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(HSC)的后代致力于沿着特定的细胞谱系分化，最终获得终末分化细胞的特征。在过去的几十年中，HSC的鉴定和分离有了很大的进步，然而，调控干细胞谱系承诺和分化的机制仍然不清楚。稳态造血系统的发育、维持和功能依赖于对细胞凋亡途径的严密调控。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，这意味着细胞死亡因子是潜在的疾病起始者和治疗干预的靶点。最近的研究强调了带有caspase募集结构域的凋亡抑制因子(ARC)在人类恶性肿瘤中的重要性，然而，ARC在正常造血中的功能还没有被研究过。我们假设ARC在正常的血液干细胞和髓系分化中起关键作用。利用ARC缺陷(ARC-/-)小鼠模型，我们将研究ARC在造血中的作用。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。对ARC-/-脾的观察显示，脾肿大，髓系细胞呈三系扩张，巨核细胞增多，幼稚细胞增多，与骨髓增殖和髓外造血相一致。弧形/非弧形骨髓显示网织蛋白染色增加，细胞数量减少，与骨髓纤维化一致。ARC-/-小鼠的造血学发现与Ph-MPN类似于原发性骨髓纤维化。进一步的分析显示ARC-/-骨髓中的HSC间隔室扩大，体外实验显示在甲基纤维素集落实验中HSC的分化能力发生改变。此外，ARC-/-小鼠的骨髓增生性疾病可以移植到致死性照射的同种受者身上。本项目旨在阐明ARC在HSC功能、髓系分化和Ph-MPN中的作用。为了确定ARC在干细胞和祖细胞的谱系承诺以及髓系分化和Ph-MPN细胞方面的特征，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。

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.