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Fusion-negative rhabdomyosarcoma (FN-RMS) is the most common soft tissue sarcoma of childhood arising from undifferentiated skeletal muscle cells from uncertain origin. Currently used therapies are poorly tumor-specific and fail to tackle the molecular machinery underlying the tumorigenicity and uncontrolled proliferation of FN-RMS. We and other groups recently found that microRNAs (miRNA) network contributes to myogenic epigenetic memory and can influence pluripotent stem cell commitments. Here, we used the previously identified promyogenic miRNAs and tailored it to the murine FN-RMS. Subsequently, we addressed the effects of miRNAs in vivo by performing syngeneic transplant of pre-treated FN-RMS cell line in C57Bl/6 mice. miRNA pre-treatment affects murine FN-RMS cell proliferation in vivo as showed by bioluminescence imaging analysis, resulting in better muscle performances as highlighted by treadmill exhaustion tests. In conclusion, in our study we identified a novel miRNA combination tackling the anti-myogenic features of FN-RMS by reducing proliferation and described novel antitumorigenic therapeutic targets that can be further explored for future pre-clinical applications. ispartof: FRONTIERS IN PHYSIOLOGY vol:12 ispartof: location:Switzerland status: published

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis., Rhabdomyosarcomas are tumours blocked in myogenic differentiation, which despite the expression of master muscle regulatory factors, including MYOD, are unable to differentiate. Here, the authors show that SNAI2 is upregulated by MYOD through super enhancers, binds to MYOD target enhancers, and arrests differentiation.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy, and surgery, clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors, resembling embryonic developing skeletal muscle. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle such as the prostate, bladder, salivary gland, biliary tree, and omentum. Previously, we described that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of a presumed adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice, gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human embryonal RMS (ERMS). With the short latency and anatomically restricted tumor location in the neck, we sought to leverage this model to explore the cell of origin of ERMS. Lineage tracing experiments identified aP2-Cre labeled cells are distinctly nonmyogenic and were identified as endothelial cells found in the interstitium between muscle fibers. We illustrate that aP2-Cre is not expressed in the quiescent or activated muscle stem cells or satellite cells. Expression of oncogenic SmoM2 with aP2-Cre results in proliferation and expansion of the aP2-Cre labeled muscle interstitial endothelial cells and myogenic transdifferentiation resulting in ERMS. Activation of the hedgehog pathway in aP2-Cre labeled endothelial progenitors results in expression of skeletal muscle specification factors specific in the head and neck development, including TBX1, PITX2, TCF21, and MSC. We illustrate that endothelium and skeletal muscle within the head and neck arise from KDR (VEFGR2)-expressing progenitors. Hedgehog pathway activation in committed KDR+ endothelial progenitors results in Tbx1 expression and subsequent MYOD1 expression driving a partially myogenic program characteristic of ERMS. Our work identifies reprogramming cell fate as a mechanism of transformation in pediatric sarcoma and illustrates that it is dangerous to assume the cell of origin from the characteristics of the tumor cell. Citation Format: Catherine J. Drummond, Kathrine E. Gadek, Madeline Bush, Matthew R. Garcia, Mark E. Hatley. Developmental reprogramming via Hedgehog pathway activation in nonmyogenic endothelial progenitors drives fusion-negative rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR01.

