is expected to grow at a significant CAGR due to the expansion of product offerings by key players. Europe is expected to have the second large share in the global rheumatoid arthritis stem cell therapy market throughout the forecast period.

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Allogeneic Mesenchymal Stem Cell Segment Is Expected To Lead In the Global Rheumatoid Arthritis Stem Cell Therapy Market over the Forecast Period,...

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Pigs can breathe through their butts. Can humans? – Livescience.com

By daniellenierenberg

Mice, rats and pigs all share a secret superpower: They can all use their intestines to breathe, and scientists discovered this by pumping oxygen up the animals' butts.

Why run such experiments, you ask? The research team wanted to find a potential alternative to mechanical ventilation, a medical treatment where a machine pushes air into a patient's lungs through the windpipe. Ventilators deliver oxygen to the lungs and help remove carbon dioxide from the blood, but the machines aren't always available.

Early in the COVID-19 pandemic, for example, hospitals faced a severe shortage of ventilators, The New York Times reported. Although doctors can also use a technique called extracorporeal membrane oxygenation (ECMO), where blood is pumped out of the body and reoxygenated with a machine, the procedure carries inherent risks, such as bleeding and blood clots; and it's often less readily available than ventilators, according to Mayo Clinic.

Related: The 10 weirdest medical cases in the animal kingdom

In search of another solution, the study authors drew inspiration from aquatic animals like sea cucumbers and freshwater fish called loaches (Misgumus anguillicandatus), which use their intestines for respiration. It was unclear whether humans and other mammals have similar capabilities, although some scientists attempted to answer that question in the 1950s and 1960s.

"We initially looked at a mouse model system to see if we could deliver oxygen gas intra-anusly," said senior author Dr. Takanori Takebe, a professor at the Tokyo Medical and Dental University and a director at the Center for Stem Cell and Organoid Research and Medicine at Cincinnati Children's Hospital Medical Center.

"Every time we performed experiments, we were quite surprised," Takebe told Live Science.

Without intestinal ventilation, mice placed in a low-oxygen environment survived for only about 11 minutes; with ventilation into their anuses, 75% survived for 50 minutes, thanks to an infusion of oxygen that reached their hearts. The team then tried using oxygenated liquid, rather than gas, in mice, rats and pigs, and they found similarly promising results. The team noted that more work still needs to be done to see if the approach is safe and effective in humans, according to a paper on their findings published May 14 in the journal Med.

"The pandemic has highlighted the need to expand options for ventilation and oxygenation in critical illness, and this niche will persist even as the pandemic subsides," as there will be times when mechanical ventilation is unavailable or inadequate on its own, Dr. Caleb Kelly, a clinical fellow and physician-scientist at Yale School of Medicine, wrote in a commentary of the study. If, after further evaluation, intestinal ventilation eventually becomes common practice in intensive care units, this new study "will be marked by historians as a key scientific contribution," he wrote.

Before starting their experiments in rodents, the team got very familiar with loach guts. The fish take in oxygen mostly through their gills, but occasionally, when exposed to low-oxygen conditions, loaches instead use a portion of their intestines for gas exchange, Takebe said. In fact, in response to the lack of oxygen, the structure of gut tissues near the anus changes such that the density of nearby blood vessels increases and secretion of fluids related to digestion decreases.

These subtle changes allow loaches to "suck up the oxygen more efficiently," Takebe said. In addition, the outermost lining of the loach gut the epithelium is very thin, meaning oxygen can easily permeate the tissue to reach the blood vessels beneath, he added. To simulate this structure in their mouse models, the team thinned out the gut epithelium of the rodents using chemicals and various mechanical procedures.

They then placed the mice under extremely low-oxygen conditions and used a tube to pump oxygen gas up the animals' bums and into their large intestines.

Related: 8 bizarre animal surprises from 'True or Poo' Can you tell fact from myth?

Compared with mice whose gut epithelium had not been thinned, the mice with thin epitheliums survived significantly longer in the experiment with most surviving 50 minutes as compared with about 18 minutes. Again, mice not given any oxygen only survived for about 11 minutes. In addition to surviving longer, the group with thinned-out gut linings showed signs that they were no longer starved for oxygen; they stopped gasping for air or showing signs of cardiac arrest, and the oxygen pressure in their major blood vessels improved.

Although this initial experiment suggested that oxygen could pass through the intestine and into circulation, thinning out the gut epithelium would likely not be feasible in human patients, Takebe said.

Particularly in critically ill patients, "I think additional damage to the gut would be really dangerous, for the treatment perspective," Takebe said. But "over the course of the experiments, we realized that even the intact gut has some, not really efficient, but some capacity to exchange the gas," he noted, meaning there may be a way to introduce oxygen through the gut without first thinning out the tissues.

So in another experiment, rather than using oxygen gas, the team tried perfluorodecalin (PFD), a liquid fluorocarbon that can be infused with a large amount of oxygen. The liquid is already used in people, such as for use in the lungs of infants with severe respiratory distress, the authors noted in their report.

The liquid also acts as a surfactant a substance that reduces surface tension; since a surfactant lines the air sacs of the lungs and helps boost gas exchange in the organ, PFD may fulfill a similar purpose in the intestines, Takebe said.

Much like in the oxygen-gas experiments, the oxygenated PFD rescued mice from the effects of being placed in a low-oxygen chamber, enabling the rodents to meander about their cage more than mice not given the treatment. After just one injection of 0.03 ounces (1 milliliter) of the liquid, the rodents' improvements persisted for about 60 minutes.

"We are not quite sure why this improvement is persisting much longer than the original expectations," Takebe noted, as the authors expected the effects to wear off in just a couple minutes. "But the observation is really reproducible and very robust."

Related: Gasp! 11 surprising facts about the respiratory system

The team then moved on to a pig model of respiratory failure, where they placed pigs on ventilators and only provided a low level of oxygen and then injected PDF into the pigs' posteriors with a long tube. Compared with pigs not given the PFD treatment, pigs given PFD improved in terms of the oxygen saturation of their blood, and the color and warmth returned to their skin. A 13.5 oz (400 ml) infusion sustained these improvements for about 18 to 19 minutes, and the team found that they could give additional doses to the pigs without noticeable side effects.

The team also tested the safety of repeat dosing in rats and found that, while their oxygen levels rose, the animals showed no notable side effects, markers of organ damage or stray PFD lingering in their cells.

Following this success in animal models, Takebe said that his team hopes to start a clinical trial of the treatment in humans sometime next year. They would likely start by testing the safety of the approach in healthy volunteers and beginning to work out what dose levels would be reasonable, he said. However, to make the jump from animals to human patients, the team will need to address a number of critical questions.

For instance, the treatment could potentially stimulate the vagus nerve a long nerve that connects the gut and brain so trial organizers should likely be on the lookout for side effects like falling blood pressure or fainting, Takebe noted. Also, the lower gut contains relatively little oxygen compared with other organs in the body, he added. The community of bacteria and viruses that live in the gut are adapted to these low-oxygen conditions, and a sudden infusion of oxygen might disrupt those microbes, he said.

"The consequence of reversing this so-called 'physiologic hypoxia' is unknown," Kelly noted in his commentary, echoing Takebe's sentiments. In humans, it will be important to determine how many doses of oxygenated liquid could be safely administered into the gut without causing unintended changes to the intestinal environment, he wrote.

In addition, the animal models in the study don't fully reflect what critically ill patients experience during respiratory failure, a condition that often coincides with infection, inflammation and low blood flow, Kelly noted. So there may be additional factors to consider in critically ill patients that weren't relevant in rodents and pigs. And depending on a given patient's condition, they may need a higher or lower dose of PFD all of these fine details will need to be carefully assessed in future trials, Takebe said.

Originally published on Live Science.

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Pigs can breathe through their butts. Can humans? - Livescience.com

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Merck Announces Phase 3 KEYNOTE-522 Trial Met Dual Primary Endpoint of Event-Free Survival (EFS) in Patients With High-Risk Early-Stage…

By daniellenierenberg

KENILWORTH, N.J.--(BUSINESS WIRE)--Merck & Co. (NYSE: MRK), known as MSD outside the United States and Canada, today announced positive results from the pivotal neoadjuvant/adjuvant Phase 3 KEYNOTE-522 trial investigating KEYTRUDA, Mercks anti-PD-1 therapy, in combination with chemotherapy as pre-operative (neoadjuvant) treatment and then continuing as a single agent (adjuvant) treatment after surgery. KEYNOTE-522 met its dual primary endpoint of event-free survival (EFS) for the treatment of patients with high-risk early-stage triple-negative breast cancer (TNBC). Based on an interim analysis conducted by the independent Data Monitoring Committee (DMC), neoadjuvant KEYTRUDA plus chemotherapy followed by adjuvant KEYTRUDA as monotherapy showed a statistically significant and clinically meaningful improvement in EFS compared with neoadjuvant chemotherapy alone. As previously communicated, KEYNOTE-522 met its other dual primary endpoint of pathological complete response (pCR). The safety profile of KEYTRUDA in this trial was consistent with that observed in previously reported studies; no new safety signals were identified.

KEYTRUDA is the first immunotherapy to show positive results for event-free survival in patients with high-risk early-stage TNBC, a particularly aggressive form of breast cancer, said Dr. Roy Baynes, senior vice president and head of global clinical development, chief medical officer, Merck Research Laboratories. The improvement in pathological complete response rates initially observed following pre-operative treatment was encouraging, and now that we are seeing the data mature after four years to include a statistically significant improvement in event-free survival, we look forward to working with the FDA and other global authorities to bring this new option to patients as quickly as possible. We are grateful to the study participants who are critical to our efforts to advance potential treatment options for patients with TNBC.

An analysis of pCR from KEYNOTE-522 was presented at the European Society for Medical Oncology (ESMO) 2019 Congress and published in the New England Journal of Medicine. Findings showed a statistically significant increase in pCR for KEYTRUDA plus chemotherapy versus chemotherapy alone as neoadjuvant therapy in patients with early-stage TNBC, regardless of PD-L1 status. As previously announced, the company received a Complete Response Letter (CRL) from the FDA in March 2021 regarding Mercks supplemental Biologics License Application (sBLA) seeking approval for KEYTRUDA for the treatment of patients with high-risk early-stage TNBC based on these pCR data and early interim EFS findings. The CRL followed the FDAs Oncologic Drugs Advisory Committee meeting that voted 10-0 that a regulatory decision should be deferred until further data were available from KEYNOTE-522.

