Pre-Leukemic Hematopoietic Stem Cells and Clonal Evolution in Human AML

描述（由申请人提供）：急性髓系白血病（AML）是一种侵袭性骨髓恶性肿瘤，其特征是未成熟的髓系细胞在成熟和功能上有缺陷。在美国，AML每年影响1.3万名成年人，其中大多数年龄在65岁以上。即使采用化疗和/或同种异体移植等标准积极治疗，5年总生存率也在30-40%之间，65岁以上患者的生存率更低。这种较差的总生存率主要是由于化疗耐药和/或复发的疾病，往往难以接受额外的治疗。在过去的5年中，下一代DNA测序已被应用于表征人类AML基因组，并在识别大多数复发突变基因方面取得了巨大成功。重要的是，这些基因组研究表明，大多数AML病例与多个基因的突变有关，通常以不同的等位基因频率发生，表明其具有复杂的克隆结构和发育历史。这些发现提出了许多重要的问题，其中之一是多重突变是如何在造血细胞的单克隆谱系中积累的。正常的造血是由造血干细胞（HSC）发起和维持的细胞层次结构，造血干细胞产生中间祖细胞，最终形成血液中的所有分化细胞。在髓细胞分化中，HSC是唯一的长寿命自我更新群体，因为所有其他细胞和祖细胞的寿命有限，通常在几天或几周内测量。从这些考虑，已经提出了一个模型，即白血病基因突变必须在HSC中连续积累。因此，含有一些（但不是全部）白血病基因突变的HSC，称为白血病前期HSC，在诊断时必须持续存在，并可能导致疾病复发。最近，在一小部分原发病例中报道了支持人类AML中白血病前HSC克隆进化模型的证据。本研究旨在进一步研究人类AML的白血病前期HSC，假设白血病基因突变在HSC克隆中积累，并且这些白血病前期HSC克隆对疾病的发病和复发至关重要。第一个目的是从分子定义的诊断性AML病例的多样性中确定AML中白血病前HSC的克隆进化，并通过结合下一代DNA测序、单细胞基因分型和功能性异种移植测定来确定克隆进化是否存在共同模式。第二个目的是通过体外和体内试验确定白血病前突变对HSC自我更新、增殖、分化和化学敏感性的影响。第三个目标是在单个AML患者的治疗和反应过程中识别和跟踪白血病前HSC，包括临床缓解和临床复发，目的是确定这些细胞是否有助于复发性疾病。最终，对白血病前HSC和相应的创始突变的研究将促进我们对白血病发生的理解，并将对开发新的靶向治疗AML疗法至关重要。

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow characterized by the accumulation of immature myeloid cells defective in their maturation and function. AML affects 13,000 adults annually in the United States, most of them over the age of 65. Even with standard aggressive treatments with chemotherapy and/or allogeneic transplantation, five-year overall survival is between 30-40%, and much lower for those over age 65. This poor overall survival rate is primarily due to chemotherapy-resistant and/or relapsed disease that is often refractory to additional therapies. Over the last 5 years, next generation DNA sequencing has been applied to characterizing human AML genomes with great success in identifying most recurrently mutated genes. Importantly, these genomic studies have demonstrated that most cases of AML are associated with mutations in multiple genes, often occurring with different allelic frequencies, suggesting a complex clonal architecture and developmental history. These findings raise many important questions, one of which is how multiple mutations accumulate in a single clonal lineage of hematopoietic cells. Normal hematopoiesis is organized as a cellular hierarchy initiated and maintained by hematopoietic stem cells (HSC) that give rise to intermediate progenitors and eventually all the differentiated cells of the blood. In myeloid differentiation, HSC are the only long-lived self-renewing population, as all other cells and progenitors have a limited lifespan typically measured in days or weeks. From these considerations, a model has been proposed that leukemogenic mutations must serially accumulate in HSC. Therefore, HSC containing some, but not all, leukemogeneic mutations, termed pre-leukemic HSC, must persist at diagnosis and may contribute to relapsed disease. Recently, evidence supporting this model of clonal evolution of pre-leukemic HSC in human AML was reported for a small set of primary patient cases. This proposal aims to further investigate pre-leukemic HSC in human AML based on the hypothesis that leukemogenic mutations accumulate in clones of HSC and that these pre-leukemic HSC clones are critical for disease pathogenesis and relapse. The first aim is to determine the clonal evolution of pre-leukemic HSC in AML from a diversity of molecularly-defined diagnostic AML cases, and determine if there are common patterns of clonal evolution using a combination of next generation DNA sequencing, single cell genotyping, and functional xenotransplantation assays. The second aim is to determine the effect of pre-leukemic mutations on HSC self-renewal, proliferation, differentiation, and chemosensitivity using both in vitro and in vivo assays. The third aim is to identify and track pre-leukemic HSC during the course of treatment and response in individual AML patients including both clinical remission and clinical relapse, with the goal of determining if such cells contribute to relapsed disease. Ultimately, investigation of pre-leukemic HSC and the corresponding founding mutations will advance our understanding of leukemogenesis, and will be critical to the development of novel curative targeted AML therapies.