Aggressive Soft-tissue Malignancy Research Articles

A phase 2 study of alrizomadlin, a novel MDM2/p53 inhibitor, in combination with pembrolizumab for treatment of patients with malignant peripheral nerve sheath tumor (MPNST).

e14627 Background: MPNST is a rare and aggressive soft tissue malignancy accounting for 10% of all soft tissue sarcomas. This disease is characterized by high risk of local recurrence and distant metastasis, and there is no current effective standard of care treatment option for patients with MPNST, with the only curative option of complete resection. Therefore, further treatment options are warranted. Investigational mouse double minute 2 (MDM2) inhibitor alrizomadlin restores TP53 function, activating p53-mediated apoptosis in tumor cells. Alrizomadlin also functions as a host immunomodulator and may restore antitumor activity in patients with MPNST that progressed on PD-1/PD-L1 inhibitors. Methods: This US/Australian multicenter trial evaluated alrizomadlin combined with PD-1 inhibitor pembrolizumab in patients with MPNST that progressed on available therapy or in those for whom therapy was unavailable. ECOG performance status was between 0-2 for eligible patients. Alrizomadlin 150 mg was administered orally every other day for 2 consecutive weeks, with 1 week off, and pembrolizumab 200 mg intravenously for 30 minutes on Day 1 of a 21-day cycle. Results: As of December 12, 2022, preliminary clinical results are reported for 24 patients with MPNST, of whom 16 were male, with a median (range) age of 35 (14-73) years. Treatment (alrizomadlin or pembrolizumab)-related adverse events of any grade (>10%) were reported in 18 patients. They included nausea (41.7%), vomiting and anemia (37.5% each), fatigue and diarrhea (33.3% each), thrombocytopenia (25.0%), and decreased leukocyte and lymphocyte counts (12.5% each). A total of 12.5% of patients reported treatment-related serious adverse events, including colitis, abdominal pain, diarrhea, and pericardial effusion. Among17 efficacy evaluable patients, the clinical benefit rate, as defined by the combination of overall response rate and stable disease for ≥ 4 cycles, was 53% per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v.1.1) and iRECIST. The median (range) treatment duration was 10 (4-20) cycles. Conclusions: Alrizomadlin combined with pembrolizumab is well tolerated and demonstrates meaningful clinical benefit in patients with MPNST for whom standard of care therapy is unavailable. Internal study identifiers: APG-115-US-002. Clinical trial information: NCT03611868 .

The importance of fusion protein activity in Ewing sarcoma and the cell intrinsic and extrinsic factors that regulate it: A review.

Accumulating evidence shows that despite clonal origins tumors eventually become complex communities comprised of phenotypically distinct cell subpopulations. This heterogeneity arises from both tumor cell intrinsic programs and signals from spatially and temporally dynamic microenvironments. While pediatric cancers usually lack the mutational burden of adult cancers, they still exhibit high levels of cellular heterogeneity that are largely mediated by epigenetic mechanisms. Ewing sarcomas are aggressive bone and soft tissue malignancies with peak incidence in adolescence and the prognosis for patients with relapsed and metastatic disease is dismal. Ewing sarcomas are driven by a single pathognomonic fusion between a FET protein and an ETS family transcription factor, the most common of which is EWS::FLI1. Despite sharing a single driver mutation, Ewing sarcoma cells demonstrate a high degree of transcriptional heterogeneity both between and within tumors. Recent studies have identified differential fusion protein activity as a key source of this heterogeneity which leads to profoundly different cellular phenotypes. Paradoxically, increased invasive and metastatic potential is associated with lower EWS::FLI1 activity. Here, we review what is currently understood about EWS::FLI1 activity, the cell autonomous and tumor microenvironmental factors that regulate it, and the downstream consequences of these activity states on tumor progression. We specifically highlight how transcription factor regulation, signaling pathway modulation, and the extracellular matrix intersect to create a complex network of tumor cell phenotypes. We propose that elucidation of the mechanisms by which these essential elements interact will enable the development of novel therapeutic approaches that are designed to target this complexity and ultimately improve patient outcomes.