The KEYTRUDA clinical development program for TNBC encompasses several internal studies and external collaborative trials, including the ongoing studies KEYNOTE-242 and KEYNOTE-355.

Merck has an expansive clinical development program investigating KEYTRUDA in earlier lines of therapy including in neoadjuvant, adjuvant and locally advanced settings, with approximately 20 registrational studies ongoing.

About KEYNOTE-522

KEYNOTE-522 is a Phase 3, randomized, double-blind trial (ClinicalTrials.gov, NCT03036488), evaluating a regimen of neoadjuvant KEYTRUDA in combination with chemotherapy followed by adjuvant KEYTRUDA as monotherapy versus a regimen of neoadjuvant chemotherapy followed by adjuvant placebo. The dual primary endpoints are pCR and EFS. The secondary endpoints include pCR rate using alternative definitions (i.e., no invasive or noninvasive residual cancer in breast or nodes) at the time of definitive surgery, overall survival, EFS in patients whose tumors express PD-L1 (Combined Positive Score [CPS] 1), safety and patient-reported outcomes. The study enrolled 1,174 patients who were randomized 2:1 to receive either:

About Triple-Negative Breast Cancer (TNBC)

Triple-negative breast cancer is an aggressive type of breast cancer that characteristically has a high recurrence rate within the first five years after diagnosis. While some breast cancers may test positive for estrogen receptors, progesterone receptors or overexpression of human epidermal growth factor receptor 2 (HER2), TNBC tests negative for all three. Approximately 15-20% of patients with breast cancer are diagnosed with TNBC. TNBC tends to be more common in women who are younger than 40 years of age, who are African American or who have a BRCA1 mutation.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,400 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications in the U.S.

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL).

KEYTRUDA is indicated for the treatment of pediatric patients with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (CPS 10), as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

KEYTRUDA is indicated for the first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC).

Gastric Carcinoma

KEYTRUDA, in combination with trastuzumab, and fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the first-line treatment of patients with locally advanced unresectable or metastatic HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation either:

Cervical Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Triple-Negative Breast Cancer

KEYTRUDA, in combination with chemotherapy, is indicated for the treatment of patients with locally recurrent unresectable or metastatic triple-negative breast cancer (TNBC) whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test. This indication is approved under accelerated approval based on progression-free survival. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

KEYTRUDA is a monoclonal antibody that belongs to a class of drugs that bind to either the programmed death receptor-1 (PD-1) or the programmed death ligand 1 (PD-L1), blocking the PD-1/PD-L1 pathway, thereby removing inhibition of the immune response, potentially breaking peripheral tolerance and inducing immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue, can affect more than one body system simultaneously, and can occur at any time after starting treatment or after discontinuation of treatment. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions.

Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Early identification and management are essential to ensure safe use of antiPD-1/PD-L1 treatments. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue KEYTRUDA depending on severity of the immune-mediated adverse reaction. In general, if KEYTRUDA requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose adverse reactions are not controlled with corticosteroid therapy.

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis. The incidence is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.4% (94/2799) of patients receiving KEYTRUDA, including fatal (0.1%), Grade 4 (0.3%), Grade 3 (0.9%), and Grade 2 (1.3%) reactions. Systemic corticosteroids were required in 67% (63/94) of patients. Pneumonitis led to permanent discontinuation of KEYTRUDA in 1.3% (36) and withholding in 0.9% (26) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Pneumonitis resolved in 59% of the 94 patients.

Pneumonitis occurred in 8% (31/389) of adult patients with cHL receiving KEYTRUDA as a single agent, including Grades 3-4 in 2.3% of patients. Patients received high-dose corticosteroids for a median duration of 10 days (range: 2 days to 53 months). Pneumonitis rates were similar in patients with and without prior thoracic radiation. Pneumonitis led to discontinuation of KEYTRUDA in 5.4% (21) of patients. Of the patients who developed pneumonitis, 42% of these patients interrupted KEYTRUDA, 68% discontinued KEYTRUDA, and 77% had resolution.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis, which may present with diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (1.1%), and Grade 2 (0.4%) reactions. Systemic corticosteroids were required in 69% (33/48); additional immunosuppressant therapy was required in 4.2% of patients. Colitis led to permanent discontinuation of KEYTRUDA in 0.5% (15) and withholding in 0.5% (13) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Colitis resolved in 85% of the 48 patients.

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

KEYTRUDA can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.4%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 68% (13/19) of patients; additional immunosuppressant therapy was required in 11% of patients. Hepatitis led to permanent discontinuation of KEYTRUDA in 0.2% (6) and withholding in 0.3% (9) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Hepatitis resolved in 79% of the 19 patients.

KEYTRUDA with Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider monitoring more frequently as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased alanine aminotransferase (ALT) (20%) and increased aspartate aminotransferase (AST) (13%) were seen, which was at a higher frequency compared to KEYTRUDA alone. Fifty-nine percent of the patients with increased ALT received systemic corticosteroids. In patients with ALT 3 times upper limit of normal (ULN) (Grades 2-4, n=116), ALT resolved to Grades 0-1 in 94%. Among the 92 patients who were rechallenged with either KEYTRUDA (n=3) or axitinib (n=34) administered as a single agent or with both (n=55), recurrence of ALT 3 times ULN was observed in 1 patient receiving KEYTRUDA, 16 patients receiving axitinib, and 24 patients receiving both. All patients with a recurrence of ALT 3 ULN subsequently recovered from the event.

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

KEYTRUDA can cause primary or secondary adrenal insufficiency. For Grade 2 or higher, initiate symptomatic treatment, including hormone replacement as clinically indicated. Withhold KEYTRUDA depending on severity. Adrenal insufficiency occurred in 0.8% (22/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.3%) reactions. Systemic corticosteroids were required in 77% (17/22) of patients; of these, the majority remained on systemic corticosteroids. Adrenal insufficiency led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.3% (8) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Hypophysitis

KEYTRUDA can cause immune-mediated hypophysitis. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism. Initiate hormone replacement as indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Hypophysitis occurred in 0.6% (17/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.2%) reactions. Systemic corticosteroids were required in 94% (16/17) of patients; of these, the majority remained on systemic corticosteroids. Hypophysitis led to permanent discontinuation of KEYTRUDA in 0.1% (4) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Thyroid Disorders

KEYTRUDA can cause immune-mediated thyroid disorders. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism. Initiate hormone replacement for hypothyroidism or institute medical management of hyperthyroidism as clinically indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Thyroiditis occurred in 0.6% (16/2799) of patients receiving KEYTRUDA, including Grade 2 (0.3%). None discontinued, but KEYTRUDA was withheld in <0.1% (1) of patients.

Hyperthyroidism occurred in 3.4% (96/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (0.8%). It led to permanent discontinuation of KEYTRUDA in <0.1% (2) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. Hypothyroidism occurred in 8% (237/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (6.2%). It led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.5% (14) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. The majority of patients with hypothyroidism required long-term thyroid hormone replacement. The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC, occurring in 16% of patients receiving KEYTRUDA as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. The incidence of new or worsening hypothyroidism was higher in 389 adult patients with cHL (17%) receiving KEYTRUDA as a single agent, including Grade 1 (6.2%) and Grade 2 (10.8%) hypothyroidism.

Type 1 Diabetes Mellitus (DM), Which Can Present With Diabetic Ketoacidosis

Monitor patients for hyperglycemia or other signs and symptoms of diabetes. Initiate treatment with insulin as clinically indicated. Withhold KEYTRUDA depending on severity. Type 1 DM occurred in 0.2% (6/2799) of patients receiving KEYTRUDA. It led to permanent discontinuation in <0.1% (1) and withholding of KEYTRUDA in <0.1% (1). All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Immune-Mediated Nephritis With Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.1%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 89% (8/9) of patients. Nephritis led to permanent discontinuation of KEYTRUDA in 0.1% (3) and withholding in 0.1% (3) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Nephritis resolved in 56% of the 9 patients.

Immune-Mediated Dermatologic Adverse Reactions

KEYTRUDA can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome, drug rash with eosinophilia and systemic symptoms, and toxic epidermal necrolysis, has occurred with antiPD-1/PD-L1 treatments. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate nonexfoliative rashes. Withhold or permanently discontinue KEYTRUDA depending on severity. Immune-mediated dermatologic adverse reactions occurred in 1.4% (38/2799) of patients receiving KEYTRUDA, including Grade 3 (1%) and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 40% (15/38) of patients. These reactions led to permanent discontinuation in 0.1% (2) and withholding of KEYTRUDA in 0.6% (16) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 6% had recurrence. The reactions resolved in 79% of the 38 patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% (unless otherwise noted) in patients who received KEYTRUDA or were reported with the use of other antiPD-1/PD-L1 treatments. Severe or fatal cases have been reported for some of these adverse reactions. Cardiac/Vascular: Myocarditis, pericarditis, vasculitis; Nervous System: Meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy; Ocular: Uveitis, iritis and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada-like syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss; Gastrointestinal: Pancreatitis, to include increases in serum amylase and lipase levels, gastritis, duodenitis; Musculoskeletal and Connective Tissue: Myositis/polymyositis rhabdomyolysis (and associated sequelae, including renal failure), arthritis (1.5%), polymyalgia rheumatica; Endocrine: Hypoparathyroidism; Hematologic/Immune: Hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% of 2799 patients receiving KEYTRUDA. Monitor for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or Grade 2 reactions. For Grade 3 or Grade 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Fatal and other serious complications can occur in patients who receive allogeneic HSCT before or after antiPD-1/PD-L1 treatment. Transplant-related complications include hyperacute graft-versus-host disease (GVHD), acute and chronic GVHD, hepatic veno-occlusive disease after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between antiPD-1/PD-L1 treatment and allogeneic HSCT. Follow patients closely for evidence of these complications and intervene promptly. Consider the benefit vs risks of using antiPD-1/PD-L1 treatments prior to or after an allogeneic HSCT.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with an antiPD-1/PD-L1 treatment in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

Adverse Reactions

In KEYNOTE-006, KEYTRUDA was discontinued due to adverse reactions in 9% of 555 patients with advanced melanoma; adverse reactions leading to permanent discontinuation in more than one patient were colitis (1.4%), autoimmune hepatitis (0.7%), allergic reaction (0.4%), polyneuropathy (0.4%), and cardiac failure (0.4%). The most common adverse reactions (20%) with KEYTRUDA were fatigue (28%), diarrhea (26%), rash (24%), and nausea (21%).

