ARMS Cell Lines Research Articles

Abstract 5960: Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS

Abstract Alveolar rhabdomyosarcoma (aRMS), characterized by poor overall survival and limited advancements in therapy over the past four decades, poses a great challenge in childhood cancer treatment. More than 60% of high-risk aRMS is driven by the oncofusion protein PAX3-FOXO1. The direct targeting of transcription factor fusions, however, is quite difficult. Given that PAX3-FOXO1 is reported to drive disease by controlling transcriptional programs to maintain an undifferentiated myoblastic state, an alternative approach would be to target the transcriptional machinery recruited by the fusion. To identify potential novel targets in aRMS, we utilized the Broad Institute’s Cancer Dependency Map dataset, a collection of genome-wide CRISPR-Cas9 screens performed in over 1000 cancer cell line models. We focused on druggable dependencies that are selective to fusion-positive aRMS and intersected this data with PAX3-FOXO1-associated transcriptional complexes. Among these candidates, we identified CDK8 as a top selective dependency. Importantly, other components of the CDK8 kinase module in the mediator complex, such as CCNC, also scored as a strong selective dependency in fusion-positive aRMS. CDK8 expression is notably elevated in aRMS tumors compared to other pediatric tumors, and aRMS cell lines exhibit significantly higher CDK8 protein levels than non-transformed primary skeletal muscle cells. We next validated these in silico results through the application of shRNA knockdown, CRISPR knockout, and the use of CDK8 kinase inhibitory small molecules, including BI-1347, SEL-120-34A, and JH-XII-178, which demonstrated on-target activity as assessed by STAT1Ser727 phosphorylation. Both genetic loss of CDK8 function and kinase inhibition impaired aRMS cell line growth in vitro. In vivo studies demonstrated a significant inhibition of xenograft tumor growth with both doxycycline inducible CDK8-directed shRNAs and with pharmacologic inhibition of CDK8 with SEL120-34A treatment. To understand the mechanisms by which CDK8 inhibition impairs aRMS growth, we evaluated the transcriptional consequences of CDK8 inhibition by RNA-sequencing in aRMS, as well as the chromatin accessibility by ATAC-sequencing. We discovered that loss of CDK8 led to gain of chromatin accessibility and the upregulation of hallmark genes involved in myogenesis, such as MYH3, MYOG, MEF2C, and MYBPH, as well as alteration of PAX3-FOXO1 gene expression programs. We next validated this differentiation phenotype both in vitro through detection of increased myogenin staining by immunofluorescence and in vivo by visualization of myofibrils in the xenograft tumors and increased myoglobin quantified by qRT-PCR after CDK8 inhibition. In conclusion, this work identifies CDK8 as a novel and readily translatable therapeutic target in fusion-positive aRMS laying a robust foundation for further mechanistic and preclinical studies. Citation Format: Susu Zhang, Clare Malone, Brian Guedes, Kathleen Engel, Marissa Just, Kenneth Ross, Assil Fahs, Giulia Digiovanni, Diyana Granum, Rex Bentley, Leen Barbar, Christian Cerda-Smith, Elizabeth Mendes, Ozgun Erdogan, Alexander Kovach, Kristianne Oristian, Seth Zimmerman, Jack F. Shern, Kris Wood, Chris Counter, Corinne M. Linardic, Kimberly Stegmaier. Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5960.

Abstract 1558: Targeting CDK9 via the small-molecule inhibitor enitociclib as a therapeutic strategy to treat MYCN-amplified rhabdomyosarcoma and neuroblastoma in children

