Fusion-positive Rhabdomyosarcoma Research Articles

Abstract 3882: YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma

Abstract Although more than 20 years have passed since the discovery that fusion-positive rhabdomyosarcoma (FP-RMS) is driven by the chimeric fusion oncogene PAX3-FOXO1 (P3F), therapeutically tractable components of the P3F tumorigenic program have yet to be uncovered and survival rates remain dismal (5-yr overall survival <50%). YAP1 and TAZ, transcriptional co-activators of the Hippo pathway, are potent oncogenes known to mediate transcriptional addiction in malignancy. Here, we demonstrate that YAP1/TAZ complex with P3F and positively regulate P3F transcriptional activity in FP-RMS. Immunohistochemical (IHC) staining of human tissue microarrays were used to identify the relative abundance of YAP1 and TAZ in FP-RMS tumor samples. Functional interactions of YAP1/TAZ and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) was used to identify the YAP1/TAZ/P3F interactome. Experiments utilized gain- and loss-of-function vectors expressing control, wild-type YAP1/TAZ, constitutively active YAP1/TAZ (S89A/S127A), or shRNA knockdown. To identify P3F-mediated genes and pathways that are YAP1/TAZ-dependent, we used RNA-Seq and quantitative proteomics via tandem mass tag labeling. We demonstrate via IHC that YAP1/TAZ are highly abundant in FP-RMS, and through functional assays that YAP1/TAZ regulate many FP-RMS cancer phenotypes. Mechanistically, an interaction between YAP/TAZ and P3F was demonstrated via co-IPs for endogenous as well as epitope-tagged proteins. This was confirmed with IP-MS, which also revealed that YAP/TAZ and P3F share an enrichment for co-immunoprecipitated proteins involved in DNA binding and transcriptional regulation. IP-MS also demonstrated that chromatin remodeling complex proteins were enriched with TAZ immunoprecipitation. YAP1/TAZ functionally augment P3F transcriptional activity in reporter assays as well as expression of candidate P3F target genes. An unbiased approach using RNA-Seq and quantitative proteomics demonstrate that YAP1/TAZ positively regulate differential expression of P3F target genes, as well as proteins involved in cell cycle progression and pan-cancer related proteins that are typically up- or down-regulated across multiple malignancy types.In conclusion, we identify a novel complex between YAP1/TAZ and P3F, show YAP1/TAZ are positive regulators of P3F transcriptional activity, and identify the YAP1/TAZ axis as a vulnerability for P3F-transcriptional reprogramming in FP-RMS. Citation Format: Tooba Rashid, Breanne A. Burgess, Corinne M. Linardic, Michael D. Deel. YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3882.

NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency.

The NuRD complex subunit CHD4 is essential for fusion-positive rhabdomyosarcoma (FP-RMS) survival, but the mechanisms underlying this dependency are not understood. Here, a NuRD-specific CRISPR screen demonstrates that FP-RMS is particularly sensitive to CHD4 amongst the NuRD members. Mechanistically, NuRD complex containing CHD4 localizes to super-enhancers where CHD4 generates a chromatin architecture permissive for the binding of the tumor driver and fusion protein PAX3-FOXO1, allowing downstream transcription of its oncogenic program. Moreover, CHD4 depletion removes HDAC2 from the chromatin, leading to an increase and spread of histone acetylation, and prevents the positioning of RNA Polymerase 2 at promoters impeding transcription initiation. Strikingly, analysis of genome-wide cancer dependency databases identifies CHD4 as a general cancer vulnerability. Our findings describe CHD4, a classically defined repressor, as positive regulator of transcription and super-enhancer accessibility as well as establish this remodeler as an unexpected broad tumor susceptibility and promising drug target for cancer therapy.

Abstract A08: Loss of noncanonical Hippo signaling in fusion-positive alveolar rhabdomyosarcoma increases invasiveness and a dedifferentiated phenotype associated with metastasis

Abstract A hallmark of alveolar rhabdomyosarcoma (ARMS) is the presence of a chromosomal translocation encoding the PAX3-FOXO1 fusion oncogene (FP-ARMS). Patients presenting with FP-ARMS represent the subset of rhabdomyosarcoma patients with the worst prognosis, and when metastatic, survival for these children and adolescents is less than 10%. Nevertheless, standard of care has not changed in the clinic in decades due to a lack of novel effective therapeutics, and as a result, efforts are ongoing to develop better models of this disease in vivo and identify new and more predictive biomarkers. Recently, our group identified a role for Hippo/MST signaling in a genetically engineered mouse model (GEMM) of FP-ARMS that demonstrates Stk3F/F;Stk4F/F; Pax3PF/PF;Cdkn2aF/F;Myf6ICN/+ ARMS tumor-derived cells are less differentiated and more invasive in vitro as compared to Pax3PF/PF;Cdkn2aF/F; Myf6ICN/+ counterparts. Preliminary in vivo studies show that FP-ARMS GEMM tumor-derived cells form secondary metastases in both the lungs and livers of recipient animals, suggesting a new utility of the model for studies of metastatic potential. Studies of bone marrow metastasis, a lethal disease progression in PF-ARMS patients, are ongoing. Further, canonical signaling down the kinase cascade from MST1/2 to YAP1 is largely unchanged in this model, implicating instead noncanonical Hippo signaling and a potential role for MOB1, a Hippo pathway modulator implicated in mitotic exit and expressed highly in the bone marrow niche. Together, these data present an opportunity to interrogate the biology driving the predisposition of PF-RMS patients to develop metastases, and a foundation to identify novel preclinical targets. Citation Format: Kristianne M. Oristian, Lisa E.S. Crose, Napasorn Kuprasertkul, Rex C. Bentley, Yi-Tzu Lin, Nerissa Williams, David G. Kirsch, Corinne M. Linardic. Loss of noncanonical Hippo signaling in fusion-positive alveolar rhabdomyosarcoma increases invasiveness and a dedifferentiated phenotype associated with metastasis [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A08.

