RMS Cell Lines Research Articles

Abstract B10: Rhabdomyosarcoma requires MYC family genomic events to pathogenically subvert core-regulatory circuitry

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Despite multimodality therapy and trials of molecularly targeted agents, limited improvements in overall survival have been realized for patients with high-risk disease. Thus, we aimed to determine the landscape of tumor-specific gene dependencies that underlie tumorigenesis in RMS and therefore provide a valuable group of targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches, we identified a set of RMS-specific tumor dependencies involved in tumor cell growth and survival. RMS dependencies were enriched for nucleic acid binding proteins, including transcription factors (TFs). We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential TFs—MYCN, MYOD1, TCF12, SOX8, ZEB2, ZNF217, and SIX1—are members of the transcriptional core regulatory circuitry (CRC) that maintains the malignant cell state of RMS. Both c-MYC and MYCN were associated with gene and enhancer copy number increases in cell lines and primary tumors and represented strong dependencies in the RMS cell lines screened. c-MYC and MYCN function to similarly invade and regulate the CRC in respectively dependent cells. To disable the CRC, we tested A485, an inhibitor of the histone acetyltransferase enzymes involved in the establishment of super-enhancer elements that are associated with high level expression of the CRC factors. A485 caused a reversible and rapid loss of CRC factor and c-MYC and/or MYCN expression, and prolonged treatment resulted in cell cycle arrest, differentiation, and apoptosis in vitro and in vivo. This phenotype is rescued by exogenous re-expression of either c-MYC or MYCN in a manner insensitive to A485, indicating a mechanism by which these genes subvert a myogenic CRC to produce an oncogenic fate. This study defines a common set of critical dependency genes in RMS and identifies key genomic events surrounding the c-MYC and MYCN loci that lead to elevated expression and tumorigenesis. Citation Format: Adam D. Durbin, Guillaume Kugener, Mark W. Zimmerman, Chuan Yan, Neekesh V. Dharia, Elizabeth S. Frank, Xiang Chen, Ken N. Ross, Brenton Paolella, Michael Krill-Burger, David E. Root, Jesse S. Boehm, Francisca Vazquez, Andrew L. Hong, Aviad Tsherniak, David M. Langenau, William C. Hahn, Todd R. Golub, Brian J. Abraham, Richard A. Young, A. Thomas Look, Kimberly Stegmaier. Rhabdomyosarcoma requires MYC family genomic events to pathogenically subvert core-regulatory circuitry [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 B10.

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 B43: The c-Myc/AKT1/TBX3 axis is important to target in the treatment of embryonal rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS), a pediatric cancer of the skeletal muscle, is the most common malignant soft-tissue sarcoma in children and adolescents. There are two main subtypes: embryonal RMS (ERMS), which is more prevalent in children and accounts for approximately 75% of RMS cases, and alveolar RMS (ARMS), which presents in adolescents and young adults and accounts for the remaining 25% of cases. Little is known about the molecular mechanisms underpinning ERMS, and there is a need for new and effective therapeutic approaches. Here we investigate the status and role of the T-Box transcription factor 3 (TBX3), which has been well characterized for its function in development and, in particular,r the congenital disorder ulnar mammary syndrome that results from its haploinsufficiency. Conversely, TBX3 has been reported to be overexpressed in numerous cancers including a range of sarcoma subtypes and shown to contribute to oncogenic processes such as cell proliferation, migration, and invasion, in vitro and in vivo tumor-forming ability. To determine the status of TBX3 in ERMS, immunohistochemistry was performed using RMS patient tissue and Western blotting on a panel of RMS cell lines compared to normal myoblast cells. Cell culture models in which TBX3 was either stably knocked down or overexpressed were established, and in vitro and in vivo assays were performed to determine the impact of TBX3 on key hallmarks of cancer including cell proliferation (growth curves), migration (scratch motility assay), as well as tumor formation and invasion in nude mice. Our data showed high TBX3 levels in RMS patient tumor tissue sections as well as in ERMS cell lines tested. Preliminary data reveal that the mechanisms involved in upregulating TBX3 in ERMS involve c-Myc at a transcriptional level and AKT1 at a post-translational level. Importantly, TBX3 was found to promote ERMS cell proliferation and migration, anchorage-independent growth, tumor formation, and metastasis. This study describes for the first time that TBX3 is an oncogene in ERMS and identifies c-Myc/AKT1/TBX3 as an important axis that could be targeted as novel therapeutic approaches for the treatment of this cancer. Citation Format: Danica Anne Sims, Hapiloe Mabaruti Maranyane, Jade Peres, Sharon Prince. The c-Myc/AKT1/TBX3 axis is important to target in the treatment of embryonal 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 B43.