Abstract IA004: Mesenchymal progenitors and sarcomagenesis

Abstract Synovial sarcoma (SS) is an aggressive soft-tissue malignancy that can arise in any area of the body, and most frequently affects adolescents and young adults. The 10-year survival rate has been estimated at 50%. SS is characterized by a pathognomonic t(X;18)(p11.2;q11.2) translocation which produces a fusion oncogene named SS18-SSX. In mouse models, expression of SS18-SSX is sufficient to drive the development of SS-like tumors. Despite recent advancements in our understanding of SS biology, the cell of origin remains undefined. In most instances, it is likely mesenchymal in nature. We previously generated a mesenchymal progenitor (MP) specific Cre line that has enabled us to identify MPs within the embryo [MOU1], and characterize and define their lineage trajectories through development into the adult. This line has been used to generate a unique mouse model of SS where the human SS18-SSX fusion oncogene with an EGFP marker is conditionally expressed in adult MPs. This model shows 100% penetrant SS tumor formation with a median latency period of 16 weeks. Histologic analyses of tumors reveal characteristic cellular morphology and the expression of common markers of SS. Single-cell (sc) RNA-seq gene expression profiles of murine tumors recapitulate those observed in human SS samples and highlight genetic programs involved in sarcomagenesis. Using this model, we can identify and isolate neoplastic lesions prior to the development of grossly visible tumors and trace these cells through the complete transformation continuum. By comparing tumor cells to normal MPs, we aim to better understand the mechanisms underlying the SS18-SSX induced transformation of MPs. Together, this model identifies a cell of origin for SS and provides us with a unique opportunity to address the molecular and cellular drivers of SS. Citation Format: T. Michael Underhill. Mesenchymal progenitors and sarcomagenesis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA004.

Tumor suppressive functions of WNT5A in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a highly aggressive soft tissue malignancy that predominantly affects children. The main subtypes are alveolar RMS (ARMS) and embryonal RMS (ERMS) and the two show an impaired muscle differentiation phenotype. One pathway involved in muscle differentiation is WNT signaling. However, the role of this pathway in RMS is far from clear. Our recent data showed that the canonical WNT/β-Catenin pathway serves a subordinate role in RMS, whereas non-canonical WNT signaling probably is more important for this tumor entity. The present study investigated the role of WNT5A, which is the major ligand of non-canonical WNT signaling, in ERMS and ARMS. Gene expression analysis showed that WNT5A was expressed in human RMS samples and that its expression is more pronounced in ERMS. When stably overexpressed in RMS cell lines, WNT5A decreased proliferation and migration of the cells as demonstrated by BrdU incorporation and Transwell migration or scratch assay, respectively. WNT5A also decreased the self-renewal capacity and the expression of stem cell markers and modulates the levels of muscle differentiation markers as shown by sphere assay and western blot analysis, respectively. Finally, overexpression of WNT5A can destabilize active β-Catenin of RMS cells. A WNT5A knockdown has opposite effects. Together, the results suggest that WNT5A has tumor suppressive functions in RMS, which accompanies downregulation of β-Catenin.

Abstract ND09: The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers

Abstract Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [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 ND09.

MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation.

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant (P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson's clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

Ewing Sarcoma of the 9th Rib Subsequent to Pediatric Leukemia: A Case Series.

Ewing sarcoma is an aggressive malignancy of bone and soft tissue that accounts for ∼2% of cases of childhood cancer. It has been rarely reported as a secondary neoplasm. Data from the Childhood Cancer Survivor Study has evaluated secondary sarcomas in 5-year survivors of childhood cancer. We report 2 pediatric patients in northeast Pennsylvania, who developed secondary Ewing sarcoma of the 9th rib within 5 years of primary childhood leukemia diagnoses.

Abstract 3035: Identifying potential druggable targets for synovial sarcoma using comparative RNA-seq analysis