Abstract B70: Clonal evolution of chemotherapy-resistant rhabdomyosarcoma via multifocal genomic analysis of pretreatment and treatment-resistant autopsy specimens

Abstract Objective: Outcomes for patients with rhabdomyosarcoma (RMS) who have relapsed or refractory disease remain poor. Multiple large-scale tumor genomic profiling efforts have been undertaken; however, little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. Methods: To address this issue, we performed 80x whole-genome sequencing of 23 metastases and 2 pretreatment samples from two patients with lethal rhabdomyosarcoma. Multiregion and spatially separated metastases were available through our research autopsy donation program. RA 17-1 had fusion-positive alveolar RMS, while RA 17-10 had Li-Fraumeni syndrome and anaplastic embryonal RMS. Results: We observed on average 65 and 98 coding single-nucleotide variants (SNV) per sample in RA 17-1 and RA 17-10, respectively. RA 17-1 was found to have the canonical PAX03-FOX01 fusion along with MYC-N amplification, which was truncal to all samples. A somatic TP53 mutation in combination with TP53 loss of heterozygosity (LOH) was present in all metastases sequenced at autopsy. A TP53 mutation was not identified in the pretreatment biopsy sample selected for sequencing; however, TP53 immunohistochemistry demonstrated the presence of a TP53 mutation in at least 2 out of 6 pretreatment biopsy cores. In RA 17-10 each sample exhibited numerous copy number alterations and complex structural variants, including LOH of TP53, a TSC2 mutation with LOH, and MYC amplification, which were truncal to all samples. PTCH1 amplification was private to all relapse specimens in RA 17-10. RA 17-10 had more structural and copy number variations per sample than RA 17-1. Phylogenies were derived based on SNVs and demonstrated a branched evolutionary pattern for each patient. When comparing related samples’ mutational signatures, several dynamics were apparent, including the predominance of signatures 3 and 8 (DNA double-strand break repair pathways) in metastatic samples, consistent with the fact that both patients harbored TP53 mutations. Conclusion: Defining important elements of ITH within metastases remains a goal for genomic study in pediatrics, especially in patients with germline predisposition syndromes. Our findings demonstrate the dynamic nature of genomic instability processes, highlighting the importance of longitudinal sampling at the time of recurrence to define treatment effect and the changing mutational landscape in these tumors. The case of RA 17-1 demonstrates the limitations of targeted sequencing on a single sample at diagnosis, as the subclonal TP53 mutation was not originally identified on the clinical assay. TP53 mutations in fusion-positive RMS have rarely been described and likely contributed to this patient’s refractory disease course. Both RA 17-1 and RA 17-10 had more structural variants per sample then what has previously been reported for fusion-positive and fusion-negative RMS, respectively, likely highlighting the contribution of the TP53 mutation to genomic instability in each patient. Citation Format: Michael D. Kinnaman, Alvin Makohon-Moore, Nancy Bouvier, Dominik Glodzik, Max Levine, Ellie Papaemmanuil, Filemon Dela Cruz, Leonard Wexler, Andrew Kung, Christine Iacobuzio-Donahue. Clonal evolution of chemotherapy-resistant rhabdomyosarcoma via multifocal genomic analysis of pretreatment and treatment-resistant autopsy specimens [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B70.

