Inhibition of Cytochrome c-induced Caspase Activation

描述(申请人提供)：化疗药物和其他细胞损害剂诱导的细胞凋亡通常涉及细胞色素c从线粒体膜间间隙释放到细胞质，在那里它诱导APAF-1的寡聚形成“凋亡体”并激活原天冬氨酸酶9。然后激活caspase 9激活刽子手caspase3和7来分解注定要死亡的细胞。功能受损或凋亡体的形成是许多癌症的特征，包括卵巢癌、黑色素瘤、肺癌和其他癌症。我们已经证明，在表达激活的酪氨酸激酶(BCR-Ab1、TEL-PDGFR2和激活的Flt3)的白血病中，凋亡体的形成是有缺陷的。这一缺陷可追溯到白血病细胞中Hsp90的β亚型与APAF-1的紧密抑制结合，而不是正常细胞。具体来说，Hsp902在两个位点(S226/S255)的磷酸化状态的不同可能解释了白血病细胞和未转化细胞在APAF-1结合和细胞色素c诱导的caspase激活方面的差异。此外，在体外bcr-Abl转化的骨髓来源的造血干细胞中，表达模拟白血病形式的非磷酸化Hsp902变体可以抵抗细胞色素c诱导的细胞凋亡，并促进对一线治疗性伊马替尼的抵抗。这些发现表明，调节Hsp90的激酶/磷酸酶是潜在的有吸引力的治疗靶点。这项建议的目的是阐明Hsp902在白血病细胞中的磷酸化是如何被错误调节的，确定Hsp902低磷酸化如何影响APAF-1和其他Hsp902靶点，分析Hsp902低磷酸化在动物中的影响，并评估患者样本中Hsp902的磷酸化状态。这项工作的长期目标是充分了解白血病细胞凋亡体耐药的分子基础，并最终将这一知识转化为白血病的治疗剂。 公共卫生相关性：这项建议解决了将激活的酪氨酸激酶与抑制白血病的程序性细胞死亡(细胞凋亡)联系起来的分子途径。了解这些途径可能对白血病和其他表达活化酪氨酸激酶的癌症的治疗具有广泛的意义。这项工作的目标是充分了解这些信号通路，以便设计出更好的治疗白血病的药物。

DESCRIPTION (provided by applicant): Apoptotic cell death induced by chemotherapeutics and other cell-damaging agents typically involves release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm, where it induces the oligomerization of Apaf-1 to form the "apoptosome" and activate procaspase 9. Active caspase 9 then activates the executioner caspases 3 and 7 to dismantle the doomed cell. Impaired function or formation of the apoptosome is a hallmark of many cancers, including ovarian cancers, melanoma, lung cancers and others. We have demonstrated that apoptosome formation is defective in leukemias expressing activated tyrosine kinases (Bcr-Abl, Tel-PDGFR2, and activated FLT3). This defect was traced to tight inhibitory binding of the beta isoform of Hsp90 to Apaf-1 in leukemic, but not normal cells. Specifically, a difference in the phosphorylation status of Hsp902 at two sites (S226/S255) could account for the difference in Apaf-1 binding and cytochrome c-induced caspase activation between leukemic and untransformed cells. Moreover, expression of a non- phosphorylatable Hsp902 variant that mimicked the leukemic form conferred resistance to cytochrome c-induced apoptosis in untransformed cells and promoted resistance to the front-line therapeutