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Covid-19: Has Karnataka achieved herd immunity? Simultaneous triple tests will give true picture – Deccan Herald

By daniellenierenberg

At least six international studies have reported T cell reactivity against SARS-CoV-2 in 20% to 50% of people with no known exposure to the virus. Experts suggest doing three tests simultaneously: RTPCR, antibody test, and a T-cell assay, which will give a picture to policymakers if the State or the country has achieved herd immunity against SARS-CoV-2.

A type of white blood cell, T cells are part of the immune system and develop from stem cells in the bone marrow. They help protect the body from infection. Also called T lymphocyte and thymocyte.

For latest updates on coronavirus outbreak, click here

T-cell mediated immunity can be acquired due to previous exposure to other beta coronaviruses which cause the common cold. Knowing a threshold for herd immunity can allow the government to focus on that section of the population who do not have immunity. But they also caution that very few basic science labs in the country like NIMHANS, IISc, or the National Centre for Biological Sciences can do T cell assays in their labs as it is cumbersome and expensive.

Assessing how much of the population has IgG (non-neutralising antibodies), the current active Covid case burden, and T-cell induced protection, simultaneously will give a clear picture of the health of the population, with respect to Covid-19.

"Currently, we do not know when the pandemic will end. If we know how much of the population is immune, it is easier to decide how much of our resources should be allocated to fight Covid, the economy, etc. If done at the state or the sub-state level, we can understand which region needs more resources," said Dr Giridhar Babu, epidemiologist, and member of the State Covid-19 technical advisory committee (TAC).

In the serosurvey undertaken in Karnataka whose results are yet to be announced, with samples from all the eight zones of Bengaluru included, unlike the serosurveys of Delhi, Mumbai, Pune, and Punjab, Karnataka are supposed to have done all three: RTPCR, antibody, and antigen tests simultaneously in the statewide survey.

Coronavirus India update: State-wise total number of confirmed cases, deaths on October 23

Dr V Ravi, Senior Professor and Head, Neurovirology, NIMHANS, and member of State Covid-19 TAC, told DH, "T cell response assay is very cumbersome and complicated to do. Peripheral blood has to be drawn, lymphocytes separated, culture them, stimulate them with antigens, and then take a readout. It is expensive and resource-intensive. Basic science institutes like IISc, NCBS, National Institute of Immunology, ISER, some of them may have the capacity for doing it, but not the medical college laboratories."

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VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription – Science Advances

By daniellenierenberg

INTRODUCTION

Cleidocranial dysplasia (CCD) is a hereditary disease characterized by incomplete closure of the fontanelle, abnormal clavicle, short stature, and skeletal dysplasia. It has been reported that there are multiple Runx2 mutations in human CCD syndrome (1, 2). Mature osteoblasts defect and bone mineralization disorders were observed in Runx2-deficient mice. The Runx2-heterozygous mice show similar phenotypes to the CCD syndrome (24). RUNX2 triggers mesenchymal stem cells (MSCs) to differentiate into osteoblasts (3, 5). According to the skeletal pathology studies in humans and mice, it is important to accurately regulate Runx2 activity during bone formation and bone remodeling (6, 7). However, the molecular regulation of Runx2 activity remains to be further studied.

The evolutionarily conserved Hippo pathway is essential for tissue growth, organ size control, and cancer development (811). Numerous evidences revealed the important roles of Hippo components in regulating bone development and bone remodeling. YAP, the essential downstream effector of Hippo pathway, regulates multiple steps of chondrocyte differentiation during skeletal development and bone repair (12). YAP also promotes osteogenesis and suppresses adipogenic differentiation by regulating -catenin signaling (13). VGLL4, a member of the Vestigial-like family, acts as a transcriptional repressor of YAP-TEADs in the Hippo pathway (14). Our previous work found that VGLL4 suppressed lung cancer and gastric cancer progression by directly competing with YAP to bind TEADs through its two TDU (Tondu) domains (9, 15). We also found that VGLL4 played a critical role in heart valve development by regulating heart valve remodeling, maturation, and homeostasis (16). Moreover, our team found that VGLL4 regulated muscle regeneration in YAP-dependent manner at the proliferation stage and YAP-independent manner at the differentiation stage (17). Our previous studies suggest that VGLL4 plays an important role to regulate cell differentiation in multiple organs. However, the function of VGLL4 in skeletal formation and bone remodeling is unknown.

Here, we reveal the function of VGLL4 in osteoblast differentiation and bone development. Our in vivo data show that global knockout of Vgll4 results in a wide variety of skeletal defects similar to Runx2 heterozygote mice. Our in vitro studies reveal that VGLL4 deficiency strongly inhibits osteoblast differentiation. We further demonstrate that TEADs can bind to RUNX2, thereby inhibiting the transcriptional activity of RUNX2 independent of YAP binding. VGLL4 could relieve the inhibitory function of TEADs by breaking its interaction with RUNX2. In addition, deletion of VGLL4 in MSCs shows similar skeletal defects with the global Vgll4-deficient mice. Further studies show that knocking down TEADs or overexpressing RUNX2 in VGLL4-deficient osteoblasts reverses the inhibition of osteoblast differentiation.

To study the function of VGLL4 in bone, we first measured -galactosidase activity in Vgll4LacZ/+ mice (16). -Galactosidase activity was enriched in trabecular bones, cortical bones, cranial suture, and calvaria cultures (fig. S1, A to C). Furthermore, in bone marrow MSCs (BMSCs), Vgll4LacZ/+ mice displayed -galactosidase activity in osteoblast-like cells (fig. S1D). During osteoblast differentiation in vitro, osteoblast marker genes such as alkaline phosphatase (Alp) and Sp7 transcription factor (Osterix) were increased and peaked at day 7. Vgll4 showed similar trend in this process at both mRNA and protein levels (Fig. 1A and fig. S1, E and F). To further clarify the important role of VGLL4 in bone development, we used a Vgll4Vgll4-eGFP/+ reporter mouse line in which VGLL4enhanced green fluorescent protein (eGFP) fusion protein expression is under the control of the endogenous VGLL4 promoter, and GFP staining reflects VGLL4 expression pattern in skeletal tissues (16). GFP staining was performed at embryonic day 18.5, week 1, week 2, and week 4 stages. The results indicated that the VGLL4 expression level was increased during bone development (fig. S1G). In addition, VGLL4 was enriched in trabecular bones, cortical bones, chondrocytes, cranial suture, and calvaria (fig. S1, G and K to M). We then observed the colocalization of VGLL4-eGFP with markers of MSCs (CD105), osteoblasts [osteocalcin (OCN)], and chondrocytes [collagen 2a1 (Col2a1)] in long bone and calvaria (fig. S1, H to M). Next, we analyzed VGLL4 expression pattern during osteoblast development in vivo (fig. S1N), which was similar to Alp and Osterix expression patterns in mouse BMSCs of different ages. Together, both in vivo and in vitro data suggest that VGLL4 may play roles in osteoblast differentiation and bone development.

(A) Immunoblotting showed the expression profile of VGLL4 during osteoblast differentiation in C57BL/6J mouse BMSCs. Samples were collected at 0, 1, 4, 7, and 10 days after differentiation. (B) Skeletons of WT and Vgll4/ mice at postnatal day 1 (P1) were double-stained by Alizarin red/Alcian blue (n = 5). Scale bar, 5 mm. (C) Quantification of body length in (B). (D) Skull preparations from control and Vgll4/ mouse newborns were double-stained with Alizarin red and Alcian blue at P1. -QCT images of skulls were taken from control and Vgll4/ mice at P4. Scale bar, 5 mm. (E) Quantification of skull defect area in (D). (F) Clavicle preparations from control and Vgll4/ mouse newborns were double-stained with Alizarin red and Alcian blue at P1 and quantification of clavicle length. Scale bar, 5 mm. (G) Alp staining and Alizarin red staining of calvarial cells from WT and Vgll4/ mice after cultured in osteogenic medium. Scale bar, 3 mm. (H) Relative mRNA levels were quantified by RT-PCR. (I) Hematoxylin and eosin (H&E) staining of femur from WT and Vgll4/ mice at embryonic day 16.5. Scale bar, 125 m. (J) In situ hybridization for Col11 immunostaining. Scale bar, 125 m. In (C), (E), (F), and (H), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, ns, no significance; unpaired Students t test. Photo credit: Jinlong Suo, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai.

To investigate the potential function of VGLL4 in bone, we next analyzed the phenotype of Vgll4 knockout (Vgll4/) mice (16). The newborn Vgll4 knockout mice were significantly smaller and underweight compared with their control littermates (Fig. 1, B and C, and fig. S2, A and B). In particular, the membranous ossification of the skull was impaired in Vgll4/ newborns compared with the control littermates (Fig. 1, D and E). Furthermore, Vgll4 knockout mice developed a marked dwarfism phenotype with short legs and short clavicles (Fig. 1, C and F). To assess the role of VGLL4 in osteoblast differentiation, calvarial cells from Vgll4/ mice and wild-type (WT) mice were cultured in osteogenic medium. The activity of Alp in the Vgll4 deletion group was significantly reduced at the seventh day of differentiation (Fig. 1G, top) and was markedly weakened over a 14-day culture period as revealed by Alizarin red S staining (Fig. 1G, bottom). The declined osteogenesis in Vgll4 knockout cells was confirmed by the decreased expression of a series of osteogenic marker genes (Fig. 1H), including Alp, Osterix, and collagen type1 1 (Col11). In addition, in Vgll4/ mice, bone development was severely impaired with remarkable decrease in bone length and almost a complete loss of bone ossification (Fig. 1I). Consistently, immunohistochemical analysis of bone tissue sections from embryos at embryonic day 14.5 further confirmed the defects of bone formation and impaired osteoblast differentiation in Vgll4/ mice (Fig. 1J). Together, our study suggests that VGLL4 is likely to regulate MSC fate by enhancing osteoblast differentiation.

Given that the smaller size of mice is often caused by dysplasia, we also paid attention to the development of cartilage after Vgll4 deletion. As we expected, cartilage development was delayed in Vgll4-deficient mice determined by Safranin O (SO) staining (fig. S2C). Immunohistochemical analysis of collagen X (Col X) further confirmed the delay of cartilage development in Vgll4/ mice (fig. S2D). However, additional experiments would be required to determine the regulatory mechanism behind the observed chondrodysplasia. Although dwarfism was observed and trabecular bones were significantly reduced in the adult Vgll4/ mice, no significant cartilage disorder was observed by SO staining (fig. S2E). In adults, bone is undergoing continuous bone remodeling, which involves bone formation by osteoblasts and bone resorption by osteoclasts. We speculated that Vgll4 deletion might lead to decreased osteoclast activity. To distinguish this possibility, we performed histological analysis by tartrate-resistant acid phosphatase (TRAP) staining to detect osteoclast activity. The results showed that osteoclast activity was comparable between Vgll4/ mice and their control littermates (fig. S2F). Together, our results suggest that the phenotypes observed in Vgll4/ mice are mainly due to the defect of osteoblast activity.

To further explore the role of Vgll4 in the commitment of MSCs to the fate of osteoblasts, we generated Prx1-cre; Vgll4floxp/floxp mice (hereafter Vgll4prx1 mice) (fig. S3A). Prx1-Cre activity is mainly restricted to limbs and craniofacial mesenchyme cells (18, 19). Western blot analysis confirmed that VGLL4 was knocked out in BMSCs (fig. S3B). Vgll4prx1 mice survived normally after birth and had normal fertility. However, Vgll4prx1 mice exhibited marked dwarfism that was independent of sex (Fig. 2, A and B, and fig. S3C), which was similar to the phenotype of Vgll4/ mice. In particular, the membranous ossification of the skull and clavicle was also impaired in Vgll4prx1 mouse newborns compared with control littermates (Fig. 2, C to E). To assess the role of VGLL4 in osteoblast differentiation, BMSCs from Vgll4prx1 and Vgll4fl/fl mice were cultured in osteogenic medium. Markedly decreased ALP activity and mineralization were observed in Vgll4prx1 mice (Fig. 2, F and G). The declined osteogenesis in Vgll4 knockout osteoblasts was also proved by the decreased expression of a series of osteogenic marker genes, including Alp, Osterix, and Col1a1 (Fig. 2H). Normal Runx2 expression was detected in Vgll4prx1 mice (Fig. 2H). To further verify the role of VGLL4 in osteoblast differentiation, BMSCs from Vgll4fl/fl mice were infected with GFP and Cre recombinase (Cre) lentivirus and then cultured in osteogenic medium. Vgll4fl/fl BMSCs infected with Cre lentivirus showed markedly decreased ALP activity and mineralization (fig. S4A). Reduced VGLL4 expression by Cre lentivirus was confirmed by reverse transcription polymerase chain reaction (RT-PCR) (fig. S4B). The declined osteogenesis was also proved by the decreased expression of a series of osteogenic marker genes, including Alp, Osterix, and Col1a1 (fig. S4B).

(A) Skeletons of Vgll4fl/fl and Vgll4prx1 mice at P1 were double-stained by Alizarin red and Alcian blue. Scale bar, 5 mm. (B) Quantification of body length in (A) (n = 6). (C) Skull and clavicle preparation from Vgll4fl/fl and Vgll4prx1 mouse newborns were double-stained with Alizarin red and Alcian blue at P1. Scale bars, 5 mm. (D) Quantification of the defect area of skulls in (C) (n = 6). (E) Quantification of clavicle length in (C) (n = 6). (F) Alp staining and Alizarin red staining of BMSCs from Vgll4fl/fl and Vgll4prx1 mice after cultured in osteogenic medium. Scale bars, 3 mm. (G) Alp activity was measured by phosphatase substrate assay. (H) Relative mRNA levels were quantified by RT-PCR. (I) 3D -QCT images of trabecular bone (top) and cortical bone (bottom) of distal femurs. (J to N) -QCT analysis for trabecular bone volume per tissue volume (BV/TV, Tb) (J), trabecular number (Tb.N/mm) (K), trabecular thickness (Tb.Th/mm) (L), trabecular separation (Tb.Sp/mm) (M), and cortical bone thickness (Cor.Th/mm) (N). (O) Representative images of von Kossa staining of 12-week-old Vgll4fl/fl and Vgll4prx1 mice. Scale bar, 500 m. (P) Representative images of calcein and Alizarin red S labeling of proximal tibia. Scale bar, 50 m. (Q) Quantification of MAR. (R and S) ELISA analysis of serum PINP (ng ml1) and CTX-1 (ng ml1) from 10-week-old Vgll4fl/fl and Vgll4prx1 mice (n = 5). In (B), (D), (E), (G), (H), (J) to (N), and (Q) to (S), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test. Photo credit: Jinlong Suo, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai.

We next performed PCNA (proliferating cell nuclear antigen) staining and MTT assay to detect whether VGLL4 influences cell proliferation during bone development. No significant differences were found after VGLL4 deletion (fig. S5, A to C). We also did not detect significant changes of proliferation-related genes and YAP downstream genes (fig. S5, D and E). We next performed TUNEL (terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling) staining to detect whether VGLL4 influences cell apoptosis. In addition, no significant differences were found after VGLL4 deletion (fig. S5, F and G).

To further determine the function of VGLL4 in skeletal system, we did micro-quantitative computed tomography (-QCT) analysis to compare the changes in bone-related elements in the long bones of Vgll4prx1 mice and control littermates. We found that the 3-month-old Vgll4prx1 mice showed decreased bone mass per tissue volume (BV/TV) relative to age-matched control littermates (Fig. 2, I and J). Further analysis showed a reduction in trabecular number (Tb.N) of Vgll4prx1 mice compared to control mice (Fig. 2K), which was accompanied by a decrease in trabecular thickness (Tb.Th) and an increase in trabecular separation (Tb.Sp) compared to control mice (Fig. 2, L and M). Vgll4prx1 mice also showed decreased cortical bone thickness (Cor.Th) relative to the Vgll4fl/fl mice (Fig. 2N). The von Kossa staining showed reduced bone mineral deposition in 3-month-old Vgll4prx1 mice (Fig. 2O). The mineral apposition rate (MAR) was also decreased in Vgll4prx1 mice compared with control littermates by fluorescent double labeling of the mineralizing front (Fig. 2, P and Q). Consistent with the decreased bone mass in Vgll4prx1 mice, the enzyme-linked immunosorbent assay (ELISA) assay of N-terminal propeptide of type I procollagen (PINP), a marker of bone formation, revealed a reduced bone formation rate in Vgll4prx1 mice (Fig. 2R). However, the ELISA assay of C-terminal telopeptide of collagen type 1 (CTX-1), a marker of bone resorption, showed that the bone resorption rate of Vgll4prx1 mice did not change significantly (Fig. 2S). Collectively, Vgll4 conditional knockout mice mimicked the main phenotypes of the global Vgll4 knockout mice, further indicating that VGLL4 specifically regulates bone mass by promoting osteoblast differentiation.

To further determine whether the abnormal osteogenesis in Vgll4prx1 mice was caused by a primary defect in osteoblast development, we generated an osteoblast-specific Osx-cre; Vgll4floxp/floxp mice (hereafter Vgll4Osx mice) by crossing Vgll4fl/fl mice with Osx-Cre mice, a line in which Cre expression is primarily restricted to osteoblast precursors (fig. S6A) (6, 20). Vgll4Osx mice survived normally after birth and had normal fertility, but exhibited marked dwarfism in comparison with Osx-Cre mice (fig. S6, B and C), which was similar to the phenotypes of Vgll4/ and Vgll4prx1 mice. In addition, the membranous ossification of the skull and clavicle was also impaired in Vgll4Osx mice compared with control littermates (fig. S6C). -QCT analysis further confirmed the osteogenic phenotype of Vgll4Osx mice (fig. S6, D to J). Hence, the Vgll4Osx mice summarized the defects observed in the Vgll4prx1 mice, thus supporting the conclusion that VGLL4 is necessary for the differentiation and function of committed osteoblast precursors.

We next worked to figure out the mechanism how VGLL4 controls bone mass and osteoblast differentiation. The pygmy and cranial closure disorders in Vgll4/ mice were similar to that of Runx2-heterozygous mice. We therefore examined the potential interaction between VGLL4 and RUNX2. However, coimmunoprecipitation experiments did not show the interaction between VGLL4 and RUNX2 (Fig. 3A). Previous studies showed that VGLL4 could compete with YAP for binding to TEADs (9). The TEAD family contains four highly homologous proteins (8), which is involved in the regulation of myoblast differentiation and muscle regeneration (21). We determined whether the binding of VGLL4 with RUNX2 requires TEADs. Coimmunoprecipitation experiments showed that RUNX2 and TEAD14 had almost equivalent interactions (Fig. 3B). Next, we investigated whether TEADs control the transcriptional activity of Runx2. We used the 6xOSE2-luciferase reporter system that is specifically activated by RUNX2 to verify the role of TEADs (22). We performed dual-luciferase reporter assay with 6xOSE2-luciferase and Renilla in C3H10T1/2 cells, and the results showed that TEAD14 significantly inhibited the activation of 6xOSE2-luciferase induced by RUNX2 (Fig. 3C). Consistently, knockdown of TEADs by small interfering RNAs (siRNAs) markedly enhanced both basic and RUNX2-induced 6xOSE2-luciferase activity (fig. S8A). TEAD family is highly conserved, which consists of an N-terminal TEA domain and a C-terminal YAP-binding domain (YBD) (Fig. 3D) (23). Glutathione S-transferase (GST) pull-down assay revealed the direct interaction between RUNX2 and TEAD4 (Fig. 3E). Moreover, both TEA and YBD domains of TEAD4 could bind to RUNX2 (Fig. 3, F and G).

(A) Coimmunoprecipitation experiments of RUNX2 and VGLL4 in HEK-293T cells. The arrow indicated IgG heavy chain. (B) Coimmunoprecipitation experiments of RUNX2 and TEAD14 in HEK-293T cells. The arrow indicated IgG heavy chain. (C) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2 and TEAD14. Data were calculated from three independent replicates. (D) Schematic illustration of the domain organization for TEAD4, TEAD4-Nt, and TEAD4-Ct. (E) GST pull-down (PD) analysis between purified GST-RUNX2 and HIS-SUMO-TEAD4 proteins. (F) GST pull-down analysis between purified GST-RUNX2 and HIS-SUMO-TEAD4-TEA proteins. (G) Lysates from HEK-293T cells with Flag and Flag-RUNX2 expressions were incubated with recombinant GST-TEAD4-YBD protein. GST pull-down assay showed the binding between RUNX2 and TEAD4-YBD. (H) Cells isolated from WT mice were infected with TEAD lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (I) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (J) Relative mRNA levels of Alp, Col11, and Osterix were quantified by RT-PCR. (K) Cells isolated from WT mice were infected with TEAD shRNA lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (L) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (M) Relative mRNA levels of Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. (N) Relative mRNA levels of Tead1-4 were quantified by RT-PCR. In (C), (I), (J), and (L) to (N), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

To determine whether overexpression of TEAD14 affects osteoblast differentiation, BMSCs from WT mice were infected with TEAD14 lentivirus and then cultured in osteogenic medium. The activities of ALP in TEAD14 overexpression groups were significantly reduced at the seventh day of differentiation [Fig. 3, H (top) and I] and were significantly weakened by Alizarin red S staining over a 14-day culture period (Fig. 3H, bottom). The declined osteogenesis in TEAD14 overexpression cells was confirmed again by the decreased expression of a series of osteogenic marker genes, including Alp, Col11, and Osterix (Fig. 3J). Next, we blocked the total activities of TEAD14 by short hairpin RNA (shRNA) lentiviral infection (Fig. 3N). The activity of Alp in TEAD14 knockdown group was significantly increased [Fig. 3, K (top) and L]. Over a 14-day culture period, osteogenic differentiation was significantly enhanced by Alizarin red S staining (Fig. 3K, bottom). The enhanced osteogenesis in TEAD14 knockdown cells was further confirmed by elevated expression of a series of osteogenic marker genes, including Alp, Col11, and Osterix (Fig. 3M). These results suggest that TEAD14 act as repressors of RUNX2 to inhibit osteoblast differentiation.

To investigate the mechanistic role of VGLL4 in inhibiting osteoblast differentiation, we then verified whether VGLL4 could affect the interaction between TEADs and RUNX2. We found that VGLL4 reduced the interaction between RUNX2 and TEADs (Fig. 4A). To further illustrate the relationship between RUNX2/TEADs/VGLL4, we checked the interaction between RUNX2 and TEADs in the BMSC of Vgll4fl/fl mice treated with GFP or Cre lentivirus. We found that the interaction between RUNX2 and TEADs was enhanced in Cre-treated cells (Fig. 4B). We noticed that there were conserved binding sites of RUNX2 (5-AACCAC-3) and TEAD (5-CATTCC-3) in the promoter regions of Alpi, Osx, and Col1a1, which are three target genes of RUNX2 (17, 24). We performed TEAD4 and RUNX2 chromatin immunoprecipitation (ChIP) assays in BMSCs. The results indicated that both TEAD4 and RUNX2 bound on Alp, Osx, and Col1a1 promoters (fig. S7, A to I). VGLL4 was a transcriptional cofactor, which could not bind DNA directly. We have demonstrated that VGLL4 promoted RUNX2 activity by competing for its binding to TEADs. Consistently, VGLL4 partially blocked TEADs-repressed transcriptional activity of RUNX2 (Fig. 4C). However, overexpression of VGLL4 in TEADs knockdown cells showed no marked change on RUNX2-induced 6xOSE2-luciferase activity compared with TEAD knockdown (fig. S8B). We then asked whether loss of VGLL4-induced disorders of osteoblast differentiation is related to TEADs. We knocked down TEADs by lentiviral infection in Vgll4-deficient BMSCs and then induced these cells for osteogenic differentiation. The differentiation disorders caused by VGLL4 deletion were restored after TEAD knockdown (Fig. 4, D to F). These data supported that VGLL4 released the inhibition of TEADs on RUNX2, thereby promoting osteoblast differentiation.