Abstract A47: BMI1 constitutes a novel therapeutic vulnerability in fusion-positive rhabdomyosarcoma

Abstract Background: Despite intense efforts within pediatric oncology, novel, effective therapy for alveolar rhabdomyosarcoma, known as fusion-positive rhabdomyosarcoma (FP RMS) given the PAX-FOXO1 fusions characteristic of the disease, remains unrealized. Like many pediatric tumors, FP RMS displays a quiet genomic landscape, when focusing on the coding genome. However, the epigenome plays key roles in shaping tumor aggression, and previous studies have demonstrated that FP RMS is specifically enriched for methylation of Polycomb target genes, suggesting that Polycomb complexes may be deregulated and could constitute novel therapeutic targets. We hypothesized that BMI1, a key member of the Polycomb family and a tractable therapeutic target, represents a novel therapeutic vulnerability in FP RMS. Methods: We analyzed RNA and protein expression in FP RMS cell lines, patient-derived xenografts (PDXs), and human tumor specimens. We used genetic and pharmacologic approaches to manipulate BMI1 in FP RMS cells and measured effects on proliferation, cell cycle, apoptosis, and signal transduction. To examine the effect of in vivo inhibition of BMI1, we utilized xenograft models of FP RMS. Results: We examined RNA-Seq tumor datasets and tumor microarrays and demonstrated that BMI1 is robustly expressed in FP RMS tumors, PDXs, and cell line models. Next, in 2 cell line models, we depleted BMI-1 using shRNAs and siRNAs, and found that this led to striking (~70%) decreases in cell growth secondary to both G1/S phase arrest and apoptosis. Given these findings, we asked whether small-molecule inhibitors of BMI-1 mediated similar effects. We treated 4 independent FP RMS cell line models with both PTC-209 (first-generation inhibitor) and PTC-028 (orally available second-generation inhibitor with higher potency). Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in FP rhabdomyosarcoma cell line models. Similar to genetically mediated depletion, pharmacologic inhibition of BMI1 led to G1/S phase arrest and apoptosis, as demonstrated by Annexin V staining and PARP cleavage. Finally, in a xenograft model of an aggressive FP RMS, PTC-028 treatment decreased tumor growth (p=0.0005) and significantly prolonged survival by 17 days (p=0.0002). Importantly, treatment was well tolerated without evidence of toxicity or weight loss. Conclusions: BMI1 is robustly expressed in FP RMS and both genetic and pharmacologic inhibition of BMI1 lead to striking decreases in cell proliferation, with concomitant cell cycle arrest and apoptosis. BMI1 inhibition significantly decreases tumor growth, prolongs survival, and is well tolerated. Currently, we are further investigating combining BMI1 inhibition with standard chemotherapy and novel agents, as well as defining molecular mechanisms by which BMI1 exerts molecular functions in FP RMS. Targeting BMI-1 could provide a novel therapeutic option for patients with FP RMS, with potential broader implications for additional aggressive sarcomas. Citation Format: Cara E. Shields, Selma M. Cuya, Sarah Chappell, Komal Rathi, Shiv Patel, Sindhu Potlapalli, Robert W. Schnepp. BMI1 constitutes a novel therapeutic vulnerability in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A47.

Abstract B12: PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma

Abstract Chromosomal translocations drive many types of childhood cancer. Fusion-positive rhabdomyosarcoma (FP-RMS) tumors are found most frequently with a fusion between PAX3 and FOXO1, but less frequent translocation partners have been discovered, including INO80D and NCOA1. We hypothesized that all FP-RMS translocations are selecting simultaneously for (1) enhancers active in a myoblast-like epigenome and (2) protein partners with high-levels of intrinsic disorder to give PAX3 enhanced transcriptional strength. Analysis of primary tumors revealed these diverse PAX3 fusions recapitulate a near-identical transcriptome, suggesting uniform underlying molecular mechanisms. ChIP-seq evidence from cell lines and primary tumors suggested that in all FP-RMS tumors, large super-enhancer (SE) elements were present near each chosen translocation partner (distal to FOXO1, INO80D, or NCOA1). Using tools to determine the 3-D folding of chromatin (3C, 4C-seq, and HiChIP), we discovered an extensive network of hijacked FOXO1 enhancers and a SE that physically interact together and with the PAX3 promoter, only in PAX3-FOXO1 positive cells. Furthermore, pooled CRISPR tiling of cis-regulatory elements revealed special dependence on the FOXO1 SE, or certain CTCF boundary elements that facilitate enhancer interactions. ChIP-seq paired to short-term CRISPR experiments shows PAX3-FOXO1 transcription depends on an extended network of related enhancers distal to FOXO1. We find these enhancers are unique to early myoblast stages of differentiation and are bound by myogenic TFs in RMS, suggesting miswiring of normal myogenic enhancer logic. While many SE-driven genes exist in FP-RMS, most are never selected for translocation partners. We found that FOXO1, INO80D, and NCOA1 are all highly disordered proteins, a theme they hold in common despite having no amino acid sequence homology. Initial evidence suggests that PAX3-FOXO1 enables the formation of phase condensates in the nucleus that recruit high-levels of transcriptional machinery such as BRD4. Ongoing work is exploring the mechanistic chemical determinants of these interactions, and how phase condensates are manipulated by drugs that block PAX3-FOXO1’s transcriptional output. Together our studies are illuminating new paradigms for understanding how fusion transcription factors drive cancer. Citation Format: Berkley E. Gryder, Marco Wachtel, Winston Ewert, Kenneth Chang, Osama El Demerdash, Young Song, Beat W Schäfer, Christopher R. Vakoc, Javed Khan. PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B12.