Abstract Introduction: MYCN amplification is the genetic aberration most consistently associated with poor outcomes in alveolar rhabdomyosarcomas (aRMS) and neuroblastoma (NBL). It regulates key oncogenic processes including tumor cell survival, proliferation and metastasis. In aRMS, MYCN transcription is driven by the PAX3-FOXO1 gene fusion. MYCN and other short half-life mRNA transcripts depend on CDK9 to drive transcription of target genes to initiate and maintain the oncogenic program, making CDK9 blockade an effective therapeutic route against MYC-driven cancers. We have previously shown the activity and tolerability of enitociclib in tumor xenograft models. In this study, we investigated the activity of the selective small-molecule CDK9 inhibitor enitociclib (VIP152/BAY1521152) in preclinical models of pediatric MYCN-amplified aRMS and NBL. Methods: Cell-based viability assays were performed in aRMS (Rh30 and Rh41) and NBL (LAN1, SK-N-AS, SK-N-BE(2) and SK-N-MC) cell lines. Enitociclib antitumor activity was measured by Alamar Blue cell viability assay after 96 h exposure at 9 dose levels (8 nM-2 μM) with DMSO used as control. We performed combination screens of enitociclib with several FDA-approved drugs (n=215). Selected agents with IC50 values <1 μM were analyzed at constant dilution ratios of two inhibitors. Drug synergy was calculated by established methods using SynergyFinder 3.0. Target modulation was determined for RNA polymerase II (RNAPII), MYCN and apoptosis inducers caspase-3 and PARP, in addition to PAX3-FOXO1 and PLK1 protein levels in aRMS cells, by western blotting after continuous exposure with enitociclib for 24 h. Results: Significant cytotoxic activity, with IC50 values ranging from 48-182 nM and 39-123 nM was seen for aRMS cells and NBL cells, respectively. SynergyFinder found synergistic effects of enitociclib with irinotecan, carfilzomib, etoposide, bortezomib, selinexor or topotecan tested at clinically relevant concentrations in the aRMS cell line Rh41. Enitociclib induced apoptosis with cleavage of caspase-3 and PARP by western blotting in a dose- and time-dependent manner in addition to the depletion of phosphorylated RNAPII (Ser2) and MYCN protein levels. In aRMS cells, PAX3-FOXO1 fusion and PLK1 proteins were downregulated. Annexin V/propidium iodide staining confirmed a dose-dependent increase in apoptosis after enitociclib exposure. Conclusions: Our results suggest that CDK9 inhibition is potentially clinically relevant for MYCN-amplified solid tumors such as aRMS and NBL due to its significant antitumor activity and pharmacologic targetability. The data provide essential information on the distinct targets, biomarkers of activity and clinically feasible drug combinations for the development of enitociclib in clinical trials addressing an unmet need in pediatric oncology. Citation Format: Son Tran, Patrick Sipila, Melanie M. Frigault, Amy J. Johnson, Joseph Birkett, Raquel Izumi, Ahmed Hamdy, Beatrix Stelte-Ludwig, David P. Douglass, Anne-Marie Langevin, Norman J. Lacayo, Aru Narendran. Targeting CDK9 via the small-molecule inhibitor enitociclib as a therapeutic strategy to treat MYCN-amplified rhabdomyosarcoma and neuroblastoma in children [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1558.

Abstract 2362: Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

Abstract Background: Gene expression networks are perturbed in every human malignancy. One common way that this occurs is through chromosomal translocations such as the the t(2;13)(q35;q14), which creates the oncogenic transcription factor, PAX3-FOXO1, in alveolar rhabdomyosarcoma (aRMS). Identification of the direct transcriptional targets and mechanisms of control by translocated transcription factors is critical to our understanding of disease etiology and the development of targeted therapeutic approaches. However, defining the genes directly controlled by DNA binding proteins and the mechanisms of regulation at those direct target genes is challenging, because traditional genetic approaches to inactivate transcription factors cannot distinguish direct from indirect changes in gene expression. Methods: A CRISPR-based approach was used to engineer the endogenous PAX3-FOXO1 locus to incorporate the FKBP12F36V degron tag. This allowed degradation of PAX3-FOXO1 within 2 hr of treatment with the proteolysis targeting chimera, dTAG-47. We performed short time courses to analyze nascent transcription (precision nuclear run-on sequencing; PRO-seq), chromatin accessibility (ATAC-seq), and genome-wide mapping of transcriptional complexes (CUT&RUN). This identified a small cohort of high-confidence PAX3-FOXO1 gene targets and associated PAX3-FOXO1-regulated enhancer elements. In addition, we used CRISPR editing to add APEX2 and 3XFLAG tags to PAX3-FOXO1 to enable deep proteomics to identify PAX3-FOXO1-associated transcriptional complexes in aRMS cell lines. Results: Degradation of PAX3-FOXO1-FKBP12F36V led to growth inhibition, hallmarks of myogenic differentiation, and reduced growth in soft agar. PAX3-FOXO1 binding was detected at over 44,000 sites throughout the genome using CUT&RUN, and loss of chromatin bound PAX3-FOXO1 was observed within 2-4hr of PAX3-FOXO1 degradation. PRO-seq analysis defined a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Unexpectedly, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. Deep proteomic analysis showed that PAX3-FOXO1 was in close proximity to many transcriptional regulatory complexes, but appeared to coordinate the activity of these complexes at only a few hundred sites. Moreover, the continued presence of PAX3-FOXO1 at these elements was required to maintain chromatin accessibility. Conclusion: This work demonstrates the utility of using rapid degradation of endogenous transcription factors to define their direct transcription targets and the mechanisms by which these factors control gene expression. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network, and emphasizes the utility of PAX3-FOXO1 as a therapeutic target in aRMS. Citation Format: Susu Zhang, Jing Wang, Qi Liu, Hayes McDonald, Monica Bomber, Hillary Layden, Jacob Ellis, Scott Borinstein, Scott Hiebert, Kristy Stengel. Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2362.

LOXL2 promotes oncogenic progression in alveolar rhabdomyosarcoma independently of its catalytic activity

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence. Patients with the most aggressive histological variant have an unfavorable prognosis due to a high metastasis incidence. Lysyl oxidase-like 2 (LOXL2) is a lysyl oxidase, member of a family of extracellular matrix (ECM) crosslinking enzymes that recently have emerged as important regulators of tumor progression and metastasis. We report that LOXL2 is overexpressed in RMS, suggesting a potential role for LOXL2 in RMS oncogenic progression. Consistently, transient and stable LOXL2 knockdown decreased cell migratory and invasive capabilities in two ARMS cell lines. Furthermore, introduction of LOXL2 in RMS non-expressing cells using wild type or mutated (catalytically inactive) constructs resulted in increased cell migration, cell invasion and number and incidence of spontaneous lung metastasis in vivo, independently of its catalytic activity. To further study the molecular mechanism associated with LOXL2 expression, a pull-down assay on LOXL2-transfected cells was performed and analyzed by mass spectrometry. The intermediated filament protein vimentin was validated as a LOXL2-interactor. Thus, our results suggest an oncogenic role of LOXL2 in RMS by regulating cytoskeleton dynamics and cell motility capabilities.