(A) Coimmunoprecipitation experiments of RUNX2, TEADs, and VGLL4 in HEK-293T cells. The arrow indicated IgG heavy chain. (B) Coimmunoprecipitation experiments of RUNX2 and TEADs in BMSCs cells of Vgll4fl/fl mice treated with GFP and Cre lentivirus. (C) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEADs, and VGLL4. (D) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and TEAD shRNA lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (E) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (F) Relative mRNA levels of Vgll4, Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. In (B), (D), and (E), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

YAP, the key transcription cofactor in the Hippo pathway, has been widely reported in regulating bone development and bone mass (12, 13). VGLL4, a previously identified YAP antagonist, directly competes with YAP for binding to TEADs (9). Therefore, we suspected that the inhibition of RUNX2 transcriptional activity caused by VGLL4 deletion might be dependent on YAP. To this end, we validated the role of YAP by 6xOSE2-luciferase reporter system. The data showed that YAP promoted RUNX2 activity in a dose-dependent manner (Fig. 5A). Moreover, TEAD4 significantly inhibited 6xOSE2-luciferase activity induced by YAP (Fig. 5B). TEAD4Y429H, a mutation that impairs the interaction between TEAD4 and YAP/TAZ (Fig. 5C) (25), did not promote 3xSd-luciferase activity induced by YAP (Fig. 5D). We found that both TEAD and TEAD4Y429H could interact with RUNX2 (Fig. 5E), and both TEAD4 and TEAD4Y429H could inhibit the activity of RUNX2 in a dose-dependent manner (Fig. 5, F and G). Restoring the expression of both TEAD4 and TEAD4Y429H could reverse the increased osteoblast differentiation in TEAD knockdown BMSCs (Fig. 5, H and I). Furthermore, overexpression of TEAD1 could further inhibit osteogenic differentiation of BMSCs after YAP knockdown (Fig. 5J). Together, these data suggest that the inhibition of RUNX2 activity by TEADs is independent of YAP binding.

(A) Effects of YAP on Runx2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, YAP, and TEAD4. (C) Schematic illustration of TEAD4 and TEAD4Y429H mutation. (D) 3xSd-luciferase activity was determined in HEK-293T cells cotransfected with YAP, TEAD4, and TEAD4Y429H. (E) Coimmunoprecipitation experiments of RUNX2, TEAD4, and TEAD4Y429H in HEK-293T cells. The arrow indicated IgG heavy chain. (F) Effects of TEAD4 on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (G) Effects of TEAD4Y429H on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (H) Cells isolated from WT mice were infected with GFP or TEAD shRNAs, TEAD4, or TEAD4Y429H lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (I) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (J) Relative mRNA levels of Runx2, Alp, Col11, Osterix, Tead1, and Yap were quantified by RT-PCR. In (A), (B), (D), (F), (G), (I), and (J), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

We next examined how VGLL4 breaks the interaction between RUNX2 and TEADs. It has been reported that VGLL4 relies on its own two TDU domains to interact with TEADs (9), and VGLL4 HF4A mutation can disrupt the interaction between VGLL4 and TEADs (15). We hypothesized that VGLL4 competes with RUNX2 for TEAD1 binding depending on its TDU domain. On the basis of these previous studies, we performed coimmunoprecipitation experiments and found that VGLL4 HF4A abolished the interaction between VGLL4 and TEAD1 but did not affect the interaction between TEAD1 and RUNX2 (Fig. 6A). VGLL4 partially rescued the inhibition of RUNX2 transcriptional activity by TEAD1; however, VGLL4 HF4A lost this function (Fig. 6B). We then overexpressed TEAD1 by lentivirus infection in primary calvarial cells and found that the transcriptional level of Alp was significantly inhibited. This inhibition was released by overexpressing VGLL4 but not VGLL4 HF4A (Fig. 6C). To further verify the specific regulation of RUNX2 activity by VGLL4, we performed a coimmunoprecipitation experiment with low and high doses of VGLL4 and VGLL4 HF4A. The results showed that the TEAD1-RUNX2 interaction was gradually repressed along with an increasing dose of VGLL4 but not VGLL4 HF4A (Fig. 6D). Similarly, the inhibition of RUNX2 transcriptional activity by TEAD1 was gradually released with an increasing dose of VGLL4 but not VGLL4 HF4A (Fig. 6E). Super-TDU, a peptide mimicking VGLL4, could also reduce the interaction between purified RUNX2 and TEAD4 proteins (Fig. 6F). Thus, these findings suggest that VGLL4 TDU domain competes with RUNX2 for TEADs binding to release RUNX2 transcriptional activity.

(A) Coimmunoprecipitation experiments of RUNX2, TEAD1, VGLL4, and VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, VGLL4, VGLL4 HF4A, and TEAD1 (n = 3). (C) RT-PCR analysis of Alp expression in calvarial cells. Cells isolated from WT mice were infected with GFP, TEAD1, VGLL4, or VGLL4 HF4A lentivirus. (D) Coimmunoprecipitation experiments of RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (E) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A. (F) Competitive GST pull-down assay to detect the effect of VGLL4 Super-TDU on the interaction between RUNX2 and TEAD4. (G) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and RUNX2 lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (H) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (I) Relative mRNA levels of Vgll4, Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. (J) Schematic model of VGLL4/TEADs/RUNX2 in regulating osteogenic differentiation. In (B), (C), (E), (H), and (I), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

Furthermore, we overexpressed RUNX2 by lentivirus infection in Vgll4 knockout BMSCs during osteogenic differentiation, and we found that RUNX2 could significantly restore the osteogenic differentiation disorder caused by Vgll4 deletion (Fig. 6, G to I). Together, these data suggest a genetic interaction between VGLL4/TEADs/RUNX2 and provide evidences that RUNX2 overexpression rescues osteogenic differentiation disorders caused by VGLL4 deletion.

Collectively, our study demonstrates the important roles of VGLL4 in osteoblast differentiation, bone development, and bone homeostasis. In the early stage of osteoblast differentiation, TEADs interact with RUNX2 to inhibit its transcriptional activity in a YAP bindingindependent manner. During differentiation progress, VGLL4 expression gradually increases to dissociate the interaction between TEADs and RUNX2, thereby releasing the inhibition of RUNX2 transcriptional activity by TEADs and promoting osteoblasts differentiation (Fig. 6J).

Accumulating evidences have suggested that the Hippo pathway plays key roles in regulating organ size and tissue homeostasis (8, 10). However, the transcription factors TEADs have not been reported in skeletal development and bone-related diseases. VGLL4 functions as a new tumor suppressor gene, which has been reported to negatively regulate the YAP-TEADs transcriptional complex. Our previous studies show that VGLL4 plays important roles in many tissue homeostasis and organ development, such as heart and muscle (16, 17). In this study, we provide evidences to show that VGLL4 can break TEADs-mediated transcriptional inhibition of RUNX2 to promote osteoblast differentiation and bone development independent of YAP binding.

Overall, our studies establish the Vgll4-specific knockout mouse model in the skeletal system. We show that VGLL4 deletion in MSCs leads to abnormal osteogenic differentiation with delayed skull closure and reduced bone mass. Our data also reveal that VGLL4 deletion leads to chondrodysplasia. Recent researches identified that chondrocytes have the ability to transdifferentiate into osteoblasts (2628), suggesting the possibility that loss of VGLL4 might reduce or delay the pool of chondrocytes that differentiate into osteoblasts. We identify that VGLL4 regulates the RUNX2-TEADs transcriptional complex to control osteoblast differentiation and bone development. TEADs can bind to RUNX2 and inhibit its transcriptional activity in a YAP bindingindependent manner. Recent studies pointed out that reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2 (29); however, we found that TEAD4-Y429H, a mutation at the binding site of TAZ and TEAD (25, 30, 31), can still significantly inhibit the activity of RUNX2. Therefore, we consider that the way TEAD regulates RUNX2 may not depend on TAZ regulation. Further research found that VGLL4, but not VGLL4 HF4A, can alleviate the inhibition by influencing the binding between RUNX2 and TEADs. It is possible that VGLL4 might influence the structure organization of the RUNX2-TEAD complex to some extent. Structural information may be required to answer this question and may provide more insights into the mechanism of VGLL4 in osteogenic differentiation.

Previous studies showed that mutations in RUNX2 cause CCD and Runx2+/ mice show a CCD-like phenotype. However, many patients with CCD do not have RUNX2 mutations. Our study may provide clues to the pathogenesis of these patients. A significant reduction of bone mass was observed in the adult mice, suggesting that VGLL4 and TEADs might be drug targets for treatment of cranial closure disorders and osteoporosis. In addition, further investigation of the clinical correlation of VGLL4 and cleidocranial dysplasia in a larger cohort will provide more accurate information for bone research. Our work also provides clues to researchers who are studying the roles of VGLL4 in tumors or other diseases. RUNX2 is highly expressed in breast and prostate cancer cells. RUNX2 contributes to tumor growth in bone and the accompanying osteolytic diseases (32). The regulation of RUNX2 transcriptional activity by TEADs and VGLL4 is likely to play essential roles in tumor, bone metastasis, and osteolytic diseases. Our work may provide clues to researchers who are studying the role of VGLL4 in bone tumors.

We demonstrate that TEADs are involved in regulating osteoblast differentiation by overexpressing and knocking down the TEAD family in vitro. However, the exact roles of TEADs in vivo need to be further confirmed by generation of TEAD1/2/3/4 conditional knockout mice. In the follow-up work, we will continue to study the mechanism of TEADs in skeletal development and bone diseases. Overall, although there are still some shortcomings, our work has greatly contributed to understand the TEADs regulation of RUNX2 activity.

Our work defines the role of VGLL4 in regulating osteoblast differentiation and bone development, and identifies that TEADs function as repressors of RUNX2 to inhibit osteoblast differentiation. We propose a model that VGLL4 dissociates the combination between TEADs and RUNX2. It is not clear whether VGLL4 is also involved in regulating other transcription factors or signaling pathways in the process of osteoblast differentiation and bone development. If that is the case, how to achieve cooperation will be another interesting issue worthy of further study.

Vgll4Lacz/+ mice, Vgll4 knockout (Vgll4/) mice, Vgll4Vgll4-eGFP/+ mice, and Vgll4 conditional knockout (Vgll4fl/fl) mice were generated as previously described (16, 17), and Vgll4fl/fl mice were crossed with the Prx1-Cre and Osx-Cre strain to generate Vgll4prx1 and Vgll4Osx mice. All mice analyzed were maintained on the C57BL/6 background. All mice were monitored in a specific pathogenfree environment and treated in strict accordance with protocols approved by the Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

The following antibodies were used: anti-Osterix antibody (1:1000; Santa Cruz Biotechnology, SC133871), anti-RUNX2 antibodies (1:1000; Santa Cruz Biotechnology, SC-390351 and SC-10758), anti-Flag antibody (1:5000; Sigma-Aldrich, F-3165), anti-HA (hemagglutinin) antibody (1:2000; Santa Cruz Biotechnology, SC-7392), anti-HA antibody (1:1000; Sangon Biotech, D110004), anti-MYC antibody (1:1000; ABclonal Technology, AE010), anti-PCNA antibody (1:1000; Santa Cruz Biotechnology, SC-56), rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology, SC-2027), mouse IgG (Sigma-Aldrich, I5381), anti-VGLL4 antibody (1:1000; ABclonal, A18248), anti-TEAD1 antibody (1:1000; ABclonal, A6768), anti-TEAD2 antibody (1:1000; ABclonal, A15594), anti-TEAD3 antibody (1:1000; ABclonal, A7454), anti-TEAD4 antibody (1:1000; Abcam, ab58310), and antipan-TEAD (1:1000; Cell Signaling Technology, 13295).

Cells were cultured at 37C in humidified incubators containing an atmosphere of 5% CO2. Human embryonic kidney (HEK)293T cells were maintained in Dulbeccos Modified Eagle Medium (DMEM) (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) solution. C3H10T1/2 cells were maintained in -minimum essential medium (-MEM) (Corning, Corning, NY) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) solution. To induce differentiation of BMSC into osteoblasts, cells were cultured in -MEM containing 10% FBS, l-ascorbic acid (50 g/ml), and -glycerophosphate (1080 mg/ml). The osteoblast differentiation level assay was performed following a previously published method (33). To quantitate Alp activity, cells incubated with Alamar Blue to calculate cell numbers and then incubated with phosphatase substrate (Sigma-Aldrich, St. Louis, MO) dissolved in 6.5 mM Na2CO3, 18.5 mM NaHCO3, and 2 mM MgCl2 after washing by phosphate-buffered saline (PBS). Alp activity was then read with a luminometer (Envision). Bone nodule formation was stained with Alizarin red S solution (1 mg/ml; pH 5.5) after 14 days of induction.

We collected femurs and tibias from mice and flushed out the bone marrow cells with 10% FBS in PBS. All nuclear cells were seeded (2 106 cells per dish) in 100-mm culture dishes (Corning) and incubated at 37C under 5% CO2 conditions. After 48 hours, nonadherent cells were washed by PBS and adherent cells were cultured in -MEM (Corning, Corning, NY) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) solution for an additional 5 days. Mouse BMSCs in passage one were used in this study.

Total RNA was isolated from cells with TRIzol reagent (T9424, Sigma-Aldrich), and first-strand complementary DNA (cDNA) was synthesized from 0.5 g of total RNA using the PrimeScript RT Reagent Kit (PR037A, TaKaRa). The real-time RT-PCR was performed with the Bio-Rad CFX96 System. Gene expression analysis from RT-PCR was quantified relative to Hprt.

C3H10T1/2 cells were seeded overnight at 1 105 cells per well into a 12-well plate and transfected by PEI (polyethylenimine linear) with a luciferase reporter plasmid along with various expression constructs, as indicated. All wells were supplemented with control empty expression vector plasmids to keep the total amount of DNA constant. At 36 to 48 hours after transfection, the cells were harvested and subjected to dual-luciferase reporter assays according to the manufacturers protocol (Promega).

293T cells were seeded at 1 107 cells per 10-cm dish and cultured overnight. At 36 to 48 hours after transfection with PEI, cells were harvested and washed with cold PBS following experimental treatments. Then, cells were lysed with EBC buffer [50 mM tris (pH 7.5), 120 mM NaCl, and 0.5% NP-40] containing protease inhibitor cocktail (1:100; MedChem Express, HY-K0010). After ultrasonication, lysates were subjected to immunoprecipitation with anti-Flag antibodies (M2, Sigma-Aldrich) at 4C overnight, followed by washing in lysis buffer, SDSpolyacrylamide gel electrophoresis (PAGE), and immunoblotting with the indicated antibody.

RUNX2 and TEAD4-YBD were cloned into pGEX-4T-1-GST vector and expressed in Escherichia coli BL21 (DE3) cells. TEAD4 and TEAD4-TEA were cloned into HT-pET-28a-HIS-SUMO vector and expressed in E. coli BL21 (DE3) cells. The two TDU domains of VGLL4 were cloned into HT-pET-28a-MBP vector and expressed in E. coli BL21 (DE3) cells. VGLL4 Super-TDU was designed as previously described (15). GST, HIS-SUMO, and MBP-fused proteins were purified by affinity chromatography as previously described (17). The input and output samples were loaded to SDS-PAGE and detected by Western blotting.

CalceinAlizarin red S labeling measuring bone formation rate was performed as previously described (33).

Preparation of skeletal tissue and -QCT analysis were performed as previously described (34). The mouse femurs isolated from age- and sex-matched mice were skinned and fixed in 70% ethanol. Scanning was performed with the -QCT SkyScan 1176 System (Bruker Biospin). The mouse femurs were scanned at a 9-m resolution for quantitative analysis. Three-dimensional (3D) images were reconstructed using a fixed threshold.

ChIP experiments were carried out in BMSCs according to a standard protocol. The cell lysate was sonicated for 20 min (30 s on, 30 s off), and chromatin was divided into fragments ranging mainly from 200 to 500 base pairs in length. Immunoprecipitation was then performed using antibodies against TEAD4 (Abcam, ab58310), RUNX2 (Santa Cruz Biotechnology, SC-10758), and normal IgG. The DNA immunoprecipitated by the antibodies was detected by RT-PCR. The primers used were as follows: Alp-OSE2-ChIP-qPCR-F (5-GTCTCCTGCCTGTGTTTCCACAGTG-3), Alp-OSE2-ChIP-qPCR-R (5-GAAGACGCCTGCTCTGTGGACTAGAG-3), Alp-TBS-ChIP-qPCR-F (5-CCTTGCATGTAAATGGTGGACATGG-3), Alp-TBS-ChIP-qPCR-R (5-TATCATAGTCACTGAGCACTCTCTTGCG-3), Osx-OSE2-ChIP-qPCR-F (5-TTAACTGCCAAGCCATCGCTCAAG-3), Osx-OSE2-ChIP-qPCR-R (5-CCTCTATGTGTGTATGTGTGTTTACCAAACATC-3), Osx-TBS-ChIP-qPCR-F (5-ATGCCAAGAGATCCCTCATTAGGGAC-3), Osx-TBS-ChIP-qPCR-R (5-AGCTTGGTGAGCACAGCAAAGACAC-3), Col1a1-TBS/OSE2-Chip-qPCR-F (5-CTCAGCCTCAGAGCTGTTATTTATTAGAAAGG-3), and Col1a1-TBS/OSE2-Chip-qPCR-R (5-TTAATCTGATTAGAACCTATCAGCTAAGCAGATG-3). TBS indicated TEAD binding sites.

Mouse TEAD1, TEAD2, TEAD3, and TEAD4 siRNAs and the control siRNA were synthesized from Shanghai Gene Pharma Co. Ltd., Shanghai, China. siRNA oligonucleotides were transfected in C3H10T1/2 by Lipofectamine RNAiMAX (Invitrogen) following the manufacturers instructions. Two pairs of siRNAs were used to perform experiments.

Hematoxylin and eosin stain and immunohistochemistry were performed as previously described (7). Tissue sections were used for TRAP staining according to the standard protocol. Tissues were fixed in 4% paraformaldehyde for 48 hours and incubated in 15% DEPC (diethyl pyrocarbonate)EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin and sectioned at 7 m. Immunofluorescence was performed as previously described (33). Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 hour and then stained overnight with anti-PCNA antibody (SC-56). Donkey anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) was used as secondary antibodies. DAPI (4,6-diamidino-2-phenylindole) (Sigma-Aldrich, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with a Leica SP5 and SP8 confocal microscope. For embryonic mice, 5-mm tissue sections were used for immunohistochemistry staining, DIG-labeled in situ hybridization (Roche), and immunohistochemical staining (Dako).

TUNEL staining for apoptosis testing was performed as provided by Promega (G3250).

MTT assay for cell viability was performed as provided by Thermo Fisher Scientific.

We determined serum concentrations of PINP using the Mouse PINP EIA Kit (YX-160930M) according to the instructions provided. In addition, we determined serum concentrations of CTX-1 using the Mouse CTX-1 EIA Kit (YX-032033M) according to the instructions provided.

Tissue sections were used for SO staining according to the standard protocol. After paraffin sections were dewaxed into water, they were acidified with 1% acetic acid for 10 s and then fast green for 2 min, acidified with 1% acetic acid for 10 s, stained with SO for 3 min and 95% ethanol for 5 s, and dried and sealed with neutral glue.

Statistical analysis was performed by unpaired, two-tailed Students t test for comparison between two groups using GraphPad Prism Software. A P value of less than 0.05 was considered statistically significant.

Acknowledgments: We thank A. McMahon (Harvard University, Boston, MA) for providing the Prx1-Cre mouse line. We thank the cell biology core facility and the animal core facility of Shanghai Institute of Biochemistry and Cell Biology for assistance. Funding: This work was supported by the National Natural Science Foundation of China (nos. 81725010, 31625017, 81672119, and 31530043), National Key Research and Development Program of China (2017YFA0103601 and 2019YFA0802001), Strategic Priority Research Program of Chinese Academy of Sciences (XDB19000000), Shanghai Leading Talents Program, Science and Technology Commission of Shanghai Municipality (19ZR1466300), and Youth Innovation Promotion Association CAS (2018004). Author contributions: Z.W., L.Z., and W.Z. conceived and supervised the study. J.S. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. X.F. made the constructs, performed the in vitro pull-down assay and ChIP experiments, analyzed the data, and revised the manuscript. L.Z. and Z.W. provided genetic strains of mice. J.S. and Z.W. bred and analyzed Vgll4/ mice. J.L. and J.W. cultured the cells and made the constructs. W.Z., L.Z., X.F., and Z.W. edited the manuscript. 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. Additional data related to this paper may be requested from the authors.

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VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription - Science Advances

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RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis – Science Advances

By daniellenierenberg

INTRODUCTION

Ewing sarcoma (EwS) is an aggressive, poorly differentiated, human tumor characterized by a chromosomal translocation involving a member of the FET family of genes (FUS, EWSR1 and TAF15) and a member of the ETS family of transcription factors, with the EWSR1-FLI1 gene fusion the most common one (1). EwS genomes present low mutation rates with FET-ETS rearrangements as the dominant genetic aberration in the majority of tumors (2). Notably, the cell of origin of EwS is still a controversial field, although human mesenchymal stem cells (hMSCs) and human neural crest stem cells are the most accepted (35).

The EWSR1-FLI1 fusion protein, which contains the transcriptional activation and RNA binding domains of EWSR1 and the DNA binding domain of FLI1, is the main driver of tumorigenesis (3, 6). The resulting fusion oncoprotein has the ability to act as an aberrant transcription factor, leading to gene activation and repression for a well-described set of genes (3, 7). A decade ago, EWSR1-FLI1 was found to bind preferentially to DNA sites containing GGAA microsatellite repeats (8, 9). Recent studies have reported that binding of EWSR1-FLI1 multimers to GGAA repeats acts as a pioneer factor and induces the formation of de novo active enhancers by recruiting the acetyl transferases CBP/p300, E2F3, and the BRG1/BRM-associated factor chromatin remodeling complex (1012). On the other hand, it was hypothesized that monomeric EWSR1-FLI1 inhibits transcription at enhancers by displacing endogenous ETS transcription factors from GGAA motifs (10). Therefore, the mechanisms by which EWSR1-FLI1 acts as either a gene activator or repressor depend on both DNA sequence and cofactors.

Several proteins from the Polycomb group (PcG) have previously been implicated in EwS tumorigenesis. PcG was first described in Drosophila melanogaster as a key regulator of Hox genes expression. PcG proteins not only prevent differentiation by repressing lineage-specific genes but also mark bivalent chromatin regions for subsequent activation. EZH2 (the enzymatic subunit of PRC2) methylates histone H3 at lysine 27 (H3K27me3), while RING1B (the enzymatic subunit of PRC1) ubiquitinates H2A at lysine 119 (H2Aub), both considered repressive histone marks (13).

