Targeting the BCL2 immune checkpoint to enhance the immunostimulatory effects of radiation in breast cancer

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SUMMARY Hormone receptor (HR)+ breast cancer (BC) causes the majority of BC-related deaths in the US, reflecting an unmet need for innovative therapeutic approaches. Indeed, resistance to standard treatments and metastatic spread remain major challenges, and novel approaches such as immune checkpoint blockers (ICBs) have shown limited efficacy so far. The long-term objective of this proposal is to mechanistically dissect the impact of BCL2, an antiapoptotic protein that favors the resistance of cancer cells to death imposed by chemotherapy and radiation therapy (RT), on the immunological configuration of treatment naïve and irradiated HR+ BCs. Specifically, this project will test the highly innovative hypothesis that BCL2 may represent a therapeutically actionable immune checkpoint because of its ability to preserve mitochondrial integrity, based on these specific aims: 1) determining the influence of BCL2 levels on the immune microenvironment of treatment-naïve human and mouse HR+ BCs; 2) defining the impact of BCL2 on the immune microenvironment of HR+ BCs responding to RT in vitro and in vivo; and 3) elucidating the value of BCL2 as a target to boost the immunostimulatory effects of RT in mouse models of HR+ BCs, including an innovative model that mimics key features of human HR+ BC. BCL2 is a particularly significant target because ~80% of HR+ BC cases overexpress BCL2, and the BCL2 inhibitor venetoclax, is approved for clinical use. To achieve our goals, diagnostic biopsies from women with HR+ BC will be evaluated by CODEX for BCL2 expression, tumor infiltration by key immune cells that regulate anticancer immunity, and expression of immunosuppressive proteins like MHC Class I and PD-L1. The impact of BCL2 on the immunological response of HR+ BC cells to RT will be interrogated in vitro, by genetic (deletion, overexpression) and pharmacological (e.g., venetoclax administration) methods coupled to flow cytometry, IF microscopy and ELISA for the assessment of key regulators of anticancer immunity. Similar genetic and pharmacological approaches will be harnessed to alter BCL2 competence and delineate the influence of BCL2 on the immunological TME of treatment-naïve and irradiated mouse HR+ BCs established in immunocompetent hosts, based on IHC, flow cytometry and single-cell RNA sequencing. In vitro studies by flow cytometry and clonogenic assays, as well as in vivo studies based on mouse HR+ BC cells growing in immunodeficient vs immunocompetent mice, will be employed to dissect the impact of BCL2 on intrinsic radiosensitivity vs immune-dependent tumor control. Finally, different combinatorial regimens involving RT and venetoclax will be investigated for efficacy (in both treatment-naïve and treatment resistance settings) and potential mechanisms of resistance in an endogenous mouse model of HR+ BCs that mimics key features of human HR+ BC. Our findings will elucidate the impact of BCL2 on the immune TME of HR+ BC and identify the best approach to inform the initiation of clinical trials testing RT plus venetoclax in women with HR+ BC, a devastating disease that still affects >200,000 and kills >25,000 new women every year in the US.