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

拟议研究计划的目的是检查分子 BCR/ABL致癌蛋白酪氨酸激酶与 磷脂酰肌醇-3激酶（PI-3 K），并研究是否 同时抑制BCR/ABL和PI-3 K具有协同抗肿瘤作用 对白血病的作用。 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。由于它们的细胞质或 膜定位，OPTK可能发挥其致癌作用， 与几种细胞质和细胞核分子合作的潜力。 先前的研究（Skorski等人，Blood，86：726，1995）已经表明， PI-3 K是胞浆BCR/ABL致癌蛋白的下游靶点 酪氨酸激酶本建议的第一和第二个目的是进一步 研究BCR/ABL和PI-3 K之间的相互作用对 在白血病表型的启动和维持中。为此目的 我们将使用BCR/ABL无功能突变体，显性失活突变体， 和P1 - 3 K的显性活性突变体。骨髓逆转录病毒感染， 体外和体内（SCID小鼠）白血病发展测定、转染 的细胞系，定点诱变和亚克隆，酶 测定（RAS、RAF、PI-3 K、MAPK、JNK），免疫沉淀，Western印迹， 蛋白质磷酸化测定将用于这些目标。 第三个目的是研究是否同时抑制BCR/ABL和 PI-3 K具有协同抗白血病作用。我们之前的研究显示 通过反义策略下调BCR/ABL表达， PI-3 K特异性抑制剂渥曼青霉素（WT）对PI-3 K的抑制作用， 抑制慢性髓性白血病细胞增殖， 影响正常的造血作用[Skorski，et al.，Blood，86：726，1995]。我们 初步数据表明，同时抑制BCR/ABL和 PI-3 K具有协同抗白血病作用。反义策略将 通过下调BCR/ABL的表达，PI-3 K的活性将被下调。 WT抑制。将进行骨髓净化实验， 同时抑制BCR/ABL和 PI-3 K在模拟所述骨髓净化条件的条件下 [Skorski等人，J.Clin.Invest.，92：194，1993]。最后，我们将测试 包括同时抑制 BCR/ABL和Pl-3 K使用我们在SCID小鼠中CML生长的体内模型 [Skorski等人，Proc. Natl. Acad. Sci. USA，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].