The canonical PRC1 complex (defined by the presence of four subunits, comprising one variant each of PCGF, PHC, CBX, and RING1) has mostly been associated with maintaining gene repression. However, increasing evidence indicates that PRC1 complexes containing RING1B have the potential for transcription activation, via their catalytic-independent association with UTX, an H3K27me3 demethylase, and p300 acetyltransferase (14, 15). With respect to EwS, it was recently shown that EZH2 blocks endothelial and neuroectodermal differentiation (16), BMI1 promotes tumorigenicity (17), and RING1B represses the nuclear factor B pathway (18). The molecular mechanisms behind the contribution of PcG to EwS have not been addressed. Notably, the GGAA repeats are significantly decorated with H3K27me3 in H1 human embryonic cell lines and human umbilical vein endothelial cells (HUVECs) (19). This is in stark contrast with the lack of H3K27me3 mark at EWSR1-FLI1 binding sites in EwS cells (10, 11), thus suggesting a different role of PcG in EwS. Last, comparison between malignant and nonmalignant tissues revealed a misregulation of PcG target genes in EwS (20). Together, these findings suggest a potential role of the PcG during the early steps of EwS pathogenesis. Here, we report that RING1B and EWSR1-FLI1 interact and colocalize at the same genomic loci. Notably, we find that RING1B is present at promoters and enhancers of actively transcribed EWSR1-FLI1 target genes. Furthermore, we demonstrate that modulation of RING1B interferes with EWSR1-FLI1 recruitment and with the expression of EWSR1-FLI1 targets, thus unveiling an interdependent cooperation between both proteins.

Human pediatric MSCs (hpMSCs) have been proposed as a plausible cell of origin for EwS (21). Nevertheless primary human endothelial HUVECs share high similarity in gene expression profiles with EwS cells (22). Thus, to investigate the potential contribution of epigenetic alteration in the initiation of EwS, we analyzed the role of epigenetic marks in these models and compared to established EwS cell lines. We first analyzed the levels of H3K27me3 and H3K4me3 in the human EwS-derived cell line A673 at several bona fide direct targets of EWSR1-FLI1 (table S1) by chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR). Promoter of genes that are transcriptionally activated by EWSR1-FLI1, such as FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11, was enriched for the H3K4me3 mark, and lacked the H3K27me3 mark, in agreement with previous data (8, 20, 23, 24) (fig. S1A). On the other hand, transcriptionally repressed genes, such as KCNA5 (25), were enriched for H3K27me3. We next compared the levels of H3K27me3 and H3K4me3 at the same loci in HUVECs and in hpMSCs. In an apparently reversed situation to the A673 EwS cell line, analysis of those promoters presented strong enrichment for H3K27me3 but not for H3K4me3 (fig. S1B). Accordingly, infection of HUVECs with the EWSR1-FLI1 oncogene (Fig. 1A) not only led to the activation of these targets (FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11) (Fig. 1B) but also decreased the levels of H3K27me3 (Fig. 1C). This demonstrates that, although H3K27me3 is not present at oncogene binding regions in EwS cell lines such as A673, these regions are repressed by PcG before oncogene expression.

(A) Western blot showing ectopic expression of EWSR1-FLI1 upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. (B) RT-qPCR determination of relative mRNA expression of EWSR1-FLI1 target genes upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. Values are normalized to TBP. (C) H3K27me3 ChIP-qPCR at EWSR1-FLI1 target gene promoters in HUVECs infected with an empty pLIV vector or EWSR1-FLI1pLIV. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input with subtracted immunoglobulin G (IgG). (D) Bar plots of chromatin state relative frequencies in the whole genome [background (BG)] and in published EWSR1-FLI1 binding sites (FLI1) for three selected cell lines. Genome segmentations were extracted from the Epigenome Roadmap Consortium. (E) Heatmap with percentages of each chromatin state in the whole genome (BG) as compared to the frequency within published EWSR1-FLI1 binding regions for indicated cell lines by grouping in 8 similar chromatin states the initial classification containing 15 (quiescent segments were excluded). Bold format indicates enrichments greater than 10%. Enrichment scores were calculated as the difference between the value in EWSR1-FLI1 and the value at the whole genome, normalized by the value at the whole genome. (F) Cell proliferation expressed as cell number in 293T, A673, SK-ES1, and A4573 cells transiently transfected with small interfering RNA (siRNA) against a control (siCTRL) or two different RING1B sequences (siRING1B#1 and #2). Error bars in (B), (C), and (F) indicate SD of three biological independent experiments. Statistical significance in (D) and (F) is as follows: ***P < 0.001 and *P < 0.05.

To explore the chromatin and transcriptional states of EWSR1-FLI1 binding sites (10), we measured the frequency of each chromatin state at these regions (26) and compared to the corresponding value obtained for the whole genome in several cell lines [HUVECs, H1, and H9 human embryonic cells, H1-derived MSCs, bone marrow (BM)derived MSCs, and adipose-derived MSCs]. This analysis indicated that EWSR1-FLI1 binding sites are overrepresented in chromatin states associated with zinc finger genes and repeats (ZNF/repeats) and active promoters (Fig. 1D and table S1). In cells with MSC origin (such as H1, adipose, and BM-derived cell lines), EWSR1-FLI1 binding sites are overrepresented in PcG weak repressed state, which represents flanking regions of H3K27me3 peaks summit (Fig. 1D and fig. S1, C and D). Similar results were obtained when we grouped chromatin states of similar categories (Fig. 1E). This suggests that EWSR1-FLI1 occupies flanking regions of H3K27me3 summit peaks in hMSC, which are considered to be the potential cell of origin for EwS.

Data from our group have revealed that the PRC1 subunit RING1B, is highly overexpressed in EwS primary tumors (18). We thus assessed whether RING1B modulates the growth rate of EwS cells as has been reported for other PcG subunits, such as EZH2 and BMI1 (16, 17). RING1B depletion caused a reduction in cell viability in the A673, SK-ES1, and, with a lesser extent, in A4573 EwS cell lines but not in the control cell line 293T (Fig. 1F and fig. S1E), suggesting that RING1B represents an epigenetic vulnerability for EwS cells.

Chan et al. (27) recently proposed that RING1B might play a role in modulating enhancer activity. Together with its role in promoter regulation, EWSR1-FLI1 has been recently reported to generate de novo enhancers (10). This led us to postulate whether EWSR1-FLI1 and RING1B might cooperate during EwS tumorigenesis. We first aimed to define the genome-wide localization of RING1B and its repressive histone mark H2Aub in the A673 cell line by chromatin immunoprecipitation sequencing (ChIP-seq). In two independent experiments, we identified 2573 and 3945 peaks of RING1B, and 26424 and 10269 peaks of H2Aub. Using differential binding analysis (DiffBind), which allows for the identification of statistically common peaks (28), we found 2459 RING1B and 5392 H2Aub significant peaks between duplicates (P < 0.05, fig. S2A), corresponding to 1264 target genes and 3013 target genes, respectively (table S2). Genomic distribution of peaks showed that RING1B is more abundant in intergenic regions, whereas H2Aub is mainly located in promoters (Fig. 2A). Moreover, 38% of RING1B peaks were found at intergenic regions with respect to 21.5% of H2Aub peaks, and 29.2% of RING1B peaks were in promoters with respect to 40.5% of H2Aub peaks, further supporting the potential role of RING1B at enhancers. We then categorized peaks for RING1B, H2Aub, and EWSR1-FLI1 in active or poised enhancers, and in active or poised promoters, based on H3K27me3, H3K4me3, H3K27ac, and H3K4me1 (29). To complement the above data, we performed a ChIP-seq analysis using a different antibody directed against FLI1 (fig. S2B and table S2). We found that an important fraction of RING1B peaks (35%) and EWSR1-FLI1 (46%) are located at transcriptionally active enhancers and promoters of A673 cells (Fig. 2B, left). On the other hand, as expected, 35% of RING1B peaks and 37% of H2Aub peaks showed a preference for transcriptionally repressed regulatory regions (Fig. 2B, left). We then intersected the list of genes associated to RING1B and H2Aub peaks with published data of EWSR1-FLI1 target genes in A673 cells, producing a common set of 162 genes (fig. S2C and table S3). Comparing this set with 386 genes containing only RING1B and H2Aub or the group of 324 EWSR1-FLI1/RING1B genes without H2Aub confirmed that the presence of EWSR1-FLI1 correlated with higher level of transcription (P < 1016; fig. S2D, left). Functional analysis of the common gene set of 324 EWSR1-FLI1/RING1B genes (table S3) returned Gene Ontology (GO) categories related to chondrocyte and neuronal differentiation (fig. S2D, right). EWSR1-FLI1/RING1B/H2Aub genes were also enriched in neuronal differentiation category, while the RING1B/H2Aub genes were related to general transcription. These data suggest that RING1B is a positive regulator of a specific set of genes implicated in EwS and that this activity is independent of its canonical repressive mark.

(A) Pie chart showing genomic distribution of RING1B and H2Aub peaks relative to functional categories including promoter (2.5 kb from TSS), gene body (intragenic region not overlapping with promoter), and intergenic (rest of the genome). (B) Boxplot depicting percentage of regulatory elements (active/bivalent enhancers and promoters) in each described group. (C) Venn diagram depicting the overlap between RING1B and EWSR1-FLI1 in A673 cells at the peak level. (D) Aggregated plot showing the average ChIP-seq signal of RING1B and EWSR1-FLI1 at EWSR1-FLI1 binding sites. (E) Aggregated plots showing the average ChIP-seq signal of H3K27ac, H2Aub, and H3K27me3 in the three sets of RING1B and EWSR1-FLI1 peaks. (F) Heatmap showing RING1B, EWSR1-FLI1, H3K27ac, H2Aub, and H3K27me3 ChIP-seq signals segregating in the three sets of RING1B and EWSR1-FLI1 peaks. Top MEME motif for every group is shown. (G) University of California Santa Cruz (UCSC) genome browser ChIP-seq signal tracks for RING1B, EWSR1-FLI1, H2Aub, H3K27ac, H3K4me3, and H3K27me3 at NKX2-2, CCND1, VRK1, and CAV1 gene promoters and intergenic enhancer regions. Gray boxes represent EWSR1-FLI1 and RING1B colocalization and ES super-enhancers (SEnh; as shown at VRK1 and CAV1/2).

To fully understand the association of RING1B with transcriptional activation in EwS, we intersected EWSR1-FLI1 peaks with those of RING1B and obtained 955 common regions (Fig. 2C). Notably, intersection between H2Aub and RING1B peaks returned only 589 common peaks. Among the 955 overlapping EWSR1-FLI1/RING1B peaks, we inspected for genes containing an enhancer within 100 kb and obtained 1276 genes, of which 235 (18%) were reported to be regulated by EwS super-enhancers (table S4) (11). The common targets of RING1B and EWSR1-FLI1 sites were found within active enhancers, while the majority of RING1B peaks not overlapping with EWSR1-FLI1 were located in transcriptionally repressed regulatory elements (Fig. 2B, right). The distribution of RING1B peaks was centered on EWSR1-FLI1 binding sites (Fig. 2D), suggesting that their binding occurs at the same loci. We next assessed the distribution of H3K27ac, H2Aub, and H3K27me3 in genomic regions occupied by EWSR1-FLI1, RING1B, or shared (Fig. 2E). Common peaks were decorated with H3K27ac, lacking H2Aub (Fig. 2, E and F), and presented narrow RING1B peaks located in intergenic or intronic regions (fig. S2E, right). These data suggest that common sites likely represent enhancers. Known EWSR1-FLI1 target genes such as NKX2-2, CCND1, VRK1, or CAV1 presented an intergenic peak of RING1B, which overlaps with defined super-enhancers in the case of VRK1 and CAV1 (Fig. 2G). Intronic enhancers such as JARID2 or MYOM2 (fig. S2G) constitute the majority of the 162 common RING1B, EWSR1-FLI1, and H2Aub genes (53% of sites, fig. S2C). On the other hand, RING1B-specific peaks were associated with H3K27me3 and H2Aub (Fig. 2, E and F) and presented a broader distribution [e.g., HNF1B and TAL1 (fig. S2H)] mainly located within promoter or gene body regions (fig. S2E, left). The bivalent marks H3K4me3 and H3K27me3 decorated 63% of the 932 downstream genes associated to RING1B-specific peaks (P < 10300, table S4) (29). RING1Btranscription start sites (TSS) do not overlap with EWSR1-FLI1 and are decorated with H2K27me3 and H2Aub, while RING1B-distal sites overlap with EWSR1-FLI1 and with H3K27ac (fig. S2F).

Last, de novo motif analysis revealed that EWSR1-FLI1specific sites contained predominantly (P < 10282) one single occurrence of the canonical ETS motif GGAA (Fig. 2F). When EWSR1-FLI1 was associated with RING1B, we observed a significant enrichment for multimeric GGAA repeats (P < 101072) (10). Furthermore, RING1B-sepecific sites were enriched for CG sequence, as previously reported (P < 10176) (30). Together, we identified two major types of RING1B peaks in EwS: a prominent group with narrow peaks that colocalizes with EWSR1-FLI1 at enhancers of actively transcribed genes and a second group with broader peaks located at promoters, where RING1B is associated with H2Aub.

To further characterize RING1B binding regions (table S4), we analyzed several EWSR1-FLI1 active promoters (CAV1, FCGRT, NR0B1, CACNB2, FEZF1, and KIAA1797) and enhancers (CCND1, IGF1, CAV2, JARID2, VRK1, and NKX2-2) by ChIP-qPCR. Both groups showed enrichment for RING1B, with stronger signals at enhancers (Fig. 3A). Known repressed targets of the oncogene (e.g., IGFBP3, TGFBR2, and LOX) also showed binding of RING1B. At these repressed promoters, RING1B was accompanied by its canonical repressive mark H2Aub (fig. S3A). We also validated the occupancy of RING1B in EWSR1-FLI1activated promoters (CAV1, FCGRT, NR0B1, and FEZF1) and enhancers (CCND1, CAV2, JARID2, and VRK1) in SK-ES1 cells (fig. S3B). Similar to A673 cells, H2Aub correlated with RING1B at promoters of repressed genes (IGFBP3, TGFBR2, and LOX) (fig. S3C). Last, we observed that the PRC1 and PRC2 subunits, BMI1 and EZH2, respectively, were present at repressed promoters but not in active enhancers (fig. S3, D and E), as well as in promoters with broad peaks of RING1B concomitant with H3K27me3 and H2Aub but no EWSR1-FLI1 (e.g., TAL1, IGF1R, and HNF1B) (fig. S3F). Furthermore, genome-wide analysis demonstrated that BMI1 and CBX7 (31) subunits of the PRC1 canonical complex colocalize with RING1B only at repressed regions (TAL1) as shown in Fig. 3B, while no detectable peaks are present at active enhancers where EWSR1-FLI1 is present (VRK1). Thus, while RING1B decorates EWSR1-FLI1activated promoters and enhancers, it also maintains its canonical role at several oncogene repressed regions, as well as in a subgroup of genes with no EWSR1-FLI1.

(A) RING1B ChIP-qPCR of EWSR1-FLI1 bound active promoters, repressed promoters, and active enhancers. Control regions indicate the absence of RING1B and EWSR1-FLI1 binding at these sites. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. (B) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1, RING1B, CBX7, BMI1, H2Aub, and H3K27me3 at TAL1 promoter and VRK1 enhancer. (C) Histogram depicting percentages of activated and repressed genes in A673 and SK-ES1 cells with stable RING1B knockdown seq#2 (shRING1B#2) versus control seq#2 (shCTRL#2), with P < 0.05 and an absolute fold change (FC) > 1.25 or 1.5. (D) Western blot showing RING1B, RING1A, H2Aub, and H3K27me3 in A673 and SK-ES1 cells with either shCTRL#2 or shRING1B#2. Lamin B and histone H4 are used as loading controls. (E) Venn diagram showing intersection between differentially activated or repressed genes for EWSR1-FLI1 and RING1B in A673 cells; P < 0.05. (F) RT-qPCR determination of mRNA expression of EWSR1-FLI1 target genes with active enhancers in shCTRL and shRING1B A673 cells (#1 and #2). Values are normalized to GAPDH. (G) Same analysis as in (F) for SK-ES1 cells. Error bars in (A), (F), and (G) indicate SD of four independent biological experiments and ***P < 0.001, **P < 0.01, and *P < 0.05.

To understand whether RING1B behaves as a canonical repressor and/or activator in EwS, we analyzed the expression changes after knocking down RING1B using two different sets of short hairpin RNA (shRNA, seq#1 and seq#2; fig. S4A). The data obtained showed that 71.94 and 63.85% of genes were down-regulated in the A673 and SK-ES1 cell lines, respectively (FC < -1.5, Fig. 3C). This confirms our finding that RING1B acts predominantly as an activator, despite its presence at several EWSR1-FLI1repressed targets. Furthermore, H2Aub levels remained unchanged after RING1B knockdown (Fig. 3D), while RING1A knockdown produces a notable decrease in H2Aub levels (fig. S4B). These data suggest that RING1B main function in EwS is uncoupled from its ubiquitin ligase activity toward H2A and that RING1A is the main histone H2A mono-ubiquitin ligase. To further elucidate to what extent RING1B cooperates with EWSR1-FLI1 in transcription regulation, we intersected differentially expressed genes in RING1B knockdown cells (absolute FC > 1.25) with those affected by EWSR1-FLI1 knockdown (absolute FC > 1.5) (10), obtaining an overlap of 1078 genes. After segregating these data into down- and up-regulated genes, we found that RING1B and EWSR1-FLI1activated 229 genes and repressed 162 genes (Fig. 3E and table S5). Among the 229 activated genes, we found several developmental genes, including SOX2, SIX3, LYAR, and KIT. GO analysis showed regulation of the potassium channel and mechanisms that control actin monomers and filaments as the main categories (fig. S4C), in agreement with previous publications (25, 32). Among the activated genes, SOX2 and KIT harbored RING1B and EWSR1-FLI1 peaks in intergenic and intronic enhancer regions, respectively (fig. S4E). TGFBR2, a gene repressed by both EWSR1-FLI1 and RING1B, also contained an intronic enhancer where both proteins colocalized. Notably, the expression of known targets of EWSR1-FLI1, such as NKX2-2 or IGF1 (fig. S4D), was just below our logFC cutoff value. Nonetheless, we confirmed by reverse transcription (RT)qPCR the changes in expression levels of selected repressed and activated genes cobound by EWSR1-FLI1 and RING1B. We noticed that RING1B knockdown causes a significant reduction in the expression levels of those genes where both EWSR1-FLI1 and RING1B were co-occupying enhancer regions (Fig. 3, F and G). The expression of CAV1, NKX2-2, SOX2, IGF1, JARID2, and VRK1 was affected in stronger manner upon EWSR1-FLI1 knockdown, indicating that some cofactors could remain when RING1B is depleted (fig. S4F). The effect of RING1B knockdown was less pronounced when both proteins were enriched at promoter regions of active genes (fig. S4, G and H, left). As expected, at those genes where EWSR1-FLI1 acts as a repressor, RING1B knockdown induces a promoter reactivation (fig. S4, G and H, right). Overall, these data indicate that RING1B and EWSR1-FLI1 cooperate in gene activation, at both the promoter and enhancer levels, while RING1B retains its canonical role at those targets repressed by the oncogene. Since a large number of EWSR1-FLI1 and RING1B cotargets were not altered by RING1B knockdown, we postulate compensatory mechanism(s) or additional cofactors involved in their regulation.

Wild-type EWSR1 interacts with RING1B in the VCaP prostate cancer cell line (33). We also confirmed this interaction in SK-ES1 cells (Fig. 4A). Since RING1B and EWSR1-FLI1 are enriched at transcriptionally active regions, we next aimed to investigate whether both proteins interact. Coimmunoprecipitation experiments in HeLa cells where EWSR1-FLI13xFlag was overexpressed (34) confirmed that indeed oncogene interacts with RING1B (Fig. 4, B and C). Analysis of published mass spectrometry data demonstrated that several SWI/SNF subunits interact with RING1B (33), further supporting an active role of RING1B in EwS gene regulation. Together, our results indicate that EWSR1-FLI1 and RING1B not only colocalize at the same genomic regions but also physically interact, mainly through the EWSR1 component of the fusion protein.

(A) Western blot showing endogenous coimmunoprecipitation of RING1B with EWSR1 in the SK-ES1 cell line. (B) Western blot showing overexpression of EWSR1-FLI1-3xFlag and RING1B levels in HeLa stably transfected cells upon induction with indicated doxycycline concentrations for 24 hours. Calnexin is used as loading control. (C) Coimmunoprecipitation of RING1B with EWSR1-FLI1-3xFlag under induction conditions (0.5 g/ml). Inputs in (A) and (C) contain 10% of immunoprecipitated material and IgG is used as control. (D) Western blot showing RING1B and EWSR1-FLI1 in cytoplasm, soluble, and bound chromatin fractions in shCTRL#1 or shRING1B#1 SK-ES1 cells. Histone H4 is used as a control of bound chromatin, and GAPDH as a control of cytoplasmic fraction. Blot quantification of the same ordered samples is depicted below. (E) ChIP-qPCR analysis of FLI1, RING1B, and H3K27ac at EWSR1-FLI1activated enhancers of NKX2-2, SOX2, or IGF1 genes in shCTRL#2 and shRING1B#2 A673 cells. ENC1 is used as negative control region. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars indicate SD of three independent biological experiments. Statistical significance is as follows ***P < 0.001, **P < 0.01, and *P < 0.05. (F) Aggregated plot and boxplot showing the average ChIP-seq signal of RING1B and FLI1 peaks at RING1B and EWSR1-FLI1 binding sites, respectively, in shCTRL#2 and shRING1B#2 A673 cells. (G) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1 and RING1B in shCTRL#2 and shRING1B#2 A673 cells at SOX2 and VRK1 enhancer regions.

Next, we analyzed whether RING1B depletion affects the EWSR1-FLI1 recruitment to chromatin. As expected, after knockdown, we observed a notable reduction of RING1B in the chromatin bound fraction (Fig. 4D). EWSR1-FLI1 was also evicted from chromatin bound and enriched in the soluble chromatin fraction (Fig. 4D). We then monitored the occupancy of EWSR1-FLI1, RING1B, and H3K27ac at several enhancers (e.g., SOX2, NKX2-2, and IGF1). The data in Fig. 4E showed that upon RING1B knockdown, enrichments at those enhancers decreased to control values [immunoglobulin G (IgG) or ENC1 region]. To assess the decrease of EWSR1-FLI1 recruitment genome-wide, we performed ChIP-seq analysis of RING1B and FLI1 in shCTRL and shRING1B A673 cells. The analysis indicated that upon RING1B depletion, EWSR1-FLI1 binding to chromatin was reduced (Fig. 4, F and G). In sum, we conclude that in EwS, RING1B exerts its main role as activator by promoting recruitment of EWSR1-FLI1 to enhancer regions.

RING1B stimulates tumor growth and metastasis in melanoma, leukemia, and breast cancers (14, 27). We observed a reduction in colony number when RING1B is depleted in the SK-ES1 cell line (fig. S5A). To gain functional insight into the cancer pathways potentially modulated by RING1B, we performed gene set enrichment analysis (GSEA) by comparing SK-ES1 shCTRL versus shRING1B cells. The top 10 most significant pathways included interferon-, epithelial-to-mesenchymal transition, hedgehog signaling, and angiogenesis, with a 0.25 Q value cutoff (fig. S5B). In EwS, disruption of angiogenic pathways has been described (4, 22). Further inspection of angiogenic gene list revealed that key genes such as PDGFA, FGFR1, SLCO2A1, CXCL6, and S100A4 were down-regulated upon RING1B depletion (fig. S5C).

