(P 447) TARGETING FOCAL ADHESION KINASE AUGMENTS THE THERAPEUTIC POTENTIAL OF BROMODOMAIN INHIBITORS IN EWING SARCOMA

Objective: Metastatic and relapsed Ewing sarcoma (EwS) portends a dismal prognosis and new therapeutic strategies are critically needed. EwS is driven by FET::ETS fusions, most commonly EWS::FLI1, which causes global transcriptional dysregulation via epigenetic reprogramming. Given the central role for epigenetic dysregulation in EwS, we are investigating the bromodomain inhibitor (BETi) BMS-986158, the first BETi to be tested in children and adolescents in an ongoing Phase 1 clinical trial (NCT03936465). To ensure translational success of BMS-986158, combination strategies must be identified. In this study we seek to identify biologically rational pathways that can be targeted in combination with BMS-986158 to enhance its therapeutic efficacy.

Methods: Whole-transcriptome bulk RNA-seq was performed on three EwS cell lines exposed to 5nM (IC50 dose) BMS-986158 for 72hrs, 20 days, or vehicle. A kinase inhibitor screen that exploits polypharmacy and uses a linear regression based mathematical model to identify potential kinase targets was performed in drug-tolerant cells that emerged after exposure to BETi for 20 days. 2D dose response and synergy assays assessed cytotoxicity of single agents and combinations respectively. Synergy was determined using both Chou-Talalay and Bliss Synergy models. Invasion in a physiologic 3D matrix was assessed using EwS spheroids plated on rat tail collagen and imaged using brightfield microscopy. Subcutaneous xenograft EwS models in immunodeficient mice were used to test single agent and combinations identified as synergistic. Immunofluorescence and immunohistochemistry were used to detect protein expression in in vitro and in vivo specimens.

Results: Single agent BMS-986158 was initially cytostatic at low nanomolar IC50 but proliferation was restored by 20 days. In vivo treatment slowed xenograft tumor growth and increased survival (p-value 0.017, log-rank test), but tumors did not regress and eventually reach endpoint. Given outgrowth, in vitro drug-tolerant populations were investigated for mechanisms of escape. RNA-seq revealed global transcriptional rewiring with continued reversal of the EWS::FLI1 transcriptional signature, demonstrating on target effect despite restored proliferation. Interrogation of gene expression profiles showed that each cell line had acquired its own unique transcriptional signature, but pathway analysis of shared genes identified common programs. Top enriched terms among shared gene programs included: Hallmark-Myogenesis and EMT, Reactome- Collagen Formation, Integrin Cell Surface Interactions, and Gene Ontology- Cell Adhesion, Cell-Cell Signaling. This suggested that mesenchymal programs and integrin signaling had been reproducibly activated in BETi-tolerant cells. In parallel, a kinase inhibitor screen revealed focal adhesion kinase (FAK) to be one of four kinases that remained a dependency in the drug tolerant population. Phenotypically, drug tolerant cells had fibroblastic morphologies with increased F-actin filaments by phalloidin staining in 2D culture and significantly more invasive strands in 3D collagen assays compared to parent cells. Exposure of EwS cells to the FAK inhibitor Defactinib in combination with BMS-986158 showed that the agents were highly synergistic in both BETi-naïve and drug-tolerant populations. Cytotoxicity was enhanced in 2D, and in 3D spheroids cell viability and invasion were blocked across multiple EwS models. Combination studies are ongoing in EwS xenografts.

Conclusion: Our studies reveal that exposure of EwS cells to BETi leads to transcriptional rewiring that enables restoration of cell proliferation. Despite evidence of cell context-specificity, we observed that BET inhibition causes reproducible transition of EwS to more mesenchymal cell states which are dependent on FAK. This dependency can be therapeutically exploited by combining BETi with FAK inhibition, a combination which proved to be highly synergistic in vitro, with enhanced cytotoxic and anti-invasive properties. This work supports further investigation of FAK inhibitors as combination agents to enhance the efficacy of BETi, with the potential for translation to high-risk patients.