Investigating the role of ARC in hematopoiesis and myeloproliferative neoplasms

描述（由申请人提供）：在造血过程中，造血干细胞（HSC）的后代定向分化为沿着特定细胞谱系，最终获得成熟终末分化细胞的特征。在过去的几十年里，HSC的鉴定和分离已经取得了很大的进展，然而，调节干细胞谱系定型和分化的机制仍然不清楚。程序性细胞死亡在正常造血分化的调节中起着至关重要的作用，并且这种细胞过程在恶性血液病中经常改变。在费城染色体阴性的骨髓增生性肿瘤（Ph- MPN）中，经常观察到细胞死亡途径的失调，暗示细胞死亡蛋白是细胞凋亡的潜在因素。 疾病的发病机制和治疗干预的目标。目前，Ph-MPN的分子发病机制尚不清楚.近年来的研究表明，具有caspase募集结构域（ARC）的抗凋亡蛋白凋亡抑制因子在人类恶性肿瘤中的重要性，但ARC在正常和恶性造血中的作用尚未得到深入研究。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）中以及在Ph- MPN原发性骨髓纤维化（PMF）中的作用。 利用ARC敲除（ARC [-/-]）小鼠模型进行研究，我们发现老年ARC [-/-]小鼠表现出显著改变的血细胞计数和脾肿大，骨髓和未成熟细胞扩增与髓外造血一致。根据网硬蛋白染色，ARC [-/-]骨髓显示血窦减少和骨髓纤维化增加。进一步的分析显示骨髓HSC区室的扩增和体外甲基纤维素集落测定显示HSC分化能力的改变。ARC [-/-]小鼠的血液学结果与PMF的新型小鼠模型一致。此外，ARC [-/-]表型可移植到致死性辐照的同类受体中。我们假设ARC在小鼠和人HSPC功能中起关键作用，ARC的缺失可能有助于PMF的发病机制。 为了研究ARC在小鼠和人HSPCs的谱系定型和分化中的作用，我们将利用遗传学小鼠模型、干细胞移植、体外干细胞功能测定等方法来研究ARC在小鼠和人HSPCs谱系定型和分化中的作用。 和体内异种移植实验。为了阐明ARC在HSC中的机制，我们将使用转录谱来指导我们的研究，并确定ARC功能的关键分子通路。此外，我们将定量ARC转录水平与PMF患者的造血干细胞，以确定ARC表达是否与疾病的发病机制。PMF具有最严重的发病率和最高的死亡率的Ph-MPN和识别新的小鼠模型和潜在的治疗靶点是迫切需要的。

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.