Therapeutic targeting of Src kinase signal transduction pathways in AML

AML 中 Src 激酶信号转导通路的治疗靶向

• 批准号: 8413426
• 负责人: MARTIN CARROLL
• 金额: --
• 依托单位: PHILADELPHIA VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413426
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acute Myelocytic Leukemia; American; Binding; Biochemical; Biological Assay; Cell Line; Cell Survival; Cell surface; Cells; Cyclins; Cytostatics; Cytotoxic Chemotherapy; Cytotoxic agent; Dasatinib; Data; Development; Disease; Disease model; FDA approved; Future; Gene Expression Regulation; Gene Targeting; Genes; Goals; Growth; Hematological Disease; Hematopoietic; Human; In Vitro; Laboratories; Lead; Leukemic Cell; Life; Malignant - descriptor; Malignant Neoplasms; Mediating; Modeling; Mutation; Normal Cell; PIK3R3 gene; Pathogenesis; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Protein Analysis; Protein Tyrosine Kinase; Proteins; RUNX1 gene; Research Project Grants; Role; STAT5A gene; STAT5B gene; Sampling; Sampling Studies; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Small Interfering RNA; Testing; Therapeutic; Therapy Clinical Trials; Transfection; Tyrosine; Veterans; Work; Xenograft procedure; base; cell growth; chemotherapy; cohort; cytokine; drug testing; improved; in vivo; inhibitor/antagonist; killings; kinase inhibitor; knock-down; member; novel; novel therapeutics; outcome forecast; research study; src-Family Kinases; therapeutic target; treatment duration

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.

描述（由申请人提供）： 急性髓系白血病（AML）是一种预后不良的恶性血液疾病。 AML 的发病机制尚不完全清楚，但目前的模型表明，信号转导途径的病理性、不受控制的激活对于 AML 的发展是必要的。然而，大多数 AML 患者样本中信号通路激活的机制尚不清楚。一部分 AML 细胞的细胞表面酪氨酸激酶 Flt3 含有突变，最近的结果表明，临床上使用 Flt3 激酶抑制剂靶向 Flt3 可带来治疗益处。这些重要的发现表明，抑制 AML 中激活的信号通路是治疗该疾病的重要方法。我们采用公正的方法来识别 AML 中激活的信号分子。我们对 6 名 AML 患者样本中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 Lyn 和 Lck 可能有助于激活 AML 中的信号通路。对原发性 AML 细胞的激酶测定表明，lck 在所研究的 AML 样本中 80% 被激活，并且之前的工作已证明大多数 AML 细胞中 Lyn 被激活。为了初步确定 SFK 对于 AML 生长和生存是否是必需的，我们进行了三个实验。使用 siRNA 方法“敲低”Lyn 或 Lck 会导致 AML 集落形成活性降低。此外，在体外，Src 抑制剂达沙替尼抑制了 2/3 测试样品的生长。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 细胞病理信号传导的一个有吸引力的靶点和治疗靶点。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 各自与 STAT5 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)-STAT5 信号通路的异常信号传导。我们已经启动了该途径的功能研究。奇怪的是，STAT5 表达的减少对 AML 集落形成测定仅产生适度的影响。尽管如此，STAT5 表达的降低会导致许多基因的表达发生改变，包括通常在造血细胞中不表达的 PI3 激酶亚基（称为 PIK3R3）和细胞周期蛋白抑制剂 p21。具体目标 3 和 4 将确定 STAT5 是否是 SFK 的直接靶标以及 STAT5 在原发性 AML 细胞中的重要靶标是什么。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。