To assess the relevance of RING1B in vivo, we generated xenografts by injecting SK-ES1 shCTRL or shRING1B cells (seq#1 and seq#2) subcutaneously into athymic nude mice. Cells with reduced RING1B levels showed delayed engraftment and slower tumor growth (Fig. 5A). At 21 days after injection, tumors derived from shRING1B cells were significantly smaller than those from control cells (fig. S5D). Notably, the median survival increases from 26 days for shCTRL cells to 30 days for shRING1B seq#1 and from 20 to 27 days for shRING1B seq#2 (Fig. 5B). Immunohistochemical analyses of tumors confirmed reduced levels of RING1B, while the ES marker CD99 remained essentially unchanged (Fig. 5C and fig. S5E). Furthermore, shCTRL tumors displayed higher proliferation rates than shRING1B, as shown by Ki-67 staining (Fig. 5C).

(A) Tumor volume curve in xenografts established by subcutaneous injection of shCTRL and shRING1B#1 (n = 9 and n = 10, respectively, above) or shRING1B#2 (n = 12 both groups, below) SK-ES1 cells in athymic nude mice. (B) Kaplan-Meier xenograft survival curves in shCTRL and shRING1B SK-ES1 cells (#1 and #2). (C) Immunohistochemistry staining of EWSR1-FLI1, CD99, and RING1B on sections of tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 xenografts. Proliferation was analyzed by Ki67 immunohistochemistry; hematoxylin and eosin (H&E) was used as control. (D) Heatmap depicting fold changes in gene expression in six tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 groups. (E) RT-qPCR levels of mRNA expression for RING1B and EWSR1-FLI1 in shCTRL#1 and shRING1B#1 SK-ES1derived tumors; ***P < 0.001. (F) RT-qPCR levels of mRNA expression for genes regulated by EWSR1-FLI1/RING1B enhancers (left) and angiogenic genes (right) in shCTRL#1 and shRING1B#1 SK-ES1 derived tumors; *P < 0.05.

To better characterize xenograft derived tumors, we performed RNA sequencing (RNA-seq) of a cohort of tumors (six for each group, Fig. 5D). GSEA analysis confirmed the enrichment of angiogenic genes in the shCTRL tumors (fig. S5, F and G). Since RING1B retains its repressive function at several promoters, we hypothesized that the delay in survival and in tumor growth upon RING1B knockdown could be related to up-regulation of tumor suppressor genes (TSG). GSEA applied to 983 genes from TSG database (https://bioinfo.uth.edu/TSGene), indicated that this gene list was enriched in shCTRL phenotype, suggesting that tumor growth and survival differences observed were not due to RING1B repression of TSG (fig. S5H). The NKX2-2, SOX2, and IGF1 genes are necessary for EwS tumor proliferation (21, 23, 35). In agreement, confirmed RING1B and EWSR1-FLI1 expression reduction (Fig. 5E) is associated to down-regulation of these genes in xenograft tumors (Fig. 5F, left), as we previously shown in EwS cells (Fig. 3, F and G). Furthermore, after RING1B knockdown, we also validated down-regulation of S100A4, SLCO2A1, and VEGFA, which are main activators of angiogenic signaling pathways (Fig. 5F, right). All these data highlight the role of RING1B as an activator in EwS tumorigenesis.

Several kinases (including AURKB, MEK1, and CK2) have been reported to modulate the activating transcriptional function of RING1B (14, 15, 36). To investigate which pathway(s) regulates RING1B at active enhancers in EwS, we analyzed the expression levels of these three kinases in a publicly available database (4) comprising a cohort of 27 tumor samples and BM-MSCs. While MEK1 and CK2 were not expressed in primary tumors with respect to BM-MSCs (control), 11 of 27 EwS tumors (40%) showed higher levels of AURKB compared to control (fig. S6A). EWSR1-FLI1 directly regulates the expression of AURKB (37), as also demonstrated by AURKB down-regulation in EwS cell lines upon oncogene knockdown (fig. S6A).

AZD1152 is a specific AURKB inhibitor, with a median inhibitory concentration (IC50) of 19 nM in EwS cell lines (38). Accordingly, we observed IC50 values of 5 and 6 nM in SK-ES1 and A4573 cells, respectively; in contrast, the IC50 for A673 was 5 M, and AZD1152 had no effect on the control cell line 293T (fig. S6B). EwS cells that survived to the treatment showed an atypical phenotype, suggesting enhanced differentiation (fig. S6C). Furthermore, viability of EwS cell lines was not affected by the inhibition of RING1B E3 ubiquitin ligase activity with PRT4165 (fig. S6B). To further elucidate the effect of AZD1152 in EwS, cell death was analyzed by Annexin V staining. A 72-hour AZD1152 treatment of A673, SK-ES1, and A4573 cells led to an increase in the early and late apoptosis populations as compared to 293T cells (Fig. 6A). Analysis of cleaved PARP levels further demonstrated that AZD1152 stimulated apoptotic pathways in EwS cell lines, with SK-ES1 being the most sensitive (Fig. 6B). It is worth noting that the levels of EWSR1-FLI1 were decreased after AZD1152 treatment in SK-ES1 and A4573, yet RING1B levels were unaffected (Fig. 6B and fig. S6, D and E, right). To understand how AURKB modulates RING1B in EwS, we analyzed H2Aub levels after AZD1152 treatment. We observed increased levels of H2Aub repressive mark after AURKB inhibition, suggesting that this kinase indeed inhibits the ubiquitin ligase activity of RING1B in EwS (Fig. 6C). Furthermore, in SK-ES1 and A4573 cells, the increase in ubiquitin ligase activity correlated with decreased expression of EWSR1-FLI1 targets co-occupied by RING1B, with more pronounced effect on those genes where both proteins colocalize at the enhancer region (Fig. 6D and fig. S6, D and E, left). For the A673 cell line, higher doses were required to reach oncogene target deregulation, as expected. Next, we reasoned that AURKB should be present at those regions where it inhibits RING1B activity. Using ChIP-qPCR, we demonstrated that AURKB is enriched in active enhancers (CAV2, driving CAV1 expression, and SOX2; Fig. 6E) and promoters (NR0B1; fig. S6F). Furthermore, EWSR1-FLI1 down-regulation could be explained by the presence of RING1B at the EWSR1 promoter, which indirectly decreases upon AZD1152 incubation (fig. S6G). Although part of AZD1152 cytotoxicity might be related to reduction of EWSR1-FLI1 availability, the data presented suggest that RING1B regulation of oncogene targets is susceptible to AURKB inhibition. The translational value of this potential targetable vulnerability is the matter of ongoing work.

(A) Annexin V staining of SK-ES1, A673, and A4573 cells after treatment with AZD1152 (20 nM). 293T cells were used as a control cell line. (B) Western blot analysis of cleaved poly(ADP-ribose) polymerase (cPARP), EWSR1-FLI1, RING1B, and AURKB after treatment with 10 or 20 nM AZD1152, in the A673, SK-ES1, A4573, and 293T cell lines. Tubulin was used as loading control. (C) Western blot analysis of H2Aub and H3S10phospho (H3S10ph) in the A673, SK-ES1, and A4573 cell lines treated with 5 or 20 nM AZD1152. Histone H4 was used as loading control. (D) RT-qPCR determination of mRNA expression of target genes with RING1B/EWSR1-FLI bound enhancers in SK-ES1 and A4573 cells after treatment with 20 nM AZD1152. RPL27 was used for normalization. DMSO, dimethyl sulfoxide. (E) AURKB ChIP-qPCR at CAV2 and SOX2 EWSR1-FLI1/RING1B enhancers (above) and control regions (below). The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars in (D) and (E) indicate SD of three independent biological experiments. (F) Schematic representation illustrating the EWSR1-FLI1 recruitment by RING1B to repressed regions containing GGAA repeats. Once EWSR1-FLI1 has been recruited, additional cooperating factors such as AURKB might inhibit RING1B ubiquitin ligase activity, which, in turn, is able to participate in transcription activation.

Here, we investigated the genome-wide occupancy of RING1B in EwS. In agreement with previous data, we identified a set of regions bound by RING1B where it exerts its canonical repressive function. We also report that RING1B co-occupy together with EWSR1-FLI1 many intergenic and intronic regions decorated with H3K27ac. A strong enrichment in GGAA repeats has been described in regulatory elements where EWSR1-FLI1 binds producing active enhancers (10). The presence of GGAA repeats, as well as the H3K27ac association, indicates that cobinding of RING1B and EWSR1-FLI1 occurs in active enhancers. BMI1 or EZH2 was not found at these enhancer regions, suggesting a Polycomb-independent function for RING1B. Enhancers are key regulatory regions implicated in cell fate determination. Here, we unveiled that an aberrant transcription factor such as EWSR1-FLI1 relies on RING1B to activate enhancers, causing an altered gene expression profile, which favor cell transformation.

In accordance with RNA-seq data from melanoma and breast cancer, where a positive association of RING1B with transcription activation has been reported (14, 27), we observed in EwS cells a higher number of genes activated than repressed by RING1B. We found NKX2-2, SOX2, and IGF1 being direct targets down-regulated both in vivo and in vitro upon RING1B knockdown. In EwS, NKX2-2 and SOX2 are key players in tumorigenesis (21, 23), suggesting that modulation of their expression in vivo upon RING1B knockdown might contribute to decreased tumor volume and better survival, supporting an oncogenic role for RING1B.

Recent studies in hpMSCs have demonstrated that, before oncogene recruitment, H3K27me3 is enriched at regions where EWSR1-FLI1 could bind (39). In agreement with these data, we further demonstrate that upon EWSR1-FLI1 expression, those same regions loose H3K27me3 marks while becoming transcribed. Moreover, we report that enrichment in Polycomb repressed chromatin states is specific for H1-, adipose- and BM-derived MSCs, reinforcing hMSC as the putative cell of origin, which has already been described by other groups (4, 21). The existence of H3K27me3 repressed regions decorated only with PRC1 complex has already been described during differentiation of neural precursor cells, where RING1B and PCGF2 are retained while the PRC2 subunit Suz12 is not (40). In melanoma, CCND2 is marked with H3K27me3 before RING1B activation by phosphorylation (14). We have observed that GGAA repeats are differentially enriched in the binding motif analysis when RING1B is associated to chromatin with EWSR1-FLI1. In this scenario, given the interaction observed for RING1B and EWSR1-FLI1, it is tempting to speculate that RING1B targets EWSR1-FLI1 to specific sites. In line with this hypothesis, the reduced recruitment of EWSR1-FLI1 to chromatin (including enhancer regions, such as NKX2-2, SOX2, and IGF1) upon RING1B knockdown underlines the importance of RING1B in the initials steps of EwS tumorigenesis. Overall, our data suggest that RING1B is required for the recruitment of EWSR1-FLI1 to multimeric GGAA repeats (Fig. 6F).

We have demonstrated that RING1B is an essential partner of EWSR1-FLI1 triggering chromatin remodeling. Recent studies demonstrated the requirement of SWI/SNF, WDR5, and p300 acetyltransferase for EWSR1-FLI1induced transcription. Similarly, in synovial sarcoma, the SS18-SSX oncogenic fusion protein and the SWI/SNF complex colocalize at KDM2B-repressed target genes together with the noncanonical PRC1.1 complex to produce transcriptional active regions (41). Along the same lines, in leukemia, noncanonical PRC1.1 also targets active genes independently of H3K27me3 (42). Further mechanistic insights are needed to elucidate the contribution of PRC1.1 repressive complex in EwS, where somatic mutations in BCOR have been reported (1). The noncanonical PRC1.1 complex contains a DNA binding ZnF-CXXC domain able to target chromatin via KDM2B (43). ZNF/repeats chromatin state was statistically enriched in five of the six EwS cell lines analyzed.

Recently, different cell models have shown that the E3 ubiquitin ligase activity of RING1B is inactivated by phosphorylation (15, 36). Our results showing the recruitment of AURKB to enhancers are compatible with a model in which RING1B is unable to repress the newly formed ES enhancers, which were previously Polycomb-repressed regions. Once the oncogene binds to chromatin, RING1B would cooperate to induce transcription activation if its ubiquitin ligase activity is inhibited by phosphorylation (either directly or indirectly) (Fig. 6F). More studies are needed to clarify how oncogenic fusion proteins act as binding scaffolds to recruit a specific set of interactors to generate previously unknown functional units (such as neo-enhancers).

Inhibition of super-enhancers activity with BET inhibitors has emerged as a successful preclinical strategy in the fight against different pediatric cancers such as EwS, neuroblastoma, and rhabdomyosarcoma (4446). Inhibition of AURKB with AZD1152 increases H2Aub and decreases expression of key oncogene targets, thus suggesting that RING1B is essential for enhancer deregulation by EWSR1-FLI1. Nevertheless, as RING1B account for catalytic and noncatalytic dependencies (14), further investigation should address its clinical therapeutic implications. In agreement with our data, combined inhibition of AURKA and AURKB, as well as synergistic activity of AURKB with focal adhesion kinase inhibitors, has been described effective in EwS preclinical studies, although AURKB efficiency as single agent has not been proved (47, 48). In EwS cells, AZD1152 could affect the levels of RING1B, and this likely reverberates on the regulation of the oncogenes promoter since RING1B occupies the EWSR1 promoter (fig. S6, E and G).

In summary, we demonstrate the oncogenic dependency to high levels of RING1B in EwS. The data support a model in which RING1B plays a pivotal role for EWSR1-FLI1 recruitment to the multimeric DNA repeats. This, in turn, allows for transcriptional activation that defines the characteristic transcriptome of EwS. Given the role of RING1B in the activation of super-enhancers, which are critical elements for cell fate determination, we propose that the EwS cell of origin is predefined by high levels of RING1B.

The Ewings sarcoma cell lines A673, SK-ES1, and A4573, which carry the EWSR1-FLI1 translocation types I, II, and III, respectively, and the HEK293 cell line from human embryonic kidney infected with AgT from SV40 (293T), were cultured in RPMI 1640 media (Gibco) and supplemented with 10% fetal bovine serum, l-glutamine, and penicillin/streptomycin. Cells were cultured at 37C with 5% CO2. The A673 and SK-ES1 cell lines harboring shCTRL and shRING1B with seq#1 and seq#2 as well as A673 cell line with doxycycline inducible knockdown of EWSR1-FLI1 were previously described (11, 18). hpMSCs were isolated following published protocols (21). Ectopic expression of EWSR1-FLI1 3xFLAG C terminus in HeLa cells was induced with doxycycline (0.5 g/ml) (34).

All experiments performed with AZD1152 were incubated 72 hours, with the exception of RNA expression assays that were incubated 24 hours. For IC50 calculations, A673, SK-ES1, A4573, and 293T cell lines were seeded at 2000 cells per well in 96-well culture plates. AZD1152 and PRT4165 (Sigma-Aldrich) was added to complete growth medium; after 72 hours, cells were subjected to the ATPlite assay (PerkinElmer), and measurements were performed using a Tecan plate reader. Inhibitory concentrations were calculated using OriginPro 9.0 software.

EWSR1-FLI1 type 2 was amplified from a pSG5 vector with primers containing Bgl II and Hind III sequences (forward, 5-ggaggaaggAGATCTAATGGCGTCCACGG-3; reverse, 5-aagAAGCTTGTAGTAGCTGCCTAA-3). The PCR product was purified using an Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences). The product of the amplification was subcloned into the TOPO TA Cloning Kit for Sequencing following the manufacturers instructions. TOPO-EWSR1-FLI1 plasmid and the acceptor vector pEGFP-N1 were double digested with Bgl II and Hind III at 37C. The resulting EWSR1-FLI1 band was ligated into pEGFP-N1, and ligation product was then transformed into JM109 cells.

Target sequences for siRNA are described in table S6. Transfection of small duplexes (Sigma-Aldrich) was performed with Lipofectamine RNAiMAX and Optimem (Invitrogen), using 30 pmol when cells were 80% confluent; samples were collected after a 72-hour incubation. Transient transfections of GFP constructs or empty vector were done using FuGENE XP (Roche) with 1 to 2 g of plasmid when cells were 60% confluent; samples were collected after 48 hours. Both reagents were used according to the manufacturers recommendations.

Empty pLIV and EWSR1-FLI1pLIVexpressing lentiviruses were provided by N. Riggi (University Institute of Pathology Lausanne, Switzerland). Lentiviruses were produced in Lenti-X 293T packaging cells (Takara, Cultek) at a low passage number. For each plate, 7 g of the lentiviral plasmid, 5 g of the envelope plasmid (VSV-G), and 6 g of the packaging plasmid (PAX8) were prepared and introduced by calcium phosphate transfection, according to standard protocols. The supernatant containing lentiviruses was collected 48 hours after transfection. The HUVEC cell line was seeded at 3000 cells/cm2 and transduced with 3:1 of the lentiviral supernatant with fresh media containing Polybrene (Sigma-Aldrich) at 6 g/ml. Cells were selected with fresh growth media containing puromycin (0.3 g/ml) for 72 hours. A control dish without the transduction media was also selected with puromycin, to control for killing of nontransduced cells.

Histone extracts of cultured cells were isolated using the EpiQuick Histone Extraction kit (Epigentek) following the manufacturers instructions. Total cell extracts were prepared in IPH buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40] with EDTA-free protease inhibitor cocktail (Roche). For protein, fractionation standard protocols were used. Histone or total protein extracts were quantified by Bradford assay. Immunoprecipitation was performed with total cellular extracts incubated at 4C overnight with primary antibody. After incubation of immunoprecipitated samples on protein A/G and agarose beads (Santa Cruz Biotech), 30 to 50 g of whole protein extracts or 5 g of histones was resolved by polyacrylamide gel electrophoresis. Western blotting was performed using standard protocols. Incubation with primary antibodies was done at 4C overnight and LI-COR secondary antibodies that are detectable by near-infrared fluorescence were used for detection (table S6). Blots were scanned with an Odyssey CLx Infrared Imaging System at medium intensities.

Treated cells were fixed in 70% ethanol, stained with 25 l of propidium iodide (PI) (1 mg/ml), and 25 l of ribonuclease (RNase) (10 mg/ml), and incubated 30 min at 37C. For Annexin V binding, the Alexa Fluor 488 fluorophore kit (Invitrogen) was used for apoptotic cell detection. After culture and treatment, cells were resuspended in annexin binding buffer with 5 l of Alexa Fluor 488 Annexin V and 1 l of PI working solution (100 g/ml). After 15 min, samples were run in Gallios multicolor flow cytometer (Beckman Coulter) set up with the 3-lasers, 10 colors standard configuration. Histograms and cytograms were further analyzed with FlowJo 10.2.

Total RNA was isolated and purified from collected cells using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol. After quantification using the NanoDrop software (Thermo Fisher Scientific), RT was performed. A 1-g aliquot of each RNA sample was converted to cDNA in a reaction catalyzed by a retrotranscriptase enzyme (M-MLV Reverse Transcriptase Promega). Random primers and RNase inhibitor (RNasin Plus RNase Inhibitor, Promega) were also added to the reaction. cDNA obtained was analyzed by qPCR using SYBR Green PCR Master Mix (ABI). cDNA was amplified with specific oligonucleotides (table S6). Each cDNA sample was run in triplicate, and its levels were analyzed using the 7500 Fast PCR instrument (Applied Biosystems). To compare between different conditions studied, relative quantification of each target was normalized to a housekeeping gene. Last, data were analyzed using the comparative 2-ct method.

Gene expression microarrays were performed at the Microarray Analysis Service, Hospital del Mar Medical Research Institute (IMIM, Barcelona). RNA samples were amplified, labeled according to a GeneChip WT PLUS Reagent kit, and hybridized to Human Gene 2.0 ST (Affymetrix) in a GeneChip Hybridization Oven 640. Washing and scanning were performed using the Expression Wash, Stain, and Scan Kit and the GeneChip System of Affymetrix (GeneChip Fluidics Station 450 and GeneChip Scanner 3000 7G). After quality control, raw data were background corrected, quantile-normalized, and summarized to a gene level using the robust multichip average; a total of 48,144 transcript clusters, excluding controls, were obtained, which roughly corresponds to genes and other RNAs, such as long intergenic noncoding RNAs and microRNAs. NetAffx 36 annotations, based on the human genome 19, were used to summarize data into transcript clusters and to annotate analyzed data. Linear Models for Microarray (limma), a moderated t statistics model, was used for detecting differentially expressed genes between the conditions. All data analyses were performed in R (version 3.4.3) with R/Bioconductor packages aroma.affymetrix, Biobase, affy, limma, genefilter, ggplots, and Vennerable. Genes with a P less than 0.05 were selected as significant.

Raw sequencing reads in the fastq files were mapped with STAR version 2.6.a (49). GENCODE release 29, based on the GRCh38 reference genome, and the corresponding GTF file were used. The table of counts was obtained with featureCounts function in the package subread, version 1.6.4. The differential gene expression analysis (DEG) was assessed with voom+limma in the limma package version 3.40.2 and using R version 3.6.0. Raw library size differences between samples were treated with the weighted trimmed mean method implemented in the edgeR package. Clustering method used is Ward.D2 with correlation distances and principal components analysis. For the differential expression analysis, read counts were converted to log2 counts per million, and the mean-variance relationship was modeled with precision weights using voom approach in limma package. Raw data are accessible at the NCBI Gene Expression Omnibus (GEO) accession code GSE131286.

Intersection of DEG for A673 shRING1B knockdown with those for A673 shEWSR1-FLI1 with accession number GSE61953 (10) was obtained by calculating a delta-score as described by the authors. Absolute FC > 1.25 and 1.5 for RING1B and EWSR1-FLI1 datasets were selected, respectively. Overlaps for positive and negative gene sets were obtained using Vennerable R package and BioVenn. Functional analysis of the intersection between RING1B and EWSR1-FLI1 gene lists was performed in Enrichr. Normalized enrichment scores on A673 and SK-ES1 shRING1B versus shCTRL were obtained with GSEA using the Hallmark gene set collection. GSEA was used to analyze enrichment on the list of 983 down-regulated TSG in tumor samples versus normal tissue from TSGene database (50) (https://bioinfo.uth.edu/TSGene/). Analysis of expression levels for AURKB, CSNK2A1, and MAP2K1 were performed using information from GEO2R GSE7007 for the probes 209464_at, 212075_s_at, and 202670_at, respectively.

Immunohistochemical analyses were performed following standard techniques. The antibodies used are given in table S6. Tumors were fixed in formalin and embedded in paraffin for subsequent processing. Consecutive, sections were deparaffinized, rehydrated, and heated with Epitope Retrieval Solution (pH 6.0) (Novocastra Laboratories). Reactions were developed with Novolink Polymer Detection System (Novocastra Laboratories). Immunoreactivity was visualized by diaminobenzidine, and nuclei were counterstained with hematoxylin. Tissue was then dehydrated with alcohol, permeated with xylene, and mounted with Permount organic mounting solution (Thermo Fisher Scientific). Images were evaluated by a pathologist to select regions of interest and analyzed with the Dotslide Microscope and Olympia Software (Olympus). Similar regions of every sample were selected from every section.