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.

Abstract B066: Inhibition of farnesyl transferase by tipifarnib leads to isoform specific cell growth inhibition in HRAS-mutated human rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. A subset of RMS expresses the characteristic PAX3-FOXO1 or PAX7-FOXO1 translocations, thought to be oncogenic drivers. Among the fusion-negative subset of RMS, a substantial proportion express oncogenic mutations in RAS proteins or other elements of RAS signaling pathways. Activating RAS mutations are disproportionately seen in patients with high-risk RMS and attempts to augment combination chemotherapy have not resulted in improvement in overall survival in this poor prognosis group. Given the common finding of dysregulated RAS signaling, targeting RAS represents an attractive therapeutic approach. RAS family members require posttranslational modification for plasma membrane localization and subsequent downstream signaling. One therapeutic strategy, therefore, includes disruption of RAS protein prenylation, and thereby its membrane localization, via inhibition of farnesyl transferase (FTase). NRAS and KRAS may utilize geranylgeranyltransferase prenylation as a bypass when FTase is inhibited, but HRAS is uniquely dependent on FTase, and therefore, tumor cells with HRAS mutations may be preferentially sensitive to the effects of farnesyltransferase inhibition (FTI). We therefore tested human RMS cell lines harboring mutations in HRAS, KRAS or NRAS, and those wild type (WT) for H-, K-, and N-RAS, and found that only tumor cells with oncogenic HRAS mutations demonstrate growth inhibition and decreased ERK signaling in response to the FTI tipifarnib. Using high-throughput cell proliferation assays, dose dependent cell growth inhibition in response to treatment with tipifarnib is observed in HRAS-mutated RMS cell lines. We observe a reduction in phosphorylated MEK and ERK in HRAS mutated cell lines indicating effective RAS signaling inhibition. Activity of PI3K-mTOR pathway elements p70-S6K and RPS6 are inhibited by tipifarnib in cell lines that demonstrate an antiproliferative response to the compound. In vivo exposure to tipifarnib in HRAS-mutated RMS xenografts results in tumor growth inhibition. Therapeutic strategies targeting RAS protein prenylation may therefore provide an effective approach as an alternative to traditional chemotherapy, while improving outcomes and decreasing toxicities. Based on these data, a genomically-selected clinical trial using tipifarnib to treat pediatric patients with HRAS-mutated tumors, including RMS, would be of interest. Citation Format: Patience Obasaju, Kai Pollard, Amy Allen, Jiawan Wang, Christine Pratilas. Inhibition of farnesyl transferase by tipifarnib leads to isoform specific cell growth inhibition in HRAS-mutated human rhabdomyosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B066. doi:10.1158/1535-7163.TARG-19-B066

Abstract 3116: Targeting fibroblast growth factor receptors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma and is classified into two main histopathological subtypes: embryonal RMS (eRMS), characterized by different genomic changes or alveolar RMS (aRMS), driven by the oncogenic fusion protein PAX3-FOXO1. The significant toxicity associated with conventional chemotherapies represents a major complication in pediatric oncology. To improve current therapies, we adopted two different strategies targeting fibroblast growth factor receptors (FGFR) in RMS. FGFR1-4 are a family of transmembrane receptor tyrosine kinases. Their activation upon binding of fibroblast growth factors (FGF) triggers pro-survival and proliferative signals. Our goal is to deliver drugs specifically to the tumor site by taking advantage of FGFR4 overexpression in RMS. To this end, we will covalently link FGFR4 specific nanobodies to the surface of liposomal vincristine in order to actively target RMS cells. We have established the optimal conditions to formulate liposomes loaded with vincristine. Following nanobody phage display selection on recombinant FGFR4 we focused on the top scoring candidates. Flow cytometry analysis on FGFR4-expressing versus FGFR4 knock-out RMS cell lines showed receptor-specific binding of three nanobodies. In activation assays with the FGFR4 specific growth factor FGF19, we demonstrated that the three binding candidates also blocked downstream ERK activation in RMS cells. We will now assess their theranostic potential on drug-loaded and fluorescently labeled nanovesicles on RMS tumor cells in vitro and in xenografts in vivo. Surprisingly, we observed change in cell morphology followed by cell death upon exposure to FGF2 in a subset of cultured cells established from eRMS patient-derived xenografts. Inappropriate expression of FGFRs and FGF signaling is implicated in tumor progression and therefore our findings appear contradictory. Dose-response experiments have shown that FGFR inhibition with small molecule inhibitors completely rescued FGF2 toxicity. In contrast, however, we detected high expression levels of FGFR1, 2 and 4 as well as activating mutations of FGFR4 in FGF2-sensitive eRMS cells. Therefore, our results are of upmost clinical relevance since genetically-based drug selection could lead to an inappropriate treatment inducing tumor promoting conditions. Hence, our second goal is to further unravel the molecular mechanism underlying the toxic effect of FGF-2 in a subset of eRMS tumors to avoid potentially harmful treatments. In summary, we have identified FGFR4 specific nanobodies that bind to the receptor and block downstream signaling in RMS cells. Active drug delivery of liposomal vincristine to the tumor site has the potential to enhance the therapeutic impact and decrease side effects. Moreover, we discovered a toxic effect of FGF2 in a subgroup of eRMS patient derived xenograft cells which might open new avenues for treatment. Citation Format: Nagjie Alijaj, Sandrine Moutel, Maxim Gray, Maurizio Roveri, Gabriele Manzella, Marco Wachtel, Franck Perez, Beat Schäfer, Michele Bernasconi. Targeting fibroblast growth factor receptors in rhabdomyosarcoma [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 3116.