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3’ untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared with cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

Summary Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that histologically resembles embryonic skeletal muscle. RMS occurs throughout the body and an exclusively myogenic origin does not account for RMS occurring in sites devoid of skeletal muscle. We previously described an RMS model activating a conditional constitutively active Smoothened mutant (SmoM2) with aP2-Cre . Using genetic fate mapping, we show SmoM2 expression in Cre-expressing endothelial progenitors results in myogenic transdifferentiation and RMS. We show that endothelium and skeletal muscle within the head and neck arise from Kdr -expressing progenitors, and that hedgehog pathway activation results in aberrant expression of myogenic specification factors as a potential mechanism driving RMS genesis. These findings suggest that RMS can originate from aberrant development of non-myogenic cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy, and surgery, clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle, such as the prostate, bladder, salivary gland, biliary tree, and the omentum. Previously, we described that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human embryonal RMS (ERMS). With the short latency and anatomic restricted tumor location in the neck, we sought to leverage this model to explore the cell of origin of ERMS. Lineage tracing experiments identified aP2-Cre labeled cells are distinctly nonmyogenic and were identified as endothelial cells found in the interstitium between muscle fibers. We illustrate that aP2-Cre is not expressed in the quiescent or activated muscle stem cells or satellite cells. Expression of oncogenic SmoM2 with aP2-Cre results in proliferation and expansion of the aP2-Cre labeled muscle interstitial cells and myogenic transdifferentiation resulting in ERMS. Activation of the Hedgehog pathway in aP2-Cre labeled endothelial progenitors results in Tbx1 expression, which is a skeletal muscle specification factor in the head and neck. We illustrate that endothelium and skeletal muscle within the head and neck arise from Kdr (Vegfr2) expressing progenitors. Hedgehog pathway activation in committed KDR+ endothelial progenitors results in Tbx1 expression and subsequent Myod1 expression driving a partially myogenic program characteristic of ERMS. Our work identifies reprogramming cell fate as a mechanism of transformation in pediatric sarcoma. Citation Format: Catherine J. Drummond, Jason A. Hanna, Matthew R. Garcia, Daniel J. Devine, Alana J. Heyrana, David Finkelstein, Jerold E. Rehg, Mark E. Hatley. Hedgehog pathway drives fusion-negative rhabdomyosarcoma initiated from nonmyogenic endothelial progenitors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR12.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and despite aggressive treatment, clinical outcomes have not improved for three decades. There is a need to uncover the molecular underpinnings of RMS. Tumor location is a key prognostic indicator and although RMS occurs throughout the body, nearly 40% of all RMS occurs in the head and neck. It is unknown how the cell of origin affects location and therefore clinical outcome of RMS. Previously, we demonstrated that activation of Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under the control of the adipose protein 2 (aP2)-Cre recombinase transgene gives rise to aggressive skeletal muscle tumors in mice that resemble human fusion negative RMS (FN-RMS). Interestingly, tumors were anatomically restricted to the neck in aP2-Cre;SmoM2 mice. In this study we leverage the aP2-Cre;SmoM2 model of FN-RMS to interrogate how cell of origin affects tumor localization. By genetic fate mapping we determine that aP2-Cre labeled cells are non-myogenic and that aP2-Cre is not expressed in quiescent or activated muscle stem cells. Instead, we identify aP2-Cre expressing cells as endothelial cell progenitors within the muscle interstitium. Although aP2-Cre expressing endothelial cells were observed throughout the mouse, we observed that SmoM2 expression specifically drives embryonic expansion of aP2-Cre labeled cells only in the neck. SmoM2 expression reprograms endothelial progenitors resulting in a myogenic fate switch prior to terminal endothelial cell differentiation. We illustrate that endothelium and skeletal muscle within the head and neck arise from KDR expressing progenitors and that aP2-Cre is expressed after endothelial lineage commitment. Aberrant hedgehog activation in these aP2-Cre labeled endothelial progenitors results in TBX1 expression, a skeletal muscle specification factor in the head and neck, and subsequently MYOD1 expression, driving a partial myogenic program characteristic of FN-RMS. Further characterization of tumor cells isolated by flow cytometry revealed that FN-RMS in aP2-Cre;SmoM2 mice express TBX1, as well as PITX2, TCF21 and MSC, additional skeletal muscle specification factors in the head and neck. In contrast, PAX3, which specifies trunk and limb muscle, was not expressed in FN-RMS cells isolated from aP2-Cre;SmoM2 mice. Together, these results demonstrate that FN-RMS in the head and neck can arise from endothelial progenitor cells and suggest that aberrant activation of normal muscle development programs in non-myogenic cell types with developmental pliancy can drive location specific tumor formation. Citation Format: Catherine J. Drummond, Jason A. Hanna, Matthew R. Garcia, Daniel J. Devine, Alana J. Heyrana, David Finkelstein, Mark E. Hatley. Location specificity in fusion-negative rhabdomyosarcoma driven by cell of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3014.