Skeletal Muscle Lineage Research Articles

Gastruloids are competent to specify both cardiac and skeletal muscle lineages

Cardiopharyngeal mesoderm contributes to the formation of the heart and head muscles. However, the mechanisms governing cardiopharyngeal mesoderm specification remain unclear. Here, we reproduce cardiopharyngeal mesoderm specification towards cardiac and skeletal muscle lineages with gastruloids from mouse embryonic stem cells. By conducting a comprehensive temporal analysis of cardiopharyngeal mesoderm development and differentiation in gastruloids compared to mouse embryos, we present the evidence for skeletal myogenesis in gastruloids. We identify different subpopulations of cardiomyocytes and skeletal muscles, the latter of which most likely correspond to different states of myogenesis with “head-like” and “trunk-like” skeletal myoblasts. In this work, we unveil the potential of gastruloids to undergo specification into both cardiac and skeletal muscle lineages, allowing the investigation of the mechanisms of cardiopharyngeal mesoderm differentiation in development and how this could be affected in congenital diseases.

A Neural Basis for Mutant ATAXIN-1 Induced Respiratory Dysfunction in Mouse Models of Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1), a dominantly inherited neurodegenerative disorder caused by an expanded trinucleotide repeat in the ATAXIN-1 (ATXN1) gene, is characterized by motor dysfunction, cognitive impairment, and death from compromised swallowing and respiration. To delineate specific cell types that contribute to respiratory dysfunction, we utilized the floxed conditional knock-in f-ATXN1 146Q/2Q mouse. Whole body plethysmography during spontaneous respiration and respiratory challenge showed that f-ATXN1 146Q/2Q mice exhibit a spontaneous respiratory phenotype characterized by elevated respiratory frequency, volumes, and respiratory output. Consequently, the ability of f-ATXN1 146Q/2Q mice to increase ventilation during the challenge is impaired. To investigate the role of mutant ATXN1 expression in neural and skeletal muscle lineages, f-ATXN1 146Q/2Q mice were bred to Nestin-Cre and Acta1-Cre mice respectively. These analyses revealed that the abnormal spontaneous respiration in f-ATXN1 146Q/2Q mice involved two aspects: a behavioral phenotype in which SCA1 mice exhibit increased motor activity during respiratory testing and functional dysregulation of central respiratory control centers. Both aspects of spontaneous respiration were partially ameliorated by removing mutant ATXN1 from neural, but not skeletal muscle, cell lineages.

Myogenic Regulator Genes Responsible For Muscle Development in Farm Animals

Farm animals especially poultry species play important roles in meeting the worldwide demand for meat and are genetically improved to increase the efficiency of meat production. Improving muscle growth and development is crucial to meet customers' demands for high-quality meat. Myogenesis, fibrogenesis, and adipogenesis are components of fetal skeletal muscle formation. Myogenesis is controlled by a complex network of intrinsic and extrinsic components, usually divided into two or three stages and regulated by kinase-encoding genes and myogenic regulatory factor genes. The basic family of helix-loop-helix transcription factors, which includes MYF5, MYOD, myogenin, and MRF4, regulates the specification and differentiation of skeletal muscle cells throughout embryogenesis and postnatal myogenesis. With the discovery of the myogenic regulatory factor family, which includes the transcription factors MYF5, MYOD, Myogenin, and MRF4, it has become possible to determine the skeletal muscle lineage and understand the regulation of myogenic differentiation during development. These elements also play a role in determining the muscle satellite cell lineage that develops into the resident stem cell compartment in adult skeletal muscle. Although MYF5, MYOD, Myogenin and MRF4 play minor roles in mature muscle, they once again play an important role in directing satellite cell activity to regenerate skeletal muscle, linking the genetic regulation of development and regeneration myogenesis. An important step towards the goal of increasing meat yield and improving meat quality is the understanding and identification of these genes. This in-depth analysis discusses the function of myogenic regulatory factors in the specification of satellite cells, maturation and regeneration of skeletal muscle.

Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell.

