Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

Muscle cell identity requires Pax7-mediated lineage-specific DNA demethylation.

BACKGROUND: Skeletal muscle stem cells enable the formation, growth, maintenance, and regeneration of skeletal muscle throughout life. The regeneration process is compromised in several pathological conditions, and muscle progenitors derived from pluripotent stem cells have been suggested as a potential therapeutic source for tissue replacement. DNA methylation is an important epigenetic mechanism in the setting and maintenance of cellular identity, but its role in stem cell determination towards the myogenic lineage is unknown. Here we addressed the DNA methylation dynamics of the major genes orchestrating the myogenic determination and differentiation programs in embryonic stem (ES) cells, their Pax7-induced myogenic derivatives, and muscle stem cells in proliferating and differentiating conditions. RESULTS: Our data showed a common muscle-specific DNA demethylation signature required to acquire and maintain the muscle-cell identity. This specific-DNA demethylation is Pax7-mediated, and it is a prime event in muscle stem cells gene activation. Notably, downregulation of the demethylation-related enzyme Apobec2 in ES-derived myogenic precursors reduced myogenin-associated DNA demethylation and dramatically impaired the expression of differentiation markers and, ultimately, muscle differentiation. CONCLUSIONS: Our results underscore DNA demethylation as a key mechanism driving myogenesis and identify specific Pax7-mediated DNA demethylation signatures to acquire and maintain the muscle-cell identity. Additionally, we provide a panel of epigenetic markers for the efficient and safe generation of ES- and induced pluripotent stem cell (iPS)-derived myogenic progenitors for therapeutic applications.

Deconstruction of DNA methylation patterns during myogenesis reveals specific epigenetic events in the establishment of the skeletal muscle lineage.

The progressive restriction of differentiation potential from pluripotent embryonic stem cells (ESCs) to tissue-specific stem cells involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Skeletal muscle stem cells are required for the growth, maintenance, and regeneration of skeletal muscle. To investigate the contribution of DNA methylation to the establishment of the myogenic program, we analyzed ESCs, skeletal muscle stem cells in proliferating (myoblasts) and differentiating conditions (myotubes), and mature myofibers. About 1.000 differentially methylated regions were identified during muscle-lineage determination and terminal differentiation, mainly located in gene bodies and intergenic regions. As a whole, myogenic stem cells showed a gain of DNA methylation, while muscle differentiation was accompanied by loss of DNA methylation in CpG-poor regions. Notably, the hypomethylated regions in myogenic stem cells were neighbored by enhancer-type chromatin, suggesting the involvement of DNA methylation in the regulation of cell-type specific enhancers. Interestingly, we demonstrated the hypomethylation of the muscle cell-identity Myf5 super-enhancer only in muscle cells. Furthermore, we observed that upstream stimulatory factor 1 binding to Myf5 super-enhancer occurs upon DNA demethylation in myogenic stem cells. Taken altogether, we characterized the unique DNA methylation signature of skeletal muscle stem cells and highlighted the importance of DNA methylation-mediated regulation of cell identity Myf5 super-enhancer during cellular differentiation.

Genetic and epigenetic determinants of low dysferlin expression in monocytes.

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle, and peripheral blood monocytes. Expression in skeletal muscle and monocytes strongly correlates in healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While 18 of these individuals with protein levels in the range of 40%-64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of 18 detected missense variants in only four. Analysis of DNA methylation patterns at the DYSF locus showed no changes in methylation levels at CpG islands and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, the monocyte assay cannot be used to determine the carrier status; for this, a molecular analysis of DYSF must be performed.

Amelioration of Duchenne muscular dystrophy in mdx mice by elimination of matrix-associated fibrin-driven inflammation coupled to the αMß2 leukocyte integrin receptor.

In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α(M)ß(2)-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ(390-396A)) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α(M)ß(2) binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α(M)ß(2) blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α(M)ß(2) interactions may provide a novel strategy for DMD treatment.

Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity.