Miswired Enhancer Logic Drives a Cancer of the Muscle Lineage.

SummaryCore regulatory transcription factors (CR TFs) establish enhancers with logical ordering during embryogenesis and development. Here we report that in fusion-positive rhabdomyosarcoma, a cancer of the muscle lineage, the chief oncogene PAX3-FOXO1 is driven by a translocated FOXO1 super enhancer (SE) restricted to a late stage of myogenesis. Using chromatin conformation capture techniques, we demonstrate that the extensive FOXO1 cis-regulatory domain interacts with PAX3. Furthermore, RNA sequencing and chromatin immunoprecipitation sequencing data in tumors bearing rare PAX translocations implicate enhancer miswiring across all fusion-positive tumors. HiChIP of H3K27ac showed connectivity between the FOXO1 SE, additional intra-domain enhancers, and the PAX3 promoter. We show that PAX3-FOXO1 transcription is diminished when this network of enhancers is ablated by CRISPR. Our data reveal a hijacked enhancer network that disrupts the stepwise CR TF logic of normal skeletal muscle development (PAX3 to MYOD to MYOG), replacing it with an “infinite loop” enhancer logic that locks rhabdomyosarcoma in an undifferentiated stage.

Genomic Determinants of Clinical Outcomes in Rhabdomyosarcoma.

Increased availability of next-generation sequencing has allowed for the genomic characterization of a variety of pediatric tumors, although genomic determinants of response to treatment remain largely unknown. We sought to evaluate the genomic landscape and genomic determinants of clinical outcomes in rhabdomyosarcoma (RMS). Of 29,067 patients who underwent genomic profiling at our institution using a 468-gene oncopanel with complete records, 87 had RMS, of whom 22 were fusion positive. The 10 most common genetic alterations were associated with locoregional control (LC), disease-free survival (DFS), and overall survival (OS). Tumor mutational burden (TMB), defined as the total number of somatic nonsynonymous mutations normalized to the number of sequenced megabases, was also associated with clinical outcomes. Median age at diagnosis was 16.4 years and median follow-up, 2.1 years. Patients with fusion-negative RMS had more genomic alterations and a higher TMB than those with fusion-positive RMS (mean number of genomic alterations, 6.0 vs. 2.9; P = 0.007 and mean TMB, 2.6 vs. 1.0; P = 0.01). Genetic alterations in TP53 were associated with worse OS (P = 0.03). High TMB (defined as the top quartile ≥ 2.8) was associated with worse LC (P = 0.05), DFS (P = 0.04), and OS (P = 0.01), with significance retained on multivariable analysis after controlling for risk group, fusion status, and receipt of chemotherapy as per pediatric protocols. High TMB was associated with worse clinical outcomes in patients with RMS. With further validation, TMB and other genomic classifiers may be combined with traditional clinicopathologic risk factors to guide risk stratification and ultimately treatment decisions.

Signaling pathways in Rhabdomyosarcoma invasion and metastasis.

Rhabdomyosarcoma (RMS) is an aggressive childhood mesenchymal tumor with two major molecular and histopathologic subtypes: fusion-positive (FP)RMS, characterized by the PAX3-FOXO1 fusion protein and largely of alveolar histology, and fusion-negative (FN)RMS, the majority of which exhibit embryonal tumor histology. Metastatic disease continues to be associated with poor overall survival despite intensive treatment strategies. Studies on RMS biology have provided some insight into autocrine as well as paracrine signaling pathways that contribute to invasion and metastatic propensity. Such pathways include those driven by the PAX3-FOXO1 fusion oncoprotein in FPRMS and signaling pathways such as IGF/RAS/MEK/ERK, PI3K/AKT/mTOR, cMET, FGFR4, and PDGFR in both FP and FNRMS. In addition, specific cytoskeletal proteins, G protein coupled receptors, Hedgehog, Notch, Wnt, Hippo, and p53 pathways play a role, as do specific microRNA. Paracrine factors, including secreted proteins and RMS-derived exosomes that carry cargo of protein and miRNA, have also recently emerged as potentiallyimportant players in RMS biology. This review summarizes the known factors contributing to RMS invasion and metastasis and their implications on identifying targets for treatment and a better understanding of metastatic RMS.

A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and Notch signaling.

ABSTRACTThe development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue sarcoma of childhood, has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.

