Oncogenic Kit receptor signaling in vivo

描述（由申请人提供）：该提案的总体目标是双重的，1)继续我们在体内研究致癌Kit受体信号，重点是造血，2)开发伊马替尼耐药GIST的小鼠模型。在小鼠W位点编码的Kit受体在造血、配子发生、黑色素形成和肠道运动中起作用。正常Kit受体介导的功能包括细胞增殖、细胞存活、细胞粘附、细胞迁移、分泌反应和分化。在人类肿瘤中，Kit的致癌激活在胃肠道间质瘤（GIST）、肥大细胞增多症/肥大细胞白血病、急性骨髓性白血病、一部分黑色素瘤和一部分生殖细胞肿瘤中发挥作用。Kit受体的功能由激酶激活、受体自磷酸化以及与各种信号分子和信号级联的关联介导。受体酪氨酸激酶（如Kit）如何在胚胎发育和动物出生后的不同细胞类型中介导不同的细胞反应？不同细胞类型的致癌转化产生癌症的条件是什么。我们已经产生了Kit受体基因中含有敲入点突变的小鼠，该突变阻断Kit介导的PI 3激酶激活和信号传导或Src家族激酶，SFK，激活和信号传导。这些小鼠在配子发生和造血方面具有明显不同的表型。我们还研究了PI 3激酶和Src家族激酶信号在Kit介导的骨髓源肥大细胞（BMMC）细胞增殖、细胞凋亡抑制、细胞粘附、趋化性和分泌反应中的作用。Kit介导的PI 3-激酶信号传导对细胞增殖、细胞存活、细胞粘附、趋化和分泌反应至关重要，而Kit介导的SFK信号传导主要是负调控作用。此外，基因表达谱分析表明，阻断SFK信号传导可降低KitL诱导的BMMC中th2细胞因子基因的表达。为了研究致癌Kit激活Kit在肿瘤发生中的作用，我们制造了携带近膜结构域敲入Kit突变的小鼠，并复制了人类家族性GIST综合征（Sommer et al., 2003）。我们提出的部分研究将试图探讨PI 3-激酶、SFK和Ras信号在kit介导的肿瘤发生中的作用。此外，我们将产生和表征携带致癌伊马替尼耐药Kit等位基因的小鼠。公共卫生相关性：Kit受体在Cajal间质细胞的色素形成、造血、生殖细胞发育和胃肠道起搏器细胞中发挥作用。基于Kit突变小鼠的表型，Kit可能介导的细胞反应似乎是多种多样的，包括细胞增殖、细胞存活（抑制凋亡/细胞死亡）、细胞粘附、迁移、分泌反应和分化。此外，Kit在人类癌症中也有作用。与Kit的致癌激活相关的癌症主要包括GIST，但在肥大细胞增生症、精原细胞瘤、一小部分aml和一小部分黑色素瘤中也观察到Kit的致癌激活。Kit受体如何介导这些不同的结果？Kit受体可以激活几种不同的信号级联反应，包括磷脂酰肌醇-3激酶（PI -3激酶）和Src家族激酶（SFK）。这两种信号级联在受体酪氨酸激酶信号传导和致癌转化中起关键作用。为了研究这个问题，我们用苯丙氨酸取代Kit蛋白中的关键酪氨酸残基，对小鼠基因组中的Kit基因进行了修饰。Kit受体的这些替代突变阻断了PI - 3激酶或SFK的激活。对携带这些突变的小鼠的表型分析表明，PI 3-激酶在雄性生殖细胞发育中至关重要，但没有其他可识别的表型，而SFK突变小鼠在造血细胞系中存在缺陷，但在生殖细胞发育中没有缺陷。这些结果强调了Kit受体发挥作用的细胞环境或细胞背景的重要性，以及动物模型对于阐明受体酪氨酸激酶信号在不同细胞类型中的作用和机制至关重要。在大多数癌症中，致癌Kit受体突变发生在体细胞中。然而，在极少数情况下，在生殖细胞中获得致癌Kit突变并在种系中传播。我们利用同源重组方法将家族性GIST病例中发现的突变植入小鼠基因组，并获得了一种在种系中携带该突变的小鼠品系。这些小鼠非常忠实地再现了人类家族性GIST。这些发现表明，Kit突变是家族性和非家族性GIST发展的起始事件。其次，他们强调了突变Kit受体产生GIST而不是其他癌症的独特特异性，这意味着GIST细胞及其微环境中的细胞机制在支持肿瘤形成和肿瘤维持方面是非常独特的。这些GIST小鼠为研究致癌Kit受体信号传导机制提供了独特的机会，并为评估可能用于治疗伊马替尼耐药GIST的第二代药物提供了机会。作为该提案的一部分，我们首先研究了Kit-V558缺失突变在造血和血液恶性肿瘤中的作用。其次，我们将研究致癌信号衍生Kit突变小鼠的机制，该突变小鼠含有致癌Kit突变，但不能激活SFK或PI 3-激酶信号。如今，GIST患者已成功地接受了激酶抑制剂伊马替尼的治疗。然而，由于获得继发性Kit突变，大多数患者最终对药物治疗产生耐药性。受我们在小鼠中成功概括家族性GIST的鼓舞，作为该提案的一部分，我们将制作伊马替尼耐药GIST的小鼠模型。

DESCRIPTION (provided by applicant): The overall objective of this proposal is twofold, 1) to continue our investigations of oncogenic Kit receptor signaling in vivo with emphasis on hematopoiesis and 2) to develop mouse models for imatinib resistant GIST. The Kit receptor encoded at the murine W locus functions in hematopoiesis, gametogenesis, melanogenesis and gut motility. Normal Kit receptor mediated functions include cell proliferation, cell survival, cell adhesion, cell migration, secretory responses and differentiation. In human neoplasia oncogenic activation of Kit has roles in gastrointestinal stromal tumors (GIST), mastocytosis/mast cell leukemia, acute myelogenous leukemia, a subset of melanomas and a subset of germ cell tumors. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules and signaling cascades. How do receptor tyrosine kinases such as Kit mediate distinct cellular responses in different cell types during embryonic development and in the postnatal animal? And what are the requirements for oncogenic transformation in different cell types to produce cancer. We have produced mice containing knock-in point mutations in the Kit receptor gene in mice which block Kit mediated PI 3-kinase activation and signaling or Src family kinase, SFK, activation and signaling. These mice have distinctly different phenotypes in gametogenesis and hematopoiesis. We have also investigated the role of PI 3-kinase and Src family kinase signaling in Kit mediated cell proliferation, suppression of apoptosis, cell adhesion, chemotaxis and secretory responses in vitro in bone marrow derived mast cells (BMMC). Whereas Kit mediated PI 3-kinase signaling is critical for cell proliferation, cell survival, cell adhesion, chemotaxis and secretory responses, Kit mediated SFK signaling has mostly a negative regulatory role. Furthermore, gene expression profiling indicates that blocking SFK signaling reduces KitL induced expression of TH2-cytokine genes in BMMC. To investigate the role of oncogenic Kit activation Kit in tumorigenesis we have produced mice carrying a juxtamembrane domain