Endothelial and skeletal muscle lineages arise from common embryonic progenitors. Despite their shared developmental origin, adult endothelial cells (ECs) and muscle stem cells (MuSCs; satellite cells) have been thought to possess distinct gene signatures and signaling pathways. Here, we shift this paradigm by uncovering how adult MuSC behavior is affected by the expression of a subset of EC transcripts. We used several computational analyses including single-cell RNA-seq (scRNA-seq) to show that MuSCs express low levels of canonical EC markers in mice. We demonstrate that MuSC survival is regulated by one such prototypic endothelial signaling pathway (VEGFA-FLT1). Using pharmacological and genetic gain- and loss-of-function studies, we identify the FLT1-AKT1 axis as the key effector underlying VEGFA-mediated regulation of MuSC survival. All together, our data support that the VEGFA-FLT1-AKT1 pathway promotes MuSC survival during muscle regeneration, and highlights how the minor expression of select transcripts is sufficient for affecting cell behavior.

Abstract 1092: RUNX2 inhibition alters PAX3::FOXO1 driven fusion-positive rhabdomyosarcoma cell state and growth

Abstract Investigation: Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric cancer of skeletal muscle lineage. While the incidence of RMS is ~4.5 patients per million individuals aged <20 years, 5-year survival for high-risk children is <30%, and <8% when metastatic. The PAX3::FOXO1 (P3F) fusion gene, resulting from t(2:13), is the most prominent fusion oncogene that drives FP-RMS. P3F is a fusion transcription factor and supports FP-RMS by keeping it in a persistent myoblastic state. We found P3F functions in part by suppressing MST kinase activity in the Hippo tumor suppressor pathway, a developmental pathway involved in cell fate and organ size. To investigate how this may rewire gene expression to support FP-RMS, we performed ATAC-seq analysis of a myoblast-based model of FP-RMS in which MST was further inhibited and found the RUNX2 gene locus was more accessible. Since RUNX2 is a pioneer transcription factor known to interact with the “WW” domains of Hippo pathway members to support mesenchymal cell fate, we hypothesize that RUNX2 maintains these relationships to support FP-RMS cell state and survival. Methods & Results: Inquiry of in silico databases and previously published literature unveiled that sarcomas including RMS have a high dependency on RUNX2 and that P3F directly binds a RUNX2 enhancer. Interrogation of a proximity labeling proteomics study from our collaborators in the Counter lab found that RUNX2 was part of the protein interactome of 7/7 FP-RMS fusions including P3F. Additionally, Co-IP experiments confirmed that there is a direct protein-protein relationship between RUNX2, P3F, and TAZ. Next, we performed in vitro genetic loss of function (LOF) of RUNX2 using RNAi, which showed that suppression of RUNX2 caused cell death, growth inhibition, increased TAZ expression, and induction of both myogenic and osteogenic markers. We also performed complementary pharmacologic LOF using two available small molecules, AI-14-91 (Bushweller lab) in vitro, and CADD522 (commercially available) in vitro and in vivo. We found that these partially phenocopied the genetic LOF and that CADD522 reduces tumor volume and growth. Conclusions: RUNX2 is a vulnerability in RMS based on in silico, in vitro, and in vivo investigation, and may act to prevent osteogenic and myogenic differentiation via protein-protein relationships in the Hippo pathway. The next step will be assessing mechanistic relationships between the Hippo pathway and RUNX2, as well as the genome wide changes upon RUNX2 LOF by performing ChIP-seq, RNA-seq, and scRNA-seq experiments. Citation Format: Elizabeth A. Mendes, Audrey L. Chambers, Samantha J. Weitzel, Michael Deel, Aanandi Munshi, Christopher M. Counter, Seth Zimmerman, John Bushweller, Corinne M. Linardic. RUNX2 inhibition alters PAX3::FOXO1 driven fusion-positive rhabdomyosarcoma cell state and growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1092.

Differentiation Capacity of Porcine Skeletal Muscle-Derived Stem Cells as Intermediate Species between Mice and Humans.

Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined via in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was significantly stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs are closer to those in humans than to those in mice.

