Role of Shp2 in FLT3-ITD-Induced Leukemogenesis

描述（由申请人提供）：急性髓性白血病（AML）是一种致死性疾病，其在>65岁的个体（美国增长最快的人群）中的发病率急剧增加。令人遗憾的是，与65岁以下的患者（34.45%）相比，65岁以上的患者（4.3%）的5年生存率急剧下降。这些令人沮丧的统计数据促使科学家们研究导致AML的转化过程的分子机制，以努力开发用于这种致命疾病的新型分子靶向，有效和毒性较小的疗法。内部串联重复（ITD）是一种导致fms样酪氨酸激酶受体（FLT 3）中质膜结构域附近几个氨基酸插入或重复的框内突变，在近25%的AML患者中可见，并导致预后不良。与来自健康对照的骨髓单核细胞中的Shp 2水平相比，来自多发性成人急性白血病的原代白血病细胞标本中Shp 2蛋白酪氨酸磷酸酶的表达持续升高。然而，Shp 2如何促进骨髓性白血病发生尚不清楚。我们目前的初步研究表明，Shp 2与突变型N51-FLT 3和N73-FLT 3携带细胞中的FLT 3组成性相关，与WT FLT 3携带细胞中观察到的结果相比，Shp 2的遗传破坏和Shp 2的药理学抑制优先降低N51-FLT 3诱导的过度增殖。作为推论，由于Shp 2已显示调节造血细胞中Rho-GTP酶的Rac亚家族的活化，我们预测Rac 1和/或Rac 2也可能是FLT 3-ITD的相关效应子。因此，我们提出的初步研究结果表明，使用NSC 23766的药理学Rac抑制或Rac 2的遗传破坏导致N51-FLT 3诱导的过度增殖显著降低。基于我们的初步数据，STAT 5超活化在FLT 3-ITD诱导的白血病中的中心作用，以及报道的Shp 2和Rac 1/Rac 2促进活化的STAT 5核积累的积极作用，本申请的中心假设是，从机制上讲，Shp 2和Rac 1/Rac 2通过促进STAT 5核定位和STAT 5应答性前体蛋白的表达促进FLT 3-ITD诱导的白血病。白血病基因本申请的目的是确定Shp 2和Rac 1/Rac 2的遗传破坏对体内STAT 5的活化和核定位以及对FLT 3-ITD诱导的骨髓增生性疾病（MPD）的发展的影响，以检查Shp 2抑制剂和Rac 1抑制剂在体内AML异种移植模型中的功效，并确定FLT 3-ITD的胞膜和复制胞膜内有助于配体非依赖性增殖的胞内酪氨酸。

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.