Tumor Evolution and Resistance in Response to BET Inhibitor Combination Therapies in Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has limited treatment options beyond chemotherapy. In preclinical studies, inhibitors of BET family proteins have been found to have efficacy, but emergence of resistance remains a problem. TNBC has been shown to contain considerable intratumor heterogeneity, which serves as the substrate for tumor evolution and poses a challenge to achieving durable responses, especially with monotherapies. Therefore, we aimed to identify combination therapies that would extend the effectiveness of BET inhibition and to understand their effects on intratumor heterogeneity, as well as mechanisms of resistance. In order to rationally design combination therapies, we performed CRISPR/Cas9 and small molecule screens to identify synergistic targets for the prototypical BET inhibitor JQ1. We followed up on numerous hits in in vitro synergy studies and in vivo studies on cell-line and patient-derived xenografts. We found that targeting the cell cycle was generally efficacious with BET inhibition, and we chose palbociclib, a CDK4/6 inhibitor, and paclitaxel, a microtubule targeting chemotherapy, for further study. We used DNA barcoding to track the changes in intratumor heterogeneity during treatment with palbociclib and paclitaxel combined with JQ1. We found that the combination treatments exerted the strongest evolutionary pressure, as they selected for the smallest number of subclones. Although we detected a pre-existing RB1-mutant subpopulation that expanded in response to JQ1+palbociclib, mathematical modeling and genomic profiling suggested high rates of de novo acquired resistance and increased phenotypic heterogeneity in the resistant population, suggesting multiple mechanisms of resistance. Lastly, we found that prolonged treatment with JQ1 and palbociclib produced resistant cells that were tetraploid. Using fluorescent reporters, we investigated the dynamics and mechanisms of tetraploidization and found that JQ1 and palbociclib directly induce tetraploidy through disruption of mitosis and cytokinesis. In summary, we have identified novel combination therapies for TNBC and investigated their impact on tumor evolution, as well as their previously unrecognized effects on cell division. Our findings provide rationale for further preclinical and clinical study of combined BET and CDK4/6 inhibition and suggest new vulnerabilities in resistant cells that may inform second-line treatments following progressive disease.