In KEYNOTE-054, KEYTRUDA was permanently discontinued due to adverse reactions in 14% of 509 patients; the most common (1%) were pneumonitis (1.4%), colitis (1.2%), and diarrhea (1%). Serious adverse reactions occurred in 25% of patients receiving KEYTRUDA. The most common adverse reaction (20%) with KEYTRUDA was diarrhea (28%).

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Key Trends of Autologous Stem Cell Based Therapies Market 2021 Business Opportunities, Market Dynamics, Growth Size and Forecasts to 2026 – Clark…

By daniellenierenberg

New research report on Autologous Stem Cell Based Therapies market size by In4Research provides the latest developments in the market has attained and the latest news regarding the market and its building blocks. Some of the most accurate topics mentioned in the report include market drivers and restraints, challenges, opportunities, COVID-19 information related to the market, key players of the market, and the entire market segmentation.

This report provides data for the base year 2020 as well as the forecast period (2021-2026) along with the CAGR. This report contains detailed research of the market from the industry level to the market players and how the market is functioning at present along with all the relevant data and information. Thereby, the Autologous Stem Cell Based Therapies research report has been formulated encompassing all the key points that are present in the form of tables and graphs to make it more specific for decision-makers.

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The segmental analysis offered in the report pinpoints key opportunities available in the Autologous Stem Cell Based Therapies market through leading segments. The regional study of the Autologous Stem Cell Based Therapies market included in the report helps decision-makers to gain a sound understanding of the development of different geographical markets in recent years and going forth.

By Type:

By Applications:

By Region:

To comprehend Global Autologous Stem Cell Based Therapies market dynamics in the world mainly, the worldwide Autologous Stem Cell Based Therapies market is analyzed across major regions. A customized study by region and country can be provided considering the below splits.

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Major Companies included in the Autologous Stem Cell Based Therapies market:

The report provides in-depth information about profitable showing markets and examines the markets for the global Autologous Stem Cell Based Therapies market with business strategies of the key players and the new entering market industries are considered in detail and it presents the deep-dive eyesight of this Autologous Stem Cell Based Therapies market from 2021 to 2026 and prospective prediction market trends.

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Key Trends of Autologous Stem Cell Based Therapies Market 2021 Business Opportunities, Market Dynamics, Growth Size and Forecasts to 2026 - Clark...

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Global Autologous Stem Cell Based Therapies Market Survey Report, 2020-2027 KSU | The Sentinel Newspaper – KSU | The Sentinel Newspaper

By daniellenierenberg

From an insight perspective, this research report has focused on various levels of analysis industry trends analysis, top players analysis, company profiles, which discuss the basic views on the competitive landscape, emerging and high-growth segments of Autologous Stem Cell Based Therapies market, and high-growth regions. Besides, drivers, restraints, challenges, and opportunities pertaining to Autologous Stem Cell Based Therapies market are also predicted in this report.

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Major Participators LandscapeThese market players enjoyed broad industry coverage, outstanding operational ability, and strong financial resources. Manufacturers are focusing on product innovation, brand extension, and the introduction of new brands to cater to the preferences of consumers. Some of them will be endowed with vital future while others will show a weak growth during the prospective timeframe.Major market participators covered in our report are:US STEM CELL, INC. Med cell Europe Pluristem Therapeutics Inc Mesoblast Tigenix Brainstorm Cell Therapeutics Regeneus

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Autologous Stem Cell Based Therapies Application AbstractThe Autologous Stem Cell Based Therapies is commonly used into:Neurodegenerative Disorders Autoimmune Diseases Cardiovascular Diseases

Autologous Stem Cell Based Therapies Type AbstractBased on the basis of the type, the Autologous Stem Cell Based Therapies can be segmented into:Embryonic Stem Cell Resident Cardiac Stem Cells Umbilical Cord Blood Stem Cells

Table of Content1 Report Overview1.1 Product Definition and Scope1.2 PEST (Political, Economic, Social and Technological) Analysis of Autologous Stem Cell Based Therapies Market2 Market Trends and Competitive Landscape3 Segmentation of Autologous Stem Cell Based Therapies Market by Types4 Segmentation of Autologous Stem Cell Based Therapies Market by End-Users5 Market Analysis by Major Regions6 Product Commodity of Autologous Stem Cell Based Therapies Market in Major Countries7 North America Autologous Stem Cell Based Therapies Landscape Analysis8 Europe Autologous Stem Cell Based Therapies Landscape Analysis9 Asia Pacific Autologous Stem Cell Based Therapies Landscape Analysis10 Latin America, Middle East & Africa Autologous Stem Cell Based Therapies Landscape Analysis 11 Major Players Profile

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Major countries of North America, Europe, Asia Pacific, and the rest of the world are all exhaustive analyzed in the report. Apart from this, policy mobilization, social dynamics, development trends, and economic development in these countries are also taken into consideration.

Target Audience for this Report Autologous Stem Cell Based Therapies manufacturers Autologous Stem Cell Based Therapies traders, distributors, and suppliers Autologous Stem Cell Based Therapies industry associations Product managers, Autologous Stem Cell Based Therapies industry administrator, C-level executives of the industries Market Research and consulting firms Research & Clinical Laboratories

Report SpotlightsDetailed overview of marketChanging market dynamics in the industryIn-depth market segmentationHistorical, current and projected market size in terms of volume and valueRecent industry trends and developmentsCompetitive landscapeStrategies of key players and products offeredPotential and niche segments, geographical regions exhibiting promising growthA neutral perspective on market performanceMust-have information for market players to sustain and enhance their market footprints

About Global Market MonitorGlobal Market Monitor is a professional modern consulting company, engaged in three major business categories such as market research services, business advisory, technology consulting.We always maintain the win-win spirit, reliable quality and the vision of keeping pace with The Times, to help enterprises achieve revenue growth, cost reduction, and efficiency improvement, and significantly avoid operational risks, to achieve lean growth. Global Market Monitor has provided professional market research, investment consulting, and competitive intelligence services to thousands of organizations, including start-ups, government agencies, banks, research institutes, industry associations, consulting firms, and investment firms.ContactGlobal Market MonitorOne Pierrepont Plaza, 300 Cadman Plaza W, Brooklyn,NY 11201, USAName: Rebecca HallPhone: + 1 (347) 467 7721Email: info@globalmarketmonitor.comWeb Site: https://www.globalmarketmonitor.com

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Kaytlyn Gerbin is blazing trails in cell science and as an ultrarunner who has conquered Mount Rainier – GeekWire

By daniellenierenberg

Kaytlyn Gerbin, left, runs the Wonderland Trail around Mount Rainier. She completed the 93-mile loop in just under 19 hours. Her friend Tara Fraga helped with pacing between miles 30-55. (Ryan Thrower Photo)

When Kaytlyn Gerbin moved to Seattle 10 years ago to attend graduate school at the University of Washington, a friend took her to Kerry Park in the Queen Anne neighborhood on her first visit. The celebrated viewpoint offered Gerbin a glimpse of Mount Rainier that ignited an ongoing passion.

At the time, I had absolutely no idea there was a trail all the way around it, and didnt know the first thing that went into climbing to the summit or running even a few miles on the trails, Gerbin said. Since then, Ive climbed Rainier 10 times, and spent countless hours on the mountain and trails in that park.

Along with her drive to get to know Washington states most famous landmark more intimately, Gerbin achieved her PhD in bioengineering at UW, where her research was focused on the therapeutic and regenerative potential of cardiac cells. For the past four years shes been a scientist at Allen Institute for Cell Science, where she studies stem cells and cardiomyocytes, or cardiac muscle cells.

Our latest Geek of the Week, Gerbin is an accomplished ultrarunner, and she now knows a lot more about that trail that encircles Mount Rainier.

With COVID-19 lockdowns impacting her international race season last summer, Gerbin, a sponsored athlete for The North Face, went after the fastest known time, or FKT, for a run around the Wonderland Trail. Together with teammate Dylan Bowman of Portland and a small crew of local filmmakers, they made Summer of Wonder, a short film about the experience, which you can watch in full here:

The average thru-hiker takes 10-14 days to complete the 93-mile Wonderland Trail, with its 24,000 feet of elevation gain. Gerbin did it in 18 hours, 41 minutes, 53 seconds, and the film is a breathtaking look at her endurance feat.

Gerbins passion for running started with 3-mile commutes back and forth between her apartment, her research lab, and campus during grad school. Eventually she started trail running,essentially as a life hack to see if she could squeeze a five-day backpacking route into a weekend between experiments.

It turned out I was actually pretty good at that, and that opened up opportunities to start racing at some of the most competitive trail races in the U.S. and Europe, Gerbin said.

Shes since raced with Team USA at the Trail World Championships, reached the podium at the iconic Western States 100, and won races such as the Canary Islands Transgrancanaria and Cascade Crest 100 in Washington. She also still holds the womens self-supported FKT for the Rainier Infinity Loop (set in 2019), which combines the Wonderland Trail with two summits and descents of Mount Rainier.

Her preferred racing distance is anything between 50-100 miles long, the more elevation gain and technical the trail, the better. During peak training, Gerbin is usually hitting between 70-90 miles with over 20,000 feet of elevation gain each week. She calls the Pacific Northwest the best outdoor playground there is.

Although I love running fast, Im also really excited about pushing myself on more challenging terrain. So many of my other FKT goals and route ideas are along these lines, with more technical traveling than actual running, she said.

COVID permitting, her highest race priority this year is Ultra Trail du Mont Blanc, which is the most competitive world-stage for ultrarunning, at the end of August. The race circumnavigates Mont Blanc, passing through France, Italy, and Switzerland and covering around 105 miles and 33,000 feet of elevation gain.

While Gerbins experience as a scientist does inform her appreciation for what shes putting her body through during ultrarunning, shes equally passionate in the lab. At the Allen Institute shes seeking answers to broad questions about how cells work, including how single cells and all of their components are integrated into a functional system, while using imaging to build predictive models of cell behavior.

I get the opportunity to work with a multidisciplinary team of badass scientists, biologists, and engineers on really cool problems in cell biology, she said.

Learn more about our latest Geek of the Week, Kaytlyn Gerbin:

What do you do, and why do you do it? Science and ultrarunning for me have always come down to problem solving.

As a scientist, problem solving is inherent to experimental design, data analysis, and interpreting results. By asking hard questions, Im interested in pushing the field of cell biology forward, and challenging the current way of thinking.