The anti-tumor growth effect of a novel agent DMAMCL in rhabdomyosarcoma in vitro and in vivo

BackgroundRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children with poor survival. New treatment approaches are urgently needed to improve treatment efficacy in RMS patients. DMAMCL is a novel agent from Asteraceae family that has been tested in phase I clinical trials in adult glioma in Australia.MethodsFive RMS cell lines (RD, RH18, RH28, RH30 and RH41) were used. The in vitro anti-tumor effect of DMAMCL, alone or in combination with VCR or Epirubicin, was studied using MTS assay or IncuCyte-Zoom cell confluency assay, and further validated by xenograft-mouse model in vivo. Changes in caspase-3/7 activity, cell-cycle progression and generation of ROS after DMAMCL treatment were investigated. Bim mRNA expression was measured by RT-qPCR, and protein expressions of Bim and phosphorylated-NF-κB(p65) by Western blotting. Small interfering RNAs (siRNA) of Bim were used to study the role of Bim in DMAMCL-induced cell death.ResultsIn vitro, DMAMCL treatment induced a dose-dependent increase in cell death that could be blocked by pan-caspase-inhibitor-Z-VAD-fmk in five RMS cell lines. The percent of cells in SubG1 phase and activities of caspase-3/7 increased after DMAMCL treatment; The combination of DMAMCL with VCR or Epirubicin significantly increased cell death compared to each reagent alone. In vivo, DMAMCL(75 mg/kg or 100 mg/kg) inhibited tumor growth and prolonged survival of mice bearing xenograft RMS tumors (RD, RH18, RH30, RH41). Compared to treatment with DMAMCL or VCR, a combination of two reagents caused significant inhibition of tumor growth (RD, RH41), even after treatment termination. The expression of Bim increased at protein level after DMAMCL treatment both in vitro and in vivo. The expression of p-NF-κB(p65) had a transient increase and the generation of ROS increased after DMAMCL treatment in vitro. Transfection of Bim siRNA into RMS cells blocked the DMAMCL-induced increase of Bim and partially attenuated the DMAMCL-induced cell death.ConclusionDMAMCL had an anti-tumor growth effect in vitro and in vivo that potentially mediated by Bim, NF-κB pathway and ROS. A combination of DMAMCL with chemotherapeutic drugs significantly increased the treatment efficacy. Our study supports further clinical evaluation of DMAMCL in combination with conventional chemotherapy.

Abstract B26: Novel lncRNA regulates PAX3/FOXO1 expression and is essential for RMS tumorigenesis

Abstract Rhabdomyosarcoma (RMS) is a pediatric cancer of the developing skeletal muscle, and the mechanisms that drive this tumor are not completely understood. Despite the genetic alterations discovered in this disease, therapeutics targeting these mutations have not improved patient survival over the past 25 years. Since more than 60% of patients with metastatic disease do not survive, this creates an urgency to identify new vulnerabilities to exploit for therapeutic development. To do this we performed a comprehensive evaluation of the epigenomic landscape across 16 RMS orthotopic patient-derived xenograft tumors. We identified 41 super enhancers unique to RMS tumors. Within these, we discovered 9 novel lncRNAs of which 3 have potential cis-regulatory activity. We hypothesize that these lncRNAs are required for RMS tumorigenesis. Indeed, from preliminary knockdown analysis of one of these lncRNAs, lnc19_31, we observed rapid cell death in RMS cell lines and concomitant upregulation of apoptotic genes based on RNA-seq, such as TP53. Remarkably, we also observed more than 70 cancer consensus genes that were up- or downregulated, including genes that are frequently mutated in RMS patients such as BCOR and FGFR4. Within these we were excited to discover the loss of PAX3/FOX1 translocation, as this is a major driver of the alveolar subtype and often associated with poor prognosis. Taken together, we hypothesize that lncRNA deregulation is required for expression of key oncogenes and tumor suppressor and essential for RMS cell survival and tumorigenesis. These data will shed insight into a new underlying mechanism that drives this disease. These exciting findings present potential tumor vulnerabilities and, thus, new opportunities for future therapeutic discoveries that would otherwise be beyond our reach. Citation Format: Xiang Chen, Mireya Herrera-Herrera, Justina McEvoy. Novel lncRNA regulates PAX3/FOXO1 expression and is essential for RMS tumorigenesis [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 B26.