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood, and despite rigorous clinical trials the survival for children with high-risk RMS has not changed for three decades. RMS is subdivided into two major classes, fusion-positive (FP) and fusion-negative RMS (FN-RMS), based on the presence or absence of the PAX3-FOXO1 or PAX7-FOXO1 gene fusions. RMS occurs at locations throughout the body with nearly 40% of tumors occurring in the head and neck. Tumor location and fusion status are key prognostic factors. RMS resembles developing skeletal muscle and has been speculated to originate from genetically compromised skeletal muscle progenitors. However, the genes that control RMS development and specify location remain elusive. RMS also occurs in tissues devoid of skeletal muscle such as the urinary bladder, prostate, and biliary tree, suggesting the possibility of origins outside of the skeletal muscle lineage. Currently, the cell of origin and the factors that specify RMS location and thus prognosis are unknown. Previously, we showed that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human FN-RMS. In this model, tumorigenesis occurs with high penetrance (~80%), is early onset (by 2 months of age), and is anatomically restricted to the head and neck. Also, unlike previous RMS models, this model requires no additional background mutations, such as inactivation of p53, and drives only FN-RMS neoplasia. We illustrated that the transcriptome of the aP2-Cre;SmoM2 tumors recapitulates both other mouse FN-RMS models as well as human FN-RMS. With the short latency and anatomic restricted tumor location, we sought to leverage this model to explore the cell of origin of FN-RMS. Here we use genetic fate mapping with fluorescent reporter mice to interrogate the cell of origin of FN-RMS in the aP2-Cre;SmoM2 model. Tracing the aP2-Cre labeled cells with reporter mice illustrated labeled cells in both brown and white adipose tissue as well as a discrete population of cells lying between skeletal muscle fibers but not beneath the laminin sheath. These aP2-Cre labeled cells are distinct from Pax7-positive skeletal muscle stem cells or satellite cells and do not contribute to myotube formation in vitro or in vivo. Gene profiling of tomato positive cells isolated by FACS from the sternocleidomastoid (SCM) of aP2-Cre;R26-Tom and aP2-Cre;R26-Tom;SmoM2 revealed that these aP2-Cre labeled cells in muscle interstitium are endothelial cells. In SCM sections, aP2-Cre labeled cells colocalize with Pecam1. When compared to aP2-Cre;R26-Tom mice, the addition of oncogenic SmoM2 (aP2-Cre;R26-Tom;SmoM2) results in embryonic expansion of the aP2-labeled muscle interstitial cells and formation of FN-RMS. These expansions became positive for the myogenic regulatory factor MyoD1 and did not express Pecam1 at embryonic day 17.5. Subsequent gene expression analysis revealed that both myogenic regulatory factors and endothelial genes are upregulated in tumor cells. Skeletal muscle and endothelial cells share a common progenitor, and together these results suggest that aberrant Sonic Hedgehog signaling promotes a myogenic fate switch in aP2-Cre expressing endothelial progenitor cells that results in FN-RMS formation. Citation Format: Catherine J. Drummond, Jason A. Hanna, Matthew R. Garcia, Daniel Devine, Jennifer Peters, Victoria Frohlich, David Finkelstein, Mark E. Hatley. Fusion-negative rhabdomyosarcoma originating from endothelial progenitors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A16.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 [P3F] or PAX7-FOXO1 [P7F] resulting from 2;13 and 1;13 chromosomal translocations) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. To further examine the significance of DNA methylation, we generated genome-wide DNA methylation profiles for a new cohort of 48 RMS tumors for which we previously assessed mutation, copy number and expression status. Investigation of the RMS subsets defined by methylation clustering revealed a significant association of methylation with P3F versus P7F fusion status in the FP subset, and an association of methylation with RAS mutation status in the FN subset. Localization studies of differentially methylated probes showed these probes were not evenly distributed with respect to annotated genomic features. In particular, hypomethylated probes were enriched in FP tumors in the promoter region and in the intergenic region, whereas hypermethylated probes were enriched in these regions in FN tumors. In contrast, hypermethylated probes were enriched in FP tumors in the 3' UTR region whereas hypomethylated probes were enriched in FN tumors in these regions. In our new larger cohort of cases, there was a significant difference in the distribution of P3F binding sites between genes with and without differential methylation. Integrative analysis of P3F binding sites, promoter methylation and gene expression demonstrated that genes with P3F binding sites tended to be more highly expressed in FP tumors (compared to FN tumors) than genes without P3F binding sites regardless of promoter methylation status. Though promoter hypomethylation is most highly associated with enhanced expression among genes with P3F binding sites, the group of genes with P3F binding sites and promoter hypomethylation is small in number compared to the much larger group of genes with P3F binding sites but without promoter hypomethylation. In conclusion, these results demonstrate the interaction of these epigenetic changes with mutational alterations and transcriptional organization in RMS tumors and provide a direction for future studies of these epigenetic events. Citation Format: Wenyue Sun, Bishwanath Chatterjee, Jack F. Shern, Sivasish Sindiri, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3877. doi:10.1158/1538-7445.AM2017-3877