As an ultrarunner, its a different kind of problem solving, but I lean on the same mindset to figure out how to push my athletic limits further and faster.

One thing that always amazes me is how adaptable the human body is. My training in cell science gives me context for how all of these stressors and inputs were putting on our bodies are fundamentally happening at the single cell level, and it keeps me thinking about the cells response to external cues in my research.

Whats the single most important thing people should know about your field? Yes, I do think about science and when Im running, and no, I do not geek out on heart rate monitors and training zones and all those numbers when Im running.

Where do you find your inspiration? Im inspired by brilliant women that are pushing whats possible in both science and in sports. I think we often set boundaries for ourselves about what we think is possible, without ever letting ourselves really hit that limit. Im inspired by women who set bold goals and bring others up and along for the ride, redefining whats possible.

Whats the one piece of technology you couldnt live without, and why? My Garmin 935. I use this watch daily to track miles run, elevation gain, etc. The battery life has lasted me for 100 miles of running and ~24 hrs, but its small enough to wear every day.

Whats your workspace like, and why does it work for you? Prior to 2020, I was splitting my time between the tissue culture hood (passaging cells, differentiating cardiomyocytes, setting up experiments), conference rooms (team science and collaboration means a lot of group discussions!), and my computer for writing and analysis. Since then, Ive shifted my work to be more remote while I work on a few different manuscripts. I have an office set up at home with a window, some good tunes, plenty of coffee, and a chair for my dog to wait impatiently on.

Your best tip or trick for managing everyday work and life. (Help us out, we need it.) I have always been a to-do list person. Most mornings start with me listing out tasks (and breaking those down into many sub-tasks). I feel productive as I cross things off, and it also helps me prioritize and plan ahead to make sure I can also fit my training runs in.

Mac, Windows or Linux? Mac as a personal preference, Windows for my work computer (I do work at the Paul Allen Institute 🙂

Transporter, Time Machine or Cloak of Invisibility? Transporter. I just promise not to use it in races.

Greatest game in history: Lode Runner. I havent played it since I was a kid, but the memories of yelling at the computer with my sister frantically hitting up-down-up-down arrows make me feel like it was just yesterday.

Best gadget ever: Garmin inReach mini satellite messaging and SOS call, all in a device small enough to throw in the bottom of a pack (or shorts pocket) and forget its there. I bring this with me anytime Im headed out into the wilderness/mountains, but I hope I never need to use it.

First computer: iMac G3.

Current phone: iPhone 11.

Favorite app: I have a love/hate relationship with Strava. Ive also been using DuoLingo during the pandemic and have a strong daily streak going!

Most important technology of 2021: COVID vaccines!!

Most important technology of 2023: Advancements in remote/low-resource medical care.

Final words of advice for your fellow geeks: Most problems can be solved with more snacks and some time (works for science and running).

Twitter: @kaytlyn_gerbin

LinkedIn: Kaytlyn Gerbin

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Kaytlyn Gerbin is blazing trails in cell science and as an ultrarunner who has conquered Mount Rainier - GeekWire

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Durable B-ALL Control With Allogeneic Transplant After CAR T-Cell Therapy – Cancer Therapy Advisor

By daniellenierenberg

Children and young adults who underwent an allogeneic hematopoietic stem cell transplant (alloHSCT) after achieving complete response with CD19 CAR T-cell therapy experienced durable B-cell acute lymphoblastic leukemia (B-ALL) control, according to the results of a phase 1 trial (ClinicalTrials.gov Identifier: NCT01593696) published in the Journal of Clinical Oncology.

Although a proportion of patients who undergo CAR T-cell therapy go on to receive alloHSCT, the study authors stated that The role for [alloHSCT] following CD19-CAR T-cell therapy to improve long-term outcomes in [children and young adults] has not been examined.

The phase 1 trial evaluated 50 children and young adults with B-ALL who received CD19.28 CAR T-cell therapy. The primary objective was to determine the maximum tolerated dose of CAR T cells, toxicity, and feasibility of generating CAR T cells in the study population. In addition, this analysis retrospectively evaluated the effect of alloHSCT on survival after CAR T-cell therapy.

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At baseline, the median age was 13.5 years (range, 4.3-30.4), and 40 (80%) of the patients were male. The median number of prior regimens was 4 (range, 4.3-30.4); 22 (44%) patients had at least 1 prior HSCT, 2 (4%) had prior CD19-targeted therapy, and 5 (10%) of the patients had prior treatment with blinatumomab.

Complete response was achieved in 31 (62%) of the patients. Among these patients, 28 (90.3%) were negative for minimal residual disease. Higher rates of complete response were associated with primary refractory disease, fewer prior lines of therapy, M1 marrow, or fludarabine/cytarabine-based lymphodepletion. The median overall survival was 10.5 months (95% CI, 6.3-29.2) during a median follow-up of 4.8 years.

Of the 28 patients who achieved complete response, 21 (75%) proceeded to undergo consolidative alloHSCT. The median overall survival for these patients was 70.2 months (95% CI, 10.4-not estimable), with an event-free survival not yet reached. The rate of relapse after alloHSCT was 4.8% (95% CI, 0.3-20.3) at 12 months and 9.5% (95% CI, 1.5-26.8) at 24 months.

Any grade cytokine release syndrome (CRS) developed among 35 (70%) patients, with 9 (18%) experiencing grade 3 to 4 CRS. Of the 10 patients (20%) who developed neurotoxicity, 4 cases were severe. One cardiac arrest occurred during CRS. All patients with CRS, neurotoxicity, and cardiac arrest recovered.

The authors concluded that CD19.28 CAR T cells followed by a consolidative alloHSCT can provide long-term durable disease control in [children and young adults] with relapsed or refractory B-ALL.

Disclosure: Please see the original reference for a full disclosure of authors affiliations.

Reference

Shah NN, Lee DW, Yates B, et al. Long-term follow-up of CD19-CAR T-cell therapy in children and young adults with B-ALL. J Clin Oncol. Published online March 25, 2021. doi:org/10.1200/JCO.20.02262c

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The Physiological Challenges of Spaceflight – Cambridge Wireless

By daniellenierenberg

By Guest Blogger Rich Whittle, Bioastronautics & Human Performance Lab at Texas A&M UniversityThe recent landing of the probe Perseverance on Mars, and the excitement generated by the high-resolution images currently being broadcast back to Earth, has inevitably started people thinking about human exploration of the Red Planet. However, the challenges faced by a manned journey to Mars are much more than just technical, but reflect some fundamental aspects of human physiology. In this guest article, Rich Whittle of the Bioastronautics and Human Performance Lab at Texas A&M University, reflects on some of the key issues.

NASA has ambitious plans to begin manned exploration of Mars, although its current focus is on sending the next man and first woman to the Moon as part of the Artemis programme, which will establish a permanent human presence there in the coming decade. A key part of this latter objective is to place a spaceship called Gateway in orbit around the Moon, from which landers will take astronauts to the surface and support their activities. Gateway will also conduct a wide variety of human and scientific missions, and in particular study the physiological effects of long journeys into space, in preparation for that first manned voyage to Mars.

The human body has evolved over hundreds of thousands of years to flourish on the surface of the Earth, and it is perhaps not surprising that the stresses of spaceflight pose unique physiological and medical problems. In fact, many of the basic issues associated with spaceflight, such as hypoxia, dysbarism, acceleration, and thermal support, have been well studied through aviation and diving medicine in the years prior to spaceflight. But while the last 50 years of manned space exploration have shown that humans can adapt to space, remaining productive for up to 1 year and possibly longer, there are still many problems associated with the prolonged exposure to a unique combination of stressful stimuli including acceleration, radiation, and weightlessness. The latter condition is a critical feature of spaceflight and has significant effects on human physiology, many of which were quite unexpected at the beginning of space exploration.

Scientists have known for a long time that the human body responds in specific ways to the microgravity environment of spaceflight. For example, a person who is inactive for an extended period loses overall strength, as well as muscle and bone mass. Unsurprisingly spaceflight has a similar effect, resulting in loss of bone mineral density (BMD), and increasing the risk of bone fractures in astronauts. It is predicted that a third of astronauts will be at risk for osteoporosis during a predicted 7-month long human mission to Mars. It is however possible to compensate for this loss of muscle and bone mass using resistive exercise devices that NASA has developed to allow for more intense workouts in zero gravity.

Overall, the pathophysiological adaptive changes that occur during spaceflight, even in well-trained, highly selected, and healthy individuals, have been likened to an accelerated aging process, and are being studied in research groups around the world. My own research at Texas A&M focuses on changes to the cardiovascular system caused by the microgravity environment of spaceflight. In an upright position under the Earths standard 1G gravity, arterial blood pressure is lower above the heart and higher below the heart. But in a weightless environment the body experiences a uniform arterial pressure, which decreases the cardiac workload, and reduces the need for blood pressure regulatory mechanisms. As a result, the muscles of the heart and blood vessels begin to atrophy, and consequently some astronauts experience orthostatic intolerance, the difficulty or inability to stand because of light headedness after return to Earth. During spaceflight, cardiovascular changes are noticeable immediately after the onset of weightlessness, with astronauts exhibiting characteristically puffy faces, stuffed noses, and chicken legs, as approximately 2L of fluid is shifted from the legs towards the head.

These fluid shifts affect not only the cardiovascular system but also the brain, eyes, and other neurological functions. The apparent increase in fluid within the skull is potentially linked to a collection of pathologies of the eye known as Spaceflight Associated Neuro-ocular Syndrome (SANS). This is principally manifested through a hyperopic shift in visual acuity, which in some cases does not resolve on return to Earth.

We believe that many of these problems can be overcome through effective countermeasures during spaceflight, and are often reversible after landing. Physical exercise programs are the main countermeasure used during spaceflight to protect the cardiovascular system. The technology involved has advanced from a rowing ergometer used in the early Skylab missions, through a motorized treadmill used in the ISS. This has been recently joined by a device for performing resistive exercise, and now rowing ergometers are once again being looked at for longer duration missions to Mars due to their small footprint.

However, some astronauts have returned from the ISS with unexpectedly stiff arteries, of a magnitude expected from 10 20 years of normal aging. Arterial stiffening is often linked to an increased blood pressure and elevated risk for cardiovascular disease. Additionally, other studies have suggested that insulin resistance occurs during spaceflight, possibly due to reduced physical activity, which could lead to increased blood sugar and increased risk of developing type 2 diabetes. These results suggest that the astronauts exercise routine did not always counteract the effect of the microgravity environment and indicated that further countermeasures might be needed to help maintain astronaut health. Here at Texas A&M we are looking at both lower body negative pressure (LBNP) and artificial gravity generated through short radius centrifugation as exciting new countermeasures that could be used in long duration spaceflight.