Abstract 5618: Anti-FGFR4 antibody drug conjugate for immune therapy of rhabdomyosarcoma and hepatocellular carcinoma

Abstract Rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. It is divided in two main histological subtypes including embryonal RMS driven by RAS pathway mutations and alveolar RMS driven by a chimeric fusion gene involving PAX3 or PAX7 with FOXO1. FGFR4 is a cell surface tyrosine kinase receptor that is a critical molecule in RMS biology. In RMS it is a direct target and strongly induced by PAX3-FOXO1 as well as PAX3, and PAX7. By immunohistochemistry we found high expression of FGFR4 in RMS cancers but low expression in normal human organs. Of note FGFR4 has been reported overexpressed in hepatocellular carcinoma (HCC). We therefore hypothesized that FGFR4 will provide a rational target for immune therapy in cancers with high expression. We generated fifteen binders against FGFR4 and characterized them further as candidates for immunotherapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines with an observed avidity of 1.9nM. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 showed dose dependent binding to FGFR4 only. 3A11 was also found internalized upon binding to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and chimeric 3A11 scFvFc (mouse Fv and Human IgG1 Fc) construct was made. The chimeric form of 3A11 antibody was successfully produced in vitro and retained its specificity. To investigate FGFR4-mediated cytotoxicity in RMS cell lines, we cultured RH30 (a RMS cell line expresses FGFR4) in the presence of the chimeric 3A11 and polyclonal anti-mouse 2°ADC conjugated to Duocarmycin DM for 72 hours. Dose-dependent cytotoxicity was observed in RH30, suggesting that chimeric form of 3A11 could deliver a cytotoxic payload to FGFR4 positive cells. We are currently developing antibody drug conjugate using the cytotoxic drug, pyrrolobenzodiazepine which will be tested in vitro and in vivo models of RMS. We have thus developed a chimeric antibody that may provide effective immune therapy for cancers with high FGFR4 expression including RMS and HCC Citation Format: Adam Cheuk, Nitya Shivaprasad, Martin Skarzynski, Sivasubramanian Baskar, Peter Azorsa, Javed Khan. Anti-FGFR4 antibody drug conjugate for immune therapy of rhabdomyosarcoma and hepatocellular carcinoma [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 5618.

Abstract 4132: ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway

Abstract Alternative splicing is a process contributing to structural transcript variation and proteome diversity but is often disrupted in cancer. Like most other transcripts, the insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. The expression of these isoforms is tightly regulated during development, however there is an aberrant increase in IN-RA expression in childhood cancers like Rhabdomyo- & Osteo-sarcoma. This increased IN-RA expression is deleterious to the cells because it encodes for a receptor which has high affinity for both insulin & IGF2 growth hormones and it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks like proliferation & angiogenesis. Rhabdosarcoma is the 3rd most frequent solid tumor and the most common soft-tissue sarcoma in children. Despite improvement in the treatment for this disease, there is substantial inadequacy of less toxic alternative therapies. We found that there is a significantly increased expression of IN-RA levels in a cohort of 40 Rhabdosarcoma (RMS) patients (embryonal & alveolar) as compared to the control samples. We confirmed this increased expression in multiple RMS cell lines. Furthermore, we went on to show that cellular stress such as hypoxia increases alternative splicing to produce more IN-RA isoform. In order to get a mechanistic insight into this phenomenon, we utilized this hypoxia-inducible splicing system and performed an antisense-oligonucleotide (ASO) screen to characterize sequence elements and splicing factors involved in the regulation of IN-R splicing. We found that sequence elements flanking exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. We then performed a refined ASO walk to target the regions important for exon inclusion or exclusion and identified a region known to be a binding site for the splicing-factor CUGBP1. RMS derived cell lines almost exclusively express IN-RA but when treated with our lead ASO compound that targets this region, they show a dramatic decrease in the IN-RA expression. More importantly, the ASO treated cells exhibit a significant reduction in cell proliferation and migration. Application of ASO compound attenuates the IGF signaling pathway measured by decreased pAKT staining. Additionally, when tested in RMS xenograft models, we see decreased extravasation of blood vessels in the grafts cells transfected with the ASO that blocks IN-RA expression compared to controls. Our current data shows promising insight into how we can impede the IGF2 pathway by reducing IN-RA expression and consequently mitigate tumor hallmarks like cell-proliferation, migration and angiogenesis. The goal is to use these ASO compounds as therapeutic interventions in conjunction with already established anti-IGF1 receptor therapies to treat pediatric Rhabdomyosarcoma. Citation Format: Safiya Khurshid, Matias Montes, Brianne Sanford, Chelsea Brown, Daniel Comiskey, Frank Rigo, Peter Houghton, Dawn Chandler. ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway [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 4132.