C-Myb regulates tumorigenic potential of embryonal rhabdomyosarcoma cells

Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors, the most common sub-types are embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma. Immunohistochemical analysis revealed c-Myb expression in both eRMS and aRMS. c-Myb has been reported to be often associated with malignant human cancers. We therefore investigated the c-Myb role in RMS using cellular models of RMS. Specific suppression of c-Myb by a lentiviral vector expressing doxycycline (Dox)-inducible c-Myb shRNA inhibited proliferation, colony formation, and migration of the eRMS cell line (RD), but not of the aRMS cell line (RH30). Upon c-Myb knockdown in eRMS cells, cells accumulated in G0/G1 phase, the invasive behaviour of cells was repressed, and elevated levels of myosin heavy chain, marker of muscle differentiation, was detected. Next, we used an RD-based xenograft model to investigate the role of c-Myb in eRMS tumorigenesis in vivo. We found that Dox administration did not result in efficient suppression of c-Myb in growing tumors. However, when c-Myb-deficient RD cells were implanted into SCID mice, we observed inefficient tumor grafting and attenuation of tumor growth during the initial stages of tumor expansion. The presented study suggests that c-Myb could be a therapeutic target in embryonal rhabdomyosarcoma assuming that its expression is ablated.

The histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) as a therapeutic agent in rhabdomyosarcoma

ABSTRACT Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21Cip1 and p27Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21Cip1 and p27Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects . Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.

Abstract 4630: Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy

Abstract Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often a t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1 which is critical for tumorigenesis and cell survival. Our aim here was to identify a pharmacological combination therapy approach interfering with PAX3-FOXO1 biology at different levels. Since loss of the fusion protein results in cell death, we first aimed to pharmacologically enhance this effect. To this end, we screened aRMS tumor cells with a library of 208 drugs while simultaneously silencing PAX3-FOXO1 by shRNA. This identified the BH3-mimetic ABT-263 to sensitize aRMS cells to cell death after PAX3-FOXO1 depletion. To further characterize the cell death mechanisms we used combined shRNA and CRISPR approaches to perform a BH3 protein profiling. In accordance with identification of ABT-263 we demonstrate that aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner upon depletion of PAX3-FOXO1. In a parallel approach, and to identify drugs altering PAX3-FOXO1 protein stability, we screened the same drug library directly measuring fusion protein levels as read-out. This revealed that inhibition of Aurora kinase A negatively affects PAX3-FOXO1 protein levels. Finally, using both aRMS cell lines and patient-derived xenografts we demonstrate that combination treatment of Aurora kinase A inhibitors together with ABT-263 synergistically induces cell death and greatly slows tumor growth in vitro and in vivo. Taken together, these data show a novel functional interaction of Aurora kinase A and PAX3-FOXO1 and suggest new opportunities for targeted combination treatment of aRMS. Citation Format: Johannes Ommer, Marco Wachtel, Beat W. Schaefer. Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy [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 4630.

PO-369 KDM3A/Ets1/MCAM axis in rhabdomyosarcoma and osteosarcoma

IntroductionRhabdomyosarcoma (RMS) and Osteosarcoma (OS) are the most common primary malignant tumours of soft tissue and bones, respectively, affecting children, adolescents and young adults. RMS can be divided into two major histopathological and molecular subtypes: driver oncofusion (PAX3/7-FOXO1) positive, alveolar (ARMS), and oncofusion-negative embryonal (ERMS). Oncofusion positive ARMS carries a worse prognosis, in part due to higher propensity for metastasis. OS is also an aggressive disease with high propensity for metastasis. Patients with metastatic RMS and OS have few therapeutic options, including currently no effective targeted therapies, and typically face poor outcomes. Our laboratory has previously identified a novel, potentially druggable, tumour and metastasis-promotional, axis in Ewing Sarcoma, another aggressive bone and soft tissue cancer of childhood, involving the histone demethylase KDM3A, the Ets1 transcription factor, and the cell adhesion protein MCAM. We have additionally observed KDM3A to be overexpressed in RMS, and in a subset of OS. The aim of this study was to examine whether the KDM3A/Ets1/MCAM axis is intact in RMS and OS, and what impact it has on the biology of these diseases.Material and methodsRMS and OS cell lines were analysed for KDM3A, Ets1 and MCAM expression, and selected cell lines were subjected to stable shRNA-mediated KDM3A, Ets1 and MCAM knockdown (KD). Phenotypic effects of knockdown were analysed by clonogenic and transendothelial invasion assays. Preliminary tumour xenograft studies were also performed.Results and discussionsKDM3A is uniformly overexpressed in ERMS and ARMS cell lines and a subset of OS cell lines. KDM3A KD inhibits colony formation and transendothelial invasion in ERMS and ARMS cells and a high-expressing OS cell line. In preliminary studies KDM3A KD also inhibits ARMS tumorigenesis in an orthotopic model. Strikingly, similar to our prior studies in Ewing Sarcoma, a KDM3A/Ets1/MCAM regulatory axis is intact in RMS and OS, and MCAM KD exerts similar phenotypic effects to those achieved with KDM3A KD.ConclusionOur studies indicate that a KDM3A/Ets1/MCAM molecular axis is intact in RMS and OS, and regulates growth and invasive properties. Together with our previous studies in Ewing Sarcoma, these findings suggest that this may be an important tumour and metastasis promotional axis in all three common paediatric sarcomas, despite distinct cellular origins of these cancers.

