JAK2 signaling in erythropoiesis

描述(由申请人提供)：Janus tyroine kinase2(JAK2)在多个谱系的造血中起着重要作用，而JAK2功能获得突变V617F是骨髓增生性肿瘤(MPN)的主要决定因素。JAK2激酶抑制剂在治疗MPNS时显示出血液学毒性，呼吁采用新的治疗方法，仅针对受影响的谱系，而不针对其他谱系。由于缺乏对JAK2信号如何调节不同血细胞生成的了解，这项任务受到了阻碍。我们建议通过确定JAK2信号通路来填补这一知识空白，JAK2信号通路分别驱动红细胞生成和粒细胞生成，并描绘了JAK2用于调节红系分化的信号和转录的两条新途径。这项提议的总体目标是描述JAK2调节红细胞生成的潜在机制。目标1将描述一组新的小鼠模型；每个模型都表达不同的激活JAK2突变体，导致不同的MPN表型。我们将确定不同的JAK2突变体是否通过促进谱系特异性增殖或通过共同祖细胞间的偏向分化导致红细胞增多症或粒细胞增多症，并确定导致不同MPN表型所需的下游信号通路。我们还将把这些机械论的研究转化为人的环境。AIMS 2和3将剖析JAK2协调红系分化的信号和转录的两个新的分子机制，并研究它们在MPN发展中的作用。目的2将使用体外和体内实验来表征JAK2如何引导促红细胞生成素受体的内吞作用来终止信号。这一过程涉及PI3K的P85亚基的新功能，并且不依赖于PI3K的活性。这一过程中的缺陷导致了原发性和家族性先天性红细胞增多症信号的延长。在目标3中，我们将使用转录图谱和全基因组染色质免疫沉淀以及随后的高通量测序(CHIP-SEQ)来描绘一条新的途径，通过JAK2控制转录，而不是通过典型的JAK-STAT途径，而是通过磷酸化多梳抑制复合体2来调节染色质结构。这些研究的结果将填补我们对造血干细胞和祖细胞中调节正常和过度红细胞生成的信号的关键空白。这些结果也将进一步加深我们对EPO信号是如何终止的理解，并将改变JAK2如何激活红系转录网络的范式。此外，这些结果将阐明MPN生物学，并有助于设计新的、更有效的治疗剂，专门针对受影响的谱系，而不影响其他谱系。

DESCRIPTION (provided by applicant): The Janus tyrosine kinase 2 (JAK2) plays an important role in hematopoiesis of multiple lineages, and a gain-of-function JAK2 mutation, V617F, is the major determinant in myeloproliferative neoplasms (MPNs). JAK2 kinase inhibitors showed hematological toxicity in treating MPNs, calling for novel therapeutics that can target only the affected lineage while sparing others. This task is hampered by lack of understanding in how JAK2 signaling regulates the generation of different blood cells. We propose to fill this knowledge gap by determining JAK2 signaling pathways that differentially drive erythropoiesis vs. granulopoiesis, and delineate two novel pathways utilized by JAK2 to regulate signaling and transcription for erythroid differentiation. The overall goal of this proposl is to characterize mechanisms underlying how JAK2 regulates erythropoiesis. Aim 1 will characterize a novel set of murine models; each expresses a different activating JAK2 mutant that results in a distinct MPN phenotype. We will determine whether different JAK2 mutants cause erythrocytosis or granulocytosis by promoting lineage-specific proliferation or by skewing differentiation in common progenitor compartments, and identify downstream signaling pathways required therein to cause different MPN phenotypes. We will also translate these mechanistic studies into the human setting. Aims 2 and 3 will dissect two novel molecular mechanisms for JAK2 to orchestrate signaling and transcription for erythroid differentiation, and examine their contribution in MPN development. Aim 2 will use both in vitro and in vivo experiments to characterize how JAK2 directs endocytosis of the erythropoietin receptor to terminate signaling. This process involves a new function of the p85 subunit of PI3K and is PI3K kinase activity-independent. Defects in this process result in prolonged signaling in primary and familial congenital polycythemia. In Aim 3, we will employ transcriptional profiling and genome-wide chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to delineate a novel pathway whereby JAK2 controls transcription, not by the canonical JAK-STAT pathway, but by regulating chromatin structure through phosphorylating the polycomb repression complex 2. Results from these studies will fill key gaps in our understanding of signaling in hematopoietic stem and progenitor cells that regulate both normal and excessive erythropoiesis. These results will also further our understanding of how Epo signaling is terminated, and will shift the paradigm of how JAK2 activates erythroid transcriptional network. In addition, these results will shed light on MPN biology and facilitate the design of novel and more effective therapeutic agents that specifically target affected lineage without compromising other lineages.