Shivakrupa R, Bernstein Alan, Watring Nicole, Linnekin Diana
Basic Research Laboratory, Center for Cancer Research, Building 567, Room 226, National Cancer Institute-Frederick, Frederick, MD 21702, USA.
Cancer Res. 2003 Aug 1;63(15):4412-9.
The Kit receptor tyrosine kinase is critical for the growth and development of hematopoietic cells, germ cells, and the interstitial cells of Cajal. Gain-of-function mutations in codon 816 of the catalytic domain of human Kit [codon 814 of murine Kit (mKit)] are found in patients with mastocytosis, leukemia, and germ cell tumors. There are no drugs that inhibit the activity of Kit catalytic domain mutants to a greater extent than wild-type Kit. The objective of this study was to understand the biochemical mechanisms mediating mast cell transformation by this Kit mutant to identify molecular targets for pharmacological intervention. To this end, we examined signaling pathways activated in the murine mast cell line IC2 infected with either wild-type (IC2-mKit) or mutant mKit (IC2-mKit(D814Y)). In this study, we show that mKit(D814Y) is constitutively phosphorylated on tyrosine 719, and this likely results in constitutive association with activated phosphatidylinositol 3'-kinase (PI3K). In vitro growth of IC2-mKit(D814Y) cells is more sensitive to inhibition of PI3K than SCF-induced growth of IC2-mKit cells. s.c. injection of IC2-mKit(D814Y) in syngeneic mice results in mast cell tumors. To determine whether inhibition of PI3K could reduce mKit(D814Y)-mediated tumorigenicity, mice were treated with 1.5 mg/kg wortmannin three times a week. Five weeks after injection of tumor cells, a 75% reduction in tumor weight was observed when wortmannin treatments were initiated 2 days after inoculation with tumor cells. A 66% reduction occurred when treatment was initiated 2 weeks after inoculation. Treatment with wortmannin increased necrosis in the tumors, and this was associated with apoptosis. Interestingly, there was no effect on tumor vasculature. Thus, PI3K is required for survival and growth of the IC2-mKit(D814Y) mast cell line both in vitro and in vivo. These findings may provide insight into designing strategies for treatment of mastocytosis and other diseases associated with mutations in the Kit catalytic domain.
Kit受体酪氨酸激酶对造血细胞、生殖细胞和Cajal间质细胞的生长与发育至关重要。在肥大细胞增多症、白血病和生殖细胞肿瘤患者中发现了人类Kit催化结构域第816密码子（小鼠Kit [mKit]的第814密码子）的功能获得性突变。目前尚无药物能比野生型Kit更有效地抑制Kit催化结构域突变体的活性。本研究的目的是了解介导肥大细胞转化的生化机制，以确定药物干预的分子靶点。为此，我们检测了感染野生型（IC2-mKit）或突变型mKit（IC2-mKit(D814Y)）的小鼠肥大细胞系IC2中激活的信号通路。在本研究中，我们发现mKit(D814Y)的酪氨酸719持续磷酸化，这可能导致其与激活的磷脂酰肌醇3'-激酶（PI3K）持续结合。IC2-mKit(D814Y)细胞的体外生长比干细胞因子（SCF）诱导的IC2-mKit细胞生长对PI3K抑制更敏感。将IC2-mKit(D814Y)皮下注射到同基因小鼠中会导致肥大细胞瘤。为了确定抑制PI3K是否能降低mKit(D814Y)介导的致瘤性，小鼠每周接受3次1.5 mg/kg渥曼青霉素治疗。在注射肿瘤细胞5周后，在接种肿瘤细胞2天后开始用渥曼青霉素治疗时，观察到肿瘤重量减少了75%。在接种2周后开始治疗时，肿瘤重量减少了66%。用渥曼青霉素治疗增加了肿瘤中的坏死，这与细胞凋亡有关。有趣的是，对肿瘤血管没有影响。因此，PI3K对于IC2-mKit(D814Y)肥大细胞系在体外和体内的存活与生长都是必需的。这些发现可能为设计治疗肥大细胞增多症和其他与Kit催化结构域突变相关疾病的策略提供思路。