Abstract Synovial sarcoma (SS) is an aggressive soft-tissue malignancy, accounting for 10% of all soft-tissue sarcomas. These tumors can occur at any age but most often affect young adults and adolescents, developing deep in the distal extremities. The prognosis of SS tumors is poor, with a 5-year survival rate of 36-76%, a high rate of metastasis and few treatments. The purpose of this study was to identify novel overexpressed oncogenes that could serve as druggable targets for treating synovial sarcoma patients. We compared the RNA-Seq expression profiles of a cohort of 36 synovial sarcomas to our compendium of RNA-Seq expression data from 12,236 tumor samples (treehousegenomics.ucsc.edu) from pediatric and adult cancer patients. In comparing gene expression in the synovial sarcoma cohort samples against the compendium samples, gene expression outliers were defined as having expression above the gene-specific outlier threshold as defined by the Tukey's outlier method. Among the overexpression outliers, pathway enrichment analysis was used to identify common and druggable pathways, with implications for potential therapeutics for patients with SS. Our analysis identified the overexpression of members of the Sonic Hedgehog pathway in the majority of synovial sarcoma samples. For example, GLI1 expression exceeded the outlier threshold in 35 out of 36 samples. This pathway can be targeted by available small molecule inhibitors. Ongoing work focuses on evaluating the role of Sonic Hedgehog signaling in the pathogenesis of SS using pharmacological inhibition, CRISPRi studies in cell line models of the disease and nanopore sequencing. We currently have 4 patient-derived synovial sarcoma cell lines (HSSY-II, SYO-1, YAMATO, and ASKA) that we can grow in both adherent conditions and in 3D cell culture as sarcospheres. We detected the expression of the SYT-SSX fusion transcript in each of the cell lines by RT-PCR to confirm the cell lines maintained expression of the pathogenic fusion. This work has implications for using comparative tumor RNA-seq derived gene expression data for nominating novel druggable targets specific to synovial sarcoma tumors. Citation Format: Yvonne A. Vasquez, Jacob Pfeil, Letitia Mueller, Holly Beale, Alfred G. Lyle, Lauren Sanders, Katrina Learned, Ellen Kephart, Anouk van den Bout, Allison Cheney, Sahar Hosseinzadeh, Isabel Bjork, Sofie R. Salama, Olena Vaske. Identifying potential druggable targets for synovial sarcoma using comparative RNA-seq analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3035.

FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma.

The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.

Ewing Sarcoma/Peripheral Neuroectodermal Tumor of Bone and Soft Tissue in Infants: A Report from Children Cancer Hospital of Egypt

Introduction: Ewing sarcoma is an aggressive malignancy of bone and soft tissue. It is rare in children under age 5years. Ewing's sarcoma family tumors include classic Ewing’s sarcoma, primitive neuroectodermal tumour and Askin tumor ES is the second most common variety of primary bone cancer in adolescents and young adults. The treatment strategy for ES is characterized by multi-disciplinary collaboration between pediatric oncologists, medical oncologists, radiation oncologists, and orthopedic surgeons. Although the survival rate of ES patients has improved, their prognosis remains unsatisfactory, and the treatment of ES is still challenging specially in age group below 3 years. Patients and methods: A Retrospective study of children below 3 years with a diagnosis Ewing Sarcoma/PNET treated at our CCHE from July 2009 till December 2018. Results: A review of 46 patients with age ranged from 3 months to 3 years, Mean of age was 1.8. Primary site was Osseous and extraosseus in 23 infant patients for each. Main site was mainly axial in 28 cases (60.8%). Cases were treated according to COG protocol. Infantile Ewing sarcoma OS and EFS at 5-years OS were 75.5% and 66.7%. Conclusion: Patients below 3 years represent an unusually young age group. Conclusion: ES/PNET in age below 3 years is a unique age group with a different management and outcome.

Desmoplastic small round cell tumor is dependent on the EWS-WT1 transcription factor

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft-tissue malignancy with a poor overall survival and no effective therapeutic options. The tumor is believed to be dependent on the continued activity of the oncogenic EWS-WT1 transcription factor. However, the dependence of the tumor on EWS-WT1 has not been well established. In addition, there are no studies exploring the downstream transcriptional program across multiple cell lines. In this study, we have developed a novel approach to selectively silence EWS-WT1 without impacting either wild-type EWSR1 or WT1. We show a clear dependence of the tumor on EWS-WT1 in two different cell lines, BER and JN-DSCRT-1. In addition, we identify and validate important downstream target pathways commonly dysregulated in other translocation-positive sarcomas, including PRC2, mTOR, and TGFB. Surprisingly, there is striking overlap between the EWS-WT1 and EWS-FLI1 gene signatures, despite the fact that the DNA-binding domain of the fusion proteins, WT1 and FLI1, is structurally unique and classified as different types of transcription factors. This study provides important insight into the biology of this disease relative to other translocation-positive sarcomas, and the basis for the therapeutic targeting of EWS-WT1 for this disease that has limited therapeutic options.

