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

摘要 最近的科学进步使嵌合抗原受体(CAR)T细胞疗法成为复发和难治性癌症的一种促进治疗选择。在这一策略中，从患者身上提取T细胞，重新编程，然后重新注入受试者，以寻找并摧毁癌细胞。然而，并不是所有的人都对这种治疗有反应，因此需要更好地了解抗肿瘤免疫细胞功能的机制，以提高应答率。成功的治疗与CART的扩张、长期的持久性以及早期的记忆分化有关，而增强这些特性的方法可以极大地提高CAR T细胞的潜力。DNA修饰酶如甲基胞嘧啶双加氧酶TET2可能参与了T细胞分化和功能的控制，而TET2的破坏导致T细胞的增殖、寿命和早期记忆分化。然而，TET2在调节CAR T细胞命运和功能中的作用还不是很清楚。我推测，TET2通过直接改变DNA甲基化状态以及通过染色质重塑来催化和非催化地调节CAR T细胞的分化和抗肿瘤活性。TET2的催化作用是通过将5-甲基胞嘧啶(5mC)氧化为5-羟甲基胞嘧啶(5hmC)、5-甲酰胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)来诱导DNA去甲基化。然后通过被动稀释5hmC或主动切除5fC和5caC来实现去甲基化。这两个活性去甲基化途径在CART细胞中的生物学影响尚不清楚。在目标1中，我建议阐明TET2催化的甲基组改变在调节CART细胞分化和效应器功能中的作用。我推测，将5hmC转化为5fC的主动切除承诺步骤对于TET2介导的T细胞分化和抗肿瘤功能是不可或缺的。为了评估这一点，我将研究在5hmC步骤停止氧化对CAR T细胞的分化、抗肿瘤功能以及整体和特定部位DNA甲基化的影响。此外，TET2通过组蛋白修饰蛋白之间相互作用的催化和非催化机制影响染色质结构。TET2诱导的染色质结构改变对T细胞命运的作用目前还不清楚。在目标2中，我将确定TET2对CAR T细胞分化和功能调节的影响。我推测TET2通过催化和非催化活性影响局部染色质结构的变化，从而调节CAR T细胞分化和抗肿瘤功能。因此，我将通过同时敲除催化和非催化活性以及停滞的催化功能来研究消除TET2催化功能对CAR T细胞分化、抗肿瘤效果和表观遗传格局的影响。总之，这些目标将提高我们对TET2在调节T细胞介导的肿瘤控制中的功能的理解，并为安全地调节TET2活性以改善CART细胞治疗结果的策略提供信息。

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.