Gastruloids as an in vitro model of Cardiopharyngeal Mesoderm specification into skeletal and cardiac muscle lineages

Cardiopharyngeal mesoderm (CPM) contributes to the formation of the heart and head muscles. These muscles are both affected in rare congenital disorders including velocardiofacial syndrome or cardiofaciocutaneous syndrome. While the mechanisms governing CPM specification remain relatively unknown, there is a lack of an in vitro platform that would allow the differentiation of both head and heart muscles. Develop embryonic organoids from mouse embryonic stem cells (mESC), also called gastruloids, to form and specify CPM toward the cardiac and muscle lineages. Use in vitro 3D culture to develop and characterize embryonic organoid. By using single-cell RNAseq, and high-resolution imaging of single molecule FISH experiments, we confirm that the CPM is established in gastruloids, with a kinetic of gene expression that is similar to the mouse embryo. We identify different subpopulations of cardiomyocytes and skeletal muscles most likely corresponding to the different cardiac chambers and head and trunk skeletal muscles. These findings unveil the potential of mESCs-derived gastruloids to undergo CPM specification in both head and heart lineages, allowing the investigation of the mechanisms of CPM specification in development and how this could be affected in congenital disorders.

Establishment of Skeletal Myogenic Progenitors from Non-Human Primate Induced Pluripotent Stem Cells.

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.

Abstract 6736: Suppression of antigen presentation is a hallmark of pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children and young adults, accounting for approximately 350-400 new cases annually in the US. These tumors are diagnosed based on their expression of skeletal muscle lineage markers. Clinical trials testing conventional immunotherapies such as cancer vaccines or immune checkpoint blockade (ICB) therapy have found no activity in RMS, although the root causes of failure are largely unexplored. A recent study by our group found that RMS tumors have a lack of immune infiltrate, consistent with previous reports. However, we made an unexpected observation that RMS tumors had a near absence of antigen processing and presentation (APP) gene expression, a key requisite for anti-tumor CD8+ T lymphocytes. In this study, we investigate the root causes of the lack of APP in RMS and evaluate implications for immunotherapy. Methods: Publicly available DNA sequencing datasets were used to evaluate APP gene mutations in RMS tumor samples. RNAseq from reporter iPSC cell lines demarcating specific stages of skeletal muscle differentiation were used to evaluate APP gene expression during myogenesis. Surface MHC class I expression was evaluated using flow cytometry against a panel of pediatric sarcoma cell lines. RMS PDX and cell line APP protein levels were measured by western blot. Stimulation of APP in RMS cell lines was tested using IFNγ or sub-cytotoxic doses of different epigenetic therapeutics (DNMTi, HDACi, EZH2i) before analyzing surface MHC class I expression by flow cytometry. Results: RNAseq analysis of the differentiation states across the normal human skeletal muscle lineage revealed a myoblastic cell state which transiently downregulates the APP pathway. RNA expression of HLA-A,B,C genes were lower in RMS than any other pediatric extracranial solid tumor histology studied. RMS cell lines and PDX models also displayed low APP pathway gene expression by western blot and flow cytometry, with FOXO1 fusion positive RMS having a near absence of expression. RMS tumor exome sequencing data from RMS patients showed a lack of enrichment of APP gene mutations. Instead, we found that APP could be induced in nearly all samples tested using IFNγ or epigenetic stimulation with Decitabine, Entinostat, or Tazemetostat. Conclusions: We uncovered a previously unappreciated lack of antigen presentation in RMS which phenocopies myoblasts in normal skeletal muscle development. The downregulation of MHC class I antigen presentation is not due to APP pathway mutations but rather a partially reversible downregulation mirroring this specific stage of normal skeletal muscle development. Our findings suggest that pharmacological reversal of the APP pathway downregulation may enhance immunotherapies targeting MHC class I antigens in RMS. Citation Format: David Milewski, Meijie Tian, Yong Kim, Jun Wei, Javed Khan. Suppression of antigen presentation is a hallmark of pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6736.

Abstract 1680: Characterization of sensitivity to single agents and combination treatments (VAC and VI) and development of acquired resistance in childhood rhabdomyosarcoma xenograft models

