Defining malignant hematopoiesis via single-cell multi-omics

项目摘要/摘要 通过获得体细胞突变导致克隆性血液分化导致异常堆积 血液成分和临床表现为髓系疾病。研究这些体细胞突变是如何 扰乱人类造血分化的轨迹经常受到正常 造血细胞含有无法通过细胞表面标志物区分的肿瘤细胞。至 为了克服这一局限，我们开发了一种新的单细胞多组学转录本基因分型方法(GOT) 将体细胞基因类型与数千个单细胞的转录本直接联系起来的平台。因此，得到了 在祖细胞的背景下，实现了同一样本中突变细胞和野生型细胞的比较 身份，从而将突变和野生型造血的混合从限制转变为 优势。作为原理的证明，GOT应用于钙网蛋白患者的CD34+祖细胞。 突变的骨髓增生性肿瘤(MPN)，揭示了在 突变细胞，例如巨核祖细胞中强大的未折叠蛋白反应，在一个 另一方面，在干细胞丰富的群体中，核因子-KB途径也是如此。总的来说，据透露， 钙网蛋白突变的转录影响作为祖细胞身份的函数是高度可变的- 对治疗具有重大意义，因为它能够发现特定于 最早的干细胞。因此，为了证明细胞身份依赖于其他关键驱动因素突变，如 髓系疾病的一个基本概念，我将GOT应用于血小板生成素受体突变的祖细胞 从MPN样本中获得克隆多样的细胞(目标1)。接下来，为了定义细胞外在 决定因素对体细胞突变的影响，我将确定免疫生态位与钙网蛋白突变体的相互作用 和野生型祖细胞，以及免疫调节治疗对这些相互作用的影响(目的 2)。最后，我将测试这样的假设，即细胞的表观基因组先于细胞身份依赖 通过开发和应用一种新的整合体细胞的单细胞平台来实现体细胞突变效应 祖细胞染色质可及性状态的基因分型(目标3)。因此，我将定义基因， 最终导致体细胞突变临床产出的表观遗传、转录和环境因素 在人类的造血系统中。因此，这些研究将不仅揭示克隆人的基本概念 造血分化也是治疗干预的具体靶点。

PROJECT SUMMARY / ABSTRACT Clonal blood differentiation through the acquisition of somatic mutations result in abnormal accumulation of blood components and clinically manifest as myeloid disorders. The study of how these somatic mutations perturb the differentiation trajectories in human hematopoiesis is often challenged by the admixture of normal hematopoietic cells with the neoplastic cells that cannot be distinguished by cell surface markers. To overcome this limitation, we developed a novel single-cell multi-omics Genotyping of Transcriptomes (GoT) platform that directly links somatic genotypes with transcriptomes of thousands of single cells. Thus, GoT enabled the comparison of mutant and wildtype cells within the same sample in the context of progenitor identities, thereby turning the co-mingling of mutant and wildtype hematopoiesis from a limitation to an advantage. As proof of principle, GoT was applied to CD34+ progenitor cells from patients with calreticulin- mutated myeloproliferative neoplasms (MPN), revealing key pathways that were aberrantly activated in the mutant cells, such as a robust unfolded protein response in the megakaryocytic progenitors, on the one way, and NF-KB pathway in stem cell-enriched populations, on the other. Overall, GoT revealed that the transcriptional impact of calreticulin mutations is highly variable as a function of progenitor identity – which bears significant implications for therapy by enabling the discovery of targetable pathways specific to the earliest stem cells. Thus, to demonstrate the cell identity-dependency across other key driver mutations, as a fundamental concept in myeloid disorders, I will apply GoT to thrombopoietin receptor-mutated progenitor cells and to clonally-diverse cells from MPN samples (Aim 1). Next, in order to define cell extrinsic determinants of somatic mutation impact, I will determine the immune niche interactions with calreticulin-mutant and wildtype progenitor cells, as well as the impact of immunomodulatory therapy on these interactions (Aim 2). Finally, I will test the hypothesis that the cell’s epigenome precedes the cell identity-dependency of somatic mutation effects, by developing and applying a novel single-cell platform that integrates somatic genotyping with chromatin accessibility states of progenitor cells (Aim 3). Thus, I will define the genetic, epigenetic, transcriptional and environmental factors that culminate in the clinical output of somatic mutations in human hematopoiesis. These studies will, therefore, unveil not only fundamental concepts in clonal hematopoietic differentiation but also specific targets for therapeutic intervention.