Dissecting the role of hypoxia in T cell differentiation in cancer

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PROJECT SUMMARY/ABSTRACT Immunotherapeutic treatments for cancer, especially the use of monoclonal antibody mediated blockade of `checkpoint' molecules like PD-1, has changed the treatment paradigm for patients with solid tumors like melanoma. However, only a subset of patients benefit from these therapies, due to several resistance mechanisms concentrated within the tumor microenvironment (TME), the site of action of cytotoxic T cells. T cells must contend with physical barriers to infiltration, immunosuppressive cell types, the expression of co- inhibitory ligands on target cells, and a harsh metabolic environment produced by cancer cells. In addition to T cell-extrinsic immunosuppression, T cells within tumors have a distinct differentiation trajectory, resulting in acquisition of an alternative, dysfunctional fate termed exhaustion. Exhausted T cells are terminally differentiated, hypofunctional upon stimulation, and possess poor capacity to proliferate, a crucial component of immune memory. We and others have shown that exhausted T cells have severe metabolic deficiencies, and that metabolic stress within the TME, most notably hypoxia exposure, potentiates differentiation towards exhaustion. In line with this, we and others have shown that melanoma patients with more oxidative, hypoxic tumors are more likely to progress on anti-PD1. Thus, the hypoxic TME and the intrinsic functional deficiency of exhausted T cells are linked. However, how hypoxia and resultant oxidative stress alter T cell differentiation remain unclear. Our hypothesis is that hypoxia exposure promotes T cell exhaustion, by driving aberrant chromatin bivalency and loss of transcription, such that hypoxia mitigation treatments will alter T cell differentiation and support increased T cell function. AIM 1: How does hypoxia drive epigenetic changes that bias T cell differentiation and function? Hypoxia drives several cellular adaptations, including transcriptional reprogramming via HIF-1α, induction of reactive oxygen species (ROS), and metabolic shifts. We will A) use in vitro systems to identify mechanisms of hypoxia contributing to altered histone methylation and bivalency in murine T cells; and B) determine contributions of hypoxia to the T cell epigenome and explore potential mitigation strategies in murine tumor models. AIM 2: How do hypoxia reducing regimens alter intratumoral T cell differentiation in melanoma patients? We and other have shown that targeting tumor cell metabolism or angiogenesis can increase the oxygen tension within tumors in both mouse models and melanoma patients. In this Aim, we will take advantage of two investigator-initiated clinical trials in melanoma utilizing metformin or axitinib in combination with anti-PD-1, and deeply explore transcriptional, epigenetic, metabolic, and functional outcomes associated with reduction of hypoxia coincident with anti-PD-1. We expect these studies to address knowledge gaps in the fields of epigenetics, immunology, and cancer immunotherapy, uncovering how tumor hypoxia can bias T cell differentiation and response to anti-PD-1, with the goal of identifying novel targets that mitigate hypoxia driven T cell exhaustion and overcome barriers to immunotherapy for cancer.