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）的后代开始沿着特定的细胞系分化，最终获得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，调节干细胞谱系承诺和分化的机制仍然不清楚。稳态造血依赖于细胞凋亡途径的严格调控，以维持造血系统的发育、维持和功能。在费城染色体阴性骨髓增殖性肿瘤（Ph- MPN）中，经常观察到细胞死亡途径的失调，这意味着细胞死亡因子是疾病的潜在启动因子和治疗干预的靶点。最近的研究强调了带有caspase募集结构域（ARC）的凋亡抑制因子在人类恶性肿瘤中的重要性，然而，ARC在正常造血中的功能作用尚未被研究。我们假设ARC在正常造血干细胞和髓细胞分化中起关键作用。利用ARC-缺陷（ARC -/-）小鼠模型，我们将研究ARC在造血中的功能。我们的初步研究表明，老年ARC -/-小鼠表现出贫血和血小板减少症。对ARC -/-脾脏的检查显示脾肿大，骨髓细胞呈三系扩增，巨核细胞增生，未成熟细胞增加，与骨髓增生和髓外造血一致。ARC -/-骨髓显示网状蛋白染色增加，细胞数量减少，与骨髓纤维化一致。ARC -/-小鼠的造血结果与Ph- MPN类似于原发性骨髓纤维化一致。进一步分析表明，ARC -/-骨髓中HSC区室扩大，甲基纤维素集落试验显示体外实验改变了HSC分化能力。此外，在ARC -/-小鼠骨髓增生性疾病可移植到致死性辐照的同源受体。本项目旨在阐明ARC在HSC功能、髓细胞分化和Ph- MPN中的作用。为了表征ARC在干细胞和祖细胞以及髓系分化和Ph- MPN细胞的谱系承诺，我们将利用遗传小鼠模型、干细胞移植试验、Ph- MPN患者样本以及体内和体外ARC水平的恢复。为了阐明ARC的机制，我们将使用转录分析来指导我们的研究并确定对ARC功能至关重要的分子途径。

DESCRIPTION (provided by applicant): During hematopoiesis, descendants of hematopoietic stem cells (HSC) become committed to differentiate along specific cell lineages, eventually acquiring the characteristics of terminally differentiated cells. The identification and isolation f HSC has advanced substantially over the past few decades, however, the mechanisms that regulate stem cell lineage commitment and differentiation are still obscure. Steady-state hematopoiesis relies on the tight regulation of apoptosis pathways for the development, maintenance and function of the hematopoietic system. In Philadelphia chromosome negative myeloproliferative neoplasms (Ph- MPN), dysregulation of cell death pathways is frequently observed, implicating cell death factors as potential initiators of disease and targets for therapeutic intervention. Recent studies have highlighted the importance of apoptosis repressor with caspase recruitment domain (ARC) in human malignancies, however, a functional role of ARC in normal hematopoiesis has not been investigated. We hypothesize that ARC plays a critical role in normal blood stem cell and myeloid differentiation. Utilizing an ARC-deficient (ARC -/-) mouse model we will investigate ARC function in hematopoiesis. Our preliminary studies revealed that aged ARC -/- mice exhibit anemia and thrombocytopenia. Investigation of ARC -/- spleens showed splenomegaly with a tri-lineage expansion of myeloid cells, megakaryocyte hyperplasia, and an increase in immature cells consistent with myeloproliferation and extramedullary hematopoiesis. ARC -/- bone marrow revealed increased reticulin staining and reduced cell numbers consistent with marrow fibrosis. The hematopoietic findings in ARC -/- mice are consistent with a Ph- MPN resembling primary myelofibrosis. Further analysis showed expansion of the HSC compartment in ARC -/- bone marrow and in vitro assays revealed altered HSC differentiation capacity in methylcellulose colony assays. Additionally, the myeloproliferative disease in ARC -/- mice is transplantable into lethally irradiated congenic recipients. This projects aims to elucidate the role of ARC in HSC function, myeloid differentiation, and Ph- MPN. To characterize ARC in lineage commitment of stem and progenitor cells, as well as, myeloid differentiation and Ph- MPN cells, we will utilize genetic murine models, stem cell transplant assays, Ph- MPN patient samples and restoration of ARC levels in vivo and in vitro. To elucidate the mechanism of ARC, we will use transcriptional profiling to guide our investigation and identify molecular pathways critical to ARC function.