Ewing's sarcoma with distant metastasis: A brief note on management and emerging therapies.

Ewing’s sarcoma is an aggressive fatal malignancy of bones and soft-tissue. It predominantly affects the young population, with a worldwide incidence of three cases per million. The pelvis, extremities, and ribs are the most common sites. We present a case of massive Ewing’s sarcoma of the right femur with metastasis to bones and lungs. The patient was treated with chemotherapy. However, he succumbed to his illness before completion of therapy. In conclusion, Ewing’s sarcoma with distant metastasis is a high risk case with poor prognosis. Integrating novel molecular targets with conventional chemotherapeutic agents holds a promise for high-risk Ewing’s sarcoma patients.

Selective Targeting of Myoblast Fusogenic Signaling and Differentiation-Arrest Antagonizes Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue malignancy comprised histologically of skeletal muscle lineage precursors that fail to exit the cell cycle and fuse into differentiated syncytial muscle-for which the underlying pathogenetic mechanisms remain unclear. In contrast to myogenic transcription factor signaling, the molecular machinery that orchestrates the discrete process of myoblast fusion in mammals is poorly understood and unexplored in RMS. The fusogenic machinery in Drosophila, however, is understood in much greater detail, where myoblasts are divided into two distinct pools, founder cells (FC) and fusion competent myoblasts (fcm). Fusion is heterotypic and only occurs between FCs and fcms. Here, we interrogated a comprehensive RNA-sequencing database and found that human RMS diffusely demonstrates an FC lineage gene signature, revealing that RMS is a disease of FC lineage rhabdomyoblasts. We next exploited our Drosophila RMS-related model to isolate druggable FC-specific fusogenic elements underlying RMS, which uncovered the EGFR pathway. Using RMS cells, we showed that EGFR inhibitors successfully antagonized RMS RD cells, whereas other cell lines were resistant. EGFR inhibitor-sensitive cells exhibited decreased activation of the EGFR intracellular effector Akt, whereas Akt activity remained unchanged in inhibitor-resistant cells. We then demonstrated that Akt inhibition antagonizes RMS-including RMS resistant to EGFR inhibition-and that sustained activity of the Akt1 isoform preferentially blocks rhabdomyoblast differentiation potential in cell culture and in vivo. These findings point towards selective targeting of fusion- and differentiation-arrest via Akt as a broad RMS therapeutic vulnerability. SIGNIFICANCE: EGFR and its downstream signaling mediator AKT1 play a role in the fusion and differentiation processes of rhabdomyosarcoma cells, representing a therapeutic vulnerability of rhabdomyosarcoma.

Synovial sarcoma: when epigenetic changes dictate tumour development.

Synovial sarcoma is a highly aggressive soft tissue malignancy that often affects adolescents and young adults. It is associated with a unique chromosomal translocation that results in the formation and expression of the fusion gene SS18-SSX, which underlies its pathogenesis. Although SS18-SSX provides a potentially unique therapeutic target, all attempts to neutralise it have been unsuccessful thus far. When complete surgical removal of the tumour fails, therapy is limited to largely ineffective cytotoxic drug regimens. Nevertheless, recent discoveries about the mechanisms of SS18-SSX protein function have provided insight into potential alternative therapeutic strategies. SS18-SSX displays oncogenic activity through protein-protein interactions and participation in chromatin remodelling complexes. This review summarises our current understanding of the function of SS18-SSX and the mechanisms by which it alters the epigenetic landscape of permissive cells to induce transformation and the subsequent development of synovial sarcoma.