PD-L1 assessment in pediatric rhabdomyosarcoma: a pilot study

BackgroundRhabdomyosarcomas (RMSs) are the most frequent soft tissue sarcoma in children and adolescents, defined by skeletal muscle differentiation and the status of FOXO1 fusions. In pediatric malignancies, in particular RMS, scant and controversial observations are reported about PD-L1 expression as a putative biomarker and few immune checkpoint clinical trials are conducted.MethodsPD-L1 assessment was evaluated by immunohistochemistry (IHC) utilizing two anti-PDL1 antibodies, in a pilot cohort of 25 RMS. Results were confirmed in primary and commercial RMS cell lines by cytofluorimetric analysis and IHC.ResultsPD-L1 expression was detectable, by both anti-PD-L1 antibodies, in the immune contexture of immune cells infiltrating and/or surrounding the tumor, in 15/25 (60%) RMS, while absent expression was observed in neoplastic cells. Flow cytometry analysis and PD-L1 IHC of commercial and primary RMS cell lines confirmed a very small percentage of PD-L1 positive-tumor cells, under the detection limits of conventional IHC. Interestingly, increased PD-L1 expression was observed in the immune contexture of 4 RMS cases post chemotherapy compared to their matched pre-treatment samples.ConclusionHere we identify a peculiar pattern of PD-L1 expression in our RMS series with scanty positive-tumor cells detected by flow cytometry, and recurrent expression in the immune cells surrounding or infiltrating the tumor burden.

The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells

BackgroundMYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01.MethodsMYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively.ResultsMYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed.ConclusionsAn alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.

Abstract B22: Uncovering the role of lysyl oxidase-like 2 in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue malignancy in childhood and adolescence that originates as a consequence of regulatory disruption in the growth and differentiation of muscle precursor cells. Members of the lysyl oxidase family of proteins (LOX and LOXL1-4) are amine oxidases that catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. Apart from their traditional role, novel functions have been attributed to these proteins related to tumor progression. In this study we sought to unravel the potential role of this family of proteins during the progression of RMS. By qRT-PCR and Western blot, lysyl oxidase-like 2 (LOXL2) was found overexpressed in RMS cell lines compared to other sarcoma cells. Then we characterized LOXL2 in RMS cell lines by analyzing the subcellular localization by immunofluorescence and subcellular fractionation. Post-translational modifications were also analyzed: glycosylation and proteolytic processing by tunicamycin treatment and secretion by analysis of the conditioned media by Western blot. Moreover, we characterized the neoplastic phenotype after silencing LOXL2 in two RMS cell lines (RH4 and CW9019). Stable LOXL2 knockdown resulted in a decrease in their clonogenic, proliferative, and cell migratory capabilities. On the other hand, reexpression of LOXL2 in RH28 cells using wild-type or mutated (catalytically inactive) constructs resulted in an increase in cell proliferation and migration, independently of its catalytic activity. Finally, in vivo experiments using an orthotopic model of tumor generation inside the muscle will be carried out to further evaluate the tumorigenic role of LOXL2. Taking all into consideration, our results suggest an oncogenic role of LOXL2 in RMS. Citation Format: Olga Almacellas Rabaiget, Juan Huertas Martínez, Silvia Garcia Monclús, Santiago Rello Varona, David Herrero Martín, Roser López Alemany, Oscar M. Tirado. Uncovering the role of lysyl oxidase-like 2 in rhabdomyosarcoma [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 B22.