Cells were treated with 1% formaldehyde at room temperature for 10 min, and the cross-linking reaction was stop by adding 500 l glycine (1.25 M). Cells were resuspended in lysis buffer [0.1% SDS, 0.15 M NaCl, 1% Triton X-100, 1 mM EDTA, 20 mM tris (pH 8), and protease inhibitors (1 mg/ml)] and sonicated with Bioruptor Pico (Diagenode) for 10 cycles until chromatin was sheared to an average fragment length of 200 bp. After centrifugation, a small fraction of eluted chromatin was measured with Qubit. Starting with 30 g of sample, immunoprecipitation for each antibody was performed overnight (table S6); 50 l of Dynabeads Protein A (Invitrogen) was then added and incubated for 2 hours at 4C under rotation. Immunoprecipitates were washed once with TSE I [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 150 mM NaCl], TSE II [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 500 mM NaCl], and TSE III [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM tris-HCl (pH 8)] and then twice with tris-EDTA buffer. Washed pellets were eluted with 120 l of a solution of 1% SDS and 0.1 M NaHCO3. Eluted pellets were decross-linked for 5 hours at 65C and purified on 50 l of tris-EDTA buffer with the QIAquick PCR Purification Kit (Qiagen). Differences in the DNA content at each binding region (sequences in table S6) from every immunoprecipitation assay were determined by real-time PCR using the ABI 7700 sequence detection system and SYBR Green master mix protocol (Applied Biosystems). Each immunoprecipitation was done in triplicate, and PCR assays were performed using fixed amounts of input and immunoprecipitated DNA. For every amplicon, standard curves to calculate efficiency and melting curves to confirm single amplicons were obtained. The reported data represent real-time PCR values normalized to input DNA and are expressed as percentage (%) of bound/input signal.

Libraries were prepared using the NEBNext Ultra DNA Library Prep from Illumina according to the manufacturers protocol. Briefly, 5 ng of input and ChIP-enriched DNA were subjected to end repair and addition of A bases to 3 ends, ligation of adapters, and USER excision. All purification steps were performed using AgenCourt AMPure XP beads (Qiagen). Library amplification was performed by PCR using NEBNext Multiplex Oligos from Illumina. Final libraries were analyzed using Agilent high sensitivity chip to estimate the quantity and to check size distribution and then were quantified by qPCR using the KAPA Library Quantification Kit (KapaBiosystems) before amplification with Illuminas cBot. Libraries were loaded onto the flow cell sequencer 1 50 on Illuminas HiSeq 2500.

ChIP-seq samples were mapped against the hg19 human genome assembly using BowTie with the option m 1 to discard those reads that could not be uniquely mapped to just one region. A second replicate of RING1B and H2Aub was sequenced to evaluate the statistical significance of the results. Model-based analysis of ChIP-seq (MACS) was run individually on each replicate with the default parameters but with the shift size adjusted to 100 bp to perform the peak calling against the corresponding control sample (51). DiffBind was initially run over the peaks reported by MACS for each pair of replicates of the same experiment to generate a consensus set of peaks (28). Next, DiffBind was run again over each pair of replicates of the same experiment, samples and inputs, to find the peaks from the consensus set that were significantly enriched in both replicates in comparison to the corresponding controls (categories, DBA_CONDITION; block, DBA_REPLICATE; and method, DBA_DESEQ2_BLOCK). DiffBind RING1B peaks with P < 0.05 and H2Aub peaks with P < 0.05 and false discovery rate < 0.00001 were selected for further analysis. The genome distribution of each set of peaks was calculated by counting the number of peaks fitted on each class of region according to RefSeq annotations. Promoter is the region between 2.5 kb upstream and 2.5 kb downstream of the TSS. Genic regions correspond to the rest of the gene (the part that is not classified as promoter), and the rest of the genome is considered to be intergenic. Peaks that overlapped with more than one genomic feature were proportionally counted the same number of times. Each set of target genes was retrieved by matching the ChIP-seq peaks in the region 2.5 kb upstream of the TSS until the end of the transcripts as annotated in RefSeq. Reports of functional enrichments of GO categories were generated using the EnrichR tool. Aggregated plots showing the average distribution of ChIP-seq reads around the summit of each peak were generated by counting the number of reads for each region and then averaging the values for the total number of mapped reads of each sample and the total number of peaks in the particular gene set. To perform the comparison between two sets of peaks, a minimum overlap of one nucleotide was necessary to consider one match. The heatmap displaying the density of ChIP-seq reads 5 kb around the summit of each peak set were generated by counting the number of reads in this region for each individual peak and normalizing this value with the total number of mapped reads of the sample. Peaks on each ChIP heatmap were ranked by the logarithm of the average number of reads in the same genomic region. On the other hand, we separated the single peaks of RING1B into distal and TSS (5 kb around one RefSeq gene) to generate the heatmap of ChIP-seq signal strength of RING1B, EWSR1-FLI1, H3K27me3, H2Aub, and H3K27ac over the two classes of RING1B peaks detected above (distal and TSS). To build our collection of enhancers and promoters, we reanalyzed published ChIP-seq samples of H3K4me1, H3K27ac, H3K27me3, and H3K4me3 in A673 cells (10). H3K27ac and H3K27me3 peaks were used to discriminate between active or repressed regulatory regions. Promoters were defined as ChIP peaks of H3K27 found up to 2.5 kb from the TSS of one gene and enhancers on intergenic areas outside promoters or within gene introns. H3K4me3 was required to be present in promoters but absent in enhancers. We defined four classes of regulatory elements: active enhancers (H3K27ac), active promoters (H3K27ac + H3K4me3), poised enhancers (H3K27me3), and bivalent promoters (H3K27me3 + H3K4me3). The MEME-ChIP tool was used to perform motif-finding analysis of the sequences bound by each factor. The UCSC genome browser was used to generate the screenshots of each group of experiments along the manuscript (52). Raw data, genome-wide profiles, and peaks of each ChIP-seq experiment are accessible at the NCBI GEO accession code GSE131286.

We have determined the composition of 3945 EWSR1-FLI1 biding sites in terms of 15 chromatin states from the segmentations generated by Epigenome Roadmap Consortium (GEO code: GSE61953) for six different cell types: HUVECs (E122), H1 (E003) and H9 ES cells (E008), H1-derived mesenchymal stem cells (E006), BM-derived MSCs (E026), and adipose-derived MSC (E025) (26). The statistical significance of the relative frequency of each stage at every cell type was assessed in comparison to the same value measured along the whole genome, using the Fishers exact test. The R package GenomicRanges from Bioconductor was used for calculations of compositions. Next, to generate the final heatmap, we have grouped certain states for semantic similarity (active TSS category includes active and flanking active TSS states; transcription includes flanking, strong, and weak states; enhancers account for both genic and intergenic; bivalent TSS include also flanking bivalent promoters and PcG repressed include both repressed and weak repressed). Thus, the relative frequencies of the new eight states were recalculated, while quiescent state was discarded from the analysis. Last, the enrichment percentage at a particular stage was calculated as the difference between the relative frequency at the EWSR1-FLI1 ChIP-seq sites minus the relative frequency at the whole genome normalized by the relative frequency at the whole genome again.

In vivo studies were performed after the approval of the Institutional Animal Research Ethics Committee. Athymic nude mice (Envigo) were injected subcutaneously with 4 106 cells for shCTRL#seq1 and shRING1B#seq1 and 2 106 for seq#2. shCTRL cells were resuspended in 200 l of Matrigel (Becton Dickinson) with phosphate-buffered saline and injected into both flanks (5 mice n = 10 for seq#1 and 6 mice, n = 12 for seq#2). The same procedure was performed for the SK-ES1 shRING1B cell line. Tumor growth was monitored three times a week by measuring tumor volume with a digital caliper. Mice were euthanized when tumors reached a size of 2.5 cm in any dimension. Survival curves were calculated using the Kaplan-Meier method and were compared with a log-rank test. At the end of the experiment, tumors were excised; half of each specimen was frozen in liquid nitrogen for RNA extraction, and the other was fixed in 10% formalin for immunohistochemistry experiments.

Acknowledgments: We thank N. Riggi for reagents and technical advice and M. Martnez-Balbs for technical advice and critical reading of the manuscript. We also thank G. Pascual-Pasto, S. Mateo, and M. Suol for technical advice, S. Perez-Jaume for statistical advice, and L. Nonell from the Microarray Analysis Service, Hospital del Mar Medical Research Institute (IMIM, Barcelona) for technical advice. Last, we are grateful to the Band of Parents at Hospital Sant Joan de Du for supporting the overall research activities of the developmental tumor laboratory, PCCB. Funding: S.S.-M. and the project were supported by the Spanish Association Against Cancer (AECC) consolidated groups grant (GCB13131578) consortium. The project also had the support from the Asociacion Pablo Ugarte (APU). E.F.-B. was supported by the Spanish government grant, Instituto de Salud Carlos III (PI16/00245) to J.M. The work in the Di Croce laboratory was supported by grants from the Spanish of Economy, Industry and Competitiveness (MEIC) (BFU2016-75008-P), and Fundacion Vencer El Cancer (VEC). Author contributions: S.S.-M., L.D.C., and J.M. designed the study, conducted experiments, and wrote the manuscript. J.M. supervised all the work. S.S.-M., E.F.-B., M.S.-J., P.T., C.B., E.P., L.H.-P., and D.J.G.-D. performed the experiments. E.B. and S.G. performed all the bioinformatic analysis. I.H.-M., O.M.T., A.M.C., C.L., and E.. provided expertise and feedback. All authors reviewed the manuscript. 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. Additional data related to this paper may be requested from the authors.

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Catalent and BrainStorm Cell Therapeutics Announce Partnership for the Manufacture of Mesenchymal Stem Cell Platform Therapy NurOwn – GlobeNewswire

By daniellenierenberg

SOMERSET, N.J. and NEW YORK, Oct. 22, 2020 (GLOBE NEWSWIRE) -- Catalent (NYSE: CTLT), the leading global provider of advanced delivery technologies, development, and manufacturing solutions for drugs, biologics, cell and gene therapies, and consumer health products, and BrainStorm Cell Therapeutics Inc. (NASDAQ: BCLI), a leading developer of cellular therapies for neurodegenerative diseases, today announced an agreement for the manufacture of NurOwn, BrainStorms autologous cellular therapy being investigated for the treatment of amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease or motor neuron disease.

NurOwn induces mesenchymal stem cells (MSCs) to secrete high levels of neurotrophic factors (NTFs) known to promote the survival of neurons and neuroprotection. The therapy has received Fast Track status from the U.S. FDA for ALS and has also been granted Orphan Drug Status for ALS by both the FDA and the European Medicines Agency. BrainStorm is currently completing a 200-patient, double-blind, placebo-controlled, repeat-dosing NurOwn Phase 3 study in the U.S.

As part of its commitment, Catalent will undertake the transfer of the manufacturing process to, and provide future CGMP clinical supply of NurOwn from, its new, 32,000 square-foot cell therapy manufacturing facility in Houston, Texas. On completion of the clinical trials and in anticipation of potential approval of NurOwn, the companies will look to extend the partnership to include commercial supply from the Houston facility.

We are proud to have a partner in Catalent whose excellence in manufacturing quality therapies will support commercial supply of NurOwn, said Chaim Lebovits, Chief Executive Officer of BrainStorm Cell Therapeutics. We know that ALS patients are in urgent need of a new treatment option. If NurOwn is successful in the current clinical trials, this agreement will be integral to ensuring rapid access for patients.

Manja Boerman, Ph.D., President, Catalent Cell & Gene Therapy, said, Our experience in cell therapy development, and the manufacturing capabilities that our newly constructed, state-of-the-art facility in Houston offers, position us to best support BrainStorm, with its leading therapeutic candidate for ALS treatment. We look forward to partnering with BrainStorm and providing our stem cell manufacturing expertise as we work to optimize production and streamline the products path towards commercial launch.

About Catalent Cell & Gene Therapy

With deep experience in viral vector scale-up and production, Catalent Cell & Gene Therapy is a full-service partner for adeno-associated virus (AAV) and lentiviral vectors, and CAR-T immunotherapies. When it acquired MaSTherCell, Catalent added expertise in autologous and allogeneic cell therapy development and manufacturing to position it as a premier technology, development and manufacturing partner for innovators across the entire field of advanced biotherapeutics. Catalent has a global cell and gene therapy network of dedicated, large-scale clinical and commercial manufacturing facilities, and fill-finish and packaging capabilities located in both the U.S. and Europe. An experienced partner, Catalent Cell & Gene Therapy has worked with industry leaders across 70+ clinical and commercial programs.

About Catalent

Catalent is the leading global provider of advanced delivery technologies, development, and manufacturing solutions for drugs, biologics, cell and gene therapies, and consumer health products. With over 85 years serving the industry, Catalent has proven expertise in bringing more customer products to market faster, enhancing product performance and ensuring reliable global clinical and commercial product supply. Catalent employs approximately 14,000 people, including around 2,400 scientists and technicians, at more than 45 facilities, and in fiscal year 2020 generated over $3 billion in annual revenue. Catalent is headquartered in Somerset, New Jersey. For more information, visit http://www.catalent.com

More products. Better treatments. Reliably supplied.

About NurOwn

NurOwn (autologous MSC-NTF) cells represent a promising investigational therapeutic approach to targeting disease pathways important in neurodegenerative disorders. MSC-NTF cells are produced from autologous, bone marrow-derived mesenchymal stem cells (MSCs) that have been expanded and differentiated ex vivo. MSCs are converted into MSC-NTF cells by growing them under patented conditions that induce the cells to secrete high levels of neurotrophic factors. Autologous MSC-NTF cells can effectively deliver multiple NTFs and immunomodulatory cytokines directly to the site of damage to elicit a desired biological effect and ultimately slow or stabilize disease progression. BrainStorm has fully enrolled a Phase 3 pivotal trial of autologous MSC-NTF cells for the treatment of amyotrophic lateral sclerosis (ALS). BrainStorm also received U.S. FDA acceptance to initiate a Phase 2 open-label multicenter trial in progressive MS and enrollment began in March 2019.

About BrainStorm Cell Therapeutics Inc.

BrainStorm Cell Therapeutics Inc. is a leading developer of innovative autologous adult stem cell therapeutics for debilitating neurodegenerative diseases. The Company holds the rights to clinical development and commercialization of the NurOwn technology platform used to produce autologous MSC-NTF cells through an exclusive, worldwide licensing agreement. Autologous MSC-NTF cells have received Orphan Drug status designation from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of amyotrophic lateral sclerosis (ALS). BrainStorm has fully enrolled a Phase 3 pivotal trial in ALS (NCT03280056), investigating repeat-administration of autologous MSC-NTF cells at six U.S. sites supported by a grant from the California Institute for Regenerative Medicine (CIRM CLIN2-0989). The pivotal study is intended to support a filing for U.S. FDA approval of autologous MSC-NTF cells in ALS. BrainStorm also recently received U.S. FDA clearance to initiate a Phase 2 open-label multicenter trial in progressive multiple sclerosis (MS). The Phase 2 study of autologous MSC-NTF cells in patients with progressive MS (NCT03799718) completed enrollment inAugust 2020. For more information, visit the company's website at http://www.brainstorm-cell.com.

Safe-Harbor Statement

Statements in this announcement other than historical data and information, including statements regarding future clinical trial enrollment and data, constitute "forward-looking statements" and involve risks and uncertainties that could cause BrainStorm Cell Therapeutics Inc.'s actual results to differ materially from those stated or implied by such forward-looking statements. Terms and phrases such as "may", "should", "would", "could", "will", "expect", "likely", "believe", "plan", "estimate", "predict", "potential", and similar terms and phrases are intended to identify these forward-looking statements. The potential risks and uncertainties include, without limitation, BrainStorm's need to raise additional capital, BrainStorm's ability to continue as a going concern, regulatory approval of BrainStorm's NurOwn treatment candidate, the success of BrainStorm's product development programs and research, regulatory and personnel issues, development of a global market for our services, the ability to secure and maintain research institutions to conduct our clinical trials, the ability to generate significant revenue, the ability of BrainStorm's NurOwn treatment candidate to achieve broad acceptance as a treatment option for ALS or other neurodegenerative diseases, BrainStorm's ability to manufacture and commercialize the NurOwn treatment candidate, obtaining patents that provide meaningful protection, competition and market developments, BrainStorm's ability to protect our intellectual property from infringement by third parties, heath reform legislation, demand for our services, currency exchange rates and product liability claims and litigation,; and other factors detailed in BrainStorm's annual report on Form 10-K and quarterly reports on Form 10-Q available athttp://www.sec.gov. These factors should be considered carefully, and readers should not place undue reliance on BrainStorm's forward-looking statements. The forward-looking statements contained in this press release are based on the beliefs, expectations and opinions of management as of the date of this press release. We do not assume any obligation to update forward-looking statements to reflect actual results or assumptions if circumstances or management's beliefs, expectations or opinions should change, unless otherwise required by law. Although we believe that the expectations reflected in the forward-looking statements are reasonable, we cannot guarantee future results, levels of activity, performance or achievements.

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Catalent and BrainStorm Cell Therapeutics Announce Partnership for the Manufacture of Mesenchymal Stem Cell Platform Therapy NurOwn - GlobeNewswire

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World Cord Blood Day 2020 Speakers to Present Revolutionary CAR-NK Cell Therapy, Potential Treatments for Covid-19 Related MIS-C and Advantages of…

By daniellenierenberg

TUCSON, Ariz., Oct. 21, 2020 /PRNewswire/ --Registration is now open for the World Cord Blood Day 2020 virtual conference (register free on Eventbrite) featuring world renown cord blood transplant doctors and cellular therapy researchers. To be held on November 17th, the virtual conference will provide an opportunity for healthcare professionals, expectant parents, and students to learn about life-saving cord blood stem cells via a mix of livestream and on-demand sessions. The public is also invited to participate in a wide variety of free educational events being held around the globe by WCBD Official Participants (see listings on http://www.WorldCordBloodDay.org).

Attendees of the virtual conference will learn how cord blood has been used in more than 40,000 stem cell transplants since 1988 to treat over 80 life-threatening diseases including leukemia, sickle cell anemia, thalassemia, and lymphoma. Ground-breaking research will also be presented by scientists who are discovering cord blood's full potential in CAR-NK immunotherapy, the emerging field of regenerative medicine to potentially treat autism, cerebral palsy, Covid-19 related MIS-C and more. Keynote presentations will be made by Dr. Joanne Kurtzberg (Duke Department of Pediatrics, Duke Center for Autism and Brain Development), Dr. Katy Rezvani (MD Anderson Cancer Center), Dr. Jonathan Gutman (University of Colorado), Dr. Leland Metheny (Case Western Reserve University), and Monroe Burgess (Quick Specialized Healthcare Logistics). Dr. Moshe Israeli (Rabin Medical Center) will lead the opening session on HLA matching and cord blood.

In addition, a panel of industry experts will discuss how cord blood has come to the forefront during the Covid-19 pandemic. Increasingly, stem cells transplant doctors are using cord blood units collected well before the pandemic and now available for immediate use. Attendees will also hear from Dr. David Hall and Vanessa Yenson, who both beat cancer thanks to cord blood transplants.

To view the full agenda, please visit: https://www.worldcordbloodday.org/online-medical-conference-agenda-wcbd-2020.html

Organized and hosted by Save the Cord Foundation (501c3 non-profit), this year's event is officially sponsored by Quick Specialized Healthcare Logistics. "We're proud to be a sponsor of World Cord Blood Day for the fourth year in a row. This year is sure to be very informative and exciting, providing the latest information from some of the industry's top doctors and researchers. We're humbled to play a role in the research and development of cord blood derivative therapies by providing logistics supply chain solutions to cord blood, biotech and pharmaceutical companies worldwide," said David Murphy, Executive VP of Quick's Life Science Division.

Inspiring Partners this year include the Cord Blood Association (CBA), Be the Match (NMDP), World Marrow Donor Association (WMDA-Netcord), AABB Center for Cellular Therapy and Foundation for the Accreditation of Cellular Therapy (FACT).

Visit http://www.WorldCordBloodDay.org to learn how you can participate and/or host an event. Join us on social media using the hashtags: #WCBD20 and #WorldCordBloodDay.

About Save the Cord Foundation

Save the Cord Foundation (a 501c3 non-profit) was established to advance cord blood education. The Foundation provides non-commercial information to parents, health professionals and the public regarding methods for saving cord blood, as well as current applications using cord blood and the latest research. Learn more at http://www.SaveTheCordFoundation.org.

About Quick Specialized Healthcare Logistics

Quick is the trusted logistics leader serving the Healthcare and Life Science community for almost 40 years. Quick safely transports human organs and tissue for transplant or research, blood, blood products, cord blood, bone marrow, medical devices, and personalized medicine, 24/7/365. Quick's specially trained experts work with hospitals, laboratories, blood banks and medical processing canters, and utilize the safest routes to ensure integrity, temperature control and chain of custody throughout the transportation process. Learn more at http://www.quickhealthcare.aero.

Media Contact:Charis Ober[emailprotected]520-419-0269

SOURCE Save the Cord Foundation

http://www.SaveTheCordFoundation.org

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World Cord Blood Day 2020 Speakers to Present Revolutionary CAR-NK Cell Therapy, Potential Treatments for Covid-19 Related MIS-C and Advantages of...

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British Society for Haematology Guideline Update for the Diagnosis and Management of Chronic Myeloid Leukemia – Cancer Therapy Advisor

By daniellenierenberg

The development of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML).1,2 However, despite effectively inducing remission and prolonging survival in patients with CML, TKI therapy does not eradicate leukemia stem cells (LSCs), which are responsible for drug resistance, relapse, and disease progression.2 Given recent changes to the treatment paradigm, updated clinical practice guidelines are essential to ensure optimal clinical care is provided.2

The British Society for Haematology (BSH) published a guideline update for the investigation and management of CML in adults and children in the British Journal of Haematology.1 Lead author of the guidelines, Graeme Smith, MD, of St Jamess University Hospital in the United Kingdom, and coauthors, developed the evidence-based recommendations to provide clinical practitioners with clear guidance on the diagnosis and treatment of adults and children with CML (Tables 1 and 2).

Diagnosis and Key Investigations

The diagnosis of CML is established based on findings from a peripheral blood smear and bone marrow aspirate showing positivity for BCR-ABL1, and the presence of the Philadelphia (Ph) chromosome. The Ph chromosome, or a variant, is present in approximately 95% of CML cases. Other cases include a cryptic BCR-ABL1 fusion, commonly detected by reverse transcriptase polymerase chain reaction (RT-PCR), or fluorescence in situ hybridization (FISH). Additional findings from bone marrow aspirate include the presence of other cytogenetic abnormalities, including isochromosome 17q or trisomy 19, and trisomy 8, suggesting a higher risk of progression to accelerated phase or blast crisis in adults.

Table 1. Selected Recommendations by the BSH Guideline Panel on the Diagnosis of CML1

Table 2. ELTS Score Calculation1

This article originally appeared on Hematology Advisor

Excerpt from:
British Society for Haematology Guideline Update for the Diagnosis and Management of Chronic Myeloid Leukemia - Cancer Therapy Advisor

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Randomized Controlled Study Using Direct Injection of Remestemcel-L Into Inflamed Gut of Patients With Crohn’s Disease and Ulcerative Colitis -…

By daniellenierenberg

NEW YORK, Oct. 22, 2020 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO; ASX:MSB), global leader in allogeneic cellular medicines for inflammatory diseases, today announced that a randomized, controlled study of remestemcel-L delivered by an endoscope directly to the areas of inflammation and tissue injury in up to 48 patients with medically refractory Crohns disease and ulcerative colitis has commenced at Cleveland Clinic.

