Mechanisms underlying constitutive activation and oncogenic potential of mutant c-kit receptor tyrosine kinase

突变c-kit受体酪氨酸激酶的组成性激活和致癌潜力的机制

• 批准号: 11670998
• 负责人: IKEDA Hirokazu
• 金额: $ 2.24万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670998/
• 关键词: c-kit receptor tyrosine kinase; oncogenic mutation; molecular target; tyrosine kinase inhibitor; tyrophostine; natural killer; T-cell lymphoma; naturalkiller; 二量体化; ras

The c-kit receptor tyrosine kinase(KIT)plays important roles in embryonhic development, *ematopoiesis and also malignant transformation. We revealed that two point mutations, Val559-->Gly(G559)mutation in the juxtamembrane domain and Asp814-->Val(V814)mutation in the kinase domain, lead to constitutive and oncogenic activation of KIT.Since oncogenic mutants seem to alter their conformation and substrate specificity compared with wild-type(WT)KIT, they may be preferentially inhibited by some compound. Tyrophostines are a class of tyrosine kinase inhibitor either through competition for substrate on through ATP binding. When KIT-WT, KIT-G559 and KIT-V814 expression vector were transfected into an embryonic kidney cell line 293T and treated with increasing dose of tyrophostin AG1296, kinase activity of KIT-G559 protein was more effectively inhibited than that of KIT-WT, whereas that of KIT-V814 was not affected at all. When these KIT genes were introduced into a murine interleukin(IL)-3-d … More ependent cell line Ba/F3, Ba/F3-KIT-WT showed Stem Cell Factor(SCF)-dependent, while Ba/F3-KIT-G559 and Ba/F3-KIT-V814 factor-independent. AG1296 inhibited both factor-independent growth of Ba/F3-KIT-G559 and SCF-dependent growth of Ba/F3-KIT-WT in a dose dependent manner but with different potencies. AG1296 had no effect on factor-independent growth of Ba/F3-KIT-V814 or IL-3-dependent growth of Ba/F3-mock. AG1296 inhibited activation of MAP kinase more effectively in Ba/F3-KIT-G559 than in Ba/F3-KIT-WT.Furthermore, AG1296 blocked antiapoptotic activity of KIT-G559 more potently than that of KIT-WT.These results suggest that a gain-of-function KIT mutant may be used as a molecular target for cancer therapy.Sinonasal natural killer/T-cell lymphoma is one of the major constituents of lethal midline granuloma. Since natural killerT cell expresses c-kit, we examined c-kit mutation in these cases by PCR-SSCP followed by direct sequencing. Twelve single nucleotide substitution mutations were seen in 23 cases. Furthermore, seven of eight mutations(92%)in exon 17 occurred at codon 825 and three of four mutations(75%)in exon 11 occurred at codon 56l. Although the codon 825 mutation was not a gain-of-function mutation, the mechanisms of these mutations in pathogenesis are investigated. Less

c-kit受体酪氨酸激酶（KIT）在胚胎发育、造血和恶性转化中起着重要作用。我们发现两个点突变，近膜结构域的Val559- >Gly（G559）突变和激酶结构域的Asp814- >Val（V814）突变，导致KIT的组成性和致癌激活。由于与野生型（WT）KIT相比，致癌突变体似乎改变了它们的构象和底物特异性，它们可能被某些化合物优先抑制。酪氨酸是一类酪氨酸激酶抑制剂，通过竞争底物和ATP结合。将KIT-WT、KIT-G559和KIT-V814表达载体转染到胚胎肾细胞系293T中，并用增加剂量的tyrophostin AG1296处理后，KIT-G559蛋白的激酶活性比KIT-WT更有效地受到抑制，而KIT-V814蛋白的激酶活性则不受影响。将这些KIT基因导入小鼠白细胞介素(IL)-3-d…依赖性更强的细胞系Ba/F3， Ba/F3-KIT- wt表现为干细胞因子（SCF）依赖性，而Ba/F3-KIT- g559和Ba/F3-KIT- v814则表现为不依赖性。AG1296抑制Ba/F3-KIT-G559的因子依赖性生长和Ba/F3-KIT-WT的scf依赖性生长均呈剂量依赖性，但作用强度不同。AG1296对Ba/F3-KIT-V814的因子依赖性生长和Ba/F3-mock的il -3依赖性生长均无影响。AG1296在Ba/F3-KIT-G559中比在Ba/F3-KIT-WT中更有效地抑制MAP激酶的激活。AG1296对KIT-G559抗凋亡活性的抑制作用强于KIT-WT。这些结果表明，功能获得性KIT突变体可能被用作癌症治疗的分子靶点。鼻窦自然杀伤/ t细胞淋巴瘤是致死性中线肉芽肿的主要成分之一。由于自然杀伤ert细胞表达c-kit，我们通过PCR-SSCP和直接测序检测了这些病例中的c-kit突变。23例出现12个单核苷酸替代突变。此外，外显子17的8个突变中有7个（92%）发生在密码子825上，外显子11的4个突变中有3个（75%）发生在密码子56l上。虽然密码子825突变不是一个功能获得突变，但研究了这些突变的发病机制。少

项目成果

Daino H.et al.: "Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells: possible involvement of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells"Blood. (in press).

Daino H.等人：“人类造血细胞中细胞外泛素诱导细胞凋亡：白细胞介素 6 依赖性造血细胞中蛋白酶体途径可能参与 STAT3 降解”血液。

Matsumura I.et al.: "Transcriptional regulation of cyclin D1 promoter by STAT5: its involvement in cytokine-dependent growth of hematopoietic cells"EMBO J.. 18. 1367-1377 (1999)

Matsumura I.等：“STAT5 对细胞周期蛋白 D1 启动子的转录调节：其参与造血细胞的细胞因子依赖性生长”EMBO J.. 18. 1367-1377 (1999)

Daino, H., et al.: "Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells : possible involve-ment of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells."Blood. 95. 2577-2585 (2000)

Daino, H., 等人：“人类造血细胞中胞外泛素诱导细胞凋亡：白细胞介素 6 依赖性造血细胞中蛋白酶体途径可能涉及 STAT3 降解。”血液。

Oritani K.et al.: "Both Stat3-activation and Stat3-independent BCL2 downregulation are important for interleukin-6-induced apoptosis of 1A9-M cells"Blood. 93. 1346-1354 (1999)

Oritani K.等人：“Stat3 激活和 Stat3 独立的 BCL2 下调对于白细胞介素 6 诱导的 1A9-M 细胞凋亡都很重要”血液。

Daino, H., et al: "Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells : possible involve-ment of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells."Blood. 95. 2577-2585 (2000)

Daino, H., 等人：“人类造血细胞中胞外泛素诱导细胞凋亡：白细胞介素 6 依赖性造血细胞中蛋白酶体途径可能涉及 STAT3 降解。”血液。