Abstract For childhood rhabdomyosarcoma (RMS), a tumor demonstrating characteristics of skeletal muscle lineage, survival has not changed significantly in over two decades. In a recent high-risk clinical trial (ARST0431) 25% of patients progressed during 52 weeks of chemo-radiation treatment, and 80% had tumor recurrence or progression within 24 months. Two drug modules comprise the core for treatment: VAC (vincristine, actinomycin-D, cyclophosphamide) and VI (vincristine, irinotecan). However, the molecular basis for intrinsic or acquired resistance to these combinations is poorly understood.To simulate clinical heterogeneity of RMS, we have developed 40 PDX/CDX xenograft models and using Single Mouse Testing (SMT) have characterized the sensitivity of each model to individual agents and to VAC/VI combinations. Tumor volume regression and Event-Free Survival (EFS) were used to assess chemosensitivity. To select for acquired resistance, tumor bearing mice were treated with one cycle of VAC or VI, tumor was transplanted upon regrowth when volume reached 400% of that on day 1 of treatment. The process was repeated for up to 5 cycles of therapy. Development of resistance was measured by decreased EFS and tumor progression during treatment. Parental and isogenic lines with acquired resistance were snap frozen for DNA and RNA sequencing. Sensitivity for each tumor models was assessed by EFS for treated vs untreated tumor and probability plotted against time (days) after initiating treatment. Each single agent significantly extended EFS compared to control EFS (P<0.001), consistent with known clinical activity. Of note VAC and VI combinations had greater than additive EFS (e.g. EFSVAC>>EFSvincristine + EFSactD + EFScyclophosphamide). The sensitivity of the 40 xenograft models to VAC ranged from Progressive Disease with EFS <42 days to Maintained Complete Response at 140 days. A similar range in sensitivity to VI was observed also, but the correlation between sensitivity to VAC and VI was poor (r=0.375) indicating greater sensitivity of a model to one combination than the other. Ten lines have been selected in vivo for acquired resistance to VAC and VI. Resistance was developed between cycle 2 and cycle 5 of treatment with a mean decrease in EFS of 65 +/- 15% compared to the EFS on drug cycle 1. In summary, we have characterized the sensitivity of 40 RMS xenograft models to single agents and VAC/VI combinations and have developed 20 models with acquired resistance to these combinations. Tumor tissue has been obtained from all 40 models and from 20 models with acquired drug resistance. Sequencing of these models is ongoing and preliminary results will be presented. Supported by 1 UO1 CA263981-01. Citation Format: Samson Ghilu, Peter J. Houghton, Siyuan Zheng, Raushan T. Kurmasheva. Characterization of sensitivity to single agents and combination treatments (VAC and VI) and development of acquired resistance in childhood rhabdomyosarcoma xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1680.

Loss of CRY2 promotes regenerative myogenesis by enhancing PAX7 expression and satellite cell proliferation.

The regenerative capacity of skeletal muscle is dependent on satellite cells. The circadian clock regulates the maintenance and function of satellite cells. Cryptochrome 2 (CRY2) is a critical component of the circadian clock, and its role in skeletal muscle regeneration remains controversial. Using the skeletal muscle lineage and satellite cell-specific CRY2 knockout mice (CRY2scko), we show that the deletion of CRY2 enhances muscle regeneration. Single myofiber analysis revealed that deletion of CRY2 stimulates the proliferation of myoblasts. The differentiation potential of myoblasts was enhanced by the loss of CRY2 evidenced by increased expression of myosin heavy chain (MyHC) and myotube formation in CRY2-/- cells versus CRY2+/+ cells. Immunostaining revealed that the number of mononucleated paired box protein 7 (PAX7+) cells associated with myotubes formed by CRY2-/- cells was increased compared with CRY2+/+ cells, suggesting that more reserve cells were produced in the absence of CRY2. Loss of CRY2 leads to the activation of the ERK1/2 signaling pathway and ETS1, which binds to the promoter of PAX7 to induce its transcription. CRY2 deficient myoblasts survived better in ischemic muscle. Therefore, CRY2 is essential in regulating skeletal muscle repair.

Myogenesis defects in a patient-derived iPSC model of hereditary GNE myopathy

Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy (GNEM) is an autosomal recessive distal myopathy with rimmed vacuoles typically manifesting in late adolescence/early adulthood. GNE encodes the rate-limiting enzyme in sialic acid biosynthesis, which is necessary for the proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study, we aimed to address this knowledge gap by querying the underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem-cell (iPSC) model. Control and patient-specific iPSCs were differentiated down a skeletal muscle lineage, whereby patient-derived GNEM iPSC clones were able to recapitulate key characteristics of the human pathology and further demonstrated defects in myogenic progression. Single-cell RNA sequencing time course studies revealed clear differences between control and GNEM iPSC-derived muscle precursor cells (iMPCs), while pathway studies implicated altered stress and autophagy signaling in GNEM iMPCs. Treatment of GNEM patient-derived iMPCs with an autophagy activator improved myogenic differentiation. In summary, we report an in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a contributing mechanism to the etiology of GNE myopathy.