Abstract A33: Histone demethylase KDM3A in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, adolescents, and young adults, presenting a myogenic phenotype. RMS can be divided into two major histopathologic subtypes: driver oncofusion (PAX3/7-FOXO1) positive, alveolar (ARMS), and oncofusion-negative embryonal (ERMS). Histone methylation is a key element of chromatin-based modifications that regulates a number of cellular processes, including DNA replication, DNA repair, and gene transcription. Histone demethylase gene family members (HDMs) regulate gene expression by removing methyl marks on histone tails to activate or repress transcription. KDM3A (lysine-specific demethylase 3A or JMJD1A) is a histone demethylase belonging to the Jumonji C-terminal domain (JmjC)-containing histone demethylases that selectively removes mono- and dimethyl groups from H3K9. It has been demonstrated that KDM3A is overexpressed in many kinds of cancer, such as bladder, lung, and breast cancer, and, previously shown by our laboratory, in Ewing sarcoma, contributing to carcinogenesis. We and others have identified KDM3A as overexpressed in RMS, but the functional consequences of this overexpression have not been examined. The aim of this study was to analyze the influence of KDM3A on RMS biology. Two different RMS cells lines were used, RH30 (ARMS) and RD (ERMS), to perform stable shRNA-mediated KDM3A knockdown. The protein and RNA levels were analyzed by Western blotting and RT-qPCR, respectively. The phenotypic effects of KDM3A knockdown were analyzed by proliferation assays (MTT and clonogenic assay) and cell migration (transwell migration assay). KDM3A knockdown was able to inhibit the proliferation, colony formation, and migration of RMS cell lines, demonstrating an important role in RMS biology. Citation Format: Lays M. Sobral, Paul Jedlicka. Histone demethylase KDM3A in rhabdomyosarcoma [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 A33.

Abstract B06: Targeting PI3K/mTOR in mouse rhabdomyosarcoma models driven by FGFR4 activation

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue sarcoma and while survival rates have increased over the past few decades, outcomes for intermediate and high-risk patients remain dismal. Genomic analyses show that 93% of RMS have RTK/RAS/PI3K alterations and that fibroblast growth factor receptor 4 (FGFR4) is frequently mutated or overexpressed. The point mutation V550E constitutively activates FGFR4, stimulating downstream signaling pathways. In this translational study, we demonstrate that FGFR4V550E is a potent oncogene in mouse models of RMS and that secondary tumor models can be generated following transplantation of FGFR4V550E overexpressing tumor cells into immunocompetent host mice. These models uncovered mechanisms of transformation in FGFR4V550E-driven RMS and helped identify novel compounds that inhibit RMS growth. Specifically, AKT and mTOR signaling pathways were activated in myoblasts overexpressing FGFR4V550E and similarly, FGFR4V550E overexpressing tumors and tumor-derived cells exhibited increased AKT and mTOR phosphorylation by immunoblot. Murine tumor cells overexpressing FGFR4V550E were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percent area under the dose response curve (AUC). Using this technique, FGFR4V550E overexpressing tumor cells were highly sensitive to PI3K/mTOR inhibitors. In particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4V550E tumor-derived cell and human RMS cell viability. FGFR4V550E overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC50 values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. In a preclinical study, GSK2126458 inhibited tumor growth in vivo. A statistically significant increase in disease-specific survival was observed in mice treated with GSK2126458 compared to mice treated with vehicle alone (p<0.001) or standard of care, vincristine (p<0.05). In summary, RMS driver mutations were validated in vivo and our model system was effectively used as a preclinical tumor model to identify and test therapeutic agents. Importantly, these results suggest a role for PI3K/mTOR inhibition in precision therapy regimens for RMS with FGFR4 mutations. This study provides further evidence for RMS clinical studies involving mTOR inhibitors (e.g., temsirolimus). Citation Format: Timothy McKinnon, Rosemarie Venier, Marielle Yohe, Berkley E. Gryder, Brendan Dickson, Krista Schleicher, Dariush Davani, Winnie Wei, Cynthia Guidos, Abha Gupta, Javed Khan, Rebecca Gladdy. Targeting PI3K/mTOR in mouse rhabdomyosarcoma models driven by FGFR4 activation [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 B06.