Mesoblast Chief Medical Officer Dr Fred Grossman said: Inflammation of the gut in Crohns disease and ulcerative colitis closely resembles the most severe manifestation of advanced-stage, life-threatening acute graft versus host disease (aGVHD). Mesoblasts objective is to confirm the potential for remestemcel-L to induce luminal healing and early remission in a wider spectrum of diseases with severe inflammation of the gut, in addition to steroid-refractory aGVHD.

Mesenchymal stem cells (MSCs) promote healing of inflamed gut tissue by downregulating gut mucosal effector T-cell activity and promoting regulatory T-cell formation.1 MSCs have been tested in clinical trials of Crohns disease using two different modalities: intravenous infusions of MSCs to treat the primary inflammation of Crohns disease and local injections of MSCs to treat fistulae complicating Crohns disease.

A third modality, endoscopic delivery of MSCs, has been successful in preclinical experimental models of colitis, reducing the excessive cytokine storm in the inflamed gut and resulting in tissue healing.2-3 The study at Cleveland Clinic will be the first in humans using local delivery of MSCs in the gut, and will enable Mesoblast to compare clinical outcomes using this delivery method with results from an ongoing randomized, placebo-controlled trial in patients with biologic-refractory Crohns disease where remestemcel-L was administered intravenously.

The studys lead investigator Dr Amy L. Lightner, Associate Professor of Surgery in the Department of Colon and Rectal Surgery at Cleveland Clinic, stated: We are aiming to establish a new treatment paradigm by administering remestemcel-L at one of two escalating doses, or placebo, directly to inflamed gut tissue in patients with medically refractory Crohns disease and ulcerative colitis, both highly debilitating conditions with significant, unmet medical needs.

According to recent estimates, more than three million people (1.3%) in the US alone have inflammatory bowel disease, with more than 33,000 new cases of Crohns disease and 38,000 new cases of ulcerative colitis diagnosed every year.4-6 Despite recent advances, approximately 30% of patients are primarily unresponsive to anti-TNF agents and even among responders, up to 10% will lose their response to the drug every year. Up to 80% of patients with medically-refractory Crohns disease eventually require surgical treatment of their disease,7 which can have a devastating impact on quality of life.

References1.Mayne C and Williams C. Induced and natural regulatory T cells in the development of inflammatory bowel disease. Inflamm Bowel Dis 2013; 19: 17721788.2.Molendijk I et al. Intraluminal Injection of Mesenchymal Stromal Cells in Spheroids Attenuates Experimental Colitis. Journal of Crohn's and Colitis, 2016, 9539643.Pak S eta al. Endoscopic Transplantation of Mesenchymal Stem Cell Sheets in Experimental Colitis in Rats. Scientific Reports | (2018) 8:11314 | DOI:10.1038/s41598-018-296174.CDC Facts and Figures 20155.Globaldata Pharmapoint 20186.Dahlhamer JM, MMWR Morb Mortal Wkly Rep. 2016;65(42):11661169.7.Crohns and Colitis Foundation

About Remestemcel-LMesoblasts lead product candidate, remestemcel-L, is an investigational therapy comprising culture-expanded mesenchymal stem cells derived from the bone marrow of an unrelated donor. It is administered to patients in a series of intravenous infusions. Remestemcel-L is thought to have immunomodulatory properties to counteract severe inflammatory processes by down-regulating the production of pro-inflammatory cytokines, increasing production of anti-inflammatory cytokines, and enabling recruitment of naturally occurring anti-inflammatory cells to involved tissues.

About MesoblastMesoblast Limited (Nasdaq:MESO; ASX:MSB) is a world leader in developing allogeneic (off-the-shelf) cellular medicines. The Company has leveraged its proprietary mesenchymal lineage cell therapy technology platform to establish a broad portfolio of commercial products and late-stage product candidates. Mesoblast has a strong and extensive global intellectual property (IP) portfolio with protection extending through to at least 2040 in all major markets. The Companys proprietary manufacturing processes yield industrial-scale, cryopreserved, off-the-shelf, cellular medicines. These cell therapies, with defined pharmaceutical release criteria, are planned to be readily available to patients worldwide.

Remestemcel-L is being developed for inflammatory diseases in children and adults including steroid-refractory acute graft versus host disease and moderate to severe acute respiratory distress syndrome. Mesoblast is completing Phase 3 trials for its product candidates for advanced heart failure and chronic low back pain. Two products have been commercialized in Japan and Europe by Mesoblasts licensees, and the Company has established commercial partnerships in Europe and China for certain Phase 3 assets.

Mesoblast has locations in Australia, the United States and Singapore and is listed on the Australian Securities Exchange (MSB) and on the Nasdaq (MESO). For more information, please see http://www.mesoblast.com, LinkedIn: Mesoblast Limited and Twitter: @Mesoblast

Forward-Looking StatementsThis announcement includes forward-looking statements that relate to future events or our future financial performance and involve known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to differ materially from any future results, levels of activity, performance or achievements expressed or implied by these forward-looking statements. All statements other than statements of historical fact are forward-looking statements, which are often indicated by terms such as anticipate, believe, could, estimate, expect, goal, intend, likely, look forward to, may, plan, potential, predict, project, should, will, would and similar expressions and variations thereof. We make such forward-looking statements pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws. Forward-looking statements should not be read as a guarantee of future performance or results, and actual results may differ from the results anticipated in these forward-looking statements, and the differences may be material and adverse. The risks, uncertainties and other factors that may impact our forward-looking statements include, but are not limited to: statements about the initiation, timing, progress and results of Mesoblast and its collaborators clinical studies; Mesoblast and its collaborators ability to advance product candidates into, enroll and successfully complete, clinical studies; the timing or likelihood of regulatory filings and approvals; and the pricing and reimbursement of Mesoblasts product candidates, if approved; the potential benefits of strategic collaboration agreements and Mesoblasts ability to maintain established strategic collaborations; Mesoblasts ability to establish and maintain intellectual property on its product candidates and Mesoblasts ability to successfully defend these in cases of alleged infringement. You should read this press release together with our risk factors, in our most recently filed reports with the SEC or on our website. Uncertainties and risks that may cause Mesoblasts actual results, performance or achievements to be materially different from those which may be expressed or implied by such statements, and accordingly, you should not place undue reliance on these forward-looking statements. Unless required by law, we do not undertake any obligations to publicly update or revise any forward-looking statements, whether as a result of new information, future developments or otherwise.

Release authorized by the Chief Executive.

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Some Patients With AML Secondary to MPN May Benefit From Venetoclax in Combination With a Hypomethylating Agent – Oncology Nurse Advisor

By daniellenierenberg

The combination of the BCL2 inhibitor venetoclax with a hypomethylating agent (HMA) may be a treatment option for patients who develop acute myeloid leukemia (AML) secondary to a myeloproliferative neoplasm (MPN), according to results of a small, retrospective cohort study published in Leukemia Research.1

The Philadelphia chromosome-negative MPNs, which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal disorders resulting in the proliferation of myeloid cells in the bone marrow. Both PV and ET can progress to secondary myelofibrosis which, along with PMF, can progress to secondary AML, also known as MPN-blast phase (MPN-BP).

Furthermore, MPN-BP, defined in this study as being associated with peripheral or bone marrow blasts of at least 20%, is not sensitive to intensive chemotherapy, and clinical outcomes for patients with this disease are very poor. Median overall survival is only approximately 3 to 5 months, and allogeneic hematopoietic stem cell transplantation (HSCT) is considered the only curative option for these patients.

Venetoclax in combination with an HMA, azacitidine, decitabine, or low-dose cytarabine recently received regular US Food and Drug Administration (FDA) approval for the treatment of adults with newly diagnosed AML who are at least 75 years old or unable to tolerate intensive chemotherapy.2 However, patients with MPN-BP were excluded from the VIALE-A (ClinicalTrial.gov Identifier: NCT02993523) and VIALE-C (ClinicalTrial.gov Identifier: NCT03069352) phase 3 studies evaluating venetoclax in combination with azacitadine and low-dose cytarabine, respectively, in newly diagnosed AML.

This study included 8 patients with MPN-BP and 1 with MPN-accelerated phase (MPN-AP), defined as peripheral or bone marrow blasts of 10% to 19%, which was associated with very high-risk cytogenetics, who were treated at the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai in New York, New York. Most of the patients in this cohort had relapsed/refractory disease, and had been treated with prior therapies.

A key study finding included the achievement of either a complete response (CR) or a CR associated with incomplete hematologic recovery (CRi) in 3 patients treated with the combination of venetoclax plus decitabine or azacitidine. In addition, stable disease as best response was achieved by 2 additional patients who received this treatment.

Of note, 2 of the 3 patients who achieved a CR/CRi had experienced disease relapse on prior HMA therapy.

This suggests a synergy with the combination that is not precluded by prior HMA exposure, the study authors remarked.

Perhaps more striking was the finding that when this therapeutic approach was used as a bridge to HSCT in 3 patients who achieved CR, CRi, or stable disease, all of them were alive at a median follow-up of 8.5 months compared with 4.2 months for the overall cohort.

However, high rates of grade 3 or higher bleeding and infection were observed in this patient cohort, and occurred in 5 and 7 patients, respectively.

Given the propensity for prolonged cytopenias with resultant complications, caution should be used in patients with baseline cytopenias, study authors noted.

In closing, the study authors stated, This is the largest report of venetoclax use in patients with MPN-AP/BP and suggests that this therapeutic strategy is a viable treatment option in this adverse risk group eligible for HSCT.

They further added that prospective clinical trial evaluation of combination HMA and venetoclax in MPN-BP is warranted.

Disclosures: Multiple authors declared affiliations with industry. Please refer to the original article for a full list of disclosures.

References

1. Tremblay D, Feld J, Dougherty M, et al. Venetoclax and hypomethylating agent combination therapy in acute myeloid leukemia secondary to a myeloproliferative neoplasm. Leuk Res. Published online September 22, 2020. doi:10.1016/j.leukres.2020.106456

2. U.S. Food and Drug Administration. FDA grants regular approval to venetoclax in combination for untreated acute myeloid leukemia [news release]. U.S. Food and Drug Administration; October 16, 2020. Accessed October 19, 2020. https://www.fda.gov/drugs/drug-approvals-and-databases/fda-grants-regular-approval-venetoclax-combination-untreated-acute-myeloid-leukemia

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Be Biopharma debuts with $52M to advance engineered B-cell therapies – FierceBiotech

By daniellenierenberg

You may have heard of T cells, but Aleks Radovic-Moreno, Ph.D., Be Biopharmas co-founder, president and director, is betting on B cells as the future of cell therapies.

Our mission is to develop what we see as a new class of cell medicines that have a broad new pharmacology, he said of B cells potential. We think it's a big new white space that's enabled by the rich biology of these cells.

The Cambridge, Massachusetts-based company is capitalizingearly on research by scientists at the University of Washington School of Medicine. With a $52 million series A round in the bank, it'smaking a beeline for the clinic.

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Why the enthusiasm around B cells? The wayRadovic-Moreno sees it, they'rethe cellular gadget, if you will, that's really good at making large amounts of protein, and they also traffic to where you want them to go."

When we think about it from a drug development standpoint, now you have a system that can make a protein that you want in high quantities in places where you want it to be made, he added.

B cells may also be useful for targeting specific tissues and modulating microenvironments, or [talking] to the cells that are nearby, he said.

One of the biggest challenges to bringing Be Bio to fruition was making the products themselves. Theyre harder to engineer than other cell types thanksto their intrinsic biology, Radovic-Moreno said. Theyre also hard to make correctly and in large quantities, challenges the company only recently overcame.

Those two are the final two bottlenecks that were preventing B cells from being a viable stem cell therapy modality, he said.

RELATED: Q32 debuts with $46M to 'rebalance' innate and adaptive immunity

The applications of B cells include everything from autoimmune diseases to cancer and monogenic disorders, which are caused by variation in a single gene. B-cell therapy could eliminate the need for patients with monogenic disorders who are missing proteins to get biweekly four-hour infusions.

And that's not all. It couldalso eliminate the need for bone marrow transplants in these patients, as well asthe need for a pre-therapy round of chemotherapy, otherwise known as conditioning. For cancer patients who need conditioningahead of a stem cell treatment, the regimencan be deadly up to 10% of the time.

That's extraordinary if you think about a therapy killing patients 10% of the time, Radovic-Moreno said.

Beyond pushing Be'spipeline toward the clinic, the new fundingfrom Atlas Venture, RA Capital Management, Alta Partners, Longwood Fund and other investorswill bankroll potential partnerships and build out the company's team.

The most important thing is to build a great company, hire the best people. We want to be the best B-cell engineers in the world and in history, Radovic-Moreno said. We want to fully capitalize on the timing of this, given that it's a very kind of unusual place to be in this time and age of biotech, where you're sitting right in front of this massive blue wave, big blue ocean of possibilities so big.

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Bragar Eagel & Squire, PC Reminds Investors That Class Action Lawsuits Have Been Filed Against Mesoblast, Loop Industries, Turquoise Hill…

By daniellenierenberg

NEW YORK, Oct. 21, 2020 (GLOBE NEWSWIRE) -- Bragar Eagel & Squire, P.C., a nationally recognized shareholder rights law firm, reminds investors that class actions have been commenced on behalf of stockholders of Mesoblast Limited (NASDAQ: MESO), Loop Industries, Inc. (NASDAQ: LOOP), Turquoise Hill Resources Ltd. (NYSE: TRQ), and Reata Pharmaceuticals, Inc. (NASDAQ: RETA). Stockholders have until the deadlines below to petition the court to serve as lead plaintiff. Additional information about each case can be found at the link provided.

Mesoblast Limited (NASDAQ: MESO)

Class Period: April 16, 2019 to October 1, 2020

Lead Plaintiff Deadline: December 7, 2020

Mesoblast develops allogeneic cellular medicines using its proprietary mesenchymal lineage cell therapy platform. Its lead product candidate, RYONCIL (remestemcel-L), is an investigational therapy comprising mesenchymal stem cells derived from bone marrow. In February 2018, the Company announced that remestemcel-L met its primary endpoint in a Phase 3 trial to treat children with steroid refractory acute graft versus host disease (aGVHD).

In early 2020, Mesoblast completed its rolling submission of its Biologics License Application (BLA) with the FDA to secure marketing authorization to commercialize remestemcel-L for children with steroid refractory aGVHD.

On August 11, 2020, the FDA released briefing materials for its Oncologic Drugs Advisory Committee (ODAC) meeting to be held on August 13, 2020. Therein, the FDA stated that Mesoblast provided post hoc analyses of other studies to further establish the appropriateness of 45% as the null Day-28 ORR for its primary endpoint. The briefing materials stated that, due to design differences between these historical studies and Mesoblasts submitted study, it is unclear that these study results are relevant to the proposed indication.

On this news, the Companys share price fell $6.09, or approximately 35%, to close at $11.33 per share on August 11, 2020.

On October 1, 2020, Mesoblast disclosed that it had received a Complete Response Letter (CRL) from the FDA regarding its marketing application for remestemcel-L for treatment of SR-aGVHD in pediatric patients. According to the CRL, the FDA recommended that the Company conduct at least one additional randomized, controlled study in adults and/or children to provide further evidence of the effectiveness of remestemcel-L for SR-aGVHD. The CRL also identified a need for further scientific rationale to demonstrate the relationship of potency measurements to the products biologic activity.

On this news, the Companys share price fell $6.56, or 35%, to close at $12.03 per share on October 2, 2020.

The complaint, filed on October 8, 2020, alleges that throughout the Class Period defendants made materially false and/or misleading statements, as well as failed to disclose material adverse facts about the Companys business, operations, and prospects. Specifically, defendants failed to disclose to investors: (1) that comparative analyses between Mesoblasts Phase 3 trial and three historical studies did not support the effectiveness of remestemcel-L for steroid refractory aGVHD due to design differences between the four studies; (2) that, as a result, the FDA was reasonably likely to require further clinical studies; (3) that, as a result, the commercialization of remestemcel-L in the U.S. was likely to be delayed; and (4) that, as a result of the foregoing, defendants positive statements about the Companys business, operations, and prospects were materially misleading and/or lacked a reasonable basis.

For more information on the Mesoblast class action go to: https://bespc.com/MESO

Loop Industries, Inc. (NASDAQ: LOOP)

Class Period: September 24, 2018 to October 12, 2020

Lead Plaintiff Deadline: December 14, 2020

On October 13, 2020, Hindenburg Research published a report alleging, among other things, that Loops scientists, under pressure from CEO Daniel Solomita, were tacitly encouraged to lie about the results of the companys process internally. The report also stated that Loops previous claims of breaking PET down to its base chemicals at a recovery rate of 100% were technically and industrially impossible, according to a former employee. Moreover, the report alleged that Executives from a division of key partner Thyssenkrupp, who Loop entered into a global alliance agreement with in December 2018, told us their partnership is on indefinite hold and that Loop underestimated both costs and complexities of its process.

On this news, the Companys share price fell $3.78, or over 32%, to close at $7.83 per share on October 13, 2020.

The complaint, filed on October 13, 2020, alleges that throughout the Class Period defendants made materially false and/or misleading statements, as well as failed to disclose material adverse facts about the Companys business, operations, and prospects. Specifically, defendants failed to disclose to investors: (1) that Loop scientists were encouraged to misrepresent the results of Loops purportedly proprietary process; (2) that Loop did not have the technology to break PET down to its base chemicals at a recovery rate of 100%; (3) that, as a result, the Company was unlikely to realize the purported benefits of Loops announced partnerships with Indorama and Thyssenkrupp; and (4) that, as a result of the foregoing, defendants positive statements about the Companys business, operations, and prospects were materially misleading and/or lacked a reasonable basis.

For more information on the Loop class action go to: https://bespc.com/Loop

Turquoise Hill Resources Ltd. (NYSE: TRQ)

Class Period: July 17, 2018 to July 31, 2019

Lead Plaintiff Deadline: December 14, 2020

Turquoise Hill is an international mining company focused on the operation and development of the Oyu Tolgoi copper-gold mine in Southern Mongolia (Oyu Tolgoi), which is the Companys principal and only material resource property. Turquoise Hills subsidiary, Oyu Tolgoi LLC, holds a 66% interest in Oyu Tolgoi, and the remainder is held by the Government of Mongolia.

Rio Tinto plc and Rio Tinto Limited are operated and managed together as single economic unit and engage in mining and metals operations in approximately 35 countries. Through their subsidiaries, Rio Tinto owns 50.8% of Turquoise Hill. A Rio Tinto subsidiary, Rio Tinto International Holdings, Inc. (Rio Tinto International or RTIH; and collectively with Rio Tinto plc and Rio Tinto Limited, Rio Tinto), is also the manager of the Oyu Tolgoi project, including having responsibility for its development and construction.

On July 31, 2019, Turquoise Hill issued a press release and Management Discussion & Analysis (MD&A) making further disclosures about the status of the project, including that Turquoise Hill took a $600 million impairment charge and a substantial deferred income tax recognition adjustment tied to the Oyu Tolgoi project, and that it suffered a loss in the second quarter. The next day, before the market open, Rio Tinto issued a release concerning in part the project status, including that it had also taken an impairment charge related to the Oyu Tolgoi project, of $800 million.

Following this news, on August 1, 2019, Turquoise Hills common stock price closed at $0.53 per share, down 8.62% from the prior days closing price of $0.58 per share.

The complaint, filed on October 15, 2020, alleges that throughout the Class Period defendants made materially false and misleading statements and omitted to disclose material facts regarding the Companys business and operations. Specifically, defendants made false and or misleading statements and/or failed to disclose that: (i) the progress of underground development of Oyu Tolgoi was not proceeding as planned; (ii) there were significant undisclosed underground stability issues that called into question the design of the mine, the projected cost and timing of production; (iii) the Companys publicly disclosed estimates of the cost, date of completion and dates for production from the underground mine were not achievable; (iv) the development capital required for the underground development of Oyu Tolgoi would cost substantially more than a billion dollars over what the Company had represented; and (v) Turquoise Hill would require additional financing and/or equity to complete the project.

For more information on the Turquoise Hill class action go to: https://bespc.com/TRQ

Reata Pharmaceuticals, Inc. (NASDAQ: RETA)

Class Period: October 15, 2019 to August 7, 2020

Lead Plaintiff Deadline: December 14, 2020

Reata is a clinical stage biopharmaceutical company that develops novel therapeutics for patients with serious or life-threatening diseases by targeting molecular pathways that regulate cellular metabolism and inflammation.

Among Reatas drug candidates under development is omaveloxolone, which is in Phase 2 clinical development to treat Friedreich's ataxia (FA). Following the announcement of positive data from the MOXIe Part 2 study of omaveloxolone for FA inOctober 2019, the Company represented that it would seek submission for marketing approval of omaveloxolone for the treatment of FA in the U.S. with the U.S. Food and Drug Administration (FDA).

OnAugust 10, 2020, Reata issued a press release announcing its second quarter 2020 financial results, wherein it disclosed that the FDA is not convinced that the MOXIe Part 2 results of the Company's study assessing omaveloxolone for the treatment of FA will support a single study approval without additional evidence that lends persuasiveness to the results, and that, [i]n preliminary comments for [a] meeting, the FDA stated that [Defendants] will need to conduct a second pivotal trial that confirms the mFARS [modified Friedreich's Ataxia Rating Scale] results of the MOXIe Part 2 study with a similar magnitude of effect.

On this news, Reatas stock price fell$51.79per share, or 33.16%, to close at$104.41per share onAugust 10, 2020.

The Complaint, filed on October 15, 2020, alleges that throughout the Class Period defendants made materially false and misleading statements regarding the Companys business. Specifically, defendants made false and/or misleading statements and/or failed to disclose that: (i) the MOXIe Part 2 study results were insufficient to support a single study marketing approval of omaveloxolone for the treatment of FA in the U.S. without additional evidence; (ii) as a result, it was foreseeable that the FDA would not accept marketing approval of omaveloxolone for the treatment of FA in the U.S. based on the MOXIe Part 2 study results; and (iii) as a result, the Company's public statements were materially false and misleading at all relevant times.

For more information on the Reata class action go to: https://bespc.com/REATA

About Bragar Eagel & Squire, P.C.:Bragar Eagel & Squire, P.C. is a nationally recognized law firm with offices in New York and California. The firm represents individual and institutional investors in commercial, securities, derivative, and other complex litigation in state and federal courts across the country. For more information about the firm, please visit http://www.bespc.com. Attorney advertising. Prior results do not guarantee similar outcomes.

Contact Information:Bragar Eagel & Squire, P.C.Brandon Walker, Esq. Melissa Fortunato, Esq.Marion Passmore, Esq.(212) 355-4648investigations@bespc.comwww.bespc.com

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Bragar Eagel & Squire, PC Reminds Investors That Class Action Lawsuits Have Been Filed Against Mesoblast, Loop Industries, Turquoise Hill...

