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

项目总结 急性髓系白血病(AML)多发生于65岁及以上的成年人，与年龄相关 克隆性造血。这种情况是由突变标记的克隆扩增引起的 造血干祖细胞(HSPC)。这些HSPC扩增通常以体细胞突变为特征 白血病相关基因，如表观遗传调节基因TET2。TET2突变CH是AML的危险因素， 而CH进展为AML是通过获得协作性突变，如成分活性的Flt3ITD来促进的。 然而，目前尚不清楚衰老和TET2突变是如何相互作用推动CH向白血病演变的。我们寻求 揭示CH在衰老微环境中如何演变为白血病的表观遗传学机制，为 为阻止这种进化和预防美国老龄化人口中的白血病的治疗策略奠定了基础。至 为此，我们将利用我们团队在肿瘤内异质性和计算性方面的互补专业知识 表观基因组学；衰老、癌症演变与白血病；炎症与造血干细胞(HSC) 生物学；以及小鼠HSC的增殖和功能异质性。我们在老鼠身上的初步结果表明 随着年龄的增长，HSPC表观遗传异质性增加。这个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。表观遗传学和基因工程的兴起 TET2突变的HSC中转录异质性(Tet2MT)和Flt3ITD先于白血病转化。 我们假设衰老和TET2突变协同增强了表观遗传的异质性和进化性 因此有助于克隆性扩增和白血病的发生。在目标1中，我们将确定合并的 衰老和Tet2MT对HSPC表观遗传异质性和基因调控的影响 幼龄(2-3月龄)和老年(22月龄)小鼠Tet2MT HSPC的表观基因组图谱，包括单细胞 (SC)scRNA-seq转录本，SnATAC-seq开放染色质图谱，RRBS DNA甲基组。 我们希望在旧的Tet2MT HSPC中定义与自我更新相关的基因的表观遗传配置， 安静，以及对炎症和压力的反应。在目标2中，我们将定义 Tet2MT HSPC是在老龄化背景下自适应的。我们将把基因条形码HSPC移植到年轻的或 并评估亚克隆的扩张/收缩，以确定年龄相关的选择是否适用于所有Tet2MT HSPC或仅为一个子集。我们将使用scRNA-seq和snatac-seq来定义分子特征和表观遗传学。 在老龄化背景下正向选择的条形码HSPC的异质性。在目标3中，我们将定义 允许Flt3ITD诱导的老年人转化的Tet2MT HSPC的表观遗传构型 背景。我们将Flt3ITD基因转导到年轻或老年的Tet2MT或野生型HSC中，然后将它们移植 通过基因条形码将细胞编码成幼小或老年宿主小鼠。我们将评估选定的表观遗传学特征 HSPC通过其鉴定的后代的scRNA-seq和scatac-seq。我们预计，我们的发现将改变这一范式 表观遗传可塑性如何促进躯体进化并导致老年个体癌症的发生。

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.