Abstract 2349: Sensitivity and resistance to cell cycle and IGF-1R inhibitors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults. The five-year survival rate for RMS has hardly improved over the last three decades despite intensive and toxic chemotherapy, radiotherapy with surgery. Therefore, novel treatment approaches are required to change these outcomes. RMS has two major subtypes, embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS, the more aggressive subtype, is characterized by translocations that fuse two transcription factor-encoding genes; creating novel PAX3/PAX7-FOXO1 fusion proteins. The PAX3-FOXO1 is highly expressed in the G2 phase of the cell cycle, allowing the cell to divide following a sustained checkpoint arrest despite DNA damage induced by chemotherapy, suggesting that PAX3-FOXO1 may enhance the survival of tumor cells in response to chemotherapy. Many cell cycle regulators are altered in RMS, including CDK2, CDK4 and p53. Furthermore, RMS cells are highly dependent on the insulin-like growth factor -1 receptor (IGF-1R) signaling, however, IGF-1R targeting was not successful in the clinic. Therefore, targeting key cell cycle regulators individually or in combination with IGF-1R inhibition may expand the available therapeutic options for RMS. Purpose: The goal of the present study was to investigate the cytotoxicity of 15 small molecule inhibitors targeting the IGF-1R and cell cycle regulators in RMS cell lines and to determine potential mechanisms of drug sensitivity or resistance. Methods and Results: Seven RMS cell lines including ERMS and ARMS were studied. The IC50 values were determined for the following targeting compounds: linsitinib, BMS-754807 and picropodophyllin (PPP) (IGF-1R), riobociclib and palbociclib (CDK4/6), dinaciclib and flavopiridol (pan CDK inhibitors), BS-181HCL (CDK7), MK-1775 (WEE-1), MK-8776 (CHK1), alisertib (AURKA) and volasertib (PLK1). The most potent compounds with IC50< 10 nM were dinaciclib and volasertib. The ARMS cell lines were resistant to alisertib in comparison to the ERMS cell lines. Most cell lines were sensitive to flavopiridol, MK-1775, BMS-754807 and PPP with IC50< 100 nM, and relatively resistant to BS-181 HCL, linsitinib, R0-3306 and MK-8776 with IC50 (1-70 μM). Palbociclib and BMS-754807 showed a synergistic effect in some RMS cell lines. Ongoing studies are focusing on determining the mechanisms of interaction of these two compounds through studying cell cycle, apoptosis, and mRNA and protein expression of key regulators in the IGF-1R and RB pathways. Conclusion: These data demonstrate that dinaciclib, volasertib, flavopiridol, MK-1775, BMS-754807 and PPP are highly cytotoxic in RMS cell lines. The CDK4/6 inhibitor palbociclib may sensitize selected RMS cell lines to IGF-1R inhibitors. Targeting selected cell cycle regulators individually, or in combination with IGF-1R inhibitors may thus provide an efficacious treatment approach to be further validated in RMS patients with poor outcome. Citation Format: Justin Montoya, David W. Lee, Eiman Aleem. Sensitivity and resistance to cell cycle and IGF-1R inhibitors in rhabdomyosarcoma [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 2349. doi:10.1158/1538-7445.AM2017-2349

Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts.

Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes.

Heme Oxygenase-1 Controls an HDAC4-miR-206 Pathway of Oxidative Stress in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue cancer characterized by disturbed myogenic differentiation. Here we report a role for the oxidative stress response factor HO-1 in progression of RMS. We found that HO-1 was elevated and its effector target miR-206 decreased in RMS cell lines and clinical primary tumors of the more aggressive alveolar phenotype (aRMS). In embryonal RMS (eRMS), HO-1 expression was induced by Pax3/7-FoxO1, an aRMS hallmark oncogene, followed by a drop in miR-206 levels. Inhibition of HO-1 by tin protoporphyrin (SnPP) or siRNA downregulated Pax3/7-FoxO1 target genes and induced a myogenic program in RMS. These effects were not mediated by altered myoD expression; instead, cells with elevated HO-1 produced less reactive oxygen species, resulting in nuclear localization of HDAC4 and miR-206 repression. HO-1 inhibition by SnPP reduced growth and vascularization of RMS tumors in vivo accompanied by induction of miR-206. Effects of SnPP on miR-206 expression and RMS tumor growth were mimicked by pharmacologic inhibition of HDAC. Thus, HO-1 inhibition activates an miR-206-dependent myogenic program in RMS, offering a novel therapeutic strategy for treatment of this malignancy. Cancer Res; 76(19); 5707-18. ©2016 AACR.