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Potential impact of Covid-19 on Rheumatoid Arthritis Stem Cell Therapy Market Growth and Demand, Concludes Fact.MR – The Cloud Tribune

By daniellenierenberg

The globalRheumatoid Arthritis Stem Cell Therapy marketstudy presents an all in all compilation of the historical, current and future outlook of the market as well as the factors responsible for such a growth. With SWOT analysis, the business study highlights the strengths, weaknesses, opportunities and threats of each Rheumatoid Arthritis Stem Cell Therapy market player in a comprehensive way. Further, the Rheumatoid Arthritis Stem Cell Therapy market report emphasizes the adoption pattern of the Rheumatoid Arthritis Stem Cell Therapy across various industries.Request Sample Reporthttps://www.factmr.com/connectus/sample?flag=S&rep_id=1001The Rheumatoid Arthritis Stem Cell Therapy market report highlights the following players:The global market for rheumatoid arthritis stem cell therapy is highly fragmented. Examples of some of the key players operating in the global rheumatoid arthritis stem cell therapy market include Mesoblast Ltd., Roslin Cells, Regeneus Ltd, ReNeuron Group plc, International Stem Cell Corporation, TiGenix and others.

The Rheumatoid Arthritis Stem Cell Therapy market report examines the operating pattern of each player new product launches, partnerships, and acquisitions has been examined in detail.Important regions covered in the Rheumatoid Arthritis Stem Cell Therapy market report include:

North America (U.S., Canada)Latin America (Mexico, Brazil)Western Europe (Germany, Italy, U.K., Spain, France, Nordic countries, BENELUX)Eastern Europe (Russia, Poland, Rest Of Eastern Europe)Asia Pacific Excluding Japan (China, India, Australia & New Zealand)JapanMiddle East and Africa (GCC, S. Africa, Rest Of MEA)

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Allogeneic Mesenchymal stem cellsBone marrow TransplantAdipose Tissue Stem Cells

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The Rheumatoid Arthritis Stem Cell Therapy market report answers important questions which include:

Which regulatory authorities have granted approval to the application of Rheumatoid Arthritis Stem Cell Therapy in Health industry?How will the global Rheumatoid Arthritis Stem Cell Therapy market grow over the forecast period?Which end use industry is set to become the leading consumer of Rheumatoid Arthritis Stem Cell Therapy by 2028?What manufacturing techniques are involved in the production of the Rheumatoid Arthritis Stem Cell Therapy?Which regions are the Rheumatoid Arthritis Stem Cell Therapy market players targeting to channelize their production portfolio?Get Full Access of the Report @https://www.factmr.com/report/1001/rheumatoid-arthritis-stem-cell-therapy-market

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What is the forecast size (revenue/volumes) of the most lucrative regional market? What is the share of the dominant product/technology segment in the Rheumatoid Arthritis Stem Cell Therapy market? What regions are likely to witness sizable investments in research and development funding? What are Covid 19 implication on Rheumatoid Arthritis Stem Cell Therapy market and learn how businesses can respond, manage and mitigate the risks? Which countries will be the next destination for industry leaders in order to tap new revenue streams? Which new regulations might cause disruption in industry sentiments in near future? Which is the share of the dominant end user? Which region is expected to rise at the most dominant growth rate? Which technologies will have massive impact of new avenues in the Rheumatoid Arthritis Stem Cell Therapy market? Which key end-use industry trends are expected to shape the growth prospects of the Rheumatoid Arthritis Stem Cell Therapy market? What factors will promote new entrants in the Rheumatoid Arthritis Stem Cell Therapy market? What is the degree of fragmentation in the Rheumatoid Arthritis Stem Cell Therapy market, and will it increase in coming years?Why Choose Fact.MR?

Fact.MR follows a multi- disciplinary approach to extract information about various industries. Our analysts perform thorough primary and secondary research to gather data associated with the market. With modern industrial and digitalization tools, we provide avant-garde business ideas to our clients. We address clients living in across parts of the world with our 24/7 service availability.

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Potential impact of Covid-19 on Rheumatoid Arthritis Stem Cell Therapy Market Growth and Demand, Concludes Fact.MR - The Cloud Tribune

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Genmab Announces IFM, HOVON and Janssen Achieve Positive Topline Results in Second Part of Phase 3 CASSIOPEIA Study of Daratumumab in Multiple Myeloma…

By daniellenierenberg

Company Announcement

Copenhagen, Denmark; October 21, 2020 Genmab A/S (Nasdaq: GMAB) announced today positive topline results from the second part of the Phase 3 CASSIOPEIA (MMY3006) study of daratumumab monotherapy as maintenance treatment versus observation (no treatment) for patients with newly diagnosed multiple myeloma eligible for autologous stem cell transplant (ASCT). The second part of the study, which is being conducted by the French Intergroupe Francophone du Myelome (IFM) in collaboration with the Dutch-Belgian Cooperative Trial Group for Hematology Oncology (HOVON) and Janssen Research & Development, LLC (Janssen), met the primary endpoint of improving progression free survival (PFS) at a pre-planned interim analysis (Hazard Ratio (HR) = 0.53 (95% CI 0.42 0.68), p < 0.0001) resulting in a 47% reduction in the risk of progression or death in patients treated with daratumumab. The safety profile observed in this study was consistent with the known safety profile of daratumumab and no new safety signals were observed.

Based on the results at the pre-planned interim analysis conducted by an Independent Data Monitoring Committee (IDMC), it was recommended to unblind the study results. Janssen Biotech, Inc., which licensed daratumumab from Genmab in 2012, plans to discuss the potential for a regulatory submission for this indication with health authorities, and plans to submit the data to an upcoming medical conference and for publication in a peer-reviewed journal.

Following the positive data from the first part of the CASSIOPEIA study, we are very pleased to see this benefit. We are appreciative of the efforts of the IFM, of HOVON and of Janssen for their work on this study, said Jan van de Winkel, Ph.D., Chief Executive Officer of Genmab.

About the CASSIOPEIA (MMY3006) StudyThis Phase 3 study is a randomized, open-label, multicenter study, conducted by the IFM in collaboration with the HOVON and Janssen, which includes 1,085 newly diagnosed subjects with previously untreated symptomatic multiple myeloma who were eligible for high dose chemotherapy and ASCT. In the first part of the study, patients were randomized to receive induction and consolidation treatment with daratumumab combined with bortezomib, thalidomide and dexamethasone (VTd) or VTd alone. The primary endpoint was the number of patients that achieved a stringent complete response (sCR). In the second part of the study, patients that achieved a response underwent a second randomization to either receive maintenance treatment of daratumumab 16 mg/kg every 8 weeks for up to 2 years versus no further treatment (observation). The primary endpoint of this part of the study is progression free survival.

About Multiple MyelomaMultiple myeloma is an incurable blood cancer that starts in the bone marrow and is characterized by an excess proliferation of plasma cells.1 Multiple myeloma is the third most common blood cancer in the U.S., after leukemia and lymphoma.2 Approximately 26,000 new patients were expected to be diagnosed with multiple myeloma and approximately 13,650 people were expected to die from the disease in the U.S. in 2018.3 Globally, it was estimated that 160,000 people were diagnosed and 106,000 died from the disease in 2018.4 While some patients with multiple myeloma have no symptoms at all, most patients are diagnosed due to symptoms which can include bone problems, low blood counts, calcium elevation, kidney problems or infections.5

About DARZALEX (daratumumab)DARZALEX (daratumumab) has become a backbone therapy in the treatment of multiple myeloma. DARZALEX intravenous infusion is indicated for the treatment of adult patients in the United States: in combination with carfilzomib and dexamethasone for the treatment of patients with relapsed/refractory multiple myeloma who have received one to three previous lines of therapy; in combination with bortezomib, thalidomide and dexamethasone as treatment for patients newly diagnosed with multiple myeloma who are eligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone, for the treatment of patients with multiple myeloma who have received at least one prior therapy; in combination with pomalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received at least two prior therapies, including lenalidomide and a proteasome inhibitor (PI); and as a monotherapy for the treatment of patients with multiple myeloma who have received at least three prior lines of therapy, including a PI and an immunomodulatory agent, or who are double-refractory to a PI and an immunomodulatory agent.6 DARZALEX is the first monoclonal antibody (mAb) to receive U.S. Food and Drug Administration (U.S. FDA) approval to treat multiple myeloma.

DARZALEX is indicated for the treatment of adult patients in Europe via intravenous infusion or subcutaneous administration: in combination with bortezomib, thalidomide and dexamethasone as treatment for patients newly diagnosed with multiple myeloma who are eligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of adult patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; for use in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone, for the treatment of adult patients with multiple myeloma who have received at least one prior therapy; and as monotherapy for the treatment of adult patients with relapsed and refractory multiple myeloma, whose prior therapy included a PI and an immunomodulatory agent and who have demonstrated disease progression on the last therapy7. Daratumumab is the first subcutaneous CD38 antibody approved in Europe for the treatment of multiple myeloma. The option to split the first infusion of DARZALEX over two consecutive days has been approved in both Europe and the U.S.

In Japan, DARZALEX intravenous infusion is approved for the treatment of adult patients: in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone for the treatment of relapsed or refractory multiple myeloma. DARZALEX is the first human CD38 monoclonal antibody to reach the market in the United States, Europe and Japan. For more information, visit http://www.DARZALEX.com.

DARZALEX FASPRO (daratumumab and hyaluronidase-fihj), a subcutaneous formulation of daratumumab, is approved in the United States for the treatment of adult patients with multiple myeloma: in combination with bortezomib, melphalan and prednisone in newly diagnosed patients who are ineligible for ASCT; in combination with lenalidomide and dexamethasone in newly diagnosed patients who are ineligible for ASCT and in patients with relapsed or refractory multiple myeloma who have received at least one prior therapy; in combination with bortezomib and dexamethasone in patients who have received at least one prior therapy; and as monotherapy, in patients who have received at least three prior lines of therapy including a PI and an immunomodulatory agent or who are double-refractory to a PI and an immunomodulatory agent.8 DARZALEX FASPRO is the first subcutaneous CD38 antibody approved in the U.S. for the treatment of multiple myeloma.

Daratumumab is a human IgG1k monoclonal antibody (mAb) that binds with high affinity to the CD38 molecule, which is highly expressed on the surface of multiple myeloma cells. Daratumumab triggers a persons own immune system to attack the cancer cells, resulting in rapid tumor cell death through multiple immune-mediated mechanisms of action and through immunomodulatory effects, in addition to direct tumor cell death, via apoptosis (programmed cell death).6,9,10,11,12

Daratumumab is being developed by Janssen Biotech, Inc. under an exclusive worldwide license to develop, manufacture and commercialize daratumumab from Genmab. A comprehensive clinical development program for daratumumab is ongoing, including multiple Phase 3 studies in smoldering, relapsed and refractory and frontline multiple myeloma settings. Additional studies are ongoing or planned to assess the potential of daratumumab in other malignant and pre-malignant diseases in which CD38 is expressed, such as amyloidosis and T-cell acute lymphocytic leukemia (ALL). Daratumumab has received two Breakthrough Therapy Designations from the U.S. FDA for certain indications of multiple myeloma, including as a monotherapy for heavily pretreated multiple myeloma and in combination with certain other therapies for second-line treatment of multiple myeloma.

About Genmab Genmab is a publicly traded, international biotechnology company specializing in the creation and development of differentiated antibody therapeutics for the treatment of cancer. Founded in 1999, the company is the creator of the following approved antibodies: DARZALEX (daratumumab, under agreement with Janssen Biotech, Inc.) for the treatment of certain multiple myeloma indications in territories including the U.S., Europe and Japan, Kesimpta (subcutaneous ofatumumab, under agreement with Novartis AG), for the treatment of adults with relapsing forms of multiple sclerosis in the U.S. and TEPEZZA (teprotumumab, under agreement with Roche granting sublicense to Horizon Therapeutics plc) for the treatment of thyroid eye disease in the U.S. A subcutaneous formulation of daratumumab, known as DARZALEX FASPRO (daratumumab and hyaluronidase-fihj) in the U.S., has been approved in the U.S. and Europe for the treatment of adult patients with certain multiple myeloma indications. The first approved Genmab created therapy, Arzerra (ofatumumab, under agreement with Novartis AG), approved for the treatment of certain chronic lymphocytic leukemia indications, is available in Japan and is also available in other territories via compassionate use or oncology access programs. Daratumumab is in clinical development by Janssen for the treatment of additional multiple myeloma indications, other blood cancers and amyloidosis. Genmab also has a broad clinical and pre-clinical product pipeline. Genmab's technology base consists of validated and proprietary next generation antibody technologies - the DuoBody platform for generation of bispecific antibodies, the HexaBody platform, which creates effector function enhanced antibodies, the HexElect platform, which combines two co-dependently acting HexaBody molecules to introduce selectivity while maximizing therapeutic potency and the DuoHexaBody platform, which enhances the potential potency of bispecific antibodies through hexamerization. The company intends to leverage these technologies to create opportunities for full or co-ownership of future products. Genmab has alliances with top tier pharmaceutical and biotechnology companies. Genmab is headquartered in Copenhagen, Denmark with sites in Utrecht, the Netherlands, Princeton, New Jersey, U.S. and Tokyo, Japan.

Contact: Marisol Peron, Corporate Vice President, Communications & Investor Relations T: +1 609 524 0065; E: mmp@genmab.com

For Investor Relations: Andrew Carlsen, Senior Director, Investor RelationsT: +45 3377 9558; E: acn@genmab.com

This Company Announcement contains forward looking statements. The words believe, expect, anticipate, intend and plan and similar expressions identify forward looking statements. Actual results or performance may differ materially from any future results or performance expressed or implied by such statements. The important factors that could cause our actual results or performance to differ materially include, among others, risks associated with pre-clinical and clinical development of products, uncertainties related to the outcome and conduct of clinical trials including unforeseen safety issues, uncertainties related to product manufacturing, the lack of market acceptance of our products, our inability to manage growth, the competitive environment in relation to our business area and markets, our inability to attract and retain suitably qualified personnel, the unenforceability or lack of protection of our patents and proprietary rights, our relationships with affiliated entities, changes and developments in technology which may render our products or technologies obsolete, and other factors. For a further discussion of these risks, please refer to the risk management sections in Genmabs most recent financial reports, which are available on http://www.genmab.com and the risk factors included in Genmabs most recent Annual Report on Form 20-F and other filings with the U.S. Securities and Exchange Commission (SEC), which are available at http://www.sec.gov. Genmab does not undertake any obligation to update or revise forward looking statements in this Company Announcement nor to confirm such statements to reflect subsequent events or circumstances after the date made or in relation to actual results, unless required by law.

Genmab A/S and/or its subsidiaries own the following trademarks: Genmab; the Y-shaped Genmab logo; Genmab in combination with the Y-shaped Genmab logo; HuMax; DuoBody; DuoBody in combination with the DuoBody logo; HexaBody; HexaBody in combination with the HexaBody logo; DuoHexaBody; HexElect; and UniBody. Arzerra and Kesimpta are trademarks of Novartis AG or its affiliates. DARZALEX and DARZALEX FASPRO are trademarks of Janssen Pharmaceutica NV. TEPEZZA is a trademark of Horizon Therapeutics plc.

1 American Cancer Society. "Multiple Myeloma Overview." Available at http://www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-what-is-multiple-myeloma.Accessed June 2016.2 National Cancer Institute. "A Snapshot of Myeloma." Available at http://www.cancer.gov/research/progress/snapshots/myeloma. Accessed June 2016. 3 Globocan 2018. United States of America Fact Sheet. Available at http://gco.iarc.fr/today/data/factsheets/840-united-states-of-america-fact-sheets.pdf.4 Globocan 2018. World Fact Sheet. Available at http://gco.iarc.fr/today/data/factsheets/populations/900-world-fact-sheets.pdf. Accessed December 2018.5 American Cancer Society. "How is Multiple Myeloma Diagnosed?" http://www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-diagnosis. Accessed June 20166 DARZALEX Prescribing information, August 2020 https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761036s029lbl.pdf Last accessed August 20207 DARZALEX Summary of Product Characteristics, available at https://www.ema.europa.eu/en/medicines/human/EPAR/darzalex Last accessed June 20208 DARZALEX FASPRO Prescribing information, May 2020. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761145s000lbl.pdf Last accessed May 20209 De Weers, M et al. Daratumumab, a Novel Therapeutic Human CD38 Monoclonal Antibody, Induces Killing of Multiple Myeloma and Other Hematological Tumors. The Journal of Immunology. 2011; 186: 1840-1848.10 Overdijk, MB, et al. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma. MAbs. 2015; 7: 311-21.11 Krejcik, MD et al. Daratumumab Depletes CD38+ Immune-regulatory Cells, Promotes T-cell Expansion, and Skews T-cell Repertoire in Multiple Myeloma. Blood. 2016; 128: 384-94.12 Jansen, JH et al. Daratumumab, a human CD38 antibody induces apoptosis of myeloma tumor cells via Fc receptor-mediated crosslinking.Blood. 2012; 120(21): abstract 2974.

Company Announcement no. 45CVR no. 2102 3884LEI Code 529900MTJPDPE4MHJ122

Genmab A/SKalvebod Brygge 431560 Copenhagen VDenmark

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Genmab Announces IFM, HOVON and Janssen Achieve Positive Topline Results in Second Part of Phase 3 CASSIOPEIA Study of Daratumumab in Multiple Myeloma...

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Jeff Bridges is one of the 85,000-plus lymphoma cases expected in the U.S. this year – MarketWatch

By daniellenierenberg

Careful, man, theres a beloved actor here.

Jeff Bridges revealed that he has lymphoma, which is the most common type of blood cancer. And this sobering news has spurred celebrities and fans to send their best wishes to the star best known for playing the Dude, the White Russiandrinking bowler and casual-wear icon from the Coen brothers 1998 cult classic, The Big Lebowski.

But the Dude abides, and Bridges suggested that his outlook looks just as promising.

As the Dude would say.. New S**T has come to light, tweeted Bridges, 70, on Monday. I have been diagnosed with Lymphoma. Although it is a serious disease, I feel fortunate that I have a great team of doctors and the prognosis is good.

Celebrities such as Cary Elwes, John Lithgow, Patricia Arquette and George Takei posted encouraging words and prayers to Bridges, who is the son of Lloyd and Dorothy Bridges, and has starred in more than 70 films including Starman, True Grit and The Last Picture Show. He won an Academy Award in 2010 for Crazy Heart, and was honored with the Cecil B. DeMille lifetime-achievement award during the 2019 Golden Globes.

And he is now one of the most high-profile cases of lymphoma, a cancer of the bodys infection-fighting lymphatic system that affects the blood and bone marrow. And more than 85,000 new cases of lymphoma are expected to be diagnosed in the U.S. this year, according to American Cancer Society data shared by the Leukemia & Lymphoma Society, with some 791,550 people currently living with lymphoma or in remission from the disease in the U.S.

Many different types of lymphoma exist, and Bridges did not share any more details about his diagnosis or treatment. But his disclosure is an opportunity to share more information about lymphoma, the risk factors and symptoms to be aware of, as well as treatment options.

What is lymphoma?

Lymphoma is a type of cancer that starts in cells that are part of the bodys immune system, specifically the lymphocytes, which are a type of white blood cell that fights germs. So these cancers can affect the blood and bone marrow, as well as the other tissues and organs that produce, store and carry white blood cells including the spleen.

Doctors still dont know what specifically causes lymphoma, but at some point a lymphocyte mutates and begins to reproduce rapidly. The mutated, abnormal cells live longer than the normal cells would, and in time, the diseased and ineffective lymphocytes outnumber the healthy cells, which causes the lymph nodes, liver and spleen to swell.

There are two main types of lymphoma, the CDC explains, including:

Hodgkin lymphoma (HL), which spreads in an orderly manner from one group of lymph nodes to another.

Non-Hodgkin lymphoma (NHL), which spreads through the lymphatic system in a non-orderly manner.

What are the symptoms?

Signs and symptoms of lymphoma may include:

These symptoms can be signs of other health conditions, of course, so its recommended that anyone experiencing them should see a doctor to determine the cause.

How is it treated?

There are many different types of lymphoma including 90 different types of non-Hodgkin lymphoma and treatment varies depending on the type and severity. Generally, lymphoma treatment involves chemotherapy, radiation therapy and immunotherapy medication. The Mayo Clinic, which is an international authority on lymphoma research, explains that the goal of treatment is to destroy as many cancer cells as possible to bring the disease into remission. A bone marrow or stem cell transplant may be performed in some cases to help rebuild healthy bone marrow after chemo and radiation has suppressed the diseased bone marrow.

Bridges didnt specify his own treatment, only saying that he is beginning treatment and will keep the public posted on his recovery.

Treatment can be very expensive, however, with almost 60% of patients covered by Medicare telling the Leukemia & Lymphoma Society in a 2019 study that they decided to delay or forego treatment, largely due to steep out-of-pocket costs. It noted that some traditional Medicare lymphoma patients getting anti-cancer therapy though infusions experienced out-of-pocket costs of more than $19,000 in their first year. And costs can extend two or three years beyond a blood cancer diagnosis.

Who is most at risk?

While children, teens and adults can all develop lymphoma, some types are more common in certain age groups. The CDC notes that rates of Hodgkin lymphoma are highest among teens and young adults (ages 15 to 39) as well as among older adults (ages 75 and older). But non-Hodgkin lymphoma becomes more common as people get older.

Men are also slightly more likely to develop lymphoma than women, the CDC adds, and white people are more likely than Black people to develop non-Hodgkin lymphoma.

Cases have also been more common in people who are immunocompromised, including those who take drugs to suppress their immune systems. And some infections such as HIV and the Epstein-Barr virus are also associated with an increased lymphoma risk.

And like many other cancers, family history has been linked with a higher risk of Hodgkin lymphoma.

What is the survival rate?

The good news is, Hodgkin lymphoma is now considered to be one of the most curable forms of cancer, according to the Leukemia & Lymphoma Society, with a five-year survival rate of 94.4% among patients younger than 45 at diagnosis. And the five-year relative survival rate for those with Hodgkin lymphoma more than doubled from 40% in whites in 1960 to 1963 (the only data available) to 88.5% for all races from 2009 to 2015.

And the five-year relative survival rate for people with non-Hodgkin lymphoma rose from 31% in whites from 1960 to 1963 (the only data available) to 74.7% for all races from 2009 to 2015.

Still, an estimated 20,910 Americans are expected to die from lymphoma this year, including 19,940 with non-Hodgkin lymphoma and 970 with Hodgkin lymphoma.

How does COVID-19 complicate things?

While the medical community is still learning about COVID-19, the general consensus is that people with cancer, who are in active cancer treatment or have previously been treated for cancer, may be at higher risk of severe illness and death if they get the coronavirus. So its important that these folks lower their risk of exposure to COVID-19 by avoiding large crowds and non-essential travel; working from home, if possible; staying at least six feet away from people outside their household; wearing a face mask when they cant socially distance; as well as washing their hands frequently, and not touching their eyes, nose or mouth.

Where can I find more information or support?

Visit the CDC and American Cancer Society pages on lymphoma.

The Mayo Clinic also outlines its lymphoma research and treatment strategies on its website.

The Leukemia & Lymphoma Society and the Lymphoma Research Foundation also provide valuable information and support.

