TET2-mediated transcriptional and epigenetic control of normal and malignant hematopoiesis

TET 2是成人髓系恶性肿瘤和正常骨髓细胞中最常见的突变基因之一。 70岁以上的人。Tet 2缺失导致造血细胞扩增增加 干/祖细胞（HSPC）和随后的小鼠骨髓恶性肿瘤的发展。 因此，TET 2突变构成了HSPC早期干预的合适靶点 克隆扩展为了在治疗上利用TET 2突变，更好地了解TET 2基因的突变。 Tet 2缺失导致癌前HSPC失调和骨髓恶性肿瘤的机制 发展至关重要。作为甲基胞嘧啶双加氧酶家族的一员，TET 2将5 mC 到5 hmC，其可以相对稳定或进一步氧化到5 fC和5caC。我们 已经报道了TET 2在其肿瘤抑制功能中的催化活性的需要， HSPC。最近，我们发现了一种新的机制，血液恶性肿瘤通过增加 TET 2结合的基因组位点的突变负荷，并且，与直觉相反， TET 2-丢失。此外，我们发现TET 2在RNA羟甲基化（hm 5C）中的新作用， RNA结合蛋白PSPC 1对小鼠靶向转录物稳定性影响 胚胎干细胞重要的是，我们发现PSPC 1及其异二聚体伴侣NONO是 两者都在HSPC中大量表达。我们的研究提出了两个关键问题TET 2 正常和恶性造血的生物学：1）获得的5 hmC如何有助于 髓系恶性肿瘤的发病机制2)TET 2的催化活性是否与 HSPCs中RNA hm 5C水平？我们假设TET 2可能通过以下途径发挥酶功能： 优先5 hmC-至-5fC/5caC氧化，导致DNA去甲基化和转录 肿瘤抑制基因的激活以及通过RNA hm 5C修饰和随后的 致癌RNA的去稳定化。我们将研究DNA-5 hmC和RNA-hm 5C的调节异常如何影响DNA-5 hmC和RNA-hm 5C的表达。 TET 2突变介导的表观遗传控制可导致恶性造血， 具体目标。目的1）确定TET 2介导的DNA的优先5 hmC-5 fC/5caC氧化 HSPCs的去甲基化及其失调在髓系恶性肿瘤发病机制中的作用 新创建的5 hmC停滞和催化失活敲入突变Tet 2小鼠模型。目标2） 确定TET 2-RUNX 1伙伴关系对TET 2基因组定位的功能意义 以及HSPC中的靶基因表达。据报道，TET 2结合配偶体RUNX 1富集于 在TET 2突变型AML中，5 hmC增加的区域，但不是在hypo-5 hmC区域。我们将学习RUNX 1 在HSPC中调节TET 2基因组占用的功能，并检查RUNX 1在HSPC中的潜在作用。 赋予TET 2在正常和非正常期间优先5 hmC至5 fC/5caC氧化中的独特功能， 恶性造血目的3）研究RBP介导的TET 2对RNA hm 5C的募集作用 HSPCs中致癌RNA的修饰和不稳定及其在血液系统中的失调 恶性肿瘤我们还将研究TET 2突变如何影响HSPCs中的PSPC 1相关性 导致恶性造血

TET2 is one of the most commonly mutated genes in adult myeloid malignancies and in normal individuals over 70 years of age. Tet2-loss leads to increased expansion of hematopoietic stem/progenitor cells (HSPCs) and subsequent development of myeloid malignancies in mice. Thus, TET2 mutations constitute a suitable target for intervention at the early stages of HSPC clonal expansion. To exploit TET2 mutations therapeutically, a better understanding of the mechanisms by which Tet2-loss leads to premalignant HSPC dysregulation and myeloid malignancy development is essential. As a member of the methylcytosine dioxygenase family, TET2 converts 5mC to 5hmC, which can be relatively stable or be further oxidized to 5fC and 5caC. We have reported the requirement of catalytic activity of TET2 in its tumor-suppressive functions in HSPCs. Recently, we identified a novel mechanism of hematological malignancy via the increased mutational burden at genomic sites where TET2 binds and, counterintuitively, with gained 5hmC upon TET2-loss. In addition, we discovered a novel role of TET2 in RNA hydroxymethylation (hm5C) and target transcripts destabilization via its partner and RNA-binding protein (RBP) PSPC1 in mouse embryonic stem cells. Importantly, we found that PSPC1 and its heterodimer partner NONO are both abundantly expressed in HSPCs. Our studies raise two critical questions on TET2 biology in normal and malignant hematopoiesis: 1) How does gained 5hmC contribute to the pathogenesis of myeloid malignancy? 2) Is catalytic activity of TET2 relevant and functional at the RNA hm5C level in HSPCs? We hypothesize that TET2 may exert enzymatic functions through preferential 5hmC- to-5fC/5caC oxidation leading to DNA demethylation and transcriptional activation of tumor suppressor genes and through RNA hm5C modification and consequent destabilization of oncogenic RNAs. We will study how dysregulation of DNA-5hmC and RNA-hm5C mediated epigenetic control upon TET2 mutations can lead to malignant hematopoiesis by three specific aims. Aim 1) Define preferential TET2-mediated 5hmC-5fC/5caC oxidation for DNA demethylation in HSPCs and its dysregulation for the pathogenesis of myeloid malignancy using our newly created 5hmC stalling and catalytically inactive knock-in mutant Tet2 mouse models. Aim 2) Determine the functional significance of the TET2-RUNX1 partnership for TET2 genomic localization and target gene expression in HSPCs. RUNX1, a TET2 binding partner, is reported to be enriched in regions of increased 5hmC, but not in hypo-5hmC regions in TET2 mutant AML. We will study RUNX1 functions in regulating TET2 genomic occupancy in HSPCs and examine the potential role of RUNX1 in imparting unique TET2 functions in preferential 5hmC-to-5fC/5caC oxidation during normal and malignant hematopoiesis. Aim 3) Investigate RBP- mediated TET2 recruitment for RNA hm5C modification and destabilization of oncogenic RNAs in HSPCs and its dysregulation in hematological malignancy. We will also investigate how mutation of TET2 may impair its PSPC1 association in HSPCs leading to malignant hematopoiesis.