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage.

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45-CD34-, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Exon skipping in genes encoding lineage-defining myogenic transcription factors in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a childhood sarcoma composed of myoblast-like cells, which suggests a defect in terminal skeletal muscle differentiation. To explore potential defects in the differentiation program, we searched for mRNA splicing variants in genes encoding transcription factors driving skeletal muscle lineage commitment and differentiation. We studied two RMS cases and identified altered splicing resulting in “skipping” the second of three exons in MYOD1. RNA-seq data from 42 tumors and additional RMS cell lines revealed exon 2 skipping in both MYOD1 and MYF5 but not in MYF6 or MYOG. Complementary molecular analysis of MYOD1 mRNA found evidence for exon skipping in five additional RMS cases. Functional studies showed that so-called MYODΔEx2 protein failed to robustly induce muscle-specific genes, and its ectopic expression conferred a selective advantage in cultured fibroblasts and an RMS xenograft. In summary, we present previously unrecognized exon skipping within MYOD1 and MYF5 in RMS, and we propose that alternative splicing can represent a mechanism to alter the function of these two transcription factors in RMS.

Abstract 1679: Single-cell profiling reveals a conserved myogenic hierarchy in pediatric rhabdomyosarcomas amenable to differentiation therapy

Abstract Rhabdomyosarcoma (RMS) is a highly aggressive pediatric soft tissue cancer, associated with the skeletal muscle lineage. Despite undoubted expression of key myogenic regulatory factors, RMS cells are blocked in a proliferative state and do not complete terminal differentiation. The extent to which the skeletal muscle lineage is represented in RMS tumors, as well as the mechanisms leading to developmental arrest, remain elusive. We therefore aimed to identify RMS subpopulations, understand why RMS cells are stalled in their differentiation path, and determine mechanisms to pharmacologically restore myogenic differentiation. Using single-cell RNA sequencing and mass cytometry analysis, we profiled cells from 14 PDX-derived primary cultures and three cell lines of alveolar (aRMS) and embryonal RMS (eRMS) subtypes. We discovered that both RMS subtypes contain different cell subpopulations distributed along the myogenic lineage. aRMS tumors recapitulate a yet unrecognized branched myogenic trajectory, where progenitor cells either commit to differentiation or give rise to actively cycling myoblasts. Following in vitro exposure to chemotherapy, aRMS cells show compositional shifts towards undifferentiated states, consistent with a model where treatment rewires cellular trajectories. To quantify aRMS cellular states, we developed an automated image-based single-cell approach and applied it to identify pro-differentiating agents among a library of >240 FDA-approved drugs. We identified the RAS pathway as an important mediator of myogenic differentiation in several aRMS cultures, demonstrating the potential for differentiation therapy. Current studies are ongoing to validate these results in vivo and to uncover drug combinations that completely overcome the differentiation block. Taken together, this study reveals possible cellular origins for RMS and identifies a potential novel treatment strategy for aRMS that targets cellular differentiation. Citation Format: Sara G. Danielli, Ermelinda Porpiglia, Andrea J. De Micheli, Ingrid Bechtold, Joana G. Marques, Stephanie Kasper, Helen M. Blau, Marco Wachtel, Beat W. Schäfer. Single-cell profiling reveals a conserved myogenic hierarchy in pediatric rhabdomyosarcomas amenable to differentiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1679.

Actin-related protein 5 functions as a novel modulator of MyoD and MyoG in skeletal muscle and in rhabdomyosarcoma.