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Jeff Bridges is one of the 85,000-plus lymphoma cases expected in the U.S. this year - MarketWatch

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The Myelofibrosis Treatment market to turn out to be Sublime between 2016 and 2022 – PRnews Leader

By daniellenierenberg

Myelofibrosis or osteomyelofibrosis is a myeloproliferative disorder which is characterized by proliferation of abnormal clone of hematopoietic stem cells. Myelofibrosis is a rare type of chronic leukemia which affects the blood forming function of the bone marrow tissue. National Institute of Health (NIH) has listed it as a rare disease as the prevalence of myelofibrosis in UK is as low as 0.5 cases per 100,000 population. The cause of myelofibrosis is the genetic mutation in bone marrow stem cells. The disorder is found to occur mainly in the people of age 50 or more and shows no symptoms at an early stage. The common symptoms associated with myelofibrosis include weakness, fatigue, anemia, splenomegaly (spleen enlargement) and gout. However, the disease progresses very slowly and 10% of the patients eventually develop acute myeloid leukemia. Treatment options for myelofibrosis are mainly to prevent the complications associated with low blood count and splenomegaly.

The global market for myelofibrosis treatment is expected to grow moderately due to low incidence of a disease. However, increasing incidence of genetic disorders, lifestyle up-gradation and rise in smoking population are the factors which can boost the growth of global myelofibrosis treatment market. The high cost of therapy will the growth of global myelofibrosis treatment market.

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The global market for myelofibrosis treatment is segmented on basis of treatment type, end user and geography:

As myelofibrosis is considered as non-curable disease treatment options mainly depend on visible symptoms of a disease. Primary stages of the myelofibrosis are treated with supportive therapies such as chemotherapy and radiation therapy. However, there are serious unmet needs in myelofibrosis treatment market due to lack of disease modifying agents. Approval of JAK1/JAK2 inhibitor Ruxolitinib in 2011 is considered as a breakthrough in myelofibrosis treatment. Stem cell transplantation for the treatment of myelofibrosis also holds tremendous potential for market growth but high cost of therapy is foreseen to limits the growth of the segment.

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On the basis of treatment type, the global myelofibrosis treatment market has been segmented into blood transfusion, chemotherapy, androgen therapy and stem cell or bone marrow transplantation. Chemotherapy segment is expected to contribute major share due to easy availability of chemotherapeutic agents. Ruxolitinib is the only chemotherapeutic agent approved by the USFDA specifically for the treatment of myelofibrosis, which will drive the global myelofibrosis treatment market over the forecast period.

Geographically, global myelofibrosis treatment market is segmented into five regions viz. North America, Latin America, Europe, Asia Pacific and Middle East & Africa. Northe America is anticipated to lead the global myelofibrosis treatment market due to comparatively high prevalence of the disease in the region.

Some of the key market players in the global myelofibrosis treatment market are Incyte Corporation, Novartis AG, Celgene Corporation, Mylan Pharmaceuticals Ulc., Bristol-Myers Squibb Company, Eli Lilly and Company, Taro Pharmaceuticals Inc., AllCells LLC, Lonza Group Ltd., ATCC Inc. and others.

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Stem Cell Therapy Market 2020-2026 | XX% CAGR Projection Over the Next Five Years, Predicts Market Research Future with Market Size & Growth Key…

By daniellenierenberg

Stem Cell Therapy Market report would come handy to understand the competitors in the market and give an insight into sales, volumes, revenues in the Stem Cell Therapy Industry & will also assists in making strategic decisions. The report also helps to decide corporate product & marketing strategies. It reduces the risks involved in making decisions as well as strategies for companies and individuals interested in the Stem Cell Therapy industry. Both established and new players in Stem Cell Therapy industries can use the report to understand the Stem Cell Therapy market.

In Global Market, the Following Companies Are Covered:

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Analysis of the Market:

Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone marrow transplant is the most widely used stem-cell therapy, but some therapies derived from umbilical cord blood are also in use.

In the last several years, global stem cell therapy market developed fast at a average growth rate of 46.81%.

Market Analysis and Insights: Global Stem Cell Therapy Market

In 2019, the global Stem Cell Therapy market size was USD 403.6 million and it is expected to reach USD 1439.9 million by the end of 2026, with a CAGR of 19.7% during 2021-2026.

Global Stem Cell Therapy Scope and Market Size

Stem Cell Therapy market is segmented by Type, and by Application. Players, stakeholders, and other participants in the global Stem Cell Therapy market will be able to gain the upper hand as they use the report as a powerful resource. The segmental analysis focuses on revenue and forecast by Type and by Application in terms of revenue and forecast for the period 2015-2026.

Segment by Type, the Stem Cell Therapy market is segmented into Autologous, Allogeneic, etc.

Segment by Application, the Stem Cell Therapy market is segmented into Musculoskeletal Disorder, Wounds & Injuries, Cornea, Cardiovascular Diseases, Others, etc.

Regional and Country-level Analysis

The Stem Cell Therapy market is analysed and market size information is provided by regions (countries).

The key regions covered in the Stem Cell Therapy market report are North America, Europe, China, Japan, Southeast Asia, India and Central & South America, etc.

The report includes country-wise and region-wise market size for the period 2015-2026. It also includes market size and forecast by Type, and by Application segment in terms of revenue for the period 2015-2026.

Competitive Landscape and Stem Cell Therapy Market Share Analysis

Stem Cell Therapy market competitive landscape provides details and data information by vendors. The report offers comprehensive analysis and accurate statistics on revenue by the player for the period 2015-2020. It also offers detailed analysis supported by reliable statistics on revenue (global and regional level) by player for the period 2015-2020. Details included are company description, major business, company total revenue and the revenue generated in Stem Cell Therapy business, the date to enter into the Stem Cell Therapy market, Stem Cell Therapy product introduction, recent developments, etc.

The major vendors include Osiris Therapeutics, NuVasive, Chiesi Pharmaceuticals, JCR Pharmaceutical, Pharmicell, Medi-post, Anterogen, Molmed, Takeda (TiGenix), etc.

This report focuses on the global Stem Cell Therapy status, future forecast, growth opportunity, key market and key players. The study objectives are to present the Stem Cell Therapy development in North America, Europe, China, Japan, Southeast Asia, India and Central & South America.

Stem Cell Therapy Market Breakdown by Types:

Stem Cell Therapy Market Breakdown by Application:

Critical highlights covered in the Global Stem Cell Therapy market include:

The information available in the Stem Cell Therapy Market report is segmented for proper understanding. The Table of contents contains Market outline, Market characteristics, Market segmentation analysis, Market sizing, customer landscape & Regional landscape. For further improving the understand ability various exhibits (Tabular Data & Pie Charts) has also been used in the Stem Cell Therapy Market report.

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In the end, Stem Cell Therapy Industry report provides the main region, market conditions with the product price,profit, capacity, production, supply, demand and market growth rateand forecast etc. This report also Present newproject SWOT analysis,investment feasibility analysis, andinvestment return analysis.

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Stem Cell Therapy Market 2020-2026 | XX% CAGR Projection Over the Next Five Years, Predicts Market Research Future with Market Size & Growth Key...

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Stem Cell Assay Market In-Depth Analysis & Forecast 2017-2025 – The Think Curiouser

By daniellenierenberg

Stem Cell Assay Market: Snapshot

Stem cell assay refers to the procedure of measuring the potency of antineoplastic drugs, on the basis of their capability of retarding the growth of human tumor cells. The assay consists of qualitative or quantitative analysis or testing of affected tissues andtumors, wherein their toxicity, impurity, and other aspects are studied.

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With the growing number of successfulstem cell therapytreatment cases, the global market for stem cell assays will gain substantial momentum. A number of research and development projects are lending a hand to the growth of the market. For instance, the University of Washingtons Institute for Stem Cell and Regenerative Medicine (ISCRM) has attempted to manipulate stem cells to heal eye, kidney, and heart injuries. A number of diseases such as Alzheimers, spinal cord injury, Parkinsons, diabetes, stroke, retinal disease, cancer, rheumatoid arthritis, and neurological diseases can be successfully treated via stem cell therapy. Therefore, stem cell assays will exhibit growing demand.

Another key development in the stem cell assay market is the development of innovative stem cell therapies. In April 2017, for instance, the first participant in an innovative clinical trial at the University of Wisconsin School of Medicine and Public Health was successfully treated with stem cell therapy. CardiAMP, the investigational therapy, has been designed to direct a large dose of the patients own bone-marrow cells to the point of cardiac injury, stimulating the natural healing response of the body.

Newer areas of application in medicine are being explored constantly. Consequently, stem cell assays are likely to play a key role in the formulation of treatments of a number of diseases.

Global Stem Cell Assay Market: Overview

The increasing investment in research and development of novel therapeutics owing to the rising incidence of chronic diseases has led to immense growth in the global stem cell assay market. In the next couple of years, the market is expected to spawn into a multi-billion dollar industry as healthcare sector and governments around the world increase their research spending.

The report analyzes the prevalent opportunities for the markets growth and those that companies should capitalize in the near future to strengthen their position in the market. It presents insights into the growth drivers and lists down the major restraints. Additionally, the report gauges the effect of Porters five forces on the overall stem cell assay market.

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Global Stem Cell Assay Market: Key Market Segments

For the purpose of the study, the report segments the global stem cell assay market based on various parameters. For instance, in terms of assay type, the market can be segmented into isolation and purification, viability, cell identification, differentiation, proliferation, apoptosis, and function. By kit, the market can be bifurcated into human embryonic stem cell kits and adult stem cell kits. Based on instruments, flow cytometer, cell imaging systems, automated cell counter, and micro electrode arrays could be the key market segments.

In terms of application, the market can be segmented into drug discovery and development, clinical research, and regenerative medicine and therapy. The growth witnessed across the aforementioned application segments will be influenced by the increasing incidence of chronic ailments which will translate into the rising demand for regenerative medicines. Finally, based on end users, research institutes and industry research constitute the key market segments.

The report includes a detailed assessment of the various factors influencing the markets expansion across its key segments. The ones holding the most lucrative prospects are analyzed, and the factors restraining its trajectory across key segments are also discussed at length.

Global Stem Cell Assay Market: Regional Analysis

Regionally, the market is expected to witness heightened demand in the developed countries across Europe and North America. The increasing incidence of chronic ailments and the subsequently expanding patient population are the chief drivers of the stem cell assay market in North America. Besides this, the market is also expected to witness lucrative opportunities in Asia Pacific and Rest of the World.

Global Stem Cell Assay Market: Vendor Landscape

A major inclusion in the report is the detailed assessment of the markets vendor landscape. For the purpose of the study the report therefore profiles some of the leading players having influence on the overall market dynamics. It also conducts SWOT analysis to study the strengths and weaknesses of the companies profiled and identify threats and opportunities that these enterprises are forecast to witness over the course of the reports forecast period.

Some of the most prominent enterprises operating in the global stem cell assay market are Bio-Rad Laboratories, Inc (U.S.), Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.K.), Hemogenix Inc. (U.S.), Promega Corporation (U.S.), Bio-Techne Corporation (U.S.), Merck KGaA (Germany), STEMCELL Technologies Inc. (CA), Cell Biolabs, Inc. (U.S.), and Cellular Dynamics International, Inc. (U.S.).

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The Anthony Nolan and NHS Stem Cell Registry – The Hippocratic Post

By daniellenierenberg

More than 2 million people have registered to become stem cell donors the UK, new figures released today reveal. The UK stem cell register had an immensely successful year in 2019/20, with 326,756 new donors added over 100,000 more than the previous year.

The UK stem cell register is known as the Anthony Nolan and NHS Stem Cell Registry and is made up of donors recruited by NHS Blood and Transplant, the Welsh Blood Service, DKMS and Anthony Nolan. The UK donor registers are urging young men, and people from black, Asian and minority ethnic backgrounds to register and ensure that all patients in need of a stem cell transplant can find a, potentially, lifesaving match.

If a patient has a condition that affects their bone marrow or blood, then a stem cell transplant may be their best chance of survival. Doctors will give new, healthy stem cells to the patient via their bloodstream, where they begin to grow and create healthy red blood cells, white blood cells and platelets.

In 2019/20 62 per cent of people who donated stem cells or bone marrow to patients in the UK were men under 30. They are the demographic most likely to be chosen to donate, but make up just 19 per cent of the UK stem cell register.

The percentage of all donors from minority ethnic backgrounds has remained steady at 13 per cent in 2019/20, highlighting the importance of raising awareness of their lifesaving potential amongst this group. Patients from Black, Asian or other minority backgrounds have a 20 per cent chance of finding the best possible stem cell match from an unrelated donor, compared to 69 per cent for northern European backgrounds.

Henny Braund, Chief Executive of Anthony Nolan, said:

Nobody could have foreseen the challenges this year would bring to building a healthy, diverse stem cell register. But weve adapted and weve innovated as patients cant wait and were thrilled that in 2020, weve collectively recruited two million donors onto the stem cell register. Each donor represents hope, a potential cure for blood cancer.

I thank colleagues and partners for their commitment helping us reach this point. I am also immensely grateful for the two million selfless individuals who signed up to the registry, making themselves available whenever they are needed.

The two million milestone means increased chances for many of finding an unrelated donor match. But were still far from our goal of finding a match for everyone who needs one.

I would urge anyone thinking of joining the stem cell register, especially young men, who are the most likely to be chosen, to do so today. You could be someones lifesaver, without you there is no cure.

Christopher Harvey, Head of the Welsh Bone Marrow Registry, said:

Its incredibly heart-warming to know there are two million people in the UK who are willing to donate stem cells should they be the match for someone in need of their potentially lifesaving donation.

We see in our roles the difference stem cells make, for lots of patients receiving stem cells is the final treatment option.

Despite this great news we still have more to do. Unfortunately, there are still patients who are unable to find a match. Thats why were committed to ensuring every patient has the best possible chance of finding that one lifesaving donor in their time of need.

Guy Parkes, Head of Stem Cell Donation & Transplantation at NHS Blood and Transplant, said:

We want all patients in need of a transplant to be able to find a lifesaving match. Each time a person joins this register it brings fresh hope to patients of a match.

This register is used by hospitals across the UK to find suitable matches for patients and it has helped to save and improve the lives of thousands of people since its creation 33 years ago its amazing that we now have over 2 million people on the register, putting the chances of matching donors to patients at a record high.

Donating stem cells is an altruistic, lifesaving act and its an amazing thing to do. We will continue to expand the UK register to help patients in need. We particularly need more young men to join.

Jonathan Pearce, CEO of DKMS UK said:

Were delighted to have reached such an amazing milestone and are grateful to those two million people who are actively registered and waiting to help give someone living with blood cancer or a blood disorder a second chance of life.

At any one time there are around 2,000 people in the UK in need of a blood stem cell transplant, so whilst we recognise this achievement it goes without saying that we need to continue to encourage everyone that can register to do so. This will help to grow the numbers and diversify the registry further in order to improve the odds for those who currently have less chance of finding a matching donor.

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The Anthony Nolan and NHS Stem Cell Registry - The Hippocratic Post

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SA becomes 2nd country to allow 16 and 17-year-olds to donate bone marrow – News24

By daniellenierenberg

"Young people today are often drivers of social change movements and we look forward to engaging them."

South African youth aged 16 and 17 will be able to make history, alongside their peers in the UK, as the worlds youngest bone marrow donors.

The South African Bone Marrow Registry (SABMR) received the nod from its Clinical Governance Committee and board members, as well as the National Health Department to allow 16 and 17-year-old teens to become bone marrow, stem-cell donors.

Recent changes in legislation and advances in stem cell donation have allowed registries to reduce the age limit of donors. South Africa now joins the UK in this move. The latter became one of the first countries to do so.

Dr Charlotte Ingram, Medical Director of the SA Bone Marrow Registry (SABMR) the largest registry in the country says it's a landmark moment as the change in joining policy will contribute to saving more lives.

"In general, young people make better donors. Research shows that younger donors are associated with better survival rates for patients following a stem cell transplant."

"It's a step towards further enhancing the registry towards a younger and more ethnically diverse pool for blood cancer patients and others in need of a bone marrow transplant."

Previously, teenagers had to wait until they were 18 to join the SA Bone Marrow Registry. Now they can join by following the same procedure as others would.

While it is not required, it is important for the SABMR to involve parents and address any questions or concerns they may have re the procedure and what it entails.

Once youth have applied online, they will be contacted to discuss the easiest way of dispatching and collecting swab kits. The only initial sample that is required is a cheek swab.

Currently, 18-25-year olds only account for 6.8% of the SABMR registry but with increased awareness of bone marrow donation among young people, the figure should increase substantially.

Read:Knowledge is key: What you need to know about the most common childhood cancer in SA

"Studies tell us that generation WE (aged 14-20) and generation Z (21-25) are a lot more self-aware, socially-responsible and globally-minded than previous generations. They are more concerned about tackling social issues and want to roll up their sleeves and make a difference. Young people today are often drivers of social change movements and we look forward to engaging them."

She says there is no greater way to help another than to potentially save a life.

"So many lives are lost if there is a delay in finding a donor match. While we have 74 000 donors on our registry, we often discover that many older donors can no longer donate stem cells as they have developed hypertension, heart disease or diabetes."

"When this happens, we have to start the search process all over again, which prolongs the agonising wait for a patient, who doesn't have time to waste."

"By opening up the donor pool to a younger audience, means doctors and donors can choose the healthiest matches that substantially increases a patients chance of survival."

For now, social media will serve as the primary channel to create awareness among youth, but physical donor drives at schools and other initiatives, which encourage collaboration between learners, peers and patients are in the pipeline for 2021.

If you are between the ages of 16 and 45 and want to become a donor, contact the SABMR on 021 447 8638 or email: donors@sabmr.co.za.

Financial donations can also be made via http://www.sabmr.co.za/donate.

Submitted to Parent24 by theSA Bone Marrow Registry

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YOUR HEALTH: Saving an unborn baby breaking apart in the womb – WQAD.com

By daniellenierenberg

DENVER A baby broken.

Most doctors gave little unborn Payton Calvillo any hope she would survive.

But through strong faith and the help of a team of medical experts, she is thriving today.

"She's a complete miracle baby," said Payton's mother, Ahna Calvillo.

When Ahna was just five months pregnant, she was told her unborn baby would probably not survive birth.

"It was pretty much a death sentence from the beginning."

Payton's bones were breaking and bending inside the womb.

"She likely had a problem where she couldn't make alkaline phosphatase properly," explained Dr. Sunil Nayak, a pediatric endocrinologist at Rocky Mountain Hospital for Children.

Alkaline phosphatase is needed for bones to grow and strengthen.

There was little anyone could do.

19 different specialists were on hand for the C-section delivery

"They even asked us the question that morning, how far do you want us to go?" Ahna remembered.

"'Do you want a ventilator on her?', you know, 'How far do you want us to prolong her life?'"

"Our ultimate hope and goal was that she would come out and breathe on her own."

"She just came out screaming," said Ahna.

"She came out crying. She breathed on her own right away. She was perfect."

Payton was diagnosed with hypophosphatasia, a disorder that weakens bones.

She was immediately placed on a new FDA-approved medicine.

"Here we are just one year later at one year of age and you see a dramatic difference in the shape," said Dr. Jared Riley, a pediatric orthopedic surgeon at Rocky Mountain Hospital.

Before the medicine, 75% of all patients died by the age of five.

Now there is a 97% chance Payton will live a normal life.

"My baby was broken and that's what I needed God to do was a miracle," said Ahna.

One was also treated with bone fragments and cultured osteoblasts, which are bone-forming cells.

"Cultured" refers to cells that are grown under specific conditions outside of the natural environment (the body) and within a laboratory.

Both patients showed significant, but incomplete improvement, although no more formal studies have been conducted.

Then, the drug teriparatide (parathyroid hormone 1-34) has been given "off-label" to several adults with HPP with metatarsal stress fractures or femoral pseudo fractures, resulting in healing.

The drug is not permitted for use in children.

More research is necessary to determine the long-term safety and effectiveness of teriparatide in the treatment of HPP.

Every year eight million babies are born with genetic disorders passed down from generation to generation.

Payton will stay on the new medication for the next few years and then doctors will re-evaluate whether she needs to continue.

Payton's family didn't even know they carried the problematic HPP gene until an ultrasound revealed it in their unborn baby.

After being genetically tested, Payton's mother and grandfather are positive.

Neither one has ever suffered from weak or broken bones.

If this story has impacted your life or prompted you or someone you know to seek or change treatments, please let us know by contacting Jim Mertens at jim.mertens@wqad.com or Marjorie Bekaert Thomas atmthomas@ivanhoe.com.

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Stem Cell Therapy Market To Observe Exponential Growth By 2020-2027 | Verified Market Research – Stock Market Vista

By daniellenierenberg

Global Stem Cell Therapy Market was valued at USD 117.66 million in 2019 and is projected to reach USD 255.37 million by 2027, growing at a CAGR of 10.97% from 2020 to 2027.

For top companies in United States, European Union and China, this report investigates and analyzes the production, value, price, market share and growth rate for the top manufacturers, key data from 2020 to 2027.

The Stem Cell Therapy market report firstly introduced the basics: definitions, classifications, applications and market overview; product specifications; manufacturing processes; cost structures, raw materials and so on. Then it analyzed the worlds main region market conditions, including the product price, profit, capacity, production, supply, demand and market growth rate and forecast etc. In the end, the Stem Cell Therapy market report introduced new project SWOT analysis, investment feasibility analysis, and investment return analysis.

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Competitive Analysis:

The competitive analysis covers key players and the innovations and business strategies undertaken by them. The report captures the best long term growth opportunities for the sector and includes the latest process and product developments. The report includes basic information of the companies along with their market position, historical background, and market capitalization and revenue. The report covers revenue figures, market growth rate, and gross profit margin of each player based on regional classification and overall market position. The report provides a separate analysis of the recent business strategies such as mergers, acquisitions, product launches, joint ventures, partnerships, and collaborations.

Key features of the Report:

Leading players of Stem Cell Therapy including:

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Market Breakdown:

The market breakdown provides market segmentation data based on the availability of the data and information. The market is segmented on the basis of types and applications.

1.Stem Cell Therapy Market, By Cell Source:

Adipose Tissue-Derived Mesenchymal Stem Cells Bone Marrow-Derived Mesenchymal Stem Cells Cord Blood/Embryonic Stem Cells Other Cell Sources

2.Stem Cell Therapy Market, By Therapeutic Application:

Musculoskeletal Disorders Wounds and Injuries Cardiovascular Diseases Surgeries Gastrointestinal Diseases Other Applications

3.Stem Cell Therapy Market, By Type:

Allogeneic Stem Cell Therapy Market, By Application Musculoskeletal Disorders Wounds and Injuries Surgeries Acute Graft-Versus-Host Disease (AGVHD) Other Applications Autologous Stem Cell Therapy Market, By Application Cardiovascular Diseases Wounds and Injuries Gastrointestinal Diseases Other Applications

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To understand the global Stem Cell Therapy market dynamics, the market is analyzed across major global regions and countries. Market Research Intellect provides customized specific regional and country-wise analysis of the key geographical regions as follows:

North America: USA, Canada, Mexico

Latin America: Argentina, Chile, Brazil, Peru, and Rest of Latin America

Europe:K., Germany, Spain, Italy, and Rest of EU

Asia-Pacific: India, China, Japan, South Korea, Australia, and Rest of APAC

Middle East & Africa: Saudi Arabia, South Africa, U.A.E., and Rest of MEA

The report considers:

Historical Years: 2017-2018

Base Year: 2019

Estimated Year: 2020

Forecast Years: 2020-2027

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Stem Cell Therapy Market To Observe Exponential Growth By 2020-2027 | Verified Market Research - Stock Market Vista

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