Role of Shp2 in FLT3-ITD-Induced Leukemogenesis

描述（由申请人提供）：急性髓性白血病（AML）是一种致命疾病，在美国65岁以上的人群中发病率急剧增加，这是增长最快的人群。遗憾的是，65岁以上患者的5年生存率（4.3%）明显低于65岁以下患者（34.45%）。这些令人沮丧的统计数据促使科学家们研究导致AML转化过程的分子机制，努力开发新的、分子靶向的、有效的、毒性较小的治疗方法，用于治疗这种致命疾病。在fms样酪氨酸激酶受体（FLT3）中，内部串联重复（ITD）是一种框架内突变，导致近膜结构域附近的几个氨基酸插入或重复，在近25%的AML患者中可见，并导致预后不良。与健康对照者骨髓单核细胞中的Shp2水平相比，来自多个成人急性白血病的原发性白血病细胞标本中Shp2蛋白酪氨酸磷酸酶的表达持续升高。然而，Shp2如何促进髓性白血病的发生尚不清楚。我们的初步研究表明，在突变的N51-FLT3-和n73 -FLT3-携带细胞中，Shp2与FLT3组成相关，并且与在WT FLT3携带细胞中观察到的相比，Shp2的遗传破坏和Shp2的药理抑制优先降低了N51-FLT3诱导的过度增殖。因此，由于Shp2已被证明可以调节造血细胞中rho - gtpase的Rac亚家族的激活，我们预测Rac1和/或Rac2也可能是FLT3-ITDs的相关效应物。与此一致，我们提出的初步研究结果表明，使用NSC23766进行Rac药理学抑制或对Rac2进行遗传破坏可显著降低n51 - flt3诱导的过度增殖。根据我们的初步数据，STAT5超激活在flt3 - itd诱导的白血病中的核心作用，以及报道的Shp2和Rac1/Rac2促进活化的STAT5核积累的积极作用，本应用的中心假设是，从机制上讲，Shp2和Rac1/Rac2通过促进STAT5核定位和STAT5应答的促白血病基因的表达，促进了flt3 - itd诱导的白血病。本申请的目的是确定Shp2和Rac1/Rac2的遗传破坏对体内STAT5的激活和核定位以及flt3 - itd诱导的骨髓增生性疾病（MPD）的发展的影响，并检查Shp2抑制剂和Rac1抑制剂在体内AML异种移植模型中的疗效。并确定FLT3-ITD近膜内的细胞内酪氨酸和复制的近膜有助于配体非依赖性增殖。

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is a lethal disease which dramatically increases in incidence in individuals >65 years of age, the fastest growing population in the United States. Regrettably, the 5 year survival rate drops dramatically in patients over 65 years (4.3%) compared to patients less than 65 years (34.45%). These dismal statistics have led scientists to investigate the molecular mechanism(s) underlying the transforming process leading to AML in an effort to develop novel, molecularly-targeted, effective, and less toxic therapies for use in this lethal disease. Internal tandem duplications (ITD), an in-frame mutation leading to insertion or duplication of several amino acids near the juxtamembrane domain, in fms-like tyrosine kinase receptor (FLT3), are seen in nearly 25% of all AML patients and confer a poor prognosis. Expression of the Shp2 protein tyrosine phosphatase is consistently elevated in primary leukemia cell specimens from multiple adult acute leukemias compared to Shp2 levels in bone marrow mononuclear cells from healthy controls. However, how Shp2 contributes to myeloid leukemogenesis is unknown. We present preliminary studies demonstrating that Shp2 is constitutively associated with FLT3 in mutant N51-FLT3- and N73-FLT3-bearing cells and that genetic disruption of Shp2 and pharmacologic inhibition of Shp2 preferentially reduces N51-FLT3-induced hyperproliferation compared to that observed in WT FLT3-bearing cells. As a corollary, since Shp2 has been shown to regulate the activation of the Rac subfamily of Rho-GTPases in hematopoietic cells, we predicted that Rac1 and/or Rac2 may also be relevant effectors of FLT3-ITDs. Consistently, we present preliminary findings demonstrating that pharmacologic Rac inhibition using NSC23766 or genetic disruption of Rac2 results in significantly reduced N51-FLT3-induced hyperproliferation. Based on our preliminary data, the central role of STAT5 hyperactivation in FLT3-ITD-induced leukemia, and the reported positive roles of Shp2 and Rac1/Rac2 promoting activated STAT5 nuclear accumulation, the central hypothesis of this application is that, mechanistically, Shp2 and Rac1/Rac2 contribute to FLT3-ITD-induced leukemia by facilitating STAT5 nuclear localization and the expression of STAT5-responsive pro-leukemogenic genes. The objectives of this application are to define the consequences of genetic disruption of Shp2 and Rac1/Rac2 on the activation and nuclear localization of STAT5 and on the development of FLT3-ITD-induced myeloproliferative disorder (MPD) in vivo, to examine the efficacy of a Shp2 inhibitor and a Rac1 inhibitor in an AML xenograft model in vivo, and to define the intracellular tyrosines within the juxtamembrane and the duplicated juxtamembrane of FLT3-ITD that contribute to ligand-independent proliferation. PUBLIC HEALTH RELEVANCE: Acute myeloid leukemia (AML) is a lethal disease which dramatically increases in incidence in individuals >65 years of age, the fastest growing population in the United States. We present studies that will examine the role of the protein tyrosine phosphatase, Shp2, and the Rac subfamily of Rho GTPases in the pathogenesis of AMLs bearing internal tandem duplications (ITDs) of the fms-like tyrosine kinase receptor (FLT3). Results from these studies are expected to identify new therapeutic targets for treating AML.