As we begin to further understand the effects of spaceflight on human physiology, scientists are now starting to study some of the underlying cellular mechanisms using model organisms, cell cultures, organs on a chip and stem cells. And because many of the observed changes seen in space, such as cardiovascular dysfunction due to inflammation, lack of exercise, intracranial hypertension, and hormonal and metabolic changes, resemble those caused by aging or illnesses, the research we conduct may have important applications on Earth. Hopefully, our push for manned exploration of the planets of the solar system will lead to tangible benefits to the health and well-being of humans on our home planet.

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5 Novel Therapies Using Synthetic Biology – Nanalyze

By daniellenierenberg

The 1997 film Gattaca promised a future where humans would be free of disease and babies born on demand with the latest upgrades, including enhanced speed, intelligence, and beauty. Much like a new Tesla Roadster. However, despite the technological predictions offered by Hollywood moviemakers, were still living in a time when synthetic biology is working hard to make a dent in the world. No designer babies in sight. And stem cell technology promised so many radical breakthroughs back in the late 1990s, including growing organs for transplants and regenerating whole body parts, but the challenge of growing whole organs has been shown to be more complex than previously believed, including technologies like 3D bioprinting and xenotransplantation.

Despite the challenges and setbacks, investors believe were living in a different time, with more money pouring into the space over the last few years:

Indeed, the science and technology behind manipulating biological matter are still promising when it comes to health and medicine, especially with the rise of CRISPR gene editing. The idea that we could potentially switch on or off genes that cause disease using a cocktail of enzymes is just fantastical. While inserting CRISPR enzymes into a live human being is a bit challenging, there are regions of the body that are easily accessible, such as the eye. In a landmark clinical trial approved by the FDA and led by Editas Medicine (EDIT) and Allergan, now owned by AbbVie (ABBV), a CRISPR-Cas9 gene therapy was administered directly to patients to remove rare mutations that can cause childhood blindness.

McKinsey is calling this emerging technological renaissance the next Bio Revolution, with advances in biological sciences being accelerated by automation and artificial intelligence. The speed at which scientists and researchers were able to sequence the genome of the Rona virus is a testament to the power of these converging technologies. McKinsey predicts that synthetic biology could have a direct economic impact of $4 trillion per year, nearly half of which will be in the domain of human health.

Lets take a look through five companies that are harnessingthe revolutionary power of synthetic biology to design new therapies and treathuman diseases.

Founded in 2017 and headquartered in Alameda, California, Scribe Therapeutics is a biotechnology startup that is producing therapeutics using custom-engineered CRISPR enzyme technology. The company has raised a whopping $120 million from the likes of Andreessen Horowitz to build out a suite of CRISPR technologies designed to treat genetic diseases. Scribe Therapeutics was co-founded by Dr. Jennifer Doudna, the UC Berkeley biochemist who discovered and developed CRISPR gene-editing technology and won the Nobel Prize in Chemistry in 2020 for her pioneering work.

The team at Scribe Therapeutics has designed its XEditing (XE) technology by evolving the native CRISPR gene-editing enzymes available to us to redesign and engineer them to suit different needs. More specifically, they want to be able to modify or silence the genes of live humans to treat genetic diseases such as Huntingtons, Parkinsons, Sickle Cell Anemia, and Amyotrophic Lateral Sclerosis (ALS). Anything your parents unwittingly handed down to you, Scribe Therapeutics is looking to treat it. The research team tests thousands of redesigned enzymes and selects those with greater editing ability, specificity, and stability compared to current enzymes. Scribe Therapeutics is starting with a pipeline of therapeutics to treat neurodegenerative diseases and has its sights set on other, less common genetic conditions down the road.

Canadian biotechnology startup Notch Therapeutics was founded in 2018 and has raised $86 million to develop immune cell therapies against pre-cancer cells. The companys cell therapies are based on induced pluripotent stem cells (iPSC), which are pre-differentiated cells with the limited capacity to transform into different mature cell lines. Based on its Engineered Thymic Niche (ETN) platform, the company is developing universally compatible stem cell-derived immune cell therapies.

Normally, human immune cells only recognize othercells found in the same individual and will target cells from other individuals,which appear foreign to the immune system. Thats why donor organs can sometimesbe rejected by the recipients body the immune system sees the organ as a foreignobject. Notch Therapeutics is designing a system where the immune cellsproduced from the stem cells will be universally recognizable by allindividuals, bypassing the need to create immune cells from pluripotent stemcells derived from each recipient. These manufactured immune cells, whichinclude T cells or natural killer cells, can be programmed to target cancercells and eliminate them from the patient.

Founded in 2016, Massachusetts-based bit.bio is a synthetic biology startup thats working on merging the world of coding with biology. The company has secured $42 million after a Series A round that was completed in June 2020. A spinout of Cambridge University, bit.bio is looking to commercialize its proprietary platform, opti-ox, which can reprogram human stem cells to do its bidding cure diseases. Touted as the Cell Coding Company, bit.bio was founded by Dr. Mark Kotter, a neurosurgeon at the University of Cambridge who studied regenerative medicine and stem cell technology.

While the ability to program mammalian stem cells has been around since 1981, the company claims it can consistently reprogram human adult cells into pluripotent stem cells, and then transform them into other mature human cells within days. Currently, stem cell technology produces a statistical mixed bag of mature, differentiated cells, some of which can have potential side effects. opti-ox uses a precise combination of transcription factors to ensure stem cells mature into cardiac, muscle, liver, kidney, or lung cells with high efficiency. The holy grail for the company is to be able to produce every cell in the human body for any cell therapy safely, on-demand, and with purities approaching 100%. And well be here, waiting for that stem cell therapy for erectile dysfunction promised by the medical community.

Founded in 2020, Delonix Bioworks is a Shanghai-based synthetic biology company designing therapeutic solutions against infectious diseases. The startup received $14 million from a Seed round just back in March. The Delonix Bioworks team is focusing its initial efforts on anti-microbial resistant (AMR) infections. The emergence of resistance in some bacteria species against common antimicrobial compounds, so has led to an increasing number of infections that are difficult to treat with conventional strategies. These superbug strains are mostly spread in hospital or clinical settings due to the overuse of antibiotics.

The company is engineering attenuated, live bacteria thatcan act as vaccines against these types of infections. By introducing reprogrammed,but weakened, bacteria to express specific antigens on the surface of theirmembrane that match those of the strains that cause AMR infections into anindividual, the individuals immune system can recognize those antigens andrespond to future infections with greater speed. Its no different from how antiviralvaccines are designed, except most vaccines introduce an attenuated or inactivatedvirus to activate the immune system instead. And for those of you who skipped highschool biology, no, this is not a mind-control scheme orchestrated by biotech companies.

Founded in 2018, Octarine Bio is a Danish synthetic biology company thats building out a pipeline for high-potency cannabinoids and psilocybin derivatives for the pharmaceutical industry. Octarine Bio has brought in $3 million after a Seed round that was also completed in March. Medical studies on psychotropic compounds have been shown to help reduce anxiety, depression, and pain, and may have the potential to serve as novel psychiatric medications. A few companies have recently emerged to commercialize existing psychedelics. Octarine Bio believes it can do better by harnessing the power of synthetic biology to engineer microorganisms to produce these psychotropic compounds with better pharmacokinetic and therapeutic effects.

Normally, natural products are produced by plant and fungal species as an ill-defined mixture. The psychoactive properties of these compounds primarily stem from only a handful of compounds because their natural concentration is much higher than other derivatives in the organic material. For example, tetrahydrocannabinol (THC) is the main psychoactive agent in marijuana while psilocybin is the one found in mushrooms from the Psilocybe and other psilocybin-producing genera. However, these are just a few out of hundreds of potential psychoactive derivatives produced by these species.

Molecular derivatives may be produced at too low of concentration to test and analyze, or the plant or mushroom may have a deactivated metabolic pathway that could lead to a superior compound. By tweaking the molecular structure of the product compounds using both synthetic biology and traditional organic chemistry, the team at Octarine Bio is creating a platform to discover new potential therapeutics that may not have been available before. Magic mushrooms are about to get an upgrade for an extra potent trip.

Much like what was said about software by Marc Andreessen back in 2011, synthetic biology is starting to eat the world. While were a long way away from a dystopian future where babies are engineered with supernatural talents, were already seeing the potential side-effects of using CRISPR on the Chinese twin girls originally to immunize them from HIV, including enhanced cognition and memory. The cure for stupid is possibly lurking in the vaults of this pioneering technology. For now, well wait and see how synthetic biology and CRISPR gene editing shape up as potential therapeutics for real diseases.

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Australian scientists discover secret switch for the heart to heal itself – The New Daily

By daniellenierenberg

Cut off a piece of a zebrafish heart, and the little creature wont be at the top of its game for a few days.

But after a month, the heart will grow back to normal and life goes on as normal.

Given that a heart attack in humans known as a myocardial infarction is akin to losing a piece of your heart (because tissue dies), scientists for years have been trying to understand how zebrafish heal themselves, with a view to replicating the process in people.

Now, scientists at the Victor Chang Cardiac Research Institute have identified the genetic switch in zebrafish that prompts heart cells to divide and multiply after a heart attack, resulting in the complete regeneration and healing of damaged heart muscle in these fish.

Dr Kazu Kikuchi, who led the research, published in Science on Friday, said he was astonished by the findings.

Our research has identified a secret switch that allows heart muscle cells to divide and multiply after the heart is injured, Dr Kikuchi said in a statement.

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It kicks in when needed and turns off when the heart is fully healed. In humans where damaged and scarred heart muscle cannot replace itself, this could be a game changer.

The researchers investigated a critical gene known as Klf1, which previously had only been identified in red blood cells.

They discovered Klf1 plays a vital role in healing damaged hearts.

The gene works by making uninjured heart muscle cells called cardiomyocytes more immature and changing their metabolic wiring, a process called dedifferentiation.

This allows them to divide and make new cells

Cardiomyocytes are the heart cells primarily involved in the contractile function of the heart that enables the pumping of blood around the body.

Ordinarily, adult mammalian hearts have a limited ability to generate new cardiomyocytes whereas zebrafish will keep making new cells until their hearts are completely healed.

