Identification of therapeutic targets in clonal hematopoiesis

项目摘要/摘要 克隆性造血(CH)的特征是血液中遗传上不同的细胞亚群的生长 是急性髓系白血病(AML)的潜在前驱状态。CH与年龄相关，发生在超过20%的 60岁以上的人，会增加血液系统恶性肿瘤和心血管疾病的风险。这个 CH中最常见的突变发生在表观遗传修饰物DNMT3A、TET2、ASXL1和IDH2以及这些相同的 在AML中发现了高变异等位基因频率的突变，这与它们作为启动突变的作用一致。CH突变 已知可诱导造血干细胞和祖细胞(HSPC)的功能变化；然而，特异性 这些突变的潜在机制尚不清楚，也缺乏针对这些突变的基因特异性治疗。 考虑到与CH相关的广泛的流行率、合并症和恶变风险，还有一个未得到满足的需求 开发可防止克隆性扩张和恶性转化的新疗法。我的长期目标是 博士研究的目的是确定CH型特异性治疗靶点。到目前为止，还没有合适的体外平台 对于原代培养小鼠HSPC已被证明是一个重大的技术挑战，防止使用无偏筛选来 确定治疗目标。如目标1所述，我的论文工作建立了体外共培养系统，该系统 维持原代小鼠HSPC，并产生与先前小鼠和人类一致的CH突变表型 学习。我们已经使用该系统对DNMT3A、TET2、ASXL1和IDH2突变的HSPC进行了CRISPR/Cas9筛选 并确定了特定于基因型的依赖关系。特别有趣的是，组蛋白赖氨酸去甲基酶Jmjd1c和 Kdm3b是在TET2和IDH2突变的HSPC中观察到的强依赖性。在特定目标1.1中，我们将审问 Jmjd1c或Kdm3b的缺失与TET2或IDH2的突变协同作用产生表观遗传学和 驱动合成致命性的转录状态。在特定目标1.2中，我们将使用临床前小鼠模型和初级 在TET2和IDH2突变体CH中靶向JMJD1C和KDM3B的患者样本 AML。我的博士后研究将继续研究CH和AML，重点略有转移到阐明 负责CH突变克隆的扩展和转化的潜在表观遗传和转录回路。 正如目标2中详细描述的，我们将应用CH和AML的小鼠模型来对单个HSPC克隆进行条形码编码，然后使用单个- 细胞转录学和表观遗传学研究，以确定允许克隆扩张和转化的因素。总的来说， 这两个项目将提供对克隆扩张和转化发生的基本机制的洞察 以及寻求缓解这种克隆性扩张/转化的潜在治疗策略。这项建议将被实施 在纪念斯隆·凯特琳癌症中心(MSK)罗斯·莱文博士的实验室里，一项最先进的癌症研究 研究所。这些联系，加上Gerstner Sloan Ketling研究生院的强大资产，将为 提供丰富的协作、技术和科学资源，以执行拟议的研究和职业发展计划。

PROJECT SUMMARY/ABSTRACT Clonal hematopoiesis (CH) is characterized by the outgrowth of a genetically distinct subpopulation of cells in the blood and is a potential precursor state to acute myeloid leukemia (AML). CH is age-associated, occurring in over 20% of individuals over age 60, and confers an increased risk of both hematological malignancy and cardiovascular disease. The most common mutations in CH occur in the epigenetic modifiers DNMT3A, TET2, ASXL1, and IDH2 and these same mutations are found at high variant allele frequencies in AML consistent with their role as initiating mutations. CH mutations are known to induce functional changes in hematopoietic stem and progenitor cells (HSPCs); however, the specific mechanisms underlying these alterations are not understood and genotype-specific therapies for these mutations are lacking. Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to develop novel therapies that can prevent clonal expansion and malignant transformation. The long-term objective of my doctoral research is to identify genotype-specific therapeutic targets in CH. To date, the lack of a suitable ex vivo platform for culturing primary murine HSPCs has proved a significant technical challenge preventing the use of unbiased screens to identify therapeutic targets. As described in Aim 1, my thesis work has established an ex vivo co-culture system that maintains primary murine HSPCs and that yields phenotypes of CH mutations consistent with prior murine and human studies. We have used this system to perform CRISPR/Cas9 screens on Dnmt3a-, Tet2-, Asxl1-, and Idh2-mutant HSPCs and have identified genotype-specific dependencies. Of particular interest, the histone lysine demethylases Jmjd1c and Kdm3b are strong dependencies observed in Tet2- and Idh2-mutant HSPCs. In Specific Aim 1.1, we will interrogate the mechanisms by which loss of Jmjd1c or Kdm3b cooperates with mutations in Tet2 or Idh2 to create an epigenetic and transcriptional state that drives synthetic lethality. In Specific Aim 1.2, we will use preclinical murine models and primary patient samples to delineate the therapeutic potential of targeting JMJD1C and KDM3B in Tet2 and Idh2-mutant CH and AML. My postdoctoral research will continue to study CH and AML with a slight switch in focus to elucidating the underlying epigenetic and transcriptional circuitry responsible for the expansion and transformation of CH-mutant clones. As detailed in Aim 2, we will apply murine models of CH and AML to barcode individual HSPC clones followed by single- cell transcriptomic and epigenetic studies to define the factors that allow for clonal expansion and transformation. Overall, these two projects will offer insights into both the basic mechanisms by which clonal expansion and transformation occur and potential therapeutic strategies seeking to mitigate this clonal expansion/transformation. This proposal will be conducted in the laboratory of Dr. Ross Levine at Memorial Sloan Kettering Cancer Center (MSK), a state-of-the-art cancer research institute. These affiliations, along with the strong assets of the Gerstner Sloan Kettering Graduate School, will provide a rich set of collaborative, technical, and scientific resources to execute the proposed research and career development plans.