PI-3K IN BCR/ABL TYROSINE KINASE-INDUCED LEUKEMIAS

拟议的研究计划的目标是检查分子 Bcr/abl癌基因蛋白酪氨酸激酶与BCR/ABL的相互作用 磷脂酰肌醇-3激酶(PI-3K)，并研究 同时抑制bcr/abl和PI-3K具有协同抗肿瘤作用 抗白血病的效果。 致癌蛋白酪氨酸激酶(OPTK)似乎启动和维持 肿瘤细胞的肿瘤表型。因为它们的细胞质或 膜定位OPTKs可能发挥其致癌作用 与几个细胞质和核分子合作的潜力。 以前的研究(Skorski等人，布拉德，86：726,1995)已经表明 PI-3K是细胞质bcr/abl癌基因蛋白的下游靶点 酪氨酸激酶。这项建议的第一和第二个目标是进一步 探讨bcr/abl与PI-3K之间的相互作用(S)的作用 在白血病表型的启动和维持中。为此目的， 我们将使用bcr/abl功能缺失突变体，显性负突变体， 和显性活性突变体Pl-3K。骨髓逆转录病毒感染， 体外和体内(SCID小鼠)白血病发育试验，转染法 在细胞系中，酶促定点突变和亚克隆 检测(RAS、RAF、PI-3K、MAPK、JNK)、免疫沉淀、Western blotting、 蛋白质磷酸化分析，将用于这些目的。 第三个目的是研究同时抑制bcr/abl和bcr/abl PI-3K具有协同抗白血病作用。我们之前的研究表明 通过反义策略下调bcr/abl的表达 其特异性抑制剂Wortmannin(WT)对PI-3K的强烈抑制 抑制慢性粒细胞白血病细胞的增殖 影响正常造血[Skorski等人，布拉德，86：726,1995]。我们的 初步数据表明同时抑制bcr/abl和 PI-3K具有协同抗白血病作用。反义策略将会 他受雇于下调BCR/ABL的表达，PI-3K的活性将是 被WT抑制。将进行骨髓净化实验，以测试 同时抑制bcr/abl和bcr/abl的抗肿瘤作用 PI-3K在模拟上述骨髓净化条件下的作用 [Skorski等人，J.Clin.Invest，92：194,1993]。最后，我们将测试 一种系统治疗的效果，包括同时抑制 Bcr/abl和pl-3K在SCID小鼠体内CML生长模型中的应用 [Skorski等人，Proc.娜塔莉。阿卡德。SCI。美国，88：2351,1994]。

The objective of the proposed research program is to examine molecular interactions between BCR/ABL oncogenic protein tyrosine kinase and phosphatidylinositol-3 kinase (PI-3K), and to investigate if the simultaneous inhibition of BCR/ABL and PI-3K has synergistic antitumor effect against the leukemia. Oncogenic protein tyrosine kinases (OPTKs) appear to initiate and maintain the neoplastic phenotype of tumor cells. Because of their cytoplasmic or membrane localization it is likely that OPTKs exert their oncogenic potential in cooperation with several cytoplasmic and nuclear molecules. Previous studies (Skorski, et al., Blood, 86:726, 1995) have indicated that PI-3K is a downstream target of cytoplasmic BCR/ABL oncogenic protein tyrosine kinase. The first and second aim of this proposal is to further investigate the role that the interaction(s) between BCR/ABL and PI-3K have in initiation and maintenance of the leukemic phenotype. For this purpose we will employ BCR/ABL function-less mutants, dominant negative mutants, and dominant active mutants of Pl-3K. Bone marrow retroviral infections, in vitro and in vivo (SCID mice) leukemia development assays, transfections of the cell lines, site-directed mutagenesis and subcloning, enzymatic assays (RAS, RAF, PI-3K, MAPK, JNK), immunoprecipitation, Western blotting, protein phosphorylation assay, will be used in these aims. The third aim is to investigate if simultaneous inhibition of BCR/ABL and PI-3K exerts synergistic antileukemia effect. Our previous studies revealed that downregulation of BCR/ABL expression by the antisense strategy or inhibition of PI-3K by its specific inhibitor wortmannin (WT), strongly inhibited proliferation of chronic myelogenous leukemia cells without affecting normal hematopoiesis [Skorski, et al., Blood, 86:726, 1995]. Our preliminary data indicate that simultaneous inhibition of both BCR/ABL and PI-3K exerts synergistic antileukemia effect. The antisense strategy will he employed to downregulate BCR/ABL expression, and PI-3K activity will be inhibited by WT. Bone marrow purging experiments will be performed to test the antitumor effectiveness of the simultaneous inhibition of BCR/ABL and PI-3K in conditions mimicking those of bone marrow purging as described [Skorski et al., J.Clin.Invest., 92:194,1993]. Finally, we will test the effects of a systemic therapy consisting of simultaneous inhibition of BCR/ABL and Pl-3K using our in vivo models of CML growth in SCID mice [Skorski et al., Proc. Natl. Acad. Sci. USA, 88:2351,1994].