Abstract 890: Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma

Abstract Reciprocal chromosomal translocations resulting in the fusion of the DNA binding domain of the transcription factor PAX3 and the transactivational domain of FOXO1 underlie most cases of alveolar rhabdomyosarcoma (aRMS). Testing of compound MTPF63 against a panel of sarcoma cell lines showed that it had an inhibitory effect on proliferation of PAX3-FOXO1 translocation-positive cells. Further evaluation of its activity in Rh4 cells transduced with a PAX3-FOXO1 luciferase reporter showed that it attenuated reporter activity. Additional confirmation for the compound's inhibitory impact on PAX3-FOXO1 activity was obtained by detecting significant attenuation in the levels of Alk1 and MyoD1 proteins whose expression is driven by PAX3-FOXO1 in aRMS cells. Cell viability assays performed under 2D and 3D culturing conditions demonstrated that MTPF63 inhibits cell growth more effectively in translocation-positive alveolar RMS than in translocation-negative embryonal RMS. A hypothesis for MTPF63's mode of action was generated using the complete pool of barcoded essential heterozygous (~1150 strains) and homozygous (~4800 strains) diploid gene deletion mutants of Saccharomyces cerevisiae to identify gene deletions that confer sensitivity to the compound. Gene ontology enrichment analysis of 255 sensitive mutants whose growth rates were significantly inhibited in the presence of the compound compared to control, not only revealed calmodulin as main target of the compound but also illustrated the compound's interference with calmodulin-dependent nuclear protein import pathway, suggesting that it might impact PAX3-FOXO1 nuclear localization. Immunostaining of nuclear-localized PAX3-FOXO1 oncoprotein in two aRMS cell lines revealed cytoplasmic localization. Immunoblot analysis of fractionated cytosolic and nuclear protein lysates for PAX3-FOXO1 in MTPF63 treated cells also showed increased amounts in the cytosolic fractions. Transient overexpression of calmodulin was shown to partially rescue the inhibitory effect of the compound on PAX3-FOXO1-driven luciferase expression in RH4 reporter cells. Taken together, the data demonstrate the involvement of calmodulin in the nuclear import of PAX3-FOXO1 and introduce calmodulin antagonists as potential therapeutics against translocation-positive childhood alveolar rhabdomyosarcoma. Citation Format: Emad Darvishi, Cheryl Lynn Thomas, Berkley Eric Gryder, John Frederick Shern, Javed Khan, Girma M Woldemichael. Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma [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 890.

The Transcriptional Coactivator TAZ Is a Potent Mediator of Alveolar Rhabdomyosarcoma Tumorigenesis.

Purpose: Alveolar rhabdomyosarcoma (aRMS) is a childhood soft tissue sarcoma driven by the signature PAX3-FOXO1 (P3F) fusion gene. Five-year survival for aRMS is <50%, with no improvement in over 4 decades. Although the transcriptional coactivator TAZ is oncogenic in carcinomas, the role of TAZ in sarcomas is poorly understood. The aim of this study was to investigate the role of TAZ in P3F-aRMS tumorigenesis.Experimental Design: After determining from publicly available datasets that TAZ is upregulated in human aRMS transcriptomes, we evaluated whether TAZ is also upregulated in our myoblast-based model of P3F-initiated tumorigenesis, and performed IHC staining of 63 human aRMS samples from tissue microarrays. Using constitutive and inducible RNAi, we examined the impact of TAZ loss of function on aRMS oncogenic phenotypes in vitro and tumorigenesis in vivo Finally, we performed pharmacologic studies in aRMS cell lines using porphyrin compounds, which interfere with TAZ-TEAD transcriptional activity.Results: TAZ is upregulated in our P3F-initiated aRMS model, and aRMS cells and tumors have high nuclear TAZ expression. In vitro, TAZ suppression inhibits aRMS cell proliferation, induces apoptosis, supports myogenic differentiation, and reduces aRMS cell stemness. TAZ-deficient aRMS cells are enriched in G2-M phase of the cell cycle. In vivo, TAZ suppression attenuates aRMS xenograft tumor growth. Preclinical studies show decreased aRMS xenograft tumor growth with porphyrin compounds alone and in combination with vincristine.Conclusions: TAZ is oncogenic in aRMS sarcomagenesis. While P3F is currently not therapeutically tractable, targeting TAZ could be a promising novel approach in aRMS. Clin Cancer Res; 24(11); 2616-30. ©2018 AACR.

