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

描述(申请人提供)：急性髓系白血病(AML)是一种侵袭性的骨髓恶性肿瘤，其特征是聚集了成熟和功能有缺陷的未成熟髓系细胞。在美国，AML每年影响13,000名成年人，其中大多数超过65岁。即使采用化疗和/或异基因移植的标准积极治疗，五年的总存活率也在30%-40%之间，65岁以上的人的存活率要低得多。总体存活率较低的主要原因是化疗耐药和/或复发的疾病，这些疾病往往对其他治疗方法难以奏效。在过去的5年里，下一代DNA测序已经被应用于人类AML基因组的表征，并在识别大多数反复突变的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，通常发生在不同的等位基因频率上，这表明了一种复杂的克隆结构和发展史。这些发现提出了许多重要的问题，其中之一是多个突变是如何在单个克隆造血细胞谱系中积累的。正常的造血是由造血干细胞(HSC)启动和维持的细胞层次结构，HSC产生中间祖细胞，最终产生血液中所有分化的细胞。在髓系分化中，HSC是唯一长寿的自我更新群体，因为所有其他细胞和祖细胞的寿命都有限，通常以几天或几周为单位。从这些考虑，已经提出了一个模型，即白血病突变必须在HSC中连续积累。因此，含有部分但不是全部白血病突变的HSC，称为白血病前期HSC，必须坚持诊断，并可能导致复发。最近，有证据支持人类AML中白血病前期HSC的克隆进化模型，用于一小部分原发病例。这一建议旨在进一步研究人类AML的白血病前HSC，基于以下假设：白血病前HSC克隆积累在HSC克隆中，并且这些白血病前HSC克隆对疾病的发病和复发至关重要。第一个目的是从各种分子定义的诊断AML病例中确定AML中白血病前HSC的克隆进化，并结合下一代DNA测序、单细胞基因分型和功能性异种移植试验来确定是否存在共同的克隆进化模式。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、分化和化疗敏感性的影响。第三个目标是在治疗过程中识别和跟踪白血病前期HSC，并跟踪单个AML患者的治疗和反应，包括临床缓解和临床复发，目的是确定这些细胞是否导致复发。最终，对白血病前HSC和相应的基础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药物至关重要。

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.