Therapeutic targeting of Src kinase signal transduction pathways in AML

描述(由申请人提供)： 急性髓系白血病(AML)是一种预后较差的恶性血液疾病。AML的发病机制尚不完全清楚，但目前的模型表明，病理性的、不受控制的信号转导通路的激活对AML的发展是必要的。然而，在大多数AML患者样本中，信号通路激活的机制(S)尚不清楚。一组AML细胞含有细胞表面酪氨酸激酶Flt3的突变，最近的研究结果表明，临床上使用Flt3激酶抑制剂靶向Flt3可以带来治疗效益。这些重要的发现表明，抑制激活的信号通路是治疗AML的重要途径。我们采取了一种无偏见的方法来识别AML中激活的信号分子。我们对6例急性髓细胞白血病患者的酪氨酸磷酸化蛋白进行了磷酸化蛋白分析。有趣的是，这一分析表明，在AML细胞中，Lyn和Lck酪氨酸激酶都被磷酸化，这表明Lyn和Lck这两个Src家族的成员可能参与了AML信号通路的激活。原代AML细胞上的激酶分析表明，在所研究的AML样本中，80%的LCK被激活，以前的工作已经证明在大多数AML细胞中LYN被激活。为了初步确定SFK是否对AML的生长和生存是必需的，我们进行了三个实验。使用siRNA方法“击倒”Lyn或Lck会导致AML集落形成活性降低。此外，在体外，Src抑制剂达沙替尼对2/3的受试样本的生长有抑制作用。在体内，使用AML异种移植模型，达沙替尼在14天的治疗期内具有细胞抑制作用。这些初步结果表明，SFK是AML细胞病理信号转导的一个有吸引力的靶点，也是一个治疗的靶点。下面的目标1和2将更详细地探讨这些想法。此外，生化分析表明，Lyn和Lck分别与STAT5共沉淀。基于这一数据，我们假设AML细胞中存在通过Src家族激酶(SFK)-STAT5信号通路的异常信号。我们已经开始了对这一途径的功能研究。奇怪的是，STAT5的表达降低对AML集落形成实验的影响不大。尽管如此，STAT5的表达减少会导致一些基因的表达发生变化，包括在造血细胞中不正常表达的PI3激酶亚基PIK3R3，以及周期蛋白抑制因子p21。特异性靶点3和4将决定STAT5是否是SFK的直接靶点，以及在原代AML细胞中STAT5的重要靶点是什么。这些互补的方法将有助于更好地了解AML的发病机制，并改进治疗方法。

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is a malignant disease of the blood with a poor prognosis. The pathogenesis of AML is incompletely understood but current models propose that pathologic, uncontrolled activation of signal transduction pathways is necessary for development of AML. However, the mechanism(s) of activation of signaling pathways in the majority of AML patient samples is not clear. A subset of AML cells contain mutations in the cell surface tyrosine kinase, Flt3, and recent results demonstrate that targeting Flt3 clinically with a Flt3 kinase inhibitor leads to therapeutic benefit. These important findings demonstrate that inhibition of activated signaling pathways in AML is an important approach to therapy of the disease. We undertook an unbiased approach to identify activated signaling molecules in AML. We performed a phospho-protein analysis of tyrosine phosphorylated proteins from 6 AML patient samples. Interestingly, this analysis demonstrated phosphorylation of both lyn and lck tyrosine kinases in AML cells suggesting that Lyn and Lck, two members of the Src family kinases (SFKs) may contribute to activation of signaling pathways in AML. Kinase assays on primary AML cells demonstrate that lck is activated in 80% of AML samples studied and previous work has demonstrated activation of Lyn in the majority of AML cells. To initially determine if SFK's are necessary for AML growth and survival we performed three experiments. "Knock-down" of either Lyn or Lck using siRNA approaches leads to decreased AML colony forming activity. Additionally, in vitro, the Src inhibitor, Dasatinib inhibited the growth of 2/3 samples tested. In vivo, using a xenotransplantation model fo AML, Dasatinib had cytostatic effects over a 14 day treatment period. These initial results demonstrate that SFKs are an attractive target for pathologic signaling in AML cells and a target for therapy. Aims 1 and 2 below will explore these ideas in more detail. Furthermore, biochemical analysis shows that Lyn and Lck each co-immunoprecipitate with STAT5. Based on this data, we hypothesize that there is aberrant signaling in AML cells through a Src family kinase (SFK)-STAT5 signaling pathway. We have initiated functional studies of this pathway. Curiously, decreased expression of STAT5 has only a modest effect on AML colony forming assay. Despite this, decreased expression of STAT5 leads to altered expression of a number of genes including a subunit of PI3 kinase not normally expressed in hematopoietic cells designated PIK3R3, and the cyclin inhibitor p21. Specific Aims 3 and 4 will determine if STAT5 is a direct target of SFK's and what the important targets of STAT5 are in primary AML cells. These complementary approaches will lead to a better understanding of the pathogenesis of AML and improved therapies.