Abstract A06: Inhibition of CD99 activity by clofarabine as a novel therapeutic for Ewing sarcoma involves a novel molecular mechanism that is different than cytarabine

Abstract Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy of unknown primary origin. ES cells express high levels of CD99, a cell surface protein routinely used as a marker antigen in the histologic diagnosis of ES. A substantial body of evidence makes CD99 an attractive therapeutic target for patients with ES. We identified two structurally similar FDA-approved nucleoside (purine) analogues, clofarabine and cladribine, as specific CD99 inhibitors in a Biacore screening experiment. In validation experiments, both drugs exhibited selective cytotoxicity toward ES cells in a panel of 14 ES vs. 28 non-ES cell lines. A membrane-impermeable derivative of clofarabine, clofarabine-5′-triphosphate, showed a similar cytotoxicity on ES cell lines, suggesting inhibition of CD99 activity on cell surface as a likely mechanism by which the drug exerts its cytotoxic action without any effect on DNA metabolism. Clofrabine also inhibited the growth of three different ES xenografts in vivo. We performed a phosphokinase array to identify the mechanism behind how clofarabine may regulate cellular functions through inhibiting CD99. Mitogen- and stress-activated kinases 1 and 2 (MSK1/2) were the most significantly activated proteins by both CD99 antibody and clofarabine. Four of the 7 proteins with increased phosphorylation were related to MSK1/2, which were MSK1/2 itself, its substrates CREB and c-Jun, and its upstream kinase ERK1/2, thus representing the most likely intracellular pathway responsible for cell death due to CD99 inhibition. Clofarabine induced a significant increase in phosphorylation levels of MSK1/2 in cohort of ES xenografts, supporting its use as a potential pharmacodynamic marker of CD99 inhibition. We compared the activity of clofarabine with cytarabine, a structurally similar pyrimidine analog that has been identified as an inhibitor of EWS/FLI1 transcriptional activity but failed in clinical trials on ES patients. Cytarabine did not activate MSK1/2 phosphorylation in ES cells, suggesting that clofarabine may function through alternative mechanisms on ES cells that are different than cytarabine. Overall, our findings suggest that clofarabine directly binds to CD99 and inhibits its oncogenic activity through a novel mechanism in ES, and therefore it is a good candidate for early-phase clinical trials in children with ES. Citation Format: Haydar Çelik, Marika Sciandra, Maria Cristina Manara, Jeffrey Torestky, Katia Scotlandi, Aykut Üren. Inhibition of CD99 activity by clofarabine as a novel therapeutic for Ewing sarcoma involves a novel molecular mechanism that is different than cytarabine [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 A06.

Abstract 3176: CD99 regulates ERK1/2-MSK1/2-CREB axis in Ewing sarcoma

Abstract Ewing sarcoma (ES) is an aggressive malignancy of bone and soft tissue that affects predominantly children and young adults with a high propensity to metastasize and poor prognosis. ES cells express high levels of a cell surface protein CD99, which is routinely used as a marker antigen in the histological diagnosis of ES, and contributes to its pathogenesis. We performed a phospho-kinase array to uncover specific cellular responses evoked by CD99 inhibition in ES cells. The treatment of ES cells with CD99 antibody or a small molecule CD99 inhibitor (clofarabine) resulted in strong activation of mitogen- and stress-activated kinases 1 and 2 (MSK1/2) as well as its substrate CREB and its upstream kinase ERK1/2. These findings were further confirmed with western blot analysis in ES cells treated with either different CD99 antibodies or clofarabine. The treatment of osteosarcoma (OS) cells with clofarabine or the forced expression of CD99 in OS cells did not change their phosphorylation levels, suggesting that CD99-mediated ERK1/2-MSK1/2-CREB signaling cascade is specific to ES cells. Interestingly, knock-down of the CD99 protein in ES cells led to a significant decrease in ERK1/2 phosphorylation without any effect on the downstream phosphorylation of MSK1/2 and CREB. Cytarabine, a structurally similar pyrimidine analog that has been identified as an inhibitor of EWS/FLI1 transcriptional activity but failed in clinical trials on ES patients, did not activate ERK1/2-MSK1/2-CREB axis unlike clofarabine in ES cells. These findings suggest that clofarabine functions through alternative mechanisms on ES cells that are different than cytarabine. Clofarabine induced a significant increase in phosphorylation levels of MSK1/2 in a small cohort of TC-71 xenografts, supporting its use as a potential pharmacodynamic marker of CD99 inhibitor activity. In conclusion, our findings suggest that CD99 regulates ERK1/2-MSK1/2-CREB axis in ES, which represents an intracellular pathway that may be responsible for CD99's biological activities in ES. Citation Format: Haydar Celik, Erin J. Conn, David V. Allegakoen, Jeff R. Petro, Jeffrey A. Toretsky, Aykut Uren. CD99 regulates ERK1/2-MSK1/2-CREB axis in Ewing sarcoma [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 3176.

