Role of Vav and Rac in KIT oncogenesis

描述（由申请人提供）：KIT是一种独特的受体，在黑色素细胞、生殖细胞、Cajal间质细胞、肥大细胞和造血干细胞中具有重要的功能作用。与KIT在这些明确组织中的重要性相一致的是，在生殖细胞瘤、胃肠道间质瘤（gist）、鼻窦淋巴瘤、急性髓性白血病（AML）和系统性肥大细胞增多症（SM）中已经描述了KIT的激活突变。这些疾病中很大一部分通常携带KIT激活环突变KITD816V。KIT的激活环突变也在核心结合因子-急性髓性白血病（CBF-AML）中被观察到，这些白血病具有t（8;21）或v（16）细胞遗传学异常，分别产生融合基因AML1- ETO或CBF2-MYH11。针对成人和儿童AML的研究表明，在携带t的CBF-AML中存在KITD816V突变（8;21），根据几个临床指标，预后会恶化。尽管gist中常见的近膜区KIT突变对酪氨酸激酶抑制剂甲磺酸伊马替尼（格列卫）的抑制很敏感；酪氨酸激酶结构域羧基末端叶的KIT突变，如KITD816V，稳定了活性形式的KIT激活环构象，从而排除了充分的伊马替尼结合来抑制酪氨酸激酶。因此，与成功使用格列卫治疗gist相比，格列卫对含有KIT激活环突变体（即KITD816V）的血液系统疾病（包括SM和CBF- AML）无效。重要的是，KIT （KITD816V）激活环突变诱导原代造血细胞转化的受体近端和/或下游信号的性质尚不清楚。我们有证据表明KITD816V（小鼠KITD814V）诱导的原发性造血干细胞和祖细胞转化导致GEF Vav/Rho GTPase Rac途径的组成性激活，并且小鼠造血特异性Vav1和/或Rac2的遗传破坏通过KITD814V消除了配体独立的生长，这使得我们假设KITD814V诱导转化的信号可能部分通过该途径的过度激活介导。此外，我们有证据表明，突变KITD814V胞内区域的酪氨酸残基会导致KITD814V诱导的配体独立生长完全丧失，这使我们假设KITD814V近膜和激酶插入区域的胞内酪氨酸可能有助于KITD814V诱导的转化。基于这些发现，本应用的中心假设是Vav/Rac通路的过度激活有助于与系统性肥大细胞增多症、AML以及其他涉及KITD814V突变的疾病相关的疾病的病因学。我们提出的研究将为Vav/Rac通路的生理学意义以及近膜和激酶插入序列在调节KITD814V诱导转化中的作用提供机制见解，目前尚无药物可用于此。公共卫生相关性：现在明确的是，激活KIT突变有助于生殖细胞肿瘤、胃肠道间质瘤（gist）、鼻窦淋巴瘤、急性髓性白血病（AML）和系统性肥大细胞增多症（SM）。我们提出的研究将提供通过激活KIT突变调节转化的信号通路的机制，目前还没有药物存在。我们的研究结果有望为AML和SM等疾病的分子治疗提供新的靶点。

DESCRIPTION (provided by applicant): KIT is a unique receptor with important functional roles in melanocytes, germ cells, interstitial cells of Cajal, mast cells, and hematopoietic stem cells. Consistent with the importance of KIT in these defined tissues, activating mutations of KIT have been described in germ cell tumors, gastrointestinal stromal tumors (GISTs), sinonasal lymphomas, acute myeloid leukemia (AML), and systemic mastocytosis (SM). A significant proportion of these diseases commonly bear the KIT activation loop mutation KITD816V. Activation loop mutations of KIT have also been observed in core binding factor-acute myeloid leukemia (CBF-AML), leukemias that bear either the t(8;21) or inv(16) cytogenetic abnormality, generating the fusion genes AML1- ETO or CBF2-MYH11, respectively. Studies examining both adult and pediatric AML have indicated that the presence of the KITD816V mutation in CBF-AML carrying t(8;21) worsens the prognosis based on several clinical indices. Although KIT mutations within the juxtamembrane region that are commonly found in GISTs are sensitive to inhibition by the tyrosine kinase inhibitor, imatinib mesylate (Gleevec); KIT mutations within the carboxy-terminal lobe of the tyrosine kinase domain, such as KITD816V, stabilizes the KIT activation loop conformation in its active form, which precludes sufficient imatinib binding for tyrosine kinase inhibition. Therefore, in contrast to successful use of Gleevec to treat GISTs, Gleevec is ineffective for the treatment of the hematologic diseases harboring the KIT activation loop mutants (i.e. KITD816V), including SM and CBF- AML. Importantly, nature of the receptor proximal and/or downstream signals by which activation loop mutations in KIT (KITD816V) induce transformation in primary hematopoietic cells are poorly defined. We have evidence to demonstrate that KITD816V (KITD814V in mice) induced transformation in primary hematopoietic stem and progenitor cells results in constitutive activation of GEF Vav/Rho GTPase Rac pathway and that genetic disruption of hematopoietic specific Vav1 and/or Rac2 in mice abrogates ligand independent growth via KITD814V, leading us to hypothesize that signals involved in KITD814V induced transformation may in part be mediated via the hyperactivation of this pathway. Furthermore, we have evidence demonstrating that mutating the tyrosine residues within the intracellular domain of KITD814V results in complete loss of KITD814V induced ligand independent growth, leading us to hypothesize that the intracellular tyrosines within the juxtamembrane and the kinase insert region of KITD814V are likely to contribute to KITD814V induced transformation. Based on these findings, the central hypothesis of this application is that hyperactivation of the Vav/Rac pathway contributes to the etiology of diseases associated with systemic mastocytosis, AML as well as other diseases involving the KITD814V mutation. Our proposed studies will provide mechanistic insight into the physiologic significance of the Vav/Rac pathway as well as the involvement of the juxtamembrane and the kinase insert sequences in regulating KITD814V induced transformation for which currently no drugs exist. PUBLIC HEALTH RELEVANCE: It is now unequivocal that activating mutations of KIT contribute to germ cell tumors, gastrointestinal stromal tumors (GISTs), sinonasal lymphomas, acute myeloid leukemia (AML), and systemic mastocytosis (SM). Our proposed studies will provide mechanistic insight into the signaling pathways that regulate transformation via an activating KIT mutation for which currently no drugs exist. Our results are expected to provide new targets for molecular therapies for the treatment of diseases such as AML and SM.