Mechanisms of TET2-dependent control of CAR T cell fate determination and antitumor function

摘要

ABSTRACT Recent scientific advancements have made chimeric antigen receptor (CAR) T cell therapy a prom1s1ng treatment option for relapsed and refractory cancers. In this strategy, T cells are extracted from the patient and reprogrammed and then reinfused into the subject to seek out and destroy cancer cells. However, not all individuals respond to this treatment and therefore a better understanding of the mechanisms that underlie antitumor immune cell function is required to improve response rates. Successful therapy has been linked to increased CART expansion, long-term persistence as well as early memory differentiation, and approaches to augment these properties could greatly enhance CAR T cell potency. DNA modifying enzymes such as the methylcytosine dioxygenase TET2 may be involved in the control of T cell differentiation and function, and TET2 disruption leads to increased T cell proliferation, longevity, and early memory differentiation. However, the role of TET2 in regulating CAR T cell fate and function is not well understood. I hypothesize that TET2 catalytically and non-catalytically regulates CAR T cell differentiation and antitumor activity by direct alteration of DNA methylation status as well as through chromatin remodeling. The catalytic function of TET2 induces active DNA demethylation through successive oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC), and 5-carboxylcytosine (5caC). Demethylation is then achieved through either passive dilution of 5hmC or active excision of 5fC and 5caC. The biological impact of these two active demethylation pathways in CART cells is not understood. In Aim 1, I propose to elucidate the role of TET2-catalyzed changes to the methylome in mediating CART cell differentiation and effector function. I postulate that the active excision commitment step converting 5hmC to 5fC is indispensable for TET2-mediated control of T cell differentiation and antitumor function. To evaluate this, I will investigate the effect of halting oxidation at the 5hmC step on differentiation, antitumor function, and global as well as site-specific DNA methylation profiles in CAR T cells. Additionally, TET2 impacts chromatin structure through both catalytic and non-catalytic mechanisms involving interactions between histone modification proteins. The role of TET2-induced changes to chromatin structure on T cell fate is also currently unknown. In Aim 2, I will determine how chromatin remodeling by TET2 impacts regulation of CAR T cell differentiation and function. I hypothesize that TET2 influences changes to local chromatin structure through catalytic and non-catalytic activities to regulate CAR T cell differentiation and antitumor function. Thus, I will investigate the effect of eliminating TET2 catalytic function on the differentiation, antitumor efficacy and the epigenetic landscape of CAR T cells compared to simultaneous knockout of both catalytic and non-catalytic activity and stalled catalytic function. Together, these aims will improve our understanding of TET2 function in regulating T cell-mediated tumor control and inform strategies to safely modulate TET2 activity to improve outcomes with CART cell therapy.