PPP2R1A regulated by PAX3/FOXO1 fusion contributes to the acquisition of aggressive behavior in PAX3/FOXO1-positive alveolar rhabdomyosarcoma.

To better characterize the oncogenic role of the PAX3-FOXO1 fusion protein in the acquisition of aggressive behavior in ARMS, we employed a proteomic approach using a PAX3-FOXO1 knockdown system in ARMS cell lines. This approach revealed a protein list consisting of 107 consistently upregulated and 114 consistently downregulated proteins that were expected to be regulated by PAX3-FOXO1 fusion protein. Furthermore, we identified 16 upregulated and 17 downregulated critical proteins based on a data-mining analysis. We also evaluated the function of PPP2R1A in ARMS cells. The PPP2R1A expression was upregulated at both the mRNA and protein levels by PAX3-FOXO1 silencing. The silencing of PPP2R1A significantly increased the cell growth of all four ARMS cells, suggesting that PPP2R1A still has a tumor suppressive function in ARMS cells; however, the native expression of PPP2R1A was low in the presence of PAX3-FOXO1. In addition, the activation of PP2A—part of which was encoded by PPP2R1A—by FTY720 treatment in ARMS cell lines inhibited cell growth. On the human phospho-kinase array analysis of 46 specific Ser/Thr or Tyr phosphorylation sites on 39 selected proteins, eNOS, AKT1/2/3, RSK1/2/3 and STAT3 phosphorylation were decreased by FTY-720 treatment. These findings suggest that PPP2R1A is a negatively regulated by PAX3-FOXO1 in ARMS. The activation of PP2A—probably in combination with kinase inhibitors—may represent a therapeutic target in ARMS. We believe that the protein expression profile associated with PAX3-FOXO1 would be valuable for discovering new therapeutic targets in ARMS.

The proprotein convertase furin is required to maintain viability of alveolar rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Success of current therapies is still limited and outcome is particularly poor for metastatic alveolar rhabdomyosarcoma (aRMS). We previously identified the proprotein convertase furin as potential target for specific drug delivery with RMS-homing peptides. Furin is a protease that converts inactive precursor proteins into bioactive proteins and peptides. In this study, we investigate the biological role of furin in aRMS progression in vitro and in vivo. Furin expression was confirmed in over 86% RMS biopsies in a tissue microarray (n=89). Inducible furin silencing in vitro led to significant impairment of cell viability and proliferation in all investigated aRMS cell lines, but not in MRC5 fibroblasts. Furthermore, the aRMS cell lines Rh3 and Rh4 revealed to be very sensitive to furin silencing, undergoing caspase-dependent cell death. Notably, furin silencing in vivo led to complete remission of established Rh4 tumors and to delayed growth in Rh30 tumors. Taken together, these findings identify furin as an important factor for aRMS progression and survival. Thus, we propose furin as a novel therapeutic target for treatment of aRMS.

Abstract 2452: Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma

Abstract Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells. Tumor exosomes contain intact and functional protein, mRNA and miRNA that may alter the cellular environment to favor tumor growth. Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, with two distinct subtypes, alveolar (ARMS) and embryonal (ERMS) histology. ARMS tumors are characterized in the majority of cases by a fusion oncoprotein PAX3-FOXO1, thought to be an initiating factor in tumorigenesis. To date, no studies have been performed to investigate the possible role or content of exosomes in paracrine signaling in RMS. In the present study, we isolated and characterized exosomes secreted from RMS cells, using 3 ERMS (fusion oncoprotein negative) and 2 ARMS (fusion oncoprotein positive) cell lines. RNA analysis showed that small RNA were enriched in RMS-derived exosomes, therefore we focused on evaluating the miRNA content. Array expression analysis showed that exosome miRNA clustered together well, and to a higher extent than cellular miRNA, in ARMS cell lines. Similarly, in the 2 ERMS cell lines that had mutant p53, exosome miRNA clustered together and the pattern of expression was very different from the corresponding cellular miRNA. ARMS and ERMS exosome-enriched miRNA were different, and only 2 miRNA were found to be common among both; putative targets are implicated in cancer and inflammation. Functionally, using in vitro assays, we found that both ERMS- and ARMS-derived exosomes significantly increased the cellular migration and invasion of normal human fibroblasts, and had a positive effect on viability and proliferation of both fibroblasts and RMS cells. In vivo, Matrigel plug assay showed that RMS-derived exosomes had a positive effect on the migration and invasion of stromal cells, suggesting a positive role on angiogenesis. We conclude that RMS-derived exosomes can exert specific paracrine effects on recipient cells, enhancing cell viability as well as invasive properties of fibroblasts and stromal cells, and therefore is likely to play a role in promoting RMS angiogenesis and metastasis. Ongoing studies are aimed at directly assessing the role of RMS-derived exosomes in clinical angiogenesis and metastasis, using in vivo murine models, in addition to dissecting the role of specific enriched miRNA on recipient cell biology. Citation Format: Sandra E. Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Raya Saab. Exosomes as mediators of paracrine signalling that promote invasive behavior in 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 2452.