Genomic Predictors of Survival Outcomes in Pediatric Sarcomas

Increased availability of next generation sequencing has allowed for the genomic characterization of a variety of pediatric tumors, although genetic determinants of response to treatment remain largely unknown. We sought to evaluate the genomic landscape and genomic predictors of survival outcomes in rhabdomyosarcoma (RMS) and Ewing sarcoma (ES). Of 29,067 patients who underwent genomic profiling at our institution using a 468-gene oncopanel with complete records, 164 had either RMS (n=87, of whom 22 were fusion-positive) or ES (n=77) For the analysis, the 10 most common genetic alterations were associated with locoregional control (LC), progression-free survival (PFS), and overall survival (OS) for each individual tumor type. Tumor mutational burden (TMB), defined as the total number of somatic non-synonymous mutations normalized to the number of sequenced megabases, was also associated with disease outcomes. Median age at diagnosis was 15.7 years and 20.6 years for RMS and ES, respectively, and median follow up was 27.1 months. The median number of alterations found on genomic sequencing for RMS and ES were 4 (range, 0-25) and 2 (range, 0-21), respectively. Patients with fusion-positive RMS had less alterations on sequencing than patients with fusion-negative RMS (median 2 versus 5, p=0.007). The most common genetic alterations in RMS included TP53 (17%), NF1 (13%), CDKN2A (11%), NRAS (11%), MYOD1 (10%) MDM2 (9%), BCOR (9%) CDKN2B (9%), CDK4 (8%), and GLI1 (7%). The most common genetic alterations in ES included TP53 (12%), STAG2 (10%), ERG (9%), ERF (6%), CDKN2A (5%), CREBBP (5%), TERT (5%), BCOR (5%), FAT1 (4%), and CCND1 (4%). The 3-year LC, PFS, and OS for RMS were 52.4%, 27.5%, and 56.1%. The 3-year LC, PFS, and OS for ES were 84.7%, 61.6%, and 82.2%. Genetic alterations in TP53 were associated with significantly worse OS in RMS (p=0.03), while genetic alterations in CDKN2A were associated with worse OS in ES (p=0.002). Higher TMB (defined as >3) was significantly associated with worse LC (p=0.05), PFS (p=0.04), and OS (p=0.01) in RMS (with significance retained when separated by fusion status). Higher TMB was also associated with worse LC (p=0.02) and PFS (p=0.009) in ES, with a trend toward worse OS (p=0.16). Higher TMB predicted for worse local control and survival outcomes in both RMS and ES. Our preliminary results must be correlated with other well-known clinicopathologic risk factors with the ultimate goal of guiding risk stratification and treatment decisions.

OLIG2 is a marker of the fusion protein-driven neurodevelopmental transcriptional signature in alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (RMS) is associated with an underlying pathogenic translocation involving either PAX3 or PAX7 and FOXO1. The presence or absence of this fusion defines the biology and clinical behavior of this subtype of RMS and its identification in tumors is relevant to prognostication and treatment planning. To further explore the unique characteristics of fusion-driven RMS, we leveraged a published gene expression data set to perform an unbiased comparison of 33 fusion-positive and 25 fusion-negative RMS cases. Our analyses revealed 1790 expressed loci with more than two-fold differential expression at a threshold of P < .05. Genes with increased expression in fusion-positive relative to fusion-negative RMS were significantly enriched for those involved in "nervous system development," "neuron differentiation," and "neurogenesis," highlighting a neurodevelopmental gene expression signature driven by the alveolar RMS-associated fusion protein. We show that neurodevelopmental genes are enriched near PAX3-FOXO1 fusion protein binding sites, suggesting a genome-wide fusion protein-mediated activation of cis regulatory elements. Among the genes with differential expression in fusion-positive versus fusion-negative RMS, we identified expression of the transcriptional regulator of motor neuron and oligodendrocyte development, OLIG2, as a marker of the fusion protein-dependent neurodevelopmental signature. Immunohistochemical analysis of a cohort of 73 RMS specimens revealed OLIG2 expression in 96.4% of fusion-positive RMS (N = 27/28), but only in 6.7% of fusion-negative RMS (N = 3/45; P < .001). The proportion of OLIG2-expressing cells in fusion-negative cases did not exceed 5%, while 92.9% of fusion-positive cases showed expression in at least 5% of cells. Our findings identify OLIG2 expression as a unique manifestation of a neurodevelopmental gene expression signature driven by the oncogenic fusion protein characteristic of alveolar RMS, which may aid in the diagnostic and prognostic distinction of fusion-positive cases.

