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

摘要 双加氧酶Tet家族通过将5-甲基胞嘧啶(5mC)依次氧化为5-甲基胞嘧啶(5mC)来介导DNA去甲基化 羟甲基-(5hmC)、5-甲酰基-(5fC)和5-羧基-胞嘧啶(5caC)。Tet酶是正常所需的 Tet功能的发展和丧失--由于突变、代谢重编程、缺氧和其他额外因素 机制经常发生在许多血液系统恶性肿瘤和实体瘤中。最近的研究表明 在大规模队列中发现调节Tet催化活性的Tet蛋白和代谢酶的突变 弥漫性大B细胞淋巴瘤(DLBCL)。然而，这些突变的临床意义 DLBCL中Tet蛋白抑制肿瘤发生的分子机制 总的来说，人们对此还没有很好的理解。 我们和其他人的研究强调，Tet活性是成熟B细胞分化所必需的； DLBCL的来源细胞。为了研究Tet功能丧失在DLBCL发病机制中的作用，我最近 产生B细胞特异性缺失TET2和TET3蛋白的小鼠。TET2和TET3在小鼠体内的缺失 B细胞导致侵袭性淋巴瘤的发展和迅速死亡。Tet缺乏症的初步分析 B细胞表现出更高的基因组不稳定性和异常二级DNA结构的显著积累 称为G-四联体(G-Quad)。G-QUADS积聚发生在B细胞扩增的早期阶段 这也是其他细胞系中与Tet缺乏症相关的特征。G-Quad结构已经被 与癌症的基因组不稳定和转录改变有关，但它们的生理功能正常 细胞及其在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Tet蛋白通过限制Tet蛋白作为肿瘤抑制因子发挥作用 G-四元结构的积累。Aim1的研究将检查G-四联体结构与TET的关联 粉防己碱功能丧失过程中的活性、基因组不稳定性和转录变化 淋巴肿大。AIM2将询问Tet蛋白限制聚集的分子机制 G-四边形结构。AIM3将重点描述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。AIM2和AIM3的研究将在R00阶段进行，并将 可能会为许多新的调查路线的出现做好准备。总之，这些研究将使用B细胞作为一种 理解Tet蛋白广泛抑瘤功能机制的模型系统 并将揭示G-QUAD结构的生物学功能的新方面。 该提案概述了K99和R00阶段的具体培训计划，以获得必要的技能集 完成拟议的研究，并成功过渡到独立的职位。在这个过程中，我 我希望通过我在一项优秀研究中的持续培训来发展一条独立的研究路线 环境，由一支优秀的指导团队提供帮助。

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.