Secreted Frizzled-Related Protein 3 (SFRP3) Is Required for Tumorigenesis of PAX3-FOXO1-Positive Alveolar Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma associated with the skeletal muscle lineage. Of the two predominant subtypes, known as embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a five-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3-FOXO1. As PAX3-FOXO1 has proven chemically intractable, this study aims to identify targetable proteins that are downstream from or cooperate with PAX3-FOXO1 to support tumorigenesis. Microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PAX3-FOXO1 revealed alteration of several Wnt pathway gene members, including secreted frizzled related protein 3 (SFRP3), a secreted Wnt pathway inhibitor. Loss-of-function using shRNAs against SFRP3 was used to interrogate the role of SFRP3 in human aRMS cell lines in vitro and conditional murine xenograft systems in vivo. The combination of SFRP3 genetic suppression and the chemotherapeutic agent vincristine was also examined. In vitro, suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. In vivo, doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold, in addition to increasing myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.

Highlights of This Issue

The majority of alveolar rhabdomyosarcomas (aRMS) are driven by the oncogenic fusion protein PAX3-FOXO1. Novel therapies are needed to target PAX3-FOXO1 or pathways supporting its tumorigenic properties. To this end, Kephart and colleagues conducted a screen for genes that collaborate with PAX3-FOXO1, and identified the secreted Wnt inhibitor SFRP3. Genetic suppression of SFRP3 in aRMS cell lines decreased proliferation and increased apoptosis and differentiation. In a murine xenograft model, suppression of SFRP3 inhibited aRMS tumor growth and sensitized tumors to vincristine, part of the chemotherapeutic backbone of RMS therapy, suggesting that targeting SFRP3 may be a promising therapeutic strategy.Hong and colleagues conducted a phase I clinical trial of lenvatinib—a multikinase inhibitor of VEGFR 1–3, FGFR 1–3, PDGFRα, RET and KIT—in patients with solid tumors. The maximum tolerated dose was 10 mg twice daily. Drug-related toxicities included hypertension (43%), fatigue (42%), proteinuria (39%), and nausea (25%). Twelve patients (15.6%) achieved partial response (PR, n = 9) or unconfirmed PR (n = 3); 19 (24.7%) achieved stable disease ≥23 weeks. The toxicity profile and antitumor activity of lenvatinib are encouraging.Little is known about miRNAs in the development and treatment of T-cell lymphoblastic lymphoma (T-LBL). Using microRNA microarrays, Qian and colleagues reported, for the first time, that the downregulation of miR-374b was frequently detected in primary T-LBL tissues, which was significantly associated with worse overall survival and increased risk of recurrence. Moreover, in vitro and in vivo studies showed that miR-374b suppressed cell tumorigenicity and promoted cellular apoptosis by inhibiting AKT signal pathway though directly repressing Wnt-16 and AKT1. Targeting miR-374b pathway may represent novel prognostic biomarkers and new therapeutic strategies to improve the therapy and survival of patients with T-LBL.Cancer-associated fibroblasts (CAFs) have been associated to recurrence and survival in colon cancer. After proteomic characterization, the authors identified LOXL2 as an upregulated protein in purified colon CAFs. High expression of LOXL2 showed a clear association, either disease-free or overall survival in colon cancer patients. In addition, it displayed a significant prognostic value in the stratification of high-risk stage II and III patients. Thus, LOXL2 shows promise as a relevant tool in clinical practice for risk classification, which could lead to a more aggressive therapy and better outcomes for stage II patients.

Abstract 499: Characterization of the cell death mechanism after silencing of PAX3-FOXO1 in alveolar rhabdomyosarcoma using a CRISPR-Cas mediated knockout approach

Abstract Chromosomal translocations leading to the generation of PAX-FOXO1 fusion oncogenes are found in about 80 percent of all alveolar Rhabdomyosarcoma (aRMS). Recent whole genome sequencing studies revealed that apart from these translocations mutation frequency is generally very low in aRMS, with no other genetic changes detected in some cases. aRMS tumor cells depend on the fusion protein and exhibit cell cycle arrest or undergo cell death upon blocking of its activity. Taken together this strongly suggests that the PAX-FOXO1 fusion represents the tumor initiating event and that it remains the most important tumorigenic factor for aRMS maintenance at later tumor stages. However, the molecular mechanisms involved in tumorigenesis downstream of PAX3-FOXO1 are only partially understood yet, including the ones involved in oncogene addiction. We therefore aimed to further characterize the molecular events leading to cell death after silencing of PAX3-FOXO1. Towards this aim, we used an inducible shRNA system to silence PAX3-FOXO1 in different aRMS cell lines. Reduction of PAX3-FOXO1 below a threshold of about 20 percent of its basal level efficiently induced Caspase-dependent cell death. CRISPR-Cas mediated knock-out of individual members of the apoptotic cascade was then used to further characterize the involved apoptotic mechanism. BH3-only proteins including Bim were found as relevant sensors detecting cellular stress after silencing of PAX3-FOXO1. This data suggests that combined interference with PAX-FOXO1 activity and anti-apoptotic Bcl-2 family members might accelerate tumor cell death. Citation Format: Marco Wachtel, Felix Niggli, Beat Schäfer. Characterization of the cell death mechanism after silencing of PAX3-FOXO1 in alveolar rhabdomyosarcoma using a CRISPR-Cas mediated knockout approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 499. doi:10.1158/1538-7445.AM2015-499