Abstract 3670: Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness

Abstract Introduction: Fusion Positive Rhabdomyosarcoma (FP-RMS), a soft tissue sarcoma of adolescents and young adults, is driven by the oncogenic transcription factor PAX3-FOXO1 (P3F). Although most patients with FP-RMS initially respond to therapy, tumors frequently become refractory or relapse, underscoring a need to target resistance mechanisms and cancer stem cells. Because P3F is inherently disordered with no viable drug binding sites, it is currently not amendable to direct inhibition. Our prior work demonstrated that transcriptional co-activators TAZ and YAP are highly abundant in human FP-RMS and mediate several cancer phenotypes including apoptosis, differentiation, proliferation, and xenograft tumor growth. TAZ/YAP are essential co-activators for TEADs and AP1, which are among the top enriched transcription factor motifs at P3F binding sites. Here, we show that TAZ/YAP complex with P3F to positively regulate P3F transcriptional activity and that TAZ/YAP mediate FP-RMS stemness. Methods: Functional interactions of TAZ/YAP and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) to identify the TAZ/YAP/P3F interactome is currently underway. For reporter assays, gain- and loss-of-function vectors expressing control, wild-type TAZ/YAP (WT), constitutively active TAZ/YAP (S89A/S127A), or shRNA knockdown (KD) were used. Epitope-tagged TAZ, YAP, and P3F were used for co-IPs and IP-MS. To investigate the role of TAZ/YAP in stemness, we evaluated the expression of stem cell genes in FP-RMS 3D-spheres expressing vector, WT, S89A/S127A, or KD. We are now performing RNA-Seq as an unbiased approach to examine genes and pathways regulating FP-RMS stemness. Results: TAZ/YAP functionally augment P3F transcriptional activity in reporter assays and also regulate expression of P3F targets. Immunoblotting of 293T cells co-expressing epitope-tagged proteins demonstrate an interaction between TAZ/YAP and P3F. A TAZ/YAP/P3F complex is also seen in co-IPs of endogenous proteins in FP-RMS cells. Serial passaging of spheres increases TAZ/YAP expression, suggesting these may be important for stem cell enrichment. Indeed, expression of SOX2, OCT4, and NANOG are TAZ/YAP-dependent, whereby their expression decreases with KD and increases with S89A/S127A. Functionally, TAZ/YAP are required for FP-RMS sphere formation in limiting dilution assays. Conclusions: These studies expand on prior work showing TAZ/YAP are potent oncoproteins in FP-RMS. We identify a novel complex between TAZ/YAP and P3F, show TAZ/YAP are positive regulators of P3F transcriptional activity, and demonstrate that TAZ/YAP regulate FP-RMS cancer cell stemness. Thus, targeting TAZ/YAP could be a vulnerability of P3F and for inhibiting the cancer stem cell population. Citation Format: Breanne A. Burgess, Katherine K. Slemmons, Corinne M. Linardic, Michael D. Deel. Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3670.

Is PAX3-FOXO1 associated with worse outcome in adults with rhabdomyosarcoma (RMS)?

e22525 Background: Rhabdomyosarcoma (RMS) is a rare soft tissue sarcoma in adults. The PAX3-FOXO1 fusion gene is associated with alveolar rhabdomyosarcoma. PAX3-FOXO1 results from a stable reciprocal translocation of chromosomes 2 and 13, which fuses in-frame the DNA binding domain of PAX3 with the transactivation domain of FOXO1. Occasionally, PAX7-FOXO1 is expressed. In children, the PAX3-FOXO1 fusion gene is associated with worse outcome. We evaluated the prognostic role of FOXO1 fusion status in adults with RMS treated in a single, large volume sarcoma centre. Methods: A retrospective review of adult RMS patients (pts) diagnosed from 1984 to 2018 was done. Information on demographics, treatment, fusion status and survival was collected. Primary favourable site was defined as tumour arising in orbit, non-bladder/prostate genitourinary system and non-parameningeal head and neck. Factors were compared using Fisher’s Exact test. Event-free survival (EFS) was estimated by the Kaplan-Meier method and compared with log rank test. FOXO1 fusion status was coded as FP (fusion positive) or FN (fusion negative). Results: Of 134 pts identified in our database, fusion testing was performed in 55 (41%). Of these, PAX3 fusion was detected in 22 (40%). PAX7 was not detected. The median age of FP and FN pts was 25 yrs (range 18, 90) and 27 yrs (range 18, 65), respectively. Gender distribution was similar between FP and FN. Favourable site was seen in 13 (60%) FP and 21 (64%) FN. Nodal disease was present in 21 (95%) FP and 21 (64%) FN (p = 0.02). Distant metastases were present in 10 (45%) FP and 9 (27%) FN (n.s.). Treatment received was as follows for FP and FN, respectively: chemotherapy (21(95%), 33(100%)), radiation (14(64%), 22(67%)) and surgery (4(18%), 17(52%)). 5-yr EFS for pts without distant metastases was 27% (CI 22.6-76.6) and 46% (CI 21.5 – 70.5) for FP and FN respectively (n.s.). Conclusions: FP and FN RMS occurs in adults of all ages. Similar to children, adults with FP are more likely to present with nodal disease. Our study did not show that fusion status was associated with poorer EFS in adult RMS, however, larger series are needed to confirm this preliminary data.

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma.

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 to 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.

