Role of Shp2 in FLT3-ITD-Induced Leukemogenesis

描述(由申请人提供)：急性髓系白血病(AML)是一种致命的疾病，其发病率在65岁的人中急剧增加，这是美国增长最快的人口。令人遗憾的是，65岁以上患者的5年生存率(4.3%)显著低于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急性髓细胞白血病转化过程的分子机制(S)，以努力开发用于这种致命疾病的新颖、分子靶向、有效和毒性较低的治疗方法。内部串联重复(ITD)是一种框内突变，导致FMS样酪氨酸激酶受体(Flt3)膜旁区域附近的几个氨基酸插入或复制，在所有AML患者中可见近25%，预后不良。在多发性成人急性白血病的原代白血病细胞标本中，Shp2蛋白酪氨酸磷酸酶的表达持续高于健康对照的骨髓单个核细胞中Shp2的水平。然而，Shp2如何在髓系白血病发生中起作用尚不清楚。我们提出的初步研究表明，Shp2与突变的N51-Flt3和N73-Flt3细胞中的Flt3存在结构性关联，与在WT Flt3细胞中观察到的相比，Shp2的基因破坏和Shp2的药物抑制优先抑制N51-Flt3诱导的过度增殖。因此，由于Shp2已被证明调节Rho-GTP酶的Rac亚家族在造血细胞中的激活，我们推测，rac1和/或rac2也可能是Flt3-ITDS的相关效应因子。我们一贯提出的初步发现表明，使用NSC23766的药理RAC抑制或rac2的基因破坏导致显著减少N51-Flt3诱导的过度增殖。根据我们的初步数据，STAT5过度激活在Flt3-ITD诱导的白血病中的中心作用，以及Shp2和rac1/rac2促进激活的STAT5核聚集的积极作用，这一应用的中心假设是，从机制上讲，Shp2和rac1/rac2通过促进STAT5核定位和STAT5反应的促白血病基因的表达而在Flt3-ITD诱导的白血病中起作用。本应用的目的是确定Shp2和rac1/rac2基因突变在体内对STAT5的激活和核定位以及对Flt3-ITD诱导的骨髓增生性疾病(MPD)发展的影响，检测Shp2和rac1抑制剂在体内AML异种移植模型中的有效性，并确定有助于配体非依赖性增殖的Flt3-ITD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.