Abstract A57: The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis

Abstract Introduction: This study aims to understand the contribution of the Wnt pathway inhibitor secreted frizzled related protein 3 (SFRP3) to alveolar rhabdomyosarcoma tumorigenesis. Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of &amp;lt;30%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, our goal is to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition. Experimental procedures: Microarray analysis using the Affymetrix U133A platform was performed to compare the transcriptomes of human skeletal muscle (HSMM) cells stably expressing either an empty vector or PF. Only genes that were differentially regulated at least three-fold were considered for further study. The microarray was validated and SFRP3 mRNA levels were assessed using RT-PCR. Lentiviral technology was used to stably express shRNAs in human PF-positive aRMS cell lines Rh28 and Rh30. Cell growth, cell proliferation, apoptosis, and cell cycle analysis were measured by MTT assay, BrdU assay, immunoblot for cleaved caspase 3, and by PI staining followed by analysis by flow cytometry, respectively. For the xenograft tumors in mice, doxycycline-inducible SFRP3 shRNAs were stably expressed in aRMS cells. These cells were injected into the flanks of SCID/beige mice to generate the tumors. Induction of the shRNAs by doxycycline administration began once the tumors were palpable. All animal work was performed under institution approved IACUC protocols. Results: In a microarray analysis of the transcriptomes of human skeletal muscle myoblasts (HSMM) with an empty vector or PF, it was noted that the Wnt pathway was preferentially represented in HSMMs with PF. Of these, SFRP3 was selected for further investigation as its mRNA levels were elevated in PF-positive aRMS cells, but not eRMS or HSMM cells. shRNAs against SFRP3 were generated and stably expressed in Rh28 and Rh30 cells, validated to suppress SFRP3, and shown to profoundly decrease cell growth (p&amp;lt;0.001). Associated with this decreased cell growth was a decrease in cell proliferation (p&amp;lt;0.001), an increase in apoptosis, and an increase in the percentage of cells at G1, suggesting a cell cycle arrest. Consistent with a G1 cell cycle arrest, shRNAs against SFRP3 also increased the levels of p21, an inhibitor of cell cycle progression. To assess the effects of SFRP3 shRNAs on tumor growth in a xenograft model of aRMS, a doxycycline-inducible system was used, allowing the shRNAs to be expressed only after the tumors formed. In this model, suppression of SFRP3 reduced tumor growth and tumor weight by more than three-fold. Further, in a pilot study, the combination of SFRP3 suppression with vincristine, a standard chemotherapeutic agent used in the treatment of aRMS, was more effective at reducing cell growth in vitro than with either treatment alone and ablated tumor growth in vivo. Conclusions: In conclusion, SFRP3 is necessary for the growth of aRMS cells both in vitro and in vivo. Inhibition of SFRP3 combined with standard of care chemotherapy should be explored further as SFRP3 is a promising new target in the treatment of PF-positive aRMS. Citation Format: Julie Kephart, Rosanne Jones, Lisa Crose, Jen-Tsan Ashley Chi, Corinne Linardic. The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A57.

Abstract LB-200: Potent antitumor activity of MLN0128, an investigational selective mTORC1/2 kinase inhibitor, in preclinical models of rhabdomyosarcoma

Abstract Phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis regulates growth and homeostasis, and is often dysregulated in rhabdomyosarcoma. Agents that target mammalian target of rapamycin complex 1 (mTORC1), one of the two multi-protein complexes comprising the serine-threonine kinase mTOR, have been disappointing in clinical trials, and novel therapeutics are urgently needed to treat patients with relapsed, refractory, or metastatic alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. It is hypothesized that modest clinical results with mTORC1 only inhibitors may be attributed to the lack of inhibition of the second mTOR complex, mTORC2, and its downstream effectors, as well as release of mTOR-mediated negative feedback of the pathway that leads to activation of receptor tyrosine kinases and feedback activation of AKT. In the present preclinical study, MLN0128, a selective and highly potent inhibitor of both mTORC1 and mTORC2, was tested for its antitumor activity in vitro and in a mouse xenograft model. Data suggest that the anti-proliferative effect of MLN-0128 was superior to rapamycin, a selective mTORC1 inhibitor, in a broad panel of ERMS and ARMS cell lines where it was found to be highly effective at low concentrations from 5 to 175 nanomolar. Western immunoblot analysis of treated cells showed inhibition of phosphorylation of mTORC1 and mTORC2 targets including phosphorylated S6 ribosomal protein (pS6), phospho-p70-S6K, phospho-AKT-S473, phospho-AKT-T308, and phospho-4E-BP1. Apoptosis in both ERMS and ARMS cell lines was confirmed by the presence of cleaved poly ADP ribose. Additionally, MLN0128 was found to have superior anti-tumor effect in a xenograft model of rhabdomyosarcoma without significant toxicity when compared with rapamycin. These pre-clinical studies support further research of MLN0128 as a potential therapeutic approach for patients with ARMS and ERMS. Citation Format: Emily Slotkin, Parag Patwardhan, Shyamprasad Vasudeva Deraje, Gary Schwartz, Tap William. Potent antitumor activity of MLN0128, an investigational selective mTORC1/2 kinase inhibitor, in preclinical models of rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2014-LB-200