Historically, cellular models have been used as a tool to study myotonic dystrophy type 1 (DM1) and the validation of therapies in said pathology. However, there is a need for in vitro models that represent the clinical heterogeneity observed in patients with DM1 that is lacking in classical models. In this study, we immortalized three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds and characterized them at the genetic, epigenetic, and molecular levels. All three cell lines display DM1 hallmarks, such as the accumulation of RNA foci, MBNL1 sequestration, splicing alterations, and reduced fusion. In addition, alterations in early myogenic markers, myotube diameter and CTCF1 DNA methylation were also found in DM1 cells. Notably, the new lines show a high level of heterogeneity in both the size of the CTG expansion and the aforementioned molecular alterations. Importantly, these immortalized cells also responded to previously tested therapeutics. Altogether, our results show that these three human DM1 cellular models are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.

uPA deficiency exacerbates muscular dystrophy in MDX mice.

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.

HDAC11: a multifaceted histone deacetylase with proficient fatty deacylase activity and its roles in physiological processes.

The histone deacetylases (HDACs) family of enzymes possess deacylase activity for histone and nonhistone proteins; HDAC11 is the latest discovered HDAC and the only member of class IV. Besides its shared HDAC family catalytical activity, recent studies underline HDAC11 as a multifaceted enzyme with a very efficient long-chain fatty acid deacylase activity, which has open a whole new field of action for this protein. Here, we summarize the importance of HDAC11 in a vast array of cellular pathways, which has been recently highlighted by discoveries about its subcellular localization, biochemical features, and its regulation by microRNAs and long noncoding RNAs, as well as its new targets and interactors. Additionally, we discuss the recent work showing the consequences of HDAC11 dysregulation in brain, skeletal muscle, and adipose tissue, and during regeneration in response to kidney, skeletal muscle, and vascular injuries, underscoring HDAC11 as an emerging hub protein with physiological functions that are much more extensive than previously thought, and with important implications in human diseases.

An Integrative Analysis of DNA Methylation Pattern in Myotonic Dystrophy Type 1 Samples Reveals a Distinct DNA Methylation Profile between Tissues and a Novel Muscle-Associated Epigenetic Dysregulation.

Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1.

HDAC11 is a novel regulator of fatty acid oxidative metabolism in skeletal muscle.

Skeletal muscle is the largest tissue in mammalian organisms and is a key determinant of basal metabolic rate and whole-body energy metabolism. Histone deacetylase 11 (HDAC11) is the only member of the class IV subfamily of HDACs, and it is highly expressed in skeletal muscle, but its role in skeletal muscle physiology has never been investigated. Here, we describe for the first time the consequences of HDAC11 genetic deficiency in skeletal muscle, which results in the improvement of muscle function enhancing fatigue resistance and muscle strength. Loss of HDAC11 had no obvious impact on skeletal muscle structure but increased the number of oxidative myofibers by promoting a glycolytic-to-oxidative muscle fiber switch. Unexpectedly, HDAC11 was localized in muscle mitochondria and its deficiency enhanced mitochondrial content. In particular, we showed that HDAC11 depletion increased mitochondrial fatty acid ß-oxidation through activating the AMP-activated protein kinase-acetyl-CoA carboxylase pathway and reducing acylcarnitine levels in vivo, thus providing a mechanistic explanation for the improved muscle strength and fatigue resistance. Overall, our data reveal a unique role of HDAC11 in the maintenance of muscle fiber-type balance and the mitochondrial lipid oxidation. These findings shed light on the mechanisms governing muscle metabolism and may have implications for chronic muscle metabolic disease management.

Loss of HDAC11 accelerates skeletal muscle regeneration in mice.