Its been known for more than a decade that cardiomyocytes become more youthful in order to regenerate and Dr Kikuchi was one of the researchers to demonstrate this.

What wasnt known was how this was made to happen.

Our new paper suggests it is Klf1 which triggers this, Dr Kikuchi said.

This isnt the same as stem cell technology. In fact, dedifferentiating cardiomyocytes has proved to be a more effective healing process than stem cells.

It all comes down to that genetic switch.

Dr Kikuchi said that when the gene was removed, the zebrafish heart lost its ability to repair itself after an injury such as a heart attack, which pinpointed it as a crucial self-healing tool.

Professor Bob Graham, head of the Institutes Molecular Cardiology and Biophysics Division, says this world-first discovery made in collaboration with the Garvan Institute of Medical Research may well transform the treatment of heart attack patients and other heart diseases.

The team has been able to find this vitally important protein that swings into action after an event like a heart attack and supercharges the cells to heal damaged heart muscle. Its an incredible discovery, Professor Graham said.

The gene may also act as a switch in human hearts. We are now hoping further research into its function may provide us with a clue to turn on regeneration in human hearts, to improve their ability to pump blood around the body.

Importantly, the team also found the Klf1 gene played no role in the early development of the heart and that its regenerative properties were only switched on after a heart injury.

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Xenobots 2.0 are Here and Still Developed With Frog Stem Cells – Unite.AI

By daniellenierenberg

The same team of biologists and computer scientists from Tufts University and the University of Vermont that created the Xenobots last year have now developed Xenobots 2.0. Last years version were novel, tiny self-healing biological machines created from frog cells, and they could navigate, push payloads, and act as a collective unit in some cases.

The new Xenobots 2.0 are life forms that can self-assemble a body from single cells. They do not require muscles to move, and they have even demonstrated recordable memory. Compared to their previous counterparts, the new bots move faster, navigate even more environments, and have longer lifespans. At the same time, they can still work together and heal themselves when damaged.

The new research was published in Science Robotics.

With the Xenobots 1.0, the millimeter-sized automations were constructed top down, with the manual placement of tissue and surgical shaping of frog skin and cardiac cells, which produces motion. With the new version of the technology, they were constructed bottom up.

Stem cells were taken from the embryos of the African frog called Xenopus laevis, and this enabled them to self-assemble and grow into spheroids. After a few days, the cells differentiated and produced cilia that moved back and forth or rotated in a specific way.

These cilia provide the new bots with a type of legs that enables them to rapidly travel across surfaces. In the biological world, cilia, or tiny hair-like projections, are often found on mucous surfaces like the lungs. They help by pushing out foreign material and pathogens, but in the Xenobots, they offer rapid locomotion.

Michael Levin is a Distinguished Professor of Biology and director of the Allen Discovery Center at Tufts University. He is the corresponding author of the study.

We are witnessing the remarkable plasticity of cellular collectives, which build a rudimentary new body that is quite distinct from their default in this case, a frog despite having a completely normal genome, said Levin. In a frog embryo, cells cooperate to create a tadpole. Here, removed from that context, we see that cells can re-purpose their genetically encoded hardware, like cilia, for new functions such as locomotion. It is amazing that cells can spontaneously take on new roles and create new body plans and behaviors without long periods of evolutionary selection for those features.

Senior scientist Doug Blackiston was co-first author of the study along with research technician Emma Lederer.

In a way, the Xenobots are constructed much like a traditional robot. Only we use cells and tissues rather than artificial components to build the shape and create predictable behavior. said Blackiston On the biology end, this approach is helping us understand how cells communicate as they interact with one another during development, and how we might better control those interactions.

Over at UVM, the scientists were developing computer simulations that modeled different shapes of the Xenobots, which helped identify any different behaviors that were exhibited in both individuals and groups. The team relied on the Deep Green supercomputer cluster at UVMs Vermont Advanced Computing Core.

Led by computer scientists and robotics expert Josh Bongard, the team came up with hundreds of thousands of environmental conditions through the use of an evolutionary algorithm. The simulations were then used to identify Xenobots that could work together in swarms to gather debris in a field of particles.

We know the task, but its not at all obvious for people what a successful design should look like. Thats where the supercomputer comes in and searches over the space of all possible Xenobot swarms to find the swarm that does the job best, says Bongard. We want Xenobots to do useful work. Right now were giving them simple tasks, but ultimately were aiming for a new kind of living tool that could, for example, clean up microplastics in the ocean or contaminants in soil.

The new version of the bots are faster and more efficient at tasks like garbage collection, and they can now cover large flat surfaces. The new upgrade also includes the ability for the Xenobot to record information.

The most impressive new feature of the technology is the ability for the bots to record memory, which can then be used to modify its actions and behaviors. The newly developed memory function was tested and the proof of concept demonstrated that it could be extended in the future to detect and record light, the presence of radioactive contamination, chemical pollutants, and more.

When we bring in more capabilities to the bots, we can use the computer simulations to design them with more complex behaviors and the ability to carry out more elaborate tasks, said Bongard. We could potentially design them not only to report conditions in their environment but also to modify and repair conditions in their environment.

The new version of the robots are also able to self-heal very efficiently, demonstrating that they are capable of closing the majority of a severe full-length laceration half their thickness within just five minutes.

The new Xenobots carry over the ability to survive up to ten days on embryonic energy stores, and their tasks can be carried out with no additional energy sources. If they are kept in various different nutrients, they can continue at full speed for months.

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Scientists Create Living Machines That Move, Heal, Remember and Work in Groups – Genetic Engineering & Biotechnology News

By daniellenierenberg

Scientists have created small, synthetic living machines that self-organize from single cells, move quickly through different environments without the need for muscle cells, can remember their experiences, heal themselves when damaged, and exhibit herd behaviors.

Earlier, scientists have developed swarms of robots from synthetic materials and moving biological systems from muscle cells grown on precisely shaped scaffolds. But until now the creation of a self-directed living machine has remained beyond reach.

Biologists and computer scientists from Tufts University and the University of Vermont have created novel, tiny self-healing living machines from frog cells (Xenopus laevis) that they call Xenobots. These can move around, push a payload, and even exhibit collective behavior in a swarm.

In an article titled A cellular platform for the development of synthetic living machinespublished in the journal Science Robotics, the researchers report a method for creating these of Xenobots from frog cells. This cellular platform can be used to study self-organization, collective behavior, and bioengineering and provide versatile, soft-body, living machines for applications in biomedicine and environmental biology.

The next version of Xenobots have been createdtheyre faster, live longer, and can now record information [Doug Blackiston, Tufts University]

We are witnessing the remarkable plasticity of cellular collectives, which build a rudimentary new body that is quite distinct from their defaultin this case, a frog despite having a completely normal genome, says Michael Levin, Distinguished Professor of Biology and director of the Allen Discovery Center at Tufts University, and corresponding author of the study. In a frog embryo, cells cooperate to create a tadpole. Here, removed from that context, we see that cells can re-purpose their genetically encoded hardware, like cilia, for new functions such as locomotion. It is amazing that cells can spontaneously take on new roles and create new body plans and behaviors without long periods of evolutionary selection for those features.

Xenobots can move around in a coordinated manner with the help of cilia present on their surface. These cilia grow through normal tissue patterning and do not require complicated architectural procedures such as scaffolding or microprinting, making the high-throughput production of Xenobots possible. And while the frog cells are organizing themselves into Xenobots, they are amenable to surgical, genetic, chemical, and optical stimulation. The researchers show that the Xenobots can maneuver through water, heal after damage and exhibit predictable collective behaviors.

The scientists also provide a proof of principle for a programmable molecular memory using a light-controlled protein that can record exposure to a specific wavelength of light.

Compared to their first edition, Xenobots 1.0, that were millimeter-sized automatons constructed in a top down approach by manual placement of tissue and surgical shaping of frog skin and cardiac cells to produce motion, this updated version of Xenobots 2.0 takes a bottom up approach. The biologists took stem cells from frog embryos and allowed them to self-assemble and grow into spheroids, where some of the cells after a few days differentiated to produce ciliatiny hair-like projections that move back and forth or rotate in a specific way. Cilia act like legs to help the new spheroidal Xenobots move rapidly across a surface.

In a way, the Xenobots are constructed much like a traditional robot. Only we use cells and tissues rather than artificial components to build the shape and create predictable behavior. Says Doug Blackiston, PhD, senior scientist and co-first author on the study with research technician, Emma Lederer. On the biology end, this approach is helping us understand how cells communicate as they interact with one another during development, and how we might better control those interactions.

Scientists at UVM ran computer simulations that modeled different shapes of the Xenobots and analyzed its effects on individual and collective behavior. Robotics expert, Joshua Bongard, PhD, and a team of computer scientists used an evolutionary algorithm on the Deep Green supercomputer cluster at UVMs Vermont Advanced Computing Core to simulate the behavior of the xenobots under numerous random environmental conditions. These simulations identified Xenobots that excelled at working together in swarms to gather large piles of debris in a field of particles.

We know the task, but its not at all obviousfor peoplewhat a successful design should look like. That is where the supercomputer comes in and searches over the space of all possible Xenobot swarms to find the swarm that does the job best, says Bongard. We want Xenobots to do useful work. Right now, were giving them simple tasks, but ultimately were aiming for a new kind of living tool that could, for example, clean up microplastics in the ocean or contaminants in soil.

Xenobots can quickly collect garbage working together in a swarm to sweep through a petri dish and gather larger piles of iron oxide particles. They can also cover large flat surfaces and travel through narrow capillaries.

The Tufts scientists engineered the Xenobots with a memory capability to record one bit of information, using a fluorescent reporter protein called EosFP that glows green but when exposed to blue light at 390nm wavelength, the protein emits red light instead.

The researchers injected the cells of the frog embryos with messenger RNA coding for the EosFP protein before the stem cells were excised to create the Xenobots so that the mature Xenobots have a built-in fluorescent switch which can record exposure to blue light.

To test the memory capacity, the investigators, allowed 10 Xenobots to swim around a surface on which one spot is illuminated with a beam of blue light. After two hours, they found that three bots emitted red light. The rest remained their original green, effectively recording the travel experience of the bots. This molecular memory in Xenobots could be harnessed to detect the presence of radioactive contamination, chemical pollutants, drugs, or a disease condition.

When we bring in more capabilities to the bots, we can use the computer simulations to design them with more complex behaviors and the ability to carry out more elaborate tasks, said Bongard. We could potentially design them not only to report conditions in their environment but also to modify and repair conditions in their environment.

