Regulators of ubiquitin signaling in malignant hematopoiesis

项目摘要/摘要 骨髓增生异常综合征(MDS)是一种异种克隆性造血干/祖细胞(HSPC) 频繁转化为急性髓系白血病(AML)的疾病。导致急性髓细胞白血病的分子事件 转化的定义很模糊，唯一的治疗方法是异基因干细胞移植，对于 很少有病人是符合条件的。治疗无效的原因之一是缺乏了解 疾病的机制。因此，对遗传学的细胞和分子基础的研究- 明确的MDS和AML亚型对新疗法的发展至关重要。染色体上的缺失 (ChR)5q(Del5q)是MDS中最常见的细胞遗传学异常，提示MDS预后不良。 AML。然而，del(5q)中单个基因的功能在很大程度上还没有被探索。我们最近报道了 TRAF与叉头相关(FHA)结构域B(TIFAB)相互作用蛋白在所有报道中都被缺失 小鼠中的del(5q)MDS/AML及其缺失导致的表型使人联想到人类MDS， 部分原因是通过TRAF6激活的异常先天免疫信号；但是，我们也观察到P53的增加 活动。为了充分阐明TIFAB依赖的P53调控机制，我们进行了蛋白质组学研究 筛选TIFAB结合蛋白。泛素特异肽酶15(USP15)，一种稳定泛素的酶 MDM2和促进p53降解，成为主要候选药物。因此，我们设计了一个多管齐下的 在我们的Tifab缺乏的MDS模型中，我们首先表征了依赖TIFAB的P53活性的方法 (目标1A)第二，揭示了TIFAB-USP15复合体调控p53的分子机制 (目标1B)。我们的发现为确定USP15在已建立的恶性肿瘤中的功能提供了理论基础 造血，这正在进行得很好，我们计划在F99阶段完成 研究计划(目标1C)。我们的初步数据表明，USP15在AML中高表达，并且是 对白血病祖细胞功能很重要，但对正常的HSPC不重要。我们将采用体内和 使用我们的Usp15-/-模型、MLL-AF9白血病模型和AML细胞系的体外方法来阐明 USP15在急性髓系白血病中的作用通过完成我的论文研究，我在 小鼠遗传学、小鼠模型、包括MDS和AML在内的血液系统恶性肿瘤、癌症生物学和 细胞机制。我学习了生化分析、结构功能研究和遗传学研究，而 熟悉筛选方法，包括RNA测序和蛋白质组学。带着我的 经过全面培训，我有能力从事博士后研究方向 阐明USP15活性的决定因素，同时利用药物发现平台开发小分子- 针对这一可药物靶点的分子抑制剂(目标2)。

Project Summary / Abstract Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem and progenitor cell (HSPC) disorders with frequent transformation to acute myeloid leukemia (AML). Molecular events leading to AML transformation are poorly defined and the only curative therapy is allogeneic stem cell transplantations, for which few patients are eligible. One of the reasons for ineffective treatments is the lack of understanding of the mechanisms of disease. Therefore, investigations into the cellular and molecular basis of genetically- defined MDS and AML subtypes are crucial to the development of novel therapies. Deletions on chromosome (chr) 5q (del5q) are the most frequent cytogenetic abnormality in MDS and are indicative of poor prognosis in AML. However, the functions of individual genes within del(5q) are largely unexplored. We recently reported that TRAF-interacting protein with forkhead-associated (FHA) domain B (TIFAB) is deleted in all reported cases of del(5q) MDS/AML and its deletion in mice results in phenotypes reminiscent of human MDS, due partly to aberrant innate immune signaling via TRAF6 activation; but, we also observed increases in p53 activity. To fully elucidate the mechanism of TIFAB-dependent regulation of p53, we performed a proteomics screen for TIFAB-binding proteins. Ubiquitin-specific peptidase 15 (USP15), a deubiquitinase that stabilizes MDM2 and enforces p53 degradation, emerged as the lead candidate. Therefore, we devised a multi-pronged approach in which we first, characterized the TIFAB-dependent p53 activity in our Tifab-deficient MDS model (Aim 1A), and second, uncovered the molecular mechanism of the TIFAB-USP15 complex in regulating p53 (Aim 1B). Our findings garnered rationale for defining the function of USP15 in established malignant hematopoiesis, which is well underway and which we propose to complete during the F99 phase of this research program (Aim 1C). Our preliminary data indicate that USP15 is highly expressed in AML and is important for leukemic progenitor function, but not normal HSPCs. We will employ a combination of in vivo and in vitro approaches using our Usp15-/- model, MLL-AF9 leukemia models, and AML cell lines to elucidate the function of USP15 in AML. In completing my dissertation research, I have acquired a strong foundation in mouse genetics, murine models, hematopoietic malignancies including MDS and AML, cancer biology, and cellular mechanisms. I have learned biochemical assays, structure-function studies, and genetic studies, while gaining familiarity with screening approaches including RNA-sequencing and proteomics. With my comprehensive training, I am positioned and highly motivated to pursue a post-doctoral research direction elucidating the determinants of USP15 activity, while utilizing drug discovery platforms to develop small- molecule inhibitors against this druggable target (Aim 2).