PROJECT SUMMARY Epigenetic reinforcement of T cell exhaustion is a well-established barrier limiting multiple modalities of T cell- based immunotherapies for cancer. This proposal will investigate the specific epigenetic events that are coupled to one of the cardinal features of T cell exhaustion; a reduction in the T cell’s proliferative potential. Notably, disruption of the epigenetic regulators DNMT3A and TET2 has been reported to preserve the proliferative capacity of CD8 T cells in the setting of both chimeric antigen receptor (CAR) T cell chronic stimulation and PD- 1 blockade-mediated rejuvenation of endogenous T cells. Importantly, mutations in these genes have also been associated with clonal expansion of hematopoietic stem cells (HSCs) which occurs in the majority of otherwise healthy older adults. Such clonal hematopoiesis of indeterminate potential (CHIP) mainly results from mutations of three epigenetic regulators (DNMT3A, TET2, ASXL1). Despite disruption of DNMT3A and TET2 producing a similar T cell phenotype, a critical distinction is that DNMT3A KO T cells maintain antigen dependent homeostatic proliferation and TET2 KO T cells exhibit a hyperproliferative phenotype. ASXL1 has yet to be characterized, but we have recently determined that disruption of ASXL1 in T cells also preserves the cell’s proliferative potential in a PD-1 blockade setting. My research program is now well-poised to dissect the specific epigenetic modifications that are established by each of these discrete regulators to better define the molecular mechanism limiting the proliferative potential of T cells as they progress down the exhaustion developmental trajectory. I propose to test the hypothesis that CH-associated gene disruptions result in specific epigenetic programs that delineate memory T cell homeostasis versus a hyperproliferative state. To define these programs, I will pursue the following aims; 1) To define epigenetic programs mediated by CH- associated epigenetic regulators that promote a homeostatic versus hyperproliferative state during persistent antigen stimulation of T cells. 2) To determine if disruption of CH-regulators among naïve versus memory human T cells results in a hyperproliferative state in chronically stimulated CAR T cells. 3) To identify whether T cells isolated from myelodysplastic syndrome (MDS) patients with mutations in CH-associated epigenetic regulators exhibit epigenetic programs consistent with heightened proliferative ability. Completion of the proposed studies will define the epigenetic checkpoint(s) regulating T cell homeostatic proliferation from uncontrolled cell growth. Importantly, successful completion of this proposal will define the underlying programs that sustain T cell proliferative capacity for the design and manufacturing of next generation CAR T cell protocols. The proposed career development and training outlined in this application, in conjunction with the experimental studies, will advance my training as a clinician scientist by further establishing a novel research program that will put me on a path to independence.

PROJECT NARRATIVE The mentored research proposed here will investigate the role of clonal hematopoiesis (CH)-associated genes in driving T cell proliferation. Specifically, we will look at disruption of three main regulators of CH (DNMT3A, TET2, ASXL1) to determine the specific epigenetic programs that delineate T cell homeostasis from a hyperproliferative state. These results will inform on the underlying programs that sustain T cell proliferative capacity and guide future efforts for the design of CAR T cells.