Abstract 2975: Synthetic lethality in synovial sarcoma: SS18-SSX fusions and DNA damage response (DDR) inhibitors

Abstract Aim: Synovial sarcoma (SS) is an aggressive soft-tissue malignancy predominantly affecting the young and characterised by pathognomonic SS18-SSX fusions. Treatment options are limited and therapeutic targeting of SS18-SSX oncoproteins has not yet been achieved. We carried out large-scale, multiplatform functional genomic screenings followed by extensive in vitro and in vivo validation studies to identify therapeutically actionable dependencies in SS with the highest translational potential. Methods: High-throughput drug sensitivity and siRNA screens including 79 clinically relevant compounds (0.5-1000nM) and siRNAs targeting 1600 different genes, respectively, were carried out in SS cell lines (SYO-1, HS-SY-II, Aska-SS, Yamato-SS and CME-1) and &amp;gt;115 cancer cell lines of other histologies in triplicate in 5-day survival assays. SS drug and siRNA sensitivity data was compared to sensitivity profiles of non-SS tumour cell lines (Mann-Whitney test). Validation inhibitory and mechanistic assays were performed with selected drugs and siRNA in SS cells, non-SS cells ectopically expressing SS18-SSX fusion proteins, and relevant other controls. Presence of replication fork stress biomarkers was assessed by (fluorescent) immunohistochemistry and DNA fibre analysis. Results: Both siRNA and drug assays identified a series of genes involved the DNA damage response (DDR) to be candidate genetic dependencies in SS. siRNA screen hits included ATR, the ATR-activating proteins RAD9A and RAD18, and the two tumour suppressor genes BRCA1 and BRCA2. The drug screen identified a dependency on two clinical PARP inhibitors (PARPi); talazoparib and rucaparib. ATR and PARP sensitivity in SS cells was comparable to that of tumour cells harbouring known molecular defects associated with sensitivity. A series of validation assays using clinical ATR (ATRi) and PARP inhibitors (PARPi) confirmed a synthetic lethal effect in SS tumour cell lines in vitro, which was confirmed for ATRi in patient-derived xenografts in vivo. Ectopic expression of SS18-SSX fusion proteins in non-SS cell lines elicited ATRi and PARPi sensitivity, which was mediated by a reduction in SS18 and SMARCB1 protein levels. ATRi sensitivity in SS was characterised by an increase in biomarkers of replication fork stress (increased yH2AX, decreased replication fork speed and increased R-loops), an apoptotic response and was found to be dependent upon Cyclin E expression. Finally, we found that combinations with cisplatin or PARP inhibitors enhanced the anti-tumour cell effect of ATRi, suggesting that either single agent ATRi or combination therapy involving ATRi might be further assessed as a candidate approach for SS treatment. Conclusions: Our integrated analysis identified an unexpected dependency of SS tumour cells on DDR mediators. Importantly, sensitivity was synthetic lethal with SS18-SSX oncoproteins and is clinically actionable. Citation Format: Emmy DG Fleuren, Sam Jones, Jessica Frankum, Chris Williamson, Malini Menon, Janet Shipley, Alan Ashworth, Winette van der Graaf, Christopher J. Lord. Synthetic lethality in synovial sarcoma: SS18-SSX fusions and DNA damage response (DDR) inhibitors [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 2975.

Clofarabine inhibits Ewing sarcoma growth through a novel molecular mechanism involving direct binding to CD99.

Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy that predominantly affects children and adolescents. CD99 is a cell surface protein that is highly expressed on ES cells and is required to maintain their malignancy. We screened small molecule libraries for binding to extracellular domain of recombinant CD99 and subsequent inhibition of ES cell growth. We identified two structurally similar FDA-approved compounds, clofarabine and cladribine that selectively inhibited the growth of ES cells in a panel of 14 ES vs. 28 non-ES cell lines. Both drugs inhibited CD99 dimerization and its interaction with downstream signaling components. A membrane-impermeable analog of clofarabine showed similar cytotoxicity in culture, suggesting that it can function through inhibiting CD99 independent of DNA metabolism. Both drugs drastically inhibited anchorage-independent growth of ES cells, but clofarabine was more effective in inhibiting growth of three different ES xenografts. Our findings provide a novel molecular mechanism for clofarabine that involves direct binding to a cell surface receptor CD99 and inhibiting its biological activities.

