Therapeutic targeting of Src kinase signal transduction pathways in AML

描述（由申请人提供）： 急性髓细胞白血病（AML）是一种预后不良的恶性血液病。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激活是AML发展所必需的。然而，大多数AML患者样本中信号传导途径激活的机制尚不清楚。AML细胞的一个亚组在细胞表面酪氨酸激酶Flt 3中含有突变，最近的结果表明，用Flt 3激酶抑制剂在临床上靶向Flt 3可产生治疗益处。这些重要的发现表明，抑制AML中活化的信号通路是治疗该疾病的重要方法。我们采用了一种无偏倚的方法来鉴定AML中活化的信号分子。我们对来自6个AML患者样品的酪氨酸磷酸化蛋白进行了磷酸化蛋白分析。有趣的是，该分析证明了AML细胞中lyn和lck酪氨酸激酶的磷酸化，表明Src家族激酶（SFK）的两个成员林恩和Lck可能有助于AML中信号传导途径的激活。对原代AML细胞的激酶测定表明，lck在所研究的80%的AML样品中被激活，并且先前的工作已经证明林恩在大多数AML细胞中被激活。为了初步确定SFK是否是AML生长和存活所必需的，我们进行了三个实验。使用siRNA方法“敲低”林恩或Lck导致AML集落形成活性降低。此外，在体外，Src抑制剂达沙替尼抑制了2/3测试样品的生长。在体内，使用AML的异种移植模型，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，SFKs是AML细胞中病理信号传导的有吸引力的靶标和治疗靶标。下文目标1和2将更详细地探讨这些想法。此外，生化分析显示林恩和Lck各自与STAT 5共免疫沉淀。基于这些数据，我们假设AML细胞中存在通过Src家族激酶（SFK）-STAT 5信号通路的异常信号传导。我们已经开始了这一途径的功能研究。奇怪的是，STAT 5的表达降低对AML集落形成测定仅具有适度的影响。尽管如此，STAT 5的表达降低导致许多基因的表达改变，包括通常在造血细胞中不表达的PI 3激酶的亚基PIK 3R 3和细胞周期蛋白抑制剂p21。具体目标3和4将确定STAT 5是否是SFK的直接靶标以及STAT 5在原代AML细胞中的重要靶标是什么。这些互补的方法将导致更好地了解AML的发病机制和改进的治疗方法。

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.