Myogenic regulatory factors (MRFs) are pivotal transcription factors in myogenic differentiation. MyoD commits cells to the skeletal muscle lineage by inducing myogenic genes through recruitment of chromatin remodelers to its target loci. This study showed that actin-related protein 5 (Arp5) acts as an inhibitory regulator of MyoD and MyoG by binding to their cysteine-rich (CR) region, which overlaps with the region essential for their epigenetic functions. Arp5 expression was faint in skeletal muscle tissues. Excessive Arp5 in mouse hind limbs caused skeletal muscle fiber atrophy. Further, Arp5 overexpression in myoblasts inhibited myotube formation by diminishing myogenic gene expression, whereas Arp5 depletion augmented myogenic gene expression. Arp5 disturbed MyoD-mediated chromatin remodeling through competition with the three-amino-acid-loop-extension-class homeodomain transcription factors the Pbx1-Meis1 heterodimer for binding to the CR region. This antimyogenic function was independent of the INO80 chromatin remodeling complex, although Arp5 is an important component of that. In rhabdomyosarcoma (RMS) cells, Arp5 expression was significantly higher than in normal myoblasts and skeletal muscle tissue, probably contributing to MyoD and MyoG activity dysregulation. Arp5 depletion in RMS partially restored myogenic properties while inhibiting tumorigenic properties. Thus, Arp5 is a novel modulator of MRFs in skeletal muscle differentiation.

BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.

Abstract 13301: Microarray Reveals Differentially Expressed Long Non-coding Ribonucleic Acids In Dysferlinopathy

Introduction: Dysferlinopathy is a type of muscular dystrophy, a group of inherited degenerative muscle disorders characterized by progressive muscle weakness. It is caused by the deficiency of dysferlin, a type II transmembrane protein highly expressed in the skeletal muscle and the heart. Dysferlin interacts with several proteins and plays a crucial role in calcium dependent membrane repair in skeletal muscles. Long non-coding RNAs (lncRNAs) are non-coding RNA transcripts greater than 200 nucleotides that participate in regulating gene expression through multiple mechanisms. Hypothesis: We hypothesized the disease process in dysferlinopathy is linked to an aberrant expression of lncRNAs. Methods: LncRNA and mRNA expression profiling between the normal and dysferlin deficient murine myoblasts (C2C12 cells) were performed using a microarray (n=3). Several differentially expressed lncRNAs were validated using qRT-PCR. Tissue expression analysis was performed for selected lncRNAs across ten mice tissues. Further bioinformatics analysis was used to explore the potential function, lncRNA-mRNA correlation and potential targets of the differentially expressed lncRNAs. Results: We found 3470 lncRNAs and 2335 mRNAs that are differentially expressed by more than two fold. A set of 13 lncRNAs were selected based on their fold change, and were validated using qRT-PCR. Six out of the 13 lncRNAs tested were predominantly expressed in either myoblasts or skeletal muscle tissue. Pathway analysis of the differentially expressed genes indicated the involvement of several signaling pathways including PI3K-Akt, FoxO, Wnt, cAMP and Hippo. The differentially expressed genes were also enriched for the GO terms, developmental process and muscle system process. Conclusion: The results thus far imply that dysferlinopathy is associated with an aberrant expression of multiple lncRNAs many of which may have a specific function in the disease process. Identification of lncRNAs that are predominantly expressed in the skeletal muscle lineage suggests a muscle specific role for these lncRNAs. We are currently working on overexpressing selected lncRNAs in the normal and dysferlin deficient cells to understand their role in regulating key cellular functions.

HER Tyrosine Kinase Family and Rhabdomyosarcoma: Role in Onset and Targeted Therapy.

Rhabdomyosarcomas (RMS) are tumors of the skeletal muscle lineage. Two main features allow for distinction between subtypes: morphology and presence/absence of a translocation between the PAX3 (or PAX7) and FOXO1 genes. The two main subtypes are fusion-positive alveolar RMS (ARMS) and fusion-negative embryonal RMS (ERMS). This review will focus on the role of receptor tyrosine kinases of the human epidermal growth factor receptor (EGFR) family that is comprised EGFR itself, HER2, HER3 and HER4 in RMS onset and the potential therapeutic targeting of receptor tyrosine kinases. EGFR is highly expressed by ERMS tumors and cell lines, in some cases contributing to tumor growth. If not mutated, HER2 is not directly involved in control of RMS cell growth but can be expressed at significant levels. A minority of ERMS carries a HER2 mutation with driving activity on tumor growth. HER3 is frequently overexpressed by RMS and can play a role in the residual myogenic differentiation ability and in resistance to signaling-directed therapy. HER family members could be exploited for therapeutic approaches in two ways: blocking the HER member (playing a driving role for tumor growth with antibodies or inhibitors) and targeting expressed HER members to vehiculate toxins or immune effectors.