Abstract 4996: Targeting FGFR4 with monoclonal antibodies as therapeutic agents for the treatment of rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with two major subtypes, embryonal (ERMS) and alveolar (ARMS), and current treatment modalities have yielded event free 5-year survival in only 30% of the patients with high-risk disease. Therefore, there is a need for novel strategies to identify and validate clinically relevant targets and therapeutics for the treatment of RMS. The fibroblast growth factor receptor 4 (FGFR4) is a very attractive therapeutic target because: 1) the FGFR4 gene is over expressed in RMS, 2) it is directly transcriptionally induced by the PAX3-FOXO1 fusion oncogene found in ARMS, 3) it is crucial for survival, proliferation, metastasis and drug resistance, 4) activating mutations in the kinase domain lead to aggressive growth and poor survival in patients with ERMS and 5) genetic or pharmacologic inhibition of FGFR4 signaling inhibited tumor growth in vitro and in vivo. Most normal human tissues do not express FGFR4 protein as determined by immunohistochemistry and electrochemiluminesence assay. Based these, we hypothesize that monoclonal antibodies (mAbs) targeting FGFR4 and their derivatives can serve as potential therapeutic agents for the treatment of RMS. We have developed a total of 15 mAbs against human FGFR4 protein from rabbit and mouse (by hybridoma technology), and from human immunoglobulin libraries (by recombinant DNA technology). These mAbs specifically react with human FGFR4 protein in an ELISA and not to the other members of the FGFR family. Flow cytometer analysis demonstrated that they specifically bound to cell surface FGFR4 protein in RMS cell lines although the FGFR4 expression levels was variable in both ARMS and ERMS cell lines. Furthermore, cell surface FGFR4 mediated internalization of the bound antibody upon incubation at 37oC for as little as 30 min and maximum internalization was observed at 2 h. Two of the mouse mAbs tested were able to mediate specific cytotoxicity in FGFR4-expressing cell lines in the presence of an anti-mouse secondary antibody conjugated to cytotoxic drugs (2oADCs), e.g. monomethyl auristatin F (MMAF) or a duocarmycin derivative (DMDM). Dose titration of the anti-FGFR4 mAbs revealed maximum killing at 100 pM indicating their robust activity in vitro, and both ARMS and ERMS cell lines were susceptible to the mAb-mediated cytotoxicity. Ongoing investigations include generation of FGFR4-targeting direct ADCs and evaluation of their efficacy in vivo. Together, these observations support the contention that anti-FGFR4 mAb can be used as a vehicle to deliver a cytotoxic payload in the form of small molecule drugs and toxins. Thus, FGFR-ADCs may serve as potential therapeutic agents for the treatment of patients with RMS, as well other FGFR4-positive cancers including breast, liver, prostate and lung. Citation Format: Sivasubramanian Baskar. Targeting FGFR4 with monoclonal antibodies as therapeutic agents for the treatment of rhabdomyosarcoma. [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 4996.

Abstract 1276: Pannexin 1 regulates rhabdomyosarcoma tumor growth: a potential novel therapeutic target

Abstract Rhabdomyosarcoma (RMS) is a skeletal muscle-derived soft tissue sarcoma for which a novel therapeutic strategy is greatly needed. RMS cells have lost the ability to terminally differentiate thus proliferating indefinitely. Promoting their redifferentiation to skeletal muscle tissue is thus a promising therapeutic approach. We have recently shown that levels of Pannexin 1 (Panx1) are below detectable limits in undifferentiated myoblasts, but become highly upregulated during their differentiation and indeed promote this process. We thus hypothesize that the levels of Panx1 are deregulated in RMS and that restoration of Panx1 levels may induce differentiation of RMS cells thereby alleviating their malignant properties. Briefly, our results demonstrate that both Panx1 transcript and protein are at very low levels in, both embryonal and alveolar RMS tumor specimens and patient-derived cell lines similar to that seen in fetal skeletal muscle tissue and undifferentiated myoblasts, respectively. In vitro functional analyses have revealed that while re-introduction of Panx1 in RMS cell lines led to their partial differentiation, it significantly inhibited their rate of proliferation and migration. Moreover, re-expression of Panx1 in RMS cell lines prevented both spheroid formation and growth, and induced RMS cell apoptosis. Based on this in vitro data, pre-clinical orthotopic xenograft studies are currently underway to assess the ability of Panx1 to inhibit tumor onset and growth, and induce tumor regression in vivo. Our preliminary results thus far have shown that Panx1 over-expression significantly suppresses RMS xenograft tumor growth. Taken together, these results indicate that restoration of Panx1 levels in RMS reduces its malignant properties. This, together with further investigation into the tumor-suppressive mechanism by which Panx1 mediates its effects, will establish Panx1 as a novel therapeutic target for RMS. Citation Format: Xiao Xiang, Stephanie Langlois, Marie-Eve St-Pierre, Jessica Barre, Tammy Le Pham, Kyle N. Cowan. Pannexin 1 regulates rhabdomyosarcoma tumor growth: a potential novel therapeutic target. [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 1276.