Role of the Class IA PI3K in Hematopoietic Stem Cell Self-Renewal

建议书摘要/摘要 成体造血干细胞(HSCs)是一种罕见而独特的干细胞群体，存在于 骨髓，在那里它们经历自我更新和分化，以维持血液系统。适当地 维持自我更新和分化之间的平衡，造血干细胞从生长因子和 趋化因子激活进化保守的肌醇磷脂3-激酶/蛋白激酶B(PI3K/AKT) 信号通路。这一途径的病理激活在癌症中经常观察到，包括白血病， 使其成为癌症治疗的理想靶点。几种PI3K抑制剂已经在临床上使用，并 为了更好地指导治疗，了解PI3K在成人HSCs中的作用是至关重要的。 造血细胞表达三种IA类催化亚型PI3K(p110α，β，δ)，它们都可以 传递生长因子和细胞因子信号。在这些亚型中，p110β是独一无二的，因为除了 通过受体酪氨酸激酶(RTK)传递生长因子信号，可直接与G蛋白相互作用 偶联受体Gβɣ亚基以转导趋化因子，并与Rac和RAB5GTP酶结合。在……里面 小鼠胚胎成纤维细胞，p110β-RAB5的相互作用被证明是重要的诱导 自噬细胞循环过程是维持HSC新陈代谢和自我调节所必需的。 更新。单个1A型PI3K亚型在成熟的造血系中具有独特的功能，但它们 对于HSCs的功能是不可缺少的。为了研究1A类PI3K在HSC中的冗余角色，我们有 建立了条件缺失p110α和p110β的三重基因敲除小鼠模型 细胞和p110δ的生殖系缺失。对这些TKO小鼠的分析显示，在失去所有三个类别1A后 异构体导致HSCs数量增加，但自我更新和分化减少，效率低下 所有成熟血统的重新繁殖。这种表型不同于任何PI3K单项的表型 同型基因敲除小鼠模型，甚至来自p110α；δ双基因敲除动物，提示p110β 异构体在HSCs中起着重要的代偿作用。此外，我的数据显示，I类PI3K的损失 导致在缺乏生长因子时自噬诱导的减少，尽管自噬仍然可以 用mTOR抑制剂雷帕霉素诱导。因此，我假设损失IA类PI3K妥协 自噬诱导，这会导致HSC代谢改变，损害HSC的健康。 拟议的研究将使用我们的PI3K TKO小鼠模型来阐明目标1中的细胞机制 用于HSC中有缺陷的自我更新。目的2将确定自噬在TKO HSC功能障碍中的作用。最后， 目标3将确定p110β的哪些结合作用对其补偿作用最重要 HSC功能。综上所述，这项研究将描绘细胞和分子机制， 1API3K支持HSC自我更新和分化。

Proposal Summary/Abstract Adult hematopoietic stem cells (HSCs) are a rare and unique population of stem cells that reside in the bone marrow, where they undergo self-renewal and differentiation to maintain the blood system. To properly maintain the balance between self-renewal and differentiation, HSCs receive signals from growth factors and chemokines to activate the evolutionarily conserved phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. Pathologic activation of this pathway is frequently observed in cancers, including leukemia, making it a desirable target for cancer treatment. Several PI3K inhibitors are already used in the clinic and to better inform therapeutic targeting, it is crucial to understand the roles of PI3K in adult HSCs. Hematopoietic cells express three Class IA catalytic isoforms of PI3K (p110α, β, δ), all of which can transduce growth factor and cytokines signals. Out of these isoforms, p110β is unique, since in addition to transducing growth factor signals through receptor tyrosine kinases (RTK), it can directly interact with G-protein coupled receptor Gβɣ subunits to transduce chemokines, and also binds to RAC and to RAB5 GTPases. In mouse embryonic fibroblasts, the p110β-RAB5 interaction was shown to be important for the induction of autophagic cellular recycling process, which is essential for the maintenance of HSC metabolism and self- renewal. Individual Class 1A PI3K isoforms have unique functions in mature hematopoietic lineages, but they are dispensable for HSCs function. To study the redundant roles of Class 1A PI3K in HSCs, we have generated a triple knockout (TKO) mouse model with conditional deletion of p110α and p110β in hematopoietic cells, and germline deletion of p110δ. Analysis of these TKO mice reveals upon the loss of all three Class1A isoforms causes an increase in HSCs numbers, but decreased self-renewal and differentiation, with inefficient repopulation of all mature blood lineages. This phenotype is different from the phenotypes of any PI3K single isoform knockout mouse model, and even from p110α;δ double knockout animals, suggesting that p110β isoform plays an important compensatory role in HSCs. Moreover, my data suggests that loss of Class I PI3K causes a decrease in autophagy induction upon growth factor deprivation, though autophagy can still be induced with the mTOR inhibitor rapamycin. Thus, I hypothesize that loss of Class IA PI3K compromises autophagy induction, which causes altered HSC metabolism and impaired HSCs fitness. The proposed studies will use our PI3K TKO mouse model to elucidate in Aim 1 the cellular mechanism for defective self-renewal in HSCs. Aim 2 will establish the roles of autophagy in TKO HSC dysfunction. Lastly, Aim 3 will determine which binding interactions of p110β are the most important for its compensatory role in HSC function. In summary, this research will delineate the cellular and molecular mechanisms by which Class 1A PI3K supports HSC self-renewal and differentiation.