The biological materials we are using have many features we would like to someday implement in the botscells can act like sensors, motors for movement, communication and computation networks, and recording devices to store information, says Levin. One thing the Xenobots and future versions of biological bots can do that their metal and plastic counterparts have difficulty doing is constructing their own body plan as the cells grow and mature, and then repairing and restoring themselves if they become damaged. Healing is a natural feature of living organisms, and it is preserved in Xenobot biology.

Cells in a biological robot can also absorb and break down chemicals and work like tiny factories, synthesizing and excreting chemicals and proteins.

Xenobots were designed to exhibit swarm activity, moving about on cilia legs. [Doug Blackiston, Tufts University]

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AgeX Therapeutics Reports Fourth Quarter and Annual 2020 Financial Results and Provides Business Update – Business Wire

By daniellenierenberg

ALAMEDA, Calif.--(BUSINESS WIRE)--AgeX Therapeutics, Inc. (AgeX; NYSE American: AGE), a biotechnology company developing therapeutics for human aging and regeneration, reported its financial and operating results for the fourth quarter and the full year ended December 31, 2020.

Recent Highlights

Liquidity and Capital Resources

Amendment to 2019 Loan Agreement

On February 10, 2021, AgeX entered into an amendment to its 2019 Loan Facility Agreement with Juvenescence Limited (Juvenescence). The Amendment extends the maturity date of loans under the agreement to February 14, 2022 and increases the amount of the loan facility by $4.0 million. All loans in excess of the initial $2.0 million that AgeX previously borrowed are subject to Juvenescences discretion.

At-the-market Offering Facility

During January 2021 AgeX entered into a sales agreement with Chardan Capital Markets LLC (Chardan) for the sale of shares of AgeX common stock in at-the-market (ATM) transactions. In accordance with the terms of the sales agreement, AgeX may offer and sell shares of common stock having an aggregate offering price of up to $12.6 million through Chardan acting as the sales agent. Through March 26, 2021, AgeX raised approximately $496,000 in gross proceeds through the sale of shares of common stock.

Going Concern Considerations

As required under Accounting Standards Update 2014-15, Presentation of Financial Statements-Going Concern (ASC 205-40), AgeX evaluates whether conditions and/or events raise substantial doubt about its ability to meet its future financial obligations as they become due within one year after the date its financial statements are issued. Based on AgeXs most recent projected cash flows, AgeX believes that its cash and cash equivalents and available sources of debt and equity capital would not be sufficient to satisfy AgeXs anticipated operating and other funding requirements for the twelve months following the filing of AgeXs Annual Report on Form 10-K for the year ended December 31, 2020. These factors raise substantial doubt regarding the ability of AgeX to continue as a going concern.

Balance Sheet Information

Cash, and cash equivalents, and restricted cash totaled $0.6 million as of December 31, 2020, as compared with $2.5 million as of December 31, 2019. Since January 1, 2021, AgeX had cash proceeds of approximately $3.2 million through loans from Juvenescence, sales of shares of AgeX common stock, and the disposition of its subsidiary LifeMap Sciences, Inc. (LifeMap Sciences) through a cash-out merger.

Fourth Quarter and Annual 2020 Operating Results

Revenues: Total Revenues for the fourth quarter of 2020 were $0.5 million. Total revenues for the year ended December 31, 2020 were $1.9 million, as compared with $1.7 million in the same period in 2019. AgeX revenue was primarily generated by its subsidiary LifeMap Sciences, Inc. which AgeX disposed of on March 15, 2021 through a cash-out merger. Revenues for the year ended December 31, 2020 also included approximately $0.3 million of allowable expenses under a research grant from the NIH as compared with $0.2 million in the same period in 2019.

Operating expenses: Operating expenses for the three months ended December 31, 2020, were $2.9 million, as reported, which was comprised of $2.5 million for AgeX and $0.4 million for LifeMap Sciences, and were $2.3 million, as adjusted, comprised of $2.0 million for AgeX and $0.3 million for LifeMap Sciences.

Operating expenses for the full year 2020 were $12.4 million, as reported, which was comprised of $10.4 million for AgeX and $2.0 million for LifeMap Sciences, and were $10.2 million, as adjusted, comprised of $8.7 million for AgeX and $1.5 million for LifeMap Sciences.

Research and development expenses for the year ended December 31, 2020 decreased by $0.9 million to $5.0 million from $5.9 million in 2019. The decrease was primarily attributable to the layoff of research and development personnel in May 2020.

General and administrative expenses for the year ended December 31, 2020 decreased by $0.7 million to $7.4 million from $8.1 million in 2019. Increases in personnel costs related to an increase in administrative staffing were offset to some extent by a decrease in noncash stock-based compensation expense, general office expense and supplies and travel related expenses with the shelter in place mandates since March 15, 2020 resulting from the COVID-19 pandemic, and the elimination of shared facilities and services fees from AgeXs former parent Lineage Cell Therapeutics, Inc. following the termination of a Shared Facilities and Services Agreement on September 30, 2019.

The reconciliation between operating expenses determined in accordance with accounting principles generally accepted in the United States (GAAP) and operating expenses, as adjusted, a non-GAAP measure, is provided in the financial tables included at the end of this press release.

Other expense, net: Net other expense for the year ended December 31, 2020 was $0.5 million, as compared with net other income of $0.3 million in the same period in 2019. The change is primarily attributable to increased amortization of deferred debt costs to interest expense following the consummation of loan agreements.

Net loss attributable to AgeX: The net loss attributable to AgeX for the year ended December 31, 2020 was $10.9 million, or ($0.29) per share (basic and diluted) compared to $12.2 million, or ($0.33) per share (basic and diluted), for the same period in 2019.

About AgeX Therapeutics

AgeX Therapeutics, Inc. (NYSE American: AGE) is focused on developing and commercializing innovative therapeutics to treat human diseases to increase healthspan and combat the effects of aging. AgeXs PureStem and UniverCyte manufacturing and immunotolerance technologies are designed to work together to generate highly defined, universal, allogeneic, off-the-shelf pluripotent stem cell-derived young cells of any type for application in a variety of diseases with a high unmet medical need. AgeX has two preclinical cell therapy programs: AGEX-VASC1 (vascular progenitor cells) for tissue ischemia and AGEX-BAT1 (brown fat cells) for Type II diabetes. AgeXs revolutionary longevity platform induced Tissue Regeneration (iTR) aims to unlock cellular immortality and regenerative capacity to reverse age-related changes within tissues. HyStem is AgeXs delivery technology to stably engraft PureStem or other cell therapies in the body. AgeX is seeking opportunities to establish licensing and collaboration arrangements around its broad IP estate and proprietary technology platforms and therapy product candidates.

For more information, please visit http://www.agexinc.com or connect with the company on Twitter, LinkedIn, Facebook, and YouTube.

Forward-Looking Statements

Certain statements contained in this release are forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Any statements that are not historical fact including, but not limited to statements that contain words such as will, believes, plans, anticipates, expects, estimates should also be considered forward-looking statements. Forward-looking statements involve risks and uncertainties. Actual results may differ materially from the results anticipated in these forward-looking statements and as such should be evaluated together with the many uncertainties that affect the business of AgeX Therapeutics, Inc. and its subsidiaries, particularly those mentioned in the cautionary statements found in more detail in the Risk Factors section of AgeXs most recent Annual Report on Form 10-K filed with the Securities and Exchange Commissions (copies of which may be obtained at http://www.sec.gov). Subsequent events and developments may cause these forward-looking statements to change. AgeX specifically disclaims any obligation or intention to update or revise these forward-looking statements as a result of changed events or circumstances that occur after the date of this release, except as required by applicable law.

AGEX THERAPEUTICS, INC. AND SUBSIDIARIES

CONSOLIDATED BALANCE SHEETS

(In thousands, except par value amounts)

December 31,

2020

2019

ASSETS

CURRENT ASSETS

Cash and cash equivalents

$

527

$

2,352

Accounts and grants receivable, net

326

363

Prepaid expenses and other current assets

1,430

1,339

Total current assets

2,283

4,054

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AgeX Therapeutics Reports Fourth Quarter and Annual 2020 Financial Results and Provides Business Update - Business Wire

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Insulin 100: How the road to a diabetes cure is yielding better treatments – News@UofT

By daniellenierenberg

The pancreas, saysGary Lewis, an endocrinologist at Toronto General Hospital and director of the Banting & Best Diabetes Centre at the University of Torontos Temerty Faculty of Medicine, is like an exquisitely sensitive and perfectly networked computer.

Second by second,he notes,the pancreassecretesjust the right amount ofinsulinor glucagontolower or raiseblood sugarintotheportal veinthat leadsdirectlyto the liver, the site of key metabolic processes. Insulingis then distributedto every tissue in the body via general circulation.

Thats one reason a cure for diabetes has proven elusive 100 years after the discovery of insulin.

Another big reason is the complexity of how the disease arises. In type 1 diabetes, the immune system destroys the insulin-producing beta cells of the pancreas, creating a life-threatening spike in blood sugar. Type 2 diabetes usually comes on more slowly, as the body becomes resistant to insulin or the pancreas cant produce enough of it.

Genetics play a role in both types. Exposure to viruses and other environmental effects may be a factor in type 1. Lifestyle factors, including weight gain and physical inactivity, are strongly linked to type 2.

The bottom line, says Lewis, is that diabetes is a multifactoral disease, and were not close to a cure.

Ask about treatments, though, and Lewis gets excited.

The last two decades have brought a plethora of clinical and research advances, from new drugs to boost and sensitize the body to insulin and promote weight loss, to lifestyle interventions that improve diet, continuous monitoring of blood sugar, long- and short-lasting insulin, better insulin pumps, pancreatic transplantsand pre-clinical stem cell and immunosuppressive therapies.

Progress on treatments has been fantastic, especially for type 2, Lewis says. Im very, very hopeful.

The distinction between treatment and cure in medicine is often unclear. And for the 3.6 million Canadians living with diabetes, the distinction matters less and lessif the goal is a full and healthy life.

Type 2 diabetes accounts for about 90 per cent of diabetes cases in Canada. Prevalence is rising, but Canadians with type 2 diabetes are living longer and have fewer diabetes-related complications.

The clinic doesnt look like it did 30 years ago, says Lewis, who mainly treats patients with type 2. We see fewer amputees, less blindness. Patients are generally healthier, and their prognosis is often excellent if they maintain their blood sugar target and other key parameters.

