Tumor suppressive functions of TET proteins in B cell malignancies: role of G-quadruplex structures

摘要 TET-双加氧酶家族通过依次将5-甲基胞嘧啶（5 mC）氧化成5-甲基胞嘧啶（5 mC）来介导DNA去甲基化。 羟甲基-（5 hmC）、5-甲酰基-（5 fC）和5-羧基-胞嘧啶（5caC）。泰特酶是正常的 由于突变、代谢重编程、缺氧和其他额外的原因，泰特功能的发展和丧失 机制经常发生在许多血液恶性肿瘤和实体瘤中。最近的研究 在一个大型队列中鉴定了调节泰特催化活性的泰特蛋白和代谢酶的突变 弥漫性大B细胞淋巴瘤（DLBCL）患者。然而，这些突变的临床意义 以及泰特蛋白抑制恶性肿瘤发展的分子机制 一般来说，这一点并没有得到很好的理解。 我们和其他人的研究强调了泰特活性是成熟B细胞分化所必需的; DLBCL的起源细胞。为了研究泰特功能丧失在DLBCL发病机制中的作用，我最近 产生TET 2和TET 3蛋白的B细胞特异性缺失的小鼠。小鼠TET 2和TET 3的缺失 B细胞引起侵袭性淋巴瘤的发展和快速死亡。TET缺乏的初步分析 B细胞显示基因组不稳定性增加和异常二级DNA结构的显著积累 称为G-四重体（G-quads）。G-四聚体的积累发生在B细胞扩增的早期阶段 并且也是与其它细胞谱系中的TET缺乏相关的特征。G-quad结构已经被 与癌症中的基因组不稳定性和转录改变有关，但它们在正常人中的生理功能 细胞和恶性细胞中的病理作用仍然知之甚少。 对于这里提出的研究，我假设泰特蛋白通过限制肿瘤的生长而发挥肿瘤抑制剂的作用。 G-quad结构的累积。Aim 1中的研究将检查G-四元结构与泰特的关联 活性、基因组不稳定性和泰特诱导的功能丧失过程中的转录改变 淋巴瘤形成Aim 2将询问泰特蛋白限制药物蓄积的分子机制。 G-quad结构。Aim 3将专注于描绘G-quads调节染色质结构的机制 并在正常和恶性B细胞中发挥作用。Aim 2和Aim 3研究将在R 00阶段进行， 可能会引发许多新的调查路线。总之，这些研究将使用B细胞作为 模型系统，以了解泰特蛋白广泛肿瘤抑制功能的机制基础 并将揭示关于G-四元结构的生物学功能的新方面。 该提案概述了K99和R 00阶段的具体培训计划，以获得必要的技能， 完成拟议的研究，并成功过渡到一个独立的位置。在这个过程中，我 我期望通过我在一个优秀的研究领域的持续培训，发展出一条独立的研究路线 环境，由一个优秀的辅导团队提供便利。

Abstract TET-family of dioxygenases mediate DNA demethylation by sequentially oxidizing 5-methylcytosine (5mC) to 5- hydroxymethyl- (5hmC), 5-formyl- (5fC) and 5-carboxyl-cytosine (5caC). TET enzymes are required for normal development and loss-of TET function due to mutations, metabolic reprogramming, hypoxia and other additional mechanisms occurs frequently in many hematological malignancies and solid tumors. Recent studies have identified mutations in TET proteins and metabolic enzymes which regulate TET catalytic activity in a large cohort of patients with Diffuse Large B-cell Lymphoma (DLBCL). However, the clinical significance of these mutations in DLBCL and the molecular mechanisms through which TET proteins suppress development of malignancies in general, is not well-understood. Studies from us and others have highlighted that TET activity is required for differentiation of mature B cells; the cell-of-origin for DLBCL. To investigate the role of TET loss-of-function in the pathogenesis of DLBCL, I recently generated mice with B-cell-specific deletion of TET2 and TET3 proteins. Deletion of TET2 and TET3 in mouse B cells caused development of aggressive lymphomas and rapid mortality. Preliminary analysis of TET-deficient B cells revealed increased genomic instability and a striking accumulation of unusual secondary DNA structures called G-quadruplexes (G-quads). The accumulation of G-quads occurred at early stages of B-cell expansion and was also a feature associated with TET-deficiency in other cellular lineages. G-quad structures have been linked to genomic instability and transcriptional alterations in cancers but their physiological functions in normal cells and pathological roles in malignant cells remain poorly understood. For the studies proposed here, I hypothesize that TET proteins function as tumor suppressors by limiting the accumulation of G-quad structures. Studies in Aim1 will examine the association of G-quad structures with TET activity, genomic instability and transcriptional alterations during TET loss-of-function induced lymphomagenesis. Aim2 will interrogate molecular mechanisms by which TET proteins limit the accumulation of G-quad structures. Aim3 will focus on delineating mechanisms by which G-quads regulate chromatin structure and function in normal and malignant B cells. Studies in Aim2 and Aim3 will be performed in R00 phase and will likely prime the emergence of many new lines of investigations. Together, these studies will use B cells as a model system to understand the mechanistic basis for the broad tumor suppressive functions of TET proteins and will reveal novel facets about the biological functions of G-quad structures. The proposal outlines a specific training plan for the K99 and R00 phases, to acquire the skillset necessary to perform the proposed studies and successfully transition to an independent position. During this process, I anticipate to develop an independent line of research through my continued training in an excellent research environment, facilitated by an outstanding mentoring team.