Role of Vav and Rac in KIT oncogenesis

摘要 KIT是一种独特的受体，在黑素细胞、生殖细胞、Cajal间质细胞， 肥大细胞和造血干细胞。与KIT在这些特定组织中的重要性一致， KIT的激活突变已经在生殖细胞肿瘤，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）。显著 这些疾病的比例通常携带KIT激活环突变KITD 816 V。活化环 在核心结合因子-急性髓性白血病（CBF-AML）中也观察到KIT的突变， 携带t（8;21）或inv（16）细胞遗传学异常的白血病，产生融合基因AML 1- ETO或CBF？-MYH 11。对成人和儿童AML的研究表明， 携带t（8;21）的CBF-AML中KITD 816 V突变的存在根据几个因素预测了预后。 临床指标尽管在GIST中常见的跨膜区域内的KIT突变 对酪氨酸激酶抑制剂甲磺酸伊马替尼（格列卫）的抑制敏感; 酪氨酸激酶结构域的羧基末端叶，如KITD 816 V，稳定KIT活化环 在其活性形式的构象，这排除了足够的伊马替尼结合酪氨酸激酶抑制。 因此，与成功使用格列卫治疗胃肠道间质瘤相反，格列卫对胃肠道间质瘤的治疗无效。 携带KIT激活环突变体（即KITD 816 V）的血液病，包括SM和CBF- 急性髓细胞白血病重要的是，激活环所通过的受体近端和/或下游信号的性质 KIT中的突变（KITD 816 V）诱导原代造血细胞的转化的定义不清楚。我们有 证明KITD 816 V（小鼠中的KITD 814 V）诱导原发性造血细胞转化的证据 干细胞和祖细胞导致GEF Vav/Rho GTCRac途径的组成性激活， 小鼠中造血特异性Vav 1和/或Rac 2的遗传破坏消除了配体非依赖性生长， KITD 814 V，导致我们假设参与KITD 814 V诱导的转化的信号可能部分地与KITD 814 V诱导的转化相关。 通过这种途径的过度激活来介导。此外，我们有证据表明， KITD 814 V胞内结构域内的酪氨酸残基导致KITD 814 V完全丧失 诱导的配体非依赖性生长，导致我们假设，细胞内的酪氨酸， KITD 814 V的质膜和激酶插入区可能有助于KITD 814 V诱导的 转型基于这些发现，本申请的中心假设是， Vav/Rac通路参与了系统性肥大细胞增多症、AML等疾病的发病 与其他涉及KITD 814 V突变的疾病一样。我们提出的研究将提供机制的见解， Vav/Rac通路的生理学意义以及质膜和膜的参与， 激酶插入序列在调节KITD 814 V诱导的转化中的作用，目前还没有针对该作用的药物。

ABSTRACT 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 CBF¿-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.