Bromodomain and extraterminal (BET) domain proteins regulate immune checkpoints through AMPK-dependent and -independent pathways in peripheral T cell subsets

Abstract

Abstract Immune checkpoint blockade unleashes anti-tumor immunity of tumor-infiltrating CD8+ T cells. Yet, many cancer patients are refractory: unknown mechanisms resolve responders from non-responders. Analysis of transcriptional networks regulating immune checkpoint genes may provide insight. We used multi-parameter FACS to explore expression of several checkpoint inhibition molecules, focusing on functional roles of 5′-Adenosine Monophosphate-activated Protein Kinase (AMPK) and BET transcriptional co-regulators (BRD2, BRD3 and BRD4), both known to be key signaling pathways disturbed in obesity and cancer, resulting in exhaustion of effector CD8+ T cells and tumor progression. Here, human primary PBMCs from normal, healthy, adult donors were activated ex vivowith anti-CD3 and anti-CD28. We then measured expression of inhibitory receptors PD1, CTLA4, TIM3 and TIGIT on T cell subsets. Small molecule inhibitors of BET protein expression, including pan-BET inhibitor JQ1 and BRD4-selective PROTAC degrader MZ1, identified BET protein-regulated targets. We found that BRD4 regulates PD1, CTLA4 and TIM3 (but not TIGIT) in both CD4+ and CD8+ T cells. Treatment with Compound C, a modulator of AMPK, defined a unique PD1 and CTLA4 network. We confirmed that T cells with elevated expression of inhibitory receptors were impaired in cytokine secretion and proliferation. We conclude that separate signaling pathways likely converge on BRD2 and/or BRD3 and/or BRD4, acting independently as effectors at each promoter. Patterns of AMPK-regulated metabolism and BET gene expression are personalized and must be considered when evaluating immune checkpoint blockade in cancer therapy. Supported by grants from the Cancer Systems Biology Consortium of the National Cancer Institute (U01CA182898,U01CA243004; PI Denis), NCI Cancer Moonshot (R01CA222170; PI Denis) and Boston University's Immunology Training Program (T32AI007309-32; PI Kepler).