Abstract B01: Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors

Abstract Over the last decades, it has become increasingly clear that tumors are characterized by inter-individual heterogeneity, which can account as one of the prominent reasons for treatment failure. To address this problem, personalized precision medicine approaches need to be applied such as comprehensive genomic profiling to identify actionable driver mutations in individual tumors. Unfortunately, genomics alone is not sufficient in tumors that are driven by mutated but otherwise undruggable targets and a typically low mutational burden. This is a characteristic of many pediatric malignancies including rhabdomyosarcoma (RMS), the most common childhood soft-tissue sarcoma. Hence, we aim to develop an in vitro drug-profiling platform to identify and prioritize treatment strategies for RMS patients. To this end, we generated a panel of patient-derived xenografts (PDXs) including some diagnostic and relapse samples. We then screened 18 different culture conditions to identify suitable parameters to establish in vitro primary cultures of PDX tumors. Interestingly, addition of fetal calf serum to cell culture media has a detrimental effect on viability of most primary RMS cell cultures (PRCCs). In contrast, defined serum-free conditions allow to grow primary cultures for several passages that closely preserve the clonal composition and phenotypic characteristics of the parental tumor, as assessed by genomic and copy number analysis. Pharmacologic profiles of PRCCs using a targeted drug library of more than 200 compounds revealed patient-specific vulnerabilities, among them an unexpected sensitivity to AKT inhibitors in some fusion-positive RMS. Interestingly, hierarchical clustering of drug sensitivities clustered PAX3-FOXO1 fusion-positive tumors together and separated them from fusion-negative RMS. Moreover, a screen to establish effective drug combinations in a highly resistant high-risk relapse sample using standard chemotherapeutics (doxorubicin, etoposide, vincristine) together with our targeted compound library revealed the BH3-only mimetic ABT-263 as the top-scoring drug capable of resensitizing recurrent PRCCs to first-line treatment. Resensitizing with ABT-263 was not a patient-specific vulnerability as it was observed in several additional PRCCs. Mechanistically, genetic loss-of-function validation experiments revealed that this occurs via blockade of the BCL-XL-MCL-1 axis and is dependent on upregulation of the BH3-only protein NOXA. Taken together, our study provides an in vitro tool kit to prioritize actionable drug targets or combinatorial options for RMS patients for whom conventional therapies are failing. Citation Format: Beat W. Schaefer, Gabriele Manzella, Michaela Roemmele, Luduo Zhang, Joëlle Tchinda, Felix Niggli, Marco Wachtel. Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors [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 B01.

Loss of MST/Hippo Signaling in a Genetically Engineered Mouse Model of Fusion-Positive Rhabdomyosarcoma Accelerates Tumorigenesis.

A hallmark of fusion-positive alveolar rhabdomyosarcoma (aRMS) is the presence of a chromosomal translocation encoding the PAX3-FOXO1 fusion oncogene. Primary cell-based modeling experiments have shown that PAX3-FOXO1 is necessary, but not sufficient for aRMS tumorigenesis, indicating additional molecular alterations are required to initiate and sustain tumor growth. Previously, we showed that PAX3-FOXO1-positive aRMS is promoted by dysregulated Hippo pathway signaling, as demonstrated by increased YAP1 expression and decreased MST activity. We hypothesized that ablating MST/Hippo signaling in a genetically engineered mouse model (GEMM) of aRMS would accelerate tumorigenesis. To this end, MST1/2-floxed (Stk3F/F;Stk4F/F ) mice were crossed with a previously established aRMS GEMM driven by conditional expression of Pax3:Foxo1 from the endogenous Pax3 locus and conditional loss of Cdkn2a in Myf6 (myogenic factor 6)-expressing cells. Compared with Pax3PF/PF;Cdkn2aF/F;Myf6ICN/+ controls, Stk3F/F;Stk4F/F;Pax3PF/PF;Cdkn2aF/F;Myf6ICN/+ animals displayed accelerated tumorigenesis (P < 0.0001) and increased tumor penetrance (88% vs. 27%). GEMM tumors were histologically consistent with aRMS. GEMM tumor-derived cell lines showed increased proliferation and invasion and decreased senescence and myogenic differentiation. These data suggest that loss of MST/Hippo signaling acts with Pax3:Foxo1 expression and Cdkn2a loss to promote tumorigenesis. The rapid onset and increased penetrance of tumorigenesis in this model provide a powerful tool for interrogating aRMS biology and screening novel therapeutics.Significance: A novel mouse model sheds light on the critical role of Hippo/MST downregulation in PAX3-FOXO1-positive rhabdomyosarcoma tumorigenesis. Cancer Res; 78(19); 5513-20. ©2018 AACR.

Abstract 4357: Comparing amplification of 12q13-q14 and 12q15 chromosomal regions across cancer types through genomic and transcriptome analysis