Histone deacetylase 11 (HDAC11) is the latest identified member of the histone deacetylase family of enzymes. It is highly expressed in brain, heart, testis, kidney, and skeletal muscle, although its role in these tissues is poorly understood. Here, we investigate for the first time the consequences of HDAC11 genetic impairment on skeletal muscle regeneration, a process principally dependent on its resident stem cells (satellite cells) in coordination with infiltrating immune cells and stromal cells. Our results show that HDAC11 is dispensable for adult muscle growth and establishment of the satellite cell population, while HDAC11 deficiency advances the regeneration process in response to muscle injury. This effect is not caused by differences in satellite cell activation or proliferation upon injury, but rather by an enhanced capacity of satellite cells to differentiate at early regeneration stages in the absence of HDAC11. Infiltrating HDAC11-deficient macrophages could also contribute to this accelerated muscle regenerative process by prematurely producing high levels of IL-10, a cytokine known to promote myoblast differentiation. Altogether, our results show that HDAC11 depletion advances skeletal muscle regeneration and this finding may have potential implications for designing new strategies for muscle pathologies coursing with chronic damage. DATABASE: Data were deposited in NCBI's Gene Expression Omnibus accessible through GEO Series accession number GSE147423.

Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1.

Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.

HIV-1 transgenic expression in mice induces selective atrophy of fast-glycolytic skeletal muscle fibers.

Human immunodeficiency virus (HIV)-induced wasting syndrome, characterized by weakness and severe loss of muscle mass, is a common condition of patients with advanced acquired immunodeficiency syndrome (AIDS). The homozygous HIV-1 transgenic mouse line Tg26 reproduces the wasting syndrome of AIDS patients, thus constituting a valid animal model to characterize the muscle phenotype induced by HIV infection. In this study, we identified a selective atrophy of fast-glycolytic myofibers in skeletal muscles of homozygous HIV-1 transgenic mice, whereas the more oxidative fiber types are spared. In agreement with this, muscles enriched in fast-glycolytic myofibers such as the extensor digitorum longus and gastrocnemius, but not those rich in oxidative fibers such as the soleus, exhibited a reduced muscle size in homozygous HIV-1 transgenic mice compared to their littermate control counterparts. Additionally, muscles of heterozygous HIV-1 transgenic mice displayed increased inflammation and blunted myofiber growth in an injury-induced muscle regeneration process. Since no myogenic intrinsic defect was observed in satellite cells from the transgenic mice, these results support the notion of an inflammation-mediated, fiber-type-specific inhibition of muscle growth in the presence of the HIV-1 transgene.

MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption.

Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A1.1 contains a macrodomain capable of binding NAD+-derived metabolites. Here we report that macroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and that myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity. We found that the metabolite-binding macrodomain was essential for sustained optimal mitochondrial function but dispensable for gene regulation. Through direct binding, macroH2A1.1 inhibits basal poly-ADP ribose polymerase 1 (PARP-1) activity and thus reduces nuclear NAD+ consumption. The resultant accumulation of the NAD+ precursor NMN allows for maintenance of mitochondrial NAD+ pools that are critical for respiration. Our data indicate that macroH2A1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD+ consumption and establishing a buffer of NAD+ precursors in differentiated cells.

DNA methylation dynamics in cellular commitment and differentiation.

DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts, revealing a more dynamic regulation than originally thought, as active DNA methylation and demethylation occur during cell fate commitment and terminal differentiation. Recent data provide insights into the contribution of different epigenetic factors, and DNA methylation in particular, to the establishment of cellular memory during embryonic development and the modulation of cell type-specific gene regulation programs to ensure proper differentiation. This review summarizes published data regarding DNA methylation changes along lineage specification and differentiation programs. We also discuss the current knowledge about DNA methylation alterations occurring in physiological and pathological conditions such as aging and cancer.

Alpha-enolase plasminogen receptor in myogenesis.

