Investigating the role of ARC in hematopoiesis and myeloproliferative neoplasms

描述(申请人提供)：在造血过程中，造血干细胞(HSCs)的后代致力于沿着特定的细胞谱系分化，最终获得成熟的终末分化细胞的特征。在过去的几十年里，造血干细胞的鉴定和分离已经取得了很大的进展，然而，调控干细胞谱系承诺和分化的机制仍然不清楚。程序性细胞死亡在调节正常的造血细胞分化中起着至关重要的作用，而这一细胞过程在血液系统恶性肿瘤中经常发生改变。在费城染色体阴性的骨髓增生性肿瘤(Ph-MPN)中，细胞死亡途径的异常经常被观察到，这意味着细胞死亡蛋白是潜在的 疾病的发病机制和治疗干预的目标。目前，Ph-MPNS的分子发病机制尚不清楚。最近的研究强调了带有半胱氨酸天冬氨酸酶招募结构域(ARC)的抗凋亡蛋白在人类恶性肿瘤中的重要性，然而，ARC在正常和恶性造血中的作用还没有得到深入的研究。我们的工作旨在确定ARC在小鼠和人类造血干细胞和祖细胞(HSPC)中的作用，以及在Ph-MPN原发骨髓纤维化(PMF)中的作用。利用ARC基因敲除(ARC[-/-])小鼠模型进行研究，我们发现老年ARC[-/-]小鼠表现出显著的血细胞计数变化和脾肿大，髓系细胞和未成熟细胞的扩张与髓外造血相一致。根据网织蛋白染色，ARC[-/-]骨髓显示血窦减少，骨髓纤维化增加。进一步分析显示，骨髓HSC间隔室扩大，体外甲基纤维素集落检测显示HSC分化能力改变。ARC[-/-]小鼠的血液学发现与一种新的PMF小鼠模型一致。此外，ARC[-/-]表型可以移植到致死性照射的同源受体中。我们推测ARC在小鼠和人类HSPC功能中起关键作用，ARC的缺失可能参与PMF的发病。为了确定ARC在小鼠和人类HSPC的谱系承诺和分化中的特征，我们将利用遗传小鼠模型、干细胞移植、体外干细胞功能分析 体内异种移植实验。为了阐明ARC在HSCs中的作用机制，我们将使用转录图谱来指导我们的研究，并确定对ARC功能至关重要的分子途径。此外，我们将定量检测PMF患者HSC中ARC转录水平，以确定ARC表达是否与疾病发病机制有关。PMF是Ph-MPN中发病率最高、死亡率最高的疾病，迫切需要寻找新的小鼠模型和潜在的治疗靶点。

DESCRIPTION (provided by applicant): During hematopoiesis, descendants of hematopoietic stem cells (HSCs) become committed to differentiate along specific cell lineages, eventually acquiring the characteristics of mature terminally differentiated cells. The identification and isolation of HSCs has advanced substantially over the past few decades, however, the mechanisms that regulate stem cell lineage commitment and differentiation are still obscure. Programmed cell death plays a crucial role in the regulation of normal hematopoietic differentiation, and this cellular process is frequently altered in hematologic malignancies. In Philadelphia chromosome negative myeloproliferative neoplasms (Ph- MPN), dysregulation of cell death pathways is frequently observed, implicating cell death proteins as potential factors in the pathogenesis of disease and targets for therapeutic intervention. Currently the molecular pathogenesis of Ph- MPNs is largely unknown. Recent studies have highlighted the importance of the anti-apoptotic protein apoptosis repressor with caspase recruitment domain (ARC) in human malignancies, however, the function of ARC in normal and malignant hematopoiesis has not been thoroughly investigated. Our work aims to identify the role of ARC in mouse and human hematopoietic stem and progenitor cells (HSPCs), and in the Ph- MPN primary myelofibrosis (PMF). Utilizing an ARC knockout (ARC [-/-]) mouse model for our investigation, we have found that aged ARC [-/-] mice exhibit significantly altered blood cell counts and splenomegaly with an expansion of myeloid and immature cells consistent with extramedullary hematopoiesis. ARC [-/-] bone marrow revealed reduced blood sinusoids and increased bone marrow fibrosis according to reticulin staining. Further analysis showed expansion of the bone marrow HSC compartment and in vitro methylcellulose colony assays revealed altered HSC differentiation capacity. The hematologic findings in ARC [-/-] mice are consistent with a novel mouse model of PMF. Additionally, the ARC [-/-] phenotype is transplantable into lethally irradiated congenic recipients. We hypothesize that ARC plays a critical role in mouse and human HSPC function and that loss of ARC may contribute to the pathogenesis of PMF. To characterize ARC in lineage commitment and differentiation of mouse and human HSPCs, we will utilize genetic murine models, stem cell transplantation, in vitro stem cell functional assays and in vivo xenotransplantation experiments. To elucidate the mechanism of ARC in HSCs, we will use transcriptional profiling to guide our investigation and identify molecular pathways critical to ARC function. Furthermore, we will quantitate ARC transcript levels in HSCs from patients with PMF to determine if ARC expression is relevant to the disease pathogenesis. PMF has the most severe morbidity and greatest mortality of the Ph-MPNs and the identification of novel mouse models and potential therapeutic targets is urgently required.