Weight loss is a cornerstone of treatments to lower blood sugar, and recent research has strengthened the link between weight reduction and type 2 diabetes management. Some people with type 2 can lose weight and control blood sugar through dietary changes and exercise alone.

Bariatric surgery is very effective for weight loss and often results in diabetes remission, although it comes with surgical risks and is expensive.

If we could prevent obesity, we could greatly reduce the incidence of type 2, Lewis says. And experiments have shown wecan get a remission withlifestyle changes, so we know what works.

The problem is broad implementation.

Ive tried to lose weight and I know how difficult it can be, especially in an environment of convenient and inexpensive calories, Lewis says. Moreover, factors such as income, education, ethnicity, access to healthy food and living conditions can make lifestyle changes that curb obesity nearly impossible.

Social determinants of health are overwhelmingly the most important influence on who gets type 2 diabetes, and how well or poorly they do with it, Lewis says.

Fortunately, dozens of new drugs for diabetes have hit the market in the last two decades.

Medications for weight loss round out the armamentarium, and some also protect against kidney damage and lower cardiac risk. Current therapies can reduce body weight up to 10 per cent, although a loss of 20 per cent or more would have a greater effect on outcomes for patients with type 2 diabetes, saysJacqueline Beaudry, an assistant professor of nutritional sciences at U of T who studies links between obesity, hormones and diet.

Beaudry is probing the biology that underpins these medications, including the gut hormones GLP-1 and GIP. They control blood glucose and reduce appetite, but scientists are unsure how.

If we could understand their mechanisms of action, we could design better drugs, Beaudry says.

For people with type 1 diabetes, continuous glucose monitors, insulin pumps and even automated closed-loopsystems that run on mobile apps to deliver insulin as-needed have radically changed the patient experience.

Sara Vasconcelos left),an assistant professor at U of Ts Institute of Biomedical Engineering, has worked withCristina Nostro (right), an associate professor in the department of physiology,and her team in the McEwen Stem Cell Institute at UHNto extend the survival and functionality of pancreatic precursor cells generatedfrom human stem cells.

Cell therapy could prove more liberating still.

University labs and biotechs are working on implantable devices that house insulin-producing cells derived from stem cells.

To that end,Cristina Nostro, an associate professor in the department of physiology in the Temerty Faculty of Medicine,and her team in the McEwen Stem Cell Institute at University Health Network recently discovered a more efficient way to generate and purify pancreatic precursor cells from human stem cells in the lab.

They have also found a way to vascularize those cells by working withSara Vasconcelos, an assistant professor at U of Ts Institute of Biomedical Engineering. Together, they have extended the survival and functionality of the cells in animal models of diabetes.

The biggest problem with these therapies is that the immune system rejects them. The same challenge currently hinders pancreas and islet transplants.

The immune system is an amazing machine, were luckyits so good, says Nostro. But its very difficult to control when it goes awry, as in autoimmune conditions.

Nostro is working with immunologists at the university on a method to protect insulin-producing beta cells from immune rejection, and she says many researchers in the field are now focused on immune-protective approaches.

Another strategy for type 1 diabetes is to tamp down the autoimmune response before the disease progresses. The idea is to prevent immune cells that damage the pancreas while the body still produces beta cells.

Groups around the world are bringing different ideas and creative approaches to treat type 1 diabetes, thats the beauty of science, says Nostro. I am very hopeful about what the future holds. Who knows? Maybe we will see hybrid technologies combining a pump and cells. We have to keep an open mind.

This story was originally published in U of T Med Magazines Insulin Issue.

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Pericardial Injection Effective, Less Invasive Way to Get Regenerative Therapies to Heart – Newswise

By daniellenierenberg

Newswise Injecting hydrogels containing stem cell or exosome therapeutics directly into the pericardial cavity could be a less invasive, less costly, and more effective means of treating cardiac injury, according to new research from North Carolina State University and the University of North Carolina at Chapel Hill.

Stem cell therapy holds promise as a way to treat cardiac injury, but delivering the therapy directly to the site of the injury and keeping it in place long enough to be effective are ongoing challenges. Even cardiac patches, which can be positioned directly over the site of the injury, have drawbacks in that they require invasive surgical methods for placement.

We wanted a less invasive way to get therapeutics to the injury site, says Ke Cheng, Randall B. Terry, Jr. Distinguished Professor in Regenerative Medicine at NCStates Department of Molecular Biomedical Sciences and professor in the NCState/UNC-Chapel Hill Joint Department of Biomedical Engineering. Using the pericardial cavity as a natural mold could allow us to create cardiac patches at the site of injury from hydrogels containing therapeutics.

In a proof-of-concept study, Cheng and colleagues from NCState and UNC-Chapel Hill looked at two different types of hydrogels one naturally derived and one synthetic and two different stem cell-derived therapeutics in mouse and rat models of heart attack. The therapeutics were delivered via intrapericardial (iPC) injection.

Via fluorescent imaging the researchers were able to see that the hydrogel spread out to form a cardiac patch in the pericardial cavity. They also confirmed that the stem cell or exosome therapeutics can be released into the myocardium, leading to reduced cell death and improved cardiac function compared to animals in the group who received only the hydrogel without therapeutics.

The team then turned to a pig model to test the procedures safety and feasibility. They delivered the iPC injections using a minimally invasive procedure that required only two small incisions, then monitored the pigs for adverse effects. They found no breathing complications, pericardial inflammation, or changes in blood chemistry up to three days post-procedure.

Our hope is that this method of drug delivery to the heart will result in less invasive, less costly procedures with higher therapeutic efficacy, Cheng says. Our early results are promising the method is safe and generates a higher retention rate of therapeutics than those currently in use. Next we will perform additional preclinical studies in large animals to further test the safety and efficacy of this therapy, before we can start a clinical trial.

I anticipate in a clinical setting in the future, iPC injection could be performed with pericardial access similar to the LARIAT procedure. In that regard, only one small incision under local anesthesia is needed on the patients chest wall, says Dr. Joe Rossi, associate professor in the division of cardiology at UNC-Chapel Hill and co-author of the paper.

The research appears inNature Communicationsand was supported by the National Institutes of Health and the American Heart Association. Dr. Thomas Caranasos, director of adult cardiac surgery at UNC-Chapel Hill, also contributed to the work.

-peake-

Note to editors: An abstract follows.

Minimally invasive delivery of therapeutic agents by hydrogel injection into the pericardial cavity for cardiac repair

DOI:10.1038/s41467-021-21682-7

Authors: Dashuai Zhu, Zhenhua Li, Ke Cheng, North Carolina State University; Thomas Caranasos, Joseph Rossi, University of North Carolina at Chapel HillPublished:March 3, 2021 inNature Communications

Abstract:Cardiac patch is an effective way to deliver therapeutics to the heart. However, such procedures are normally invasive and difficult to perform. Here, we developed and tested a method to utilize the pericardial cavity as a natural mold for in situ cardiac patch formation after intrapericardial (iPC) injection of therapeutics in biocompatible hydrogels. In rodent models of myocardial infarction (MI), we demonstrated that iPC injection is an effective and safe method to deliver hydrogels containing induced pluripotent stem cells-derived cardiac progenitor cells (iPS-CPCs) or mesenchymal stem cells (MSCs)-derived exosomes. After injection, the hydrogels formed cardiac patch-like structure in the pericardial cavity, mitigating immune response and increasing the cardiac retention of the therapeutics. With robust cardiovascular regeneration and stimulation of epicardium-derived repair, the therapies mitigated cardiac remodeling and improved cardiac functions post MI. Furthermore, we demonstrated the feasibility of minimally-invasive iPC injection in a clinically-relevant porcine model as well as in human patients. Collectively, our study establishes iPC injection as a safe and effective method to deliver therapeutics to the heart for cardiac repair.

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COVID-19 kills heart muscle cells, interferes with a patients heartbeat – Study Finds

By daniellenierenberg

ST. LOUIS, Mo. Researchers in St. Louis are providing some much needed clarity about how COVID-19 impacts the human heart. COVID has been linked to cardiovascular complications for some time, but up until now its been a mystery as to how exactly the virus is interfering with the heart. For example, does the virus actually infect the heart itself? Or does it just cause inflammation which effects the cardiovascular system? Now, a team from Washington Universitys School of Medicine says theres evidence COVID-19 infects and replicates within carriers heart muscles cells.

This is of course bad news for the heart, which experiences cell death and muscle contraction issues as a result. In pursuit of an answer, the team used stem cells to create heart tissue modeling a COVID-19 infection.

Early on in the pandemic, we had evidence that this coronavirus can cause heart failure or cardiac injury in generally healthy people, which was alarming to the cardiology community, says senior author Kory J. Lavine, MD, PhD, in a university release.

Even some college athletes who had been cleared to go back to competitive athletics after COVID-19 infection later showed scarring in the heart. There has been debate over whether this is due to direct infection of the heart or due to a systemic inflammatory response that occurs because of the lung infection, the associate professor of medicine continues. Our study is unique because it definitively shows that, in patients with COVID-19 who developed heart failure, the virus infects the heart, specifically heart muscle cells.

Researchers also used stem cells to create tissues simulating heart muscle contractions. This process helped researchers to conclude that besides just killing heart muscle cells, COVID interferes with the organs contractions.

Notably, study authors add all of this heart damage can happen to an infected individual even if they show no signs of bodily inflammation.

Inflammation can be a second hit on top of damage caused by the virus, but the inflammation itself is not the initial cause of the heart injury, Lavine explains.

While this certainly isnt the first virus to impact the heart, the research team reports nothing is status quo when it comes to COVID-19. They explain that COVID-19 evokes a unique immune response dissimilar to anything seen when other viruses make contact with the heart.

COVID-19 is causing a different immune response in the heart compared with other viruses, and we dont know what that means yet, the researcher reports. In general, the immune cells seen responding to other viruses tend to be associated with a relatively short disease that resolves with supportive care. But the immune cells we see in COVID-19 heart patients tend to be associated with a chronic condition that can have long-term consequences. These are associations, so we will need more research to understand what is happening.

Researchers further confirmed their stem cell findings when they examined the real heart tissues from four COVID-19 patients.

Even young people who had very mild symptoms can develop heart problems later on that limit their exercise capacity, Lavine concludes. We want to understand whats happening so we can prevent it or treat it. In the meantime, we want everyone to take this virus seriously and do their best to take precautions and stop the spread, so we dont have an even larger epidemic of preventable heart disease in the future.

The study is published in JACC: Basic to Translational Science.

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