Impact of aging and clonal hematopoiesis on epigenetic heterogeneity, evolvability, and leukemogenesis

项目摘要 急性髓性白血病（AML）主要发生在65岁及以上的成年人中，与年龄相关性白血病有关。 克隆造血（CH）。这种情况是由突变标记的 造血干细胞和祖细胞（HSPC）。这些HSPC扩增通常以在细胞中的体细胞突变为特征。 白血病相关基因，如表观遗传调节因子TET 2。TET 2突变CH是AML的危险因素， 并且CH向AML的进展通过获得协同突变如组成型活性FLT 3 ITD来促进。 然而，目前尚不清楚衰老和TET 2突变如何相互作用，以推动CH演变为白血病。我们寻求 揭示了CH如何在衰老微环境中演变为白血病的表观遗传机制，奠定了 为治疗策略奠定了基础，以阻止这种演变并预防美国老龄人口中的白血病。到 为此，我们将利用我们团队在肿瘤内异质性和计算方面的互补专业知识， 表观基因组学;衰老、癌症演变和白血病发生;炎症和造血干细胞（HSC） 生物学;和小鼠HSC增殖和功能异质性。我们在小鼠身上的初步结果表明， HSPC表观遗传异质性在老年时增加。这一过程发生在老化的骨髓（BM）中 以炎症和HSPC支持改变为特征的微环境。表观遗传学的兴起， 具有Tet 2突变（Tet 2 MT）和Flt 3 ITD的HSC中的转录组异质性先于白血病转化。 我们假设衰老和TET 2突变协同增强表观遗传异质性和进化性 的HSPC，从而有助于克隆扩增和白血病发生。在目标1中，我们将确定 通过检测HSPC的体细胞，探讨衰老和Tet 2 MT对HSPC表观遗传异质性和基因调控的影响 年轻（2-3个月）和老年（22个月）小鼠Tet 2 MT HSPC的表观基因组图谱，包括单细胞 (sc)通过scRNA-seq的转录组、通过snATAC-seq的开放染色质谱和通过RRBS的DNA甲基化组。 我们期望在旧Tet 2 MT HSPC中定义与自我更新基因相关的表观遗传构型， 静止以及对炎症和压力的反应。在目标2中，我们将定义表观遗传构型， Tet 2 MT HSPC在老年背景下具有适应性。我们将把基因条形码的HSPC移植到年轻人或 老年小鼠，并评估亚克隆扩增/收缩，以确定年龄依赖性选择是否适用于所有Tet 2 MT HSPC或仅为子集。我们将使用scRNA-seq和snATAC-seq来定义分子特征和表观遗传学特征。 在老化背景下积极选择的条形码HSPC的异质性。在目标3中，我们将定义 老年人中允许Flt 3 ITD诱导转化的Tet 2 MT HSPC的表观遗传构型 上下文我们将Flt 3 ITD基因导入年轻或年老的Tet 2 MT或野生型HSC中，然后移植这些细胞， 将基因条形码化的细胞植入年轻或年老的宿主小鼠。我们将评估所选患者的表观遗传特征， 通过scRNA-seq和scATAC-seq对其鉴定的后代进行HSPC。我们希望我们的发现能改变 表观遗传可塑性如何促进身体进化并导致衰老个体的癌症发生。

PROJECT SUMMARY Acute myeloid leukemia (AML) occurs mostly in adults 65 years and older and is associated with age-related clonal hematopoiesis (CH). This condition that results from the clonal expansion of mutationally-marked hematopoietic stem and progenitor cells (HSPC). These HSPC expansions often feature somatic mutations in leukemia-associated genes such as the epigenetic regulator TET2. TET2-mutant CH is a risk factor for AML, and CH progression to AML is promoted by acquiring cooperating mutations such as constitutively active FLT3ITD. Yet, it is unclear how aging and TET2 mutation interact to drive the evolution of CH to leukemia. We seek to reveal the epigenetic mechanisms for how CH evolves to leukemia in an aging microenvironment, laying the foundation for therapeutic strategies to block this evolution and prevent leukemia in the aging US population. To this end, we will leverage our team's complementary expertise in intra-tumor heterogeneity and computational epigenomics; aging, cancer evolution, and leukemogenesis; inflammation and hematopoietic stem cell (HSC) biology; and mouse HSC proliferation and functional heterogeneity. Our preliminary results in mice show that HSPC epigenetic heterogeneity increases in old age. This process occurs in an aged bone marrow (BM) microenvironment characterized by inflammation and altered HSPC support. The rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 mutation (Tet2MT) and Flt3ITD precedes leukemic transformation. We hypothesize that aging and TET2 mutation cooperatively enhance epigenetic heterogeneity and evolvability of HSPC, thus contributing to clonal expansion and leukemogenesis. In Aim 1, we will determine the combined impact of aging and Tet2MT on epigenetic heterogeneity and gene regulation in HSPC by examining the somatic epigenomic landscape of Tet2MT HSPC from young (2-3 months) and old (22 months) mice, including single-cell (sc) transcriptomes by scRNA-seq, open-chromatin profiles by snATAC-seq, and DNA methylomes by RRBS. We expect to define epigenetic configurations in old Tet2MT HSPC associated with genes in self-renewal, quiescence, and responses to inflammation and stress. In Aim 2, we will define epigenetic configurations of Tet2MT HSPC that are adaptive in the aged context. We will transplant genetically barcoded HSPC into young or old mice and assess subclone expansion/contraction to determine if an age-dependent selection is for all Tet2MT HSPC or only a subset. We will use scRNA-seq and snATAC-seq to define molecular signatures and epigenetic heterogeneity of barcoded HSPC that are positively selected in the aged context. In Aim 3, we will define the epigenetic configurations of Tet2MT HSPC that are permissive to Flt3ITD-induced transformation in the aged context. We will transduce the Flt3ITD gene into young or aged Tet2MT or wild-type HSC and then transplant these genetically barcoded cells into young or old host mice. We will assess the epigenetic profiles of the selected HSPC by scRNA-seq and scATAC-seq of their identified progeny. We expect our findings will shift the paradigm for how epigenetic plasticity promotes somatic evolution and leads to cancer initiation in aging individuals.