Abstract
Epigenetic dysregulation is a hallmark of cancer. Small molecule inhibitors such as bromodomain and extraterminal (BET) protein inhibitors developed to target epigenetic dependencies have demonstrated significant promise in preclinical models. However, clinical success with epigenetic drugs as single agents has been limited by emergence of tumor cell tolerance and escape, which often occurs due to transcriptional rewiring. Ewing sarcoma (EwS), a bone and soft tissue tumor driven by the EWS::FLI1 fusion oncoprotein, is characterized by profound oncogene-dependent enhancer reprogramming. Thus, epigenetic modifying therapies are of high therapeutic interest. In this study, we sought to elucidate how EwS cells escape BET inhibition to identify biologically informed drug combinations that could be advanced to clinical trials. As expected, EwS cells and xenografts initially responded to BMS-986158, a pharmaceutical grade BET inhibitor, but proliferation was rapidly restored. A kinase inhibitor screen showed that BMS-986158 drug tolerant persister (DTP) cells were sensitive to inhibitors of Focal Adhesion Kinase (FAK), a critical signaling node downstream of extracellular matrix (ECM) engagement. RNA sequencing revealed that DTP cells had been transcriptionally rewired and that mesenchymal signature and ECM remodeling genes were specifically upregulated. Combining BMS-986158 with the FAK inhibitor Defactinib had synergistic effects, reducing EwS cell proliferation, survival, and invasion in vitro, and significantly inhibited tumor outgrowth in vivo. Our studies identify BET and FAK inhibition as a rational combination therapy worthy of further investigation for EwS, and demonstrate that defining emergent mechanisms of epigenetic drug tolerance can identify new vulnerabilities that can be therapeutically targeted.