Plasmin is a potent extracellular protease specialized in the degradation of fibrin (fibrinolysis). Active plasmin is generated by proteolytic activation of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Alpha-enolase, although traditionally considered a glycolytic enzyme, constitutes a receptor for plasminogen on several cell types, serving to localize and promote plasminogen activation pericellularly. Localization of plasmin activity on the cell surface plays a critical role in fibrinolysis and in physiopathological processes involving extracellular matrix remodelling. Previous studies have unambiguously demonstrated that uPA-dependent plasmin generation is necessary for myogenesis in vitro and for muscle regeneration in vivo. However, the implication of alpha-enolase plasminogen receptor in myogenesis had never been investigated. This review focuses on the recently reported expression and function of alpha-enolase plasminogen receptor during myogenesis. Skeletal myoblasts express alpha-enolase plasminogen receptor, being its expression greatly induced during the differentiation process in vitro. MAb 11G1, a monoclonal antibody against anti-alpha-enolase plasminogen receptor, that inhibits plasmin generation, was able to fully abrogate myoblast fusion and differentiation. Moreover, both plasmin activity and alpha-enolase plasminogen receptor expression were significantly augmented in injury-induced regenerating muscle of wild type mice and in the dystrophic muscle of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Altogether, these results indicate that the plasminogen activation (PA) system is an important component of skeletal myogenesis in vitro and in vivo. In particular, the expression of alpha-enolase plasminogen receptor may serve to concentrate and enhance plasmin generation on the cell surface of migratory myoblasts contributing to efficient muscle repair.

The plasminogen activation system in skeletal muscle regeneration: antagonistic roles of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1).

The plasminogen activation (PA) system is an extensively used mechanism for the generation of proteolytic activity in the extracellular matrix, where it contributes to tissue remodeling in a wide range of physiopathological processes. Despite the limited information available at present on plasminogen activators, their inhibitors and cognate receptors in skeletal muscle, increasing evidence is accumulating on their important roles in the homeostasis of muscle fibers and their surrounding extracellular matrix. The development of mice deficient for the individual components of the PA system has provided an incisive approach to test the proposed muscle functions in vivo. Skeletal muscle regeneration induced by injury has been analyzed in urokinase-type plasminogen activator (uPA)-, tissue-type plasminogen activator (tPA)-, plasminogen (Plg)- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice and has demonstrated profound effects of these molecules on the fibrotic state and the inflammatory response, which contribute to muscle repair. In particular, the opposite roles of uPA and its inhibitor PAI-1 in this process are highlighted. Delineating the mechanisms by which the different plasminogen activation system components regulate tissue repair will be of potential therapeutic value for severe muscle disorders.

DNA methylation dynamics in muscle development and disease.

DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts revealing a more dynamic regulation than originally thought, since active DNA methylation and demethylation occur during cellular differentiation and tissue specification. Satellite cells are the primary stem cells in adult skeletal muscle and are responsible for postnatal muscle growth, hypertrophy, and muscle regeneration. This review outlines the published data regarding DNA methylation changes along the skeletal muscle program, in both physiological and pathological conditions, to better understand the epigenetic mechanisms that control myogenesis.

Plasmin generation dependent on alpha-enolase-type plasminogen receptor is required for myogenesis.

Plasmin is a potent extracellular protease specialized in the degradation of fibrin (fibrinolysis). Active plasmin is generated by proteolytic activation of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Alpha-enolase constitutes a receptor for plasminogen on several leukocyte cell types, serving to localize and promote plasminogen activation pericellularly. However, a role for a -enolase-type plasminogen receptor (PlgR) in myogenesis has never been demonstrated. In this study, we show that C2C12 mouse myoblasts express PlgR, being its expression greatly induced during the differentiation process. A monoclonal antibody against PIgR MAb 11G1, with cell surface-generated plasmin inhibitory abilities, was able to fully abrogate C2C12 myoblast fusion and differentiation in vitro. Moreover, both plasmin activity and PlgR expression were significantly induced in regenerating skeletal muscle in vivo, either in experimentally-injured muscle or in the dystrophic muscle of mdx mouse (an animal model of human Duchenne muscular dystrophy, DMD). The mdx muscle presents better regeneration capacities and less fibrosis than the human DMD muscle; therefore, the increase in PlgR/plasmin activity in mdx muscle suggests an important contribution of the fibrinolytic system in mdx regeneration. This study constitutes the first indication of alpha-enolase-type plasminogen receptor as an important component of skeletal myogenesis, by concentrating and enhancing plasmin generation on the cell surface.

Rb1 loss modifies but does not initiate alveolar rhabdomyosarcoma.

BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a. METHODS: We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models. RESULTS: Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression. CONCLUSIONS: Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function.