Enhancing TET activity for the treatment of hematological malignancy

项目总结 大多数急性髓系白血病(AML)患者的高龄限制了侵袭性药物的使用 化疗导致总体存活率较低。AML的替代治疗策略备受追捧。 我们的研究表明维生素C(抗坏血酸)是一种潜在的无毒治疗佐剂 急性髓系白血病的治疗。抗坏血酸是人体内一种基本的微量营养素，除了作为细胞外， 抗氧化剂，作为Ten-11(Tet)酶的直接辅因子参与。哺乳动物Tet蛋白(TET1-3) 是催化5-甲基胞嘧啶(5mC)氧化的造血系的肿瘤抑制因子 导致DNA去甲基化和基因沉默的逆转。TET2功能丧失突变是常见的 在急性髓系白血病患者中观察到，并与较差的总体预后相关。重要的是，TET2突变是 几乎总是杂合的，这表明增强剩余的TET2活性(由剩余的野生- TET2等位基因)可能是治疗TET2突变AML的一种可行的治疗策略。我们已经展示了 在抗坏血酸的细胞和动物模型中，以Tet依赖的方式，通过以下方式重新编程AML表观基因组 DNA羟甲基化增加，导致DNA去甲基化，这是异常自我更新的障碍，增加 分化，减缓白血病的进展。我们假设抗坏血酸将是一种有效的 TET2突变型AML治疗药物增强残留TET抑瘤作用的研究 目前还没有其他以Tet蛋白为靶点进行功能恢复的治疗剂。我们建议 了解生理或药理剂量的抗坏血酸如何影响TET的机制基础 活性，以及通过钠依赖的维生素C转运体或总残留Tet的摄取能力 活性影响抗坏血酸治疗效果(Aim1)。TET2突变和TET2基因突变与AML进展 将结合标准AML测试不同致癌驱动因素对抗坏血酸的敏感性 化疗，以及从我们在AML细胞中失去功能的基因筛查中获得的数据将被用于 设计合理的联合治疗策略，最大限度地发挥抗坏血酸的治疗效果 佐剂(AIM2)。最后，我们将探索提高抗坏血酸作为TET2生物利用度的方法 利用亲脂性抗坏血酸类似物的辅因子和维生素C转运体的遗传或药物调节 AML细胞(Aim3)。这项建议的目标是了解抗坏血酸的剂量和急性髓系白血病背景 治疗将是最有效的，如何将抗坏血酸与现有疗法结合起来提高治疗水平 结果和确定提高抗坏血酸生物利用度以增加Tet激活的新方法 潜力。我们相信这些研究将为抗坏血酸作为非 急性髓系白血病联合治疗中的毒性佐剂。

PROJECT SUMMARY Advanced age in the majority of acute myeloid leukemia (AML) patients limits the use of aggressive chemotherapy leading to poor overall survival. Alternative treatment strategies for AML are highly sought after. Our research has revealed vitamin C (ascorbate) to be a potential non-toxic therapeutic adjuvant for the treatment of AML. Ascorbate is an essential micronutrient in humans that, in addition to its role as a cellular antioxidant, participates as a direct cofactor of Ten-eleven (TET) enzymes. Mammalian TET proteins (TET1-3) are tumor suppressors of the hematopoietic lineage that catalyze the oxidation of 5-methylcytosine (5mC) leading to DNA demethylation and the reversal of gene silencing. TET2 loss-of-function mutations are frequently observed in AML patients and are associated with a worse overall prognosis. Importantly, TET2 mutations are almost always heterozygous, suggesting that enhancing residual TET2 activity (encoded by the remaining wild- type TET2 allele) could be a viable therapeutic strategy for the treatment of TET2-mutant AML. We have shown in cellular and animal models that ascorbate, in a TET-dependent manner, reprograms the AML epigenome by increasing DNA hydroxymethylation, leading to DNA demethylation, a block in aberrant self-renewal, increased differentiation, and slowing of leukemia progression. We hypothesize that ascorbate will be an effective therapeutic agent in the treatment of TET2 mutant AML by enhancing residual TET tumor suppressive function. There are currently no other therapeutic agents that target TET proteins for functional restoration. We propose to understand the mechanistic basis of how physiological or pharmacological doses of ascorbate influence TET activity, and whether uptake capacity through sodium-dependent vitamin C transporters or total residual TET activity influences ascorbate treatment efficacy (Aim1). AML progression in the context of TET2 mutation and diverse oncogenic drivers will be tested for sensitivity to ascorbate in combination with standard AML chemotherapy, and data already obtained from our loss of function genetic screens in AML cells will be used to design rational combinatorial treatment strategies that maximize the efficacy of ascorbate as a therapeutic adjuvant (Aim2). Finally, we will explore approaches to enhance the bioavailability of ascorbate as a TET2 cofactor using lipophilic ascorbyl analogs and genetic or pharmacological modulation of vitamin C transporters on AML cells (Aim3). The goal of this proposal is to understand the dose and AML context in which ascorbate treatment will be most efficacious, how to combine ascorbate with existing therapies to improve treatment outcome and identify novel approaches to enhance ascorbate bioavailability for increased TET-activating potential. We believe these studies will provide a strong foundation for clinical translation of ascorbate as a non- toxic adjuvant in combination therapies for the treatment of AML.