knock-in Kit mutation and reproduced human familial GIST syndrome (Sommer et al., 2003). In part our proposed studies will attempt to investigate the role of PI 3-kinase, SFK and Ras signaling in Kit-mediated tumorigenesis. Furthermore we will produce and characterize mice carrying oncogenic imatinib resistant Kit alleles. PUBLIC HEALTH RELEVANCE: The Kit receptor has roles in pigment formation, hematopoiesis, germ cell development and pacemaker cells of the gastrointestinal tract in interstitial cells of Cajal. Based on phenotypes of Kit mutant mice, the cellular responses which Kit may mediate appear to be quite diverse and include cell proliferation, cell survival (suppression of apoptosis/cell death), cell adhesion, migration, secretory responses and differentiation. Furthermore, Kit has roles in human cancer. The cancers which are associated with oncogenic activation of Kit include most importantly GIST, but oncogenic activation of Kit is also observed in mastocytosis, seminomas, a small subset of AMLs and a small subset of melanomas. How does the Kit receptor mediate these diverse outcomes? The Kit receptor is known to activate several distinct signaling cascades including phosphatidyl inositol-3 kinase (PI 3-kinase) and Src family kinases (SFK). Both signaling cascades have critical roles in receptor tyrosine kinase signaling and oncogenic transformation. To investigate this question we have modified the Kit gene in the mouse genome by substituting critical tyrosine residues in the Kit protein with phenylalanine. These substitution mutations in the Kit receptor block either PI 3-kinase or SFK activation. The analysis of the phenotypes of the mice carrying these mutations brought to light that PI 3-kinase is critical in male germ cell development, but had no other discernible phenotypes, whereas the SFK mutant mice had defects in hematopoietic cell lineages, but not in germ cell development. These results highlight the critical importance of the cellular environment or cellular context in which the Kit receptor functions and that animal models are critical for elucidating the role and mechanisms of receptor tyrosine kinase signaling in different cell types. In most cancers oncogenic Kit receptor mutations occur in somatic cells. However, in rare occasions oncogenic Kit mutations are acquired in germ cells and are transmitted in the germ line. We have engineered a mutation found in a familial GIST case into the mouse genome by using homologous recombination approaches and a mouse strain which carries this mutation in the germline has been obtained. These mice recapitulate human familial GIST quite faithfully. These findings demonstrated that the Kit mutation is the initiating event in the development of familial and presumably non-familial GIST. Second they highlight the unique specificity of the mutant Kit receptor to produce GIST and not other cancers and this implies that the cellular machinery in GIST cells and their microenvironment is quite unique in supporting tumor formation and tumor maintenance. These GIST mice provide a unique opportunity to investigate the mechanism of oncogenic Kit receptor signaling and they provide an opportunity to evaluate second generation drugs which might be useful in the treatment of imatinib resistant GIST. As part of this proposal first we are investigating the role of the oncogenic Kit-V558 deletion mutation in hematopoiesis and in hematologic malignancies. Second we will investigate the mechanism of oncogenic signaling deriving Kit mutant mice containing an oncogenic Kit mutation but which fail to activate SFK or PI 3-kinase signaling. Today patients with GIST are successfully treated with the kinase inhibitor imatinib. However most patients eventually become resistant to drug treatment as a result of the acquisition of a secondary Kit mutation. Encouraged by our success to recapitulate familial GIST in mice, as part of this proposal we will to produce mouse models for imatinib resistant GIST.