Abstract 4002: Exploring the miRNA and proteomic contents of exosomes isolated from rhabdomyosarcoma tumor cells to understand their paracrine signaling

Abstract Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, with two distinct subtypes, alveolar (ARMS) and embryonal (ERMS) histologies. The alveolar subtype is characterized by a specific translocation PAX-FOXO, the protein product of which is thought to contribute to its aggressive and metastatic behaviour. Exosomes are small membranous vesicles (30-100 nm in diameter) secreted into body fluids by multiple cell types, including tumor cells. Tumor exosomes contain intact and functional protein and microRNA that can alter the cellular environment to favor tumor growth. We hypothesize that the PAX-FOXO fusion protein results in specific effects on exosome cargo and biology, contributing to the invasive and metastatic potential of ARMS cells. Thus proteomic and molecular profiling of their exosomes may increase our understanding of the role of exosomes in sarcoma progression and may lead to discovery of useful biomarkers. In the present study, we used miRNA array profiling to identify interesting exosomal miRNA expressed commonly in two different ARMS cell lines. We also used proteomic analysis and discovered differentially expressed ARMS exosomal proteins. Importantly, functional studies revealed that normal fibroblasts acquired invasion and migration ability through molecules transported in ARMS cell-derived exosomes. Our results indicate that ARMS-derived exosomes have unique gene expression signatures,miRNA and proteomics profiles which will help our understanding of sarcoma biogenesis and that could be used as biomarkers for early diagnosis of ARMS, and targets for therapeutic intervention. Citation Format: Sandra E. Ghayad, Farah Ghamloush, Raya Saab. Exploring the miRNA and proteomic contents of exosomes isolated from rhabdomyosarcoma tumor cells to understand their paracrine signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4002. doi:10.1158/1538-7445.AM2014-4002

Abstract 3109: ALK as a valid therapeutic target for the treatment of rhabdomyosarcoma

Abstract Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor tyrosine kinase, a member of the insulin receptor family, with a tightly controlled expression pattern. Deregulated ALK expression has been linked to the development of several types of cancers. Typically, chromosomal translocations involving ALK generate constitutively expressed chimeric oncoproteins, such as NPM-ALK in anaplastic large cell lymphoma and EML4-ALK in non-small cell lung carcinoma. However, activating mutations in ALK, and increased copy number that result in aberrant gene expression, have also been observed in neuroblastoma and colorectal cancers. Based on the above observations, several small molecule ALK inhibitors have been developed and are in clinical trials for a variety of tumors expressing this kinase. This has resulted in the use of crizotinib for the treatment of EML4-ALK + lung cancers. Recently several large scale immunohistochemistry and genome sequencing studies of pediatric rhabdomyosarcoma (RMS) revealed aberrant ALK expression. It was found that ALK mRNA expression was significantly different in alveolar (ARMS) and embryonic RMS (ERMS), the two major histological subtypes in children. Thus, ALK expression might play a role in tumorigenesis for this disease, and this kinase may represent a valid therapeutic target for the treatment of RMS. To answer this question we analyzed ALK expression in a large panel of RMS cell lines, and pediatric tumor samples by quantitative PCR and immunoblotting. We also compared the sensitivity of these cells to a series of ALK inhibitors. Our results indicate that the majority of ARMS cell lines demonstrated significantly higher ALK mRNA expression as compared to ERMS, and to normal human myoblast control cells. This was confirmed by western analysis. Growth inhibition assays confirmed that all RMS lines were more sensitive to crizotinib and LDK-378 than control lines, consistent with the hypothesis that ALK is a driver of cell growth in these tumors. Based on our in vitro analysis, we suggest that ALK inhibition may represent an effective therapeutic modality for the treatment of ALK+ RMS. Preclinical studies to confirm or disprove this hypothesis are currently underway. Supported by St. Jude Children's Research Hospital and by the American Lebanese Syrian Associated Charities Citation Format: Monika Wierdl, Lyudmila Tsurkan, Viktor Tollemar, Lying Chi, Elizabeth Stewart, Michael A. Dyer, Philip M. Potter. ALK as a valid therapeutic target for the treatment of rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3109. doi:10.1158/1538-7445.AM2014-3109