Role of NF-kB in hematopoietic stem cells and leukemia-initiating cell formation

描述（由申请人提供）：经典NF-？B途径，主要涉及RelA/p65， c-Rel和p50，已在人类急性髓性白血病（AML）中观察到，其中构成性NF-？B dna结合活性已在髓细胞和CD34？造血祖细胞占所有病例的47-100%。在AML中，本构性NF-？B活性可能是激活影响PI3激酶途径的突变的直接下游结果，PI3激酶途径在大多数AML病例中也具有组成性活性，并诱导NF-？B通过Akt信号传导。在小鼠中，通过诱导缺失Akt调控磷酸酶PTEN激活Akt，可导致骨髓中造血干细胞（HSC）的快速丢失和可移植、致死性骨髓增生性疾病（MPD）和AML的发展。这些研究和其他研究表明，Akt和NF-？B区分骨髓中白血病起始细胞（LIC）和正常造血干细胞。来解决NF-？B在HSC中的激活足以诱导LIC形成并促进MPD/AML，我们表达了I？B激酶(Ikk？SS/EE)在正常HSC中使用逆转录病毒载体。Ikk?重组小鼠的分析表达SS/EE -的细胞显示构成性NF-？与活化的Akt不同，B活性促进骨髓中相对快速的HSC损失，不伴随MPD/AML的发展。NF - ?b介导的HSC损失可能是由于分化和HSC自我更新潜力的丧失，因为没有证据表明HSC凋亡增加或动员到脾脏。此外，阻断NF-？在HSC中，通过Ikk调控亚基NEMO的条件缺失激活B，导致最早的多电位祖细胞亚群（c-Kit、Sca-1）显著减少。细胞)和更原始的c-Kit - Sca-1的绝对数量增加。(KLSF)细胞。NEMO缺失也完全阻断了KLSF细胞的体外增殖，这进一步支持了NF-？B在促进最早的HSC分化事件。总之，这些结果证明了经典NF-？B通路作为AML的启动事件，不足以维持HSC或促进与AML相关的LIC的发展。本提案的主要目标是检验NF-？在正常的HSC中，B的功能是结合与第一次HSC细胞分裂相关的增殖和分化，并在其他致癌变化之后维持LIC的活力，从而在活化的NF- B存在下保持HSC的自我更新。为了验证这一假设，我们将追求以下具体目标：(1)确定NF-？B在正常HSC维持、增殖和分化中的调节作用(2)。B到Akt刺激的白血病发生，骨髓中LT-HSC丢失，白血病启动细胞形成和(3)，表征AML中发现的复发性细胞遗传学异常，包括AML1-ETO， inv（16）或NUP98-HOXA9的易位，是否可以在活化的Akt或NF- B存在下保持HSC的自我更新。

DESCRIPTION (provided by applicant): Constitutive activation of the classical NF-?B pathway, which principally involves RelA/p65, c-Rel, and p50, has been observed in human acute myeloid leukemia (AML) where constitutive NF-?B DNA-binding activity has been detected in myeloid blasts and in CD34? hematopoietic progenitor cells in 47-100% of all cases. In AML, constitutive NF-?B activity could be a direct downstream consequence of activating mutations affecting the PI3 kinase pathway, which is also constitutively active in the majority of AML cases and induces NF-?B through Akt signaling. In mice, constitutive activation of Akt through inducible deletion of the Akt regulatory phosphatase, PTEN, results in rapid hematopoietic stem cell (HSC) loss from bone marrow and development of a transplantable, lethal myeloproliferative disease (MPD) and AML. These studies and others have shown that constitutive activation of Akt and NF-?B distinguishes leukemia-initiating cells (LIC) from normal HSC in bone marrow. To address whether NF-?B activation in HSC is sufficient to induce LIC formation and promote MPD/AML, we expressed a constitutively active allele of I?B kinase beta (Ikk?SS/EE) in normal HSC using a retroviral vector. Analysis of mice reconstituted with Ikk?SS/EE -expressing cells showed that constitutive NF-?B activity promoted relatively rapid HSC loss from bone marrow that, unlike activated Akt, was not accompanied by the development of MPD/AML. NF-?B-mediated HSC loss was likely due to differentiation and loss of HSC self-renewal potential since there was not evidence of increased HSC apoptosis or mobilization to the spleen. Furthermore, blocking NF-?B activation in HSC by conditional deletion of the Ikk regulatory subunit, NEMO, resulted in a substantial decrease in one of the earliest multipotential progenitor subsets (c-Kit?Sca-1? cells) and an increase in absolute numbers of more primitive c-Kit?Sca-1? (KLSF) cells. NEMO loss also completely blocked proliferation of KLSF cells in vitro, which further supports an essential role for NF-?B in promoting the earliest HSC differentiation event. Together, these results demonstrate that constitutive activation of the classical NF-?B pathway, as an initiating event in AML, is not sufficient to maintain HSC or to promote development of LIC associated with AML. The primary goal of this proposal will be to test the central hypothesis that NF-?B functions in normal HSC to couple proliferation and differentiation associated with the first HSC cell division and to maintain viability of LIC subsequent to other oncogenic changes that preserve HSC self-renewal in the presence of activated NF-?B. To test this hypothesis, we will pursue the following specific aims: (1), Determine the function of NF-?B in the regulation of normal HSC maintenance, proliferation, and differentiation (2), Define the contribution of NF-?B to Akt-stimulated leukemogenesis, LT-HSC loss from bone marrow, and to leukemia-initiating cell formation and (3), Characterize whether recurrent cytogenetic abnormalities found in AML, including the translocations AML1-ETO, inv(16), or NUP98-HOXA9, can preserve HSC self-renewal in the presence of activated Akt or NF-?B.