Roles of TET2-dependent DNA demethylation intermediates in hematological malignancies

摘要 TET 2是成人恶性血液病中最常见的突变/缺失基因之一。Tet2 突变在具有克隆造血的健康老年个体中也是普遍的。因此，TET 2突变是 一种祖先突变事件，驱动非恶性克隆生长并促进血液学 恶性转化事实上，Tet 2在小鼠中的缺失导致HSC自我更新增加， 各种恶性血液病的发展。然而，潜在的分子机制仍然存在， 大部分未知。TET 2是一种双加氧酶，其催化5 mC逐步转化为5 hmC、5 fC， 5caC，主动DNA去甲基化的初始步骤。基因组中5 mC的氧化和去甲基化是 以复杂的方式进行管理。结果表明，5 hmC和5 fC相对稳定 在分裂和非分裂细胞的基因组DNA中的胞嘧啶修饰。TET 2可能需要其催化剂 在HSC/HPC中发挥肿瘤抑制功能的活性。我们最近发现Tet 2的缺失会导致 HSC/HPC中的超致突变性，优先发生在正常情况下获得5 hmC和TET 2的基因组位点 绑定到。TET 2的丢失会自然地去除部分5 hmC和5 fC标记，但也会产生一组新的稳定的5 hmC和5 fC标记 在HSC/HPC中的基因组DNA中持续延长的时间。然而，TET 2的生理意义 HSC/HPC调控中5 hmC/5 fC形成的缺失介导的停滞和血液病的发病机制 恶性肿瘤仍有待阐明。我们已经创建了两个新的Tet 2 5 hmC失速（T1285 E，Tet 2 E/+）， Tet 2催化失活（H1295 Y/D1297 A，Tet 2 YA/+）突变敲入小鼠模型，为我们提供了独特的 阐明TET 2催化活性和TET 2依赖性5 hmC/5 fC的特定生物学作用的工具 HSC/HPC调节和血液恶性肿瘤中的转化。在目标1中，我们将阐明生物学 Tet 2丢失相关的5 hmC和5 fC/5caC形成停滞在HSC/HPC调节中的作用， 使用催化失活的和5 hmC停滞Tet 2突变小鼠模型的血液恶性肿瘤。在Aim中 2、研究TET 2酶活性和特异性TET 2依赖性DNA去甲基化对细胞DNA甲基化的影响 中间体对HSC/HPC中基因表达调控和基因组致突变性的影响。使用WT，Tet 2-/-， Tet 2 YA/YA和Tet 2 E/E HSC/HPC，我们将：（1）进行RNA-seq以鉴定差异表达的基因 （2）绘制全基因组5 mC/5 hmC/5 fC/5caC标记;和（3）进行全外显子组测序， 识别自发突变。对这些数据集的综合分析将使我们能够确定 DEG和由TET 2催化活性丧失或5 hmC → 5 fC转化引起的突变与特异性 Tet 2丢失后HSC/HPC中的5 mC/5 hmC/5 fC/5caC改变。这些研究可能揭示了 特异性TET 2依赖性胞嘧啶种类：（1）HSC/HPC中的基因调控和基因组致突变性;（2） TET 2缺失介导的HSC/HPC失调和血液恶性肿瘤这些发现将大大 对TET 2突变的血液恶性肿瘤的新治疗策略的鉴定的影响。

Abstract TET2 is one of the most commonly mutated/deleted genes in adult hematological malignancies. TET2 mutations are also prevalent in healthy elderly individuals with clonal hematopoiesis. Thus, TET2 mutations are an ancestral mutational event that drives non-malignant clonal outgrowth and facilitates hematological malignancy transformation. Indeed, Tet2 loss in mice leads to increased HSC self-renewal and the development of various hematological malignancies. However, the underlying molecular mechanisms remain largely unknown. TET2 is a dioxygenase that catalyzes the stepwise conversion of 5mC to 5hmC, 5fC and 5caC, initial steps of active DNA demethylation. The oxidation and demethylation of 5mC in the genome are regulated in a sophisticated manner. It has been shown that 5hmC and 5fC are present as relatively stable cytosine modifications in genomic DNA of both dividing and nondividing cells. TET2 likely requires its catalytic activity to exert tumor suppressive function in HSC/HPCs. We recently showed that Tet2 loss leads to hypermutagenicity in HSC/HPCs, preferentially at genomic sites that gained 5hmC and TET2 normally binds to. TET2 loss would naturally remove part of, but also creates a new set of, stable 5hmC and 5fC marks in genomic DNA for an extended period in HSC/HPCs. However, the physiological significance of the TET2 loss-mediated stalling of 5hmC/5fC formation in HSC/HPC regulation and pathogenesis of hematological malignancies remains to be elucidated. We have created two novel Tet2 5hmC stalling (T1285E, Tet2E/+) and Tet2 catalytic-inactive (H1295Y/D1297A, Tet2YA/+) mutant knock-in mouse models, which provide us unique tools to elucidate the specific biological role of TET2 catalytic activity and TET2-dependent 5hmC5fC conversion in HSC/HPC regulation and hematological malignancies. In Aim 1, we will elucidate the biological role of Tet2 loss-associated stalling of 5hmC and 5fC/5caC formation in HSC/HPC regulation and hematological malignancies using the catalytic-inactive and 5hmC stalling Tet2 mutant mouse models. In Aim 2, we will determine the effects of TET2 enzymatic activity and specific TET2-dependent DNA demethylation intermediates on gene expression regulation and genomic mutagenicity in HSC/HPCs. Using WT, Tet2-/-, Tet2YA/YA and Tet2E/E HSC/HPCs, we will: (1) perform RNA-seq to identify the differentially expressed genes (DEGs); (2) map genome-wide 5mC/5hmC/5fC/5caC marks; and (3) perform whole-exome sequencing to identify spontaneous mutations. Integrational analysis of these data sets will allow us to determine whether the DEGs and mutations caused by loss of TET2 catalytic activity or 5hmC5fC conversion correlate with specific 5mC/5hmC/5fC/5caC alterations in HSC/HPCs upon Tet2 loss. These studies could unveil potential roles of specific TET2-dependent cytosine species in: (1) gene regulation and genomic mutagenicity in HSC/HPCs; (2) TET2 loss-mediated HSC/HPC dysregulation and hematological malignancies. These findings will greatly impact on the identification of novel therapeutic strategies for TET2-mutated hematological malignancies.