Abstract PR06: Functional genomics of CIC-DUX4 fusions in zebrafish recapitulates the spectrum of human CIC-rearranged cancers

Abstract CIC-DUX4 sarcomas are aggressive soft-tissue malignancies characterized by frequent metastases and poor survival. Histologically, CIC-DUX4 sarcomas resemble Ewing sarcomas but do not express EWS-FLI1 or other FET-ETS fusion genes typical of Ewing sarcoma. The tumors arise from chromosomal translocations that fuse the transcriptional repressor CIC with the homeodomain-containing protein DUX4, creating an aberrant transcription factor. However, the critical targets of CIC-DUX4 have been only partly described, the cell of origin and mechanism of transformation are unknown, and animal models of the disease are lacking. Owing to this poor biologic understanding, patients with CIC-DUX4 sarcomas are treated with the same regimens used for classical Ewing sarcoma, despite generally poor outcomes. To comprehensively address these problems, we are taking a cross-species comparative oncology approach via analysis of human CIC-DUX4 sarcomas and genetically engineered in vivo zebrafish models. Human CIC-DUX4 transgenes exerted strong effects on zebrafish embryonic development. Beginning at 5 weeks of age, almost 40% of injected animals developed invasive, rapidly growing tumors. Strikingly, two classes of tumors emerged: small round blue cell sarcomas developed in soft tissues including the trunk musculature and the ventral abdomen, and CNS-PNETs arose in the head. This recapitulates the spectrum of human tumors with CIC gene rearrangements. To further understand the molecular mechanisms of tumorigenesis, we performed RNASeq on zebrafish CIC-DUX4 sarcoma and zebrafish EWS-FLI1 sarcomas alongside human tumors and nonmalignant tissues. This analysis revealed pathways conserved between fish and human, including RAS-MAPK signaling, and highlighted the central importance of ETV4 in this disease. Further studies established ETV4 as a valuable new marker of the human disease. Taken together these findings demonstrate that CIC-DUX4 expression is sufficient to promote the development of tumors, give insight into the cellular origins of CIC-DUX4 tumors, and provide a valuable new animal model for translational research. Citation Format: Sarah Watson, Genevieve C. Kendall, Whitney A. Murchison, Olivier Delattre, Franck Tirode, James F. Amatruda. Functional genomics of CIC-DUX4 fusions in zebrafish recapitulates the spectrum of human CIC-rearranged cancers [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 PR06.

ATR Is a Therapeutic Target in Synovial Sarcoma

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by expression of SS18-SSX fusions, where treatment options are limited. To identify therapeutically actionable genetic dependencies in SS, we performed a series of parallel, high-throughput small interfering RNA (siRNA) screens and compared genetic dependencies in SS tumor cells with those in >130 non-SS tumor cell lines. This approach revealed a reliance of SS tumor cells upon the DNA damage response serine/threonine protein kinase ATR. Clinical ATR inhibitors (ATRi) elicited a synthetic lethal effect in SS tumor cells and impaired growth of SS patient-derived xenografts. Oncogenic SS18-SSX family fusion genes are known to alter the composition of the BAF chromatin-remodeling complex, causing ejection and degradation of wild-type SS18 and the tumor suppressor SMARCB1. Expression of oncogenic SS18-SSX fusion proteins caused profound ATRi sensitivity and a reduction in SS18 and SMARCB1 protein levels, but an SSX18-SSX1 Δ71-78 fusion containing a C-terminal deletion did not. ATRi sensitivity in SS was characterized by an increase in biomarkers of replication fork stress (increased γH2AX, decreased replication fork speed, and increased R-loops), an apoptotic response, and a dependence upon cyclin E expression. Combinations of cisplatin or PARP inhibitors enhanced the antitumor cell effect of ATRi, suggesting that either single-agent ATRi or combination therapy involving ATRi might be further assessed as candidate approaches for SS treatment. Cancer Res; 77(24); 7014-26. ©2017 AACR.