Abstract Gene amplification, or an increase in copy number of a confined region on the chromosome arm, has been identified as a critical genetic event that contributes to the development of various cancers. There is increased expression of certain genes within the amplified regions, which alters normal cell growth and survival pathways, and contributes to tumorigenesis. Although previous studies show that some regions are amplified in more than one cancer type, direct analyses comparing the size and gene composition of these amplified regions across tumor types have not been performed. In the current study, we used copy number and RNA sequencing data from our published data and The Cancer Genome Atlas (TCGA) to characterize the commonly affected 12q13-q14 and 12q15 chromosomal regions in rhabdomyosarcoma, glioblastoma multiforme, lung adenocarcinoma, and liposarcoma. Based on our analysis of copy number data from high-density single-nucleotide polymorphism arrays, we observed a 0.08 Mb common region of overlap of the 12q13-q14 amplicons and a 0.20 Mb common region of overlap of the 12q15 amplicons across these tumor types. Differential gene expression analysis between amplified and nonamplified samples showed that OS9, TSPAN31, CDK4, CYP27B1, METTL1, EEF1AKMT3, and TSFM were overexpressed by the 12q13-q14 amplicons. Similarly, MDM2 and CPM were overexpressed by the 12q15 chromosomal amplicons. In addition to the common region of overlap, regions of tumor-type specific amplification were also found in our analysis. The 12q13-q14 amplicon in fusion-positive rhabdomyosarcoma extended 0.48 Mb toward the centromere, while the 12q13-q14 amplicons in dedifferentiated liposarcoma and lung adenocarcinoma extended 0.56 Mb and 0.96 Mb, respectively, toward the telomere. For the 12q15 region, the amplicon in lung adenocarcinoma extended 1.3 Mb toward the centromere, while the amplicons in dedifferentiated liposarcoma and fusion-negative rhabdomyosarcoma extended 1.4 Mb and 1.6 Mb, respectively, toward the telomere. Gene expression analyses showed that some genes from these tumor-specific regions of amplification were preferentially overexpressed in the corresponding tumor types. For example, four genes from the fusion-positive rhabdomyosarcoma-specific 12q13-q14 amplicon (NEMP1, NAB2, SHMT2, and R3HDM2) and two genes from the lung adenocarcinoma-specific 12q15 amplicon (MDM1 and RAP1B) were specifically overexpressed in these two tumor types. Our findings indicate that, in addition to common regions of amplification across multiple tumor types, there are tumor-specific amplified regions and overexpressed genes that indicate the presence of unique features within each cancer category affecting these amplification events. Citation Format: Prasantha L. Vemu, Gregory E. Hoy, Wenyue Sun, Jack Shern, Javed Khan, Frederic G. Barr. Comparing amplification of 12q13-q14 and 12q15 chromosomal regions across cancer types through genomic and transcriptome analysis [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 4357.

PAX3-FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability.

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

Abstract 4992: Miswired super enhancer logic driving childhood sarcoma

Abstract Super enhancers (SEs) are regulatory regions with unusually large deposits of active histone marks, chromatin regulators and transcriptional coactivators. Chromosomal rearrangements allowing SEs to drive oncogene expression is an emerging mechanism in tumor biology. An aggressive myoblastic cancer of childhood, alveolar (fusion-positive) rhabdomyosarcoma (FP-RMS), universally possesses a chromosomal translocation, involving most commonly PAX3 and FOXO1, more rarely PAX7-FOXO1, and in exceptional cases novel PAX3-INO80D and PAX3-NCOA1 fusions. Patients with a PAX3-fusion frequently relapse and have low survival rates. PAX3 initiates specification of the muscle lineage, but is shut off during myogenic differentiation, which is in turn dominated by master regulators MYOD and finally MYOG. FP-RMS has the master regulators needed to trigger muscle differentiation, but are halted in an early myoblastic and thus more proliferative epigenetic state. We hypothesized that the translocations miswires regulation of the fusion oncoprotein in FP-RMS by hijacking SEs and creating new topologically associated domains (TADs) which allow for continued expression of PAX fusions, thus circumventing normal myogenic enhancer logic. Thus, we recently completed the first epigenetic landscape of FP-RMS and uncovered a strong dependence on SEs for tumor survival, with PAX3-FOXO1 being a chief determinant of SE formation in collaboration with MYOD and MYOG, and oncogene MYCN. Importantly, we discovered a key SE 300 kb distal of FOXO1 which was occupied by all four of these master regulators. Further, we found that PAX3-FOXO1 is driven by this novel translocated SE forming a key TAD structure which was necessary to directly influence PAX3 upon translocation, with CTCF analysis in FP-RMS cells confirming the predicted boundaries. The CTCF motif orientation was found to be antiparallel after the translocation event, permitting chromatin loop extrusion. We demonstrate these elements to physically interact only in the presence of the translocation by chromatin conformation followed by sequencing (4C-seq). Exon-level expression via RNAseq in primary tumors revealed that the final exon of PAX3, not involved in the translocation, was unexpressed, indicating that only the allele influenced by the FOXO1 SE is activated in patients. Finally, CRISPR/Cas9 technologies were employed to functionally interrogate the relative contributions of the enhancer elements and CTCF looping sites at TAD boundaries. Together these data suggest that these newly juxtaposed enhancer elements initiate and continually drive PAX3-FOXO1 expression, implicating that enhancer miswiring is at the heart of the oncogenic process in FP-RMS. Thus, late myogenic factors (MYOG/MYOD) are contributing to drive an early factor (PAX3), changing a “progressive” enhancer logic into an “infinite loop” enhancer logic. Citation Format: Berkley E. Gryder, Marco Wachtel, Hsien-Chao Chou, Young Song, Joana Marques, Beat Schaefer, Javed Khan. Miswired super enhancer logic driving childhood sarcoma [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 4992. doi:10.1158/1538-7445.AM2017-4992