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 or PAX7-FOXO1) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. The current study will explore these issues in a larger, separate cohort of RMS tumors and compare DNA methylation in RMS cell lines and primary tumors. METHODS: DNA methylation was examined in 48 RMS tumors (21 FP and 27 FN) as well as 10 RMS cell lines (5 FP and 5 FN) on the Illumina HumanMethylation450 BeadChip platform. RESULTS: Unsupervised clustering analysis using the most variable probes (top 1%) in the 48 RMS tumors revealed that patterns of DNA methylation segregated these tumors into two distinct subgroups; one subgroup contains all 21 FP cases along with 2 FN cases and a second subgroup contains 25 of the 27 FN cases. A principal component analysis confirmed this close association of methylation pattern and fusion status, and showed that the two “discordant” FN cases map in a region between the FP and FN clusters. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. Application of an 11-gene methylation signature developed in our earlier study classified these 48 cases into FP and FN categories with >95% accuracy. In contrast to our previous findings, there was a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Though unsupervised clustering analysis indicated that FP tumors and cell lines cluster as do the FN tumors and cell lines, a principal component analysis clarified these relationships by showing that the two groups of cell lines are located at a considerable distance from the two tumor subtypes. Analysis of the most varied probes in the tumors indicated that the vast majority of these probes are hypermethylated in all RMS cell lines. In a complementary analysis with the most variable probes (top 1%) in the cell lines, most of these probes are hypomethylated in all RMS tumors, though two smaller groups of probes show differential methylation between both groups of FP and FN samples. CONCLUSIONS: This study provides an independent validation that FP and FN RMS tumors possess distinct and characteristic methylation profiles. The enrichment of PAX3-FOXO1 binding sites in genes that are differentially methylated between these FP and FN tumors suggests that the PAX3-FOXO1 fusion protein may contribute to this methylation pattern. These analyses also indicate that RMS cell lines do not faithfully recapitulate the DNA methylation patterns that characterize primary tumors. Citation Format: Wenyue Sun, Bishwanath Chatterjee, John F. Shern, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. DNA methylation characterization of fusion-positive and fusion-negative rhabdomyosarcoma primary tumors and cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2434.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS can be parsed based on clinical outcome into two subtypes, fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN-RMS) based on the presence or absence of either PAX3-FOXO1 or PAX7-FOXO1 gene fusions. In both RMS subtypes, tumor cells show histology and a gene expression pattern resembling that of developmentally arrested skeletal muscle. Differentiation therapy is an attractive approach to embryonal tumors of childhood including RMS; however, agents to drive RMS differentiation have not entered the clinic and their mechanisms remain unclear. MicroRNA-206 (miR-206) expression increases through normal muscle development and has decreased levels in RMS compared with normal skeletal muscle. Increasing miR-206 expression drives differentiation of RMS, but the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with gene and proteomic profiling coupled with genetic rescue, we identified key miR-206 targets responsible for the FN-RMS differentiation blockade, PAX7, PAX3, NOTCH3, and CCND2. Specifically, we determined that PAX7 downregulation is necessary for miR-206-induced cell cycle exit and myogenic differentiation in FN-RMS but not in FP-RMS. Gene knockdown of targets necessary for miR-206-induced differentiation alone or in combination was not sufficient to phenocopy the differentiation phenotype from miR-206, thus illustrating that miR-206 replacement offers the ability to modulate a complex network of genes responsible for the developmental arrest in FN-RMS. Genetic deletion of miR-206 in a mouse model of FN-RMS accelerated and exacerbated tumor development, indicating that both in vitro and in vivo miR-206 acts as a tumor suppressor in FN-RMS at least partially through downregulation of PAX7. Collectively, our results illustrate that miR-206 relieves the differentiation arrest in FN-RMS and suggests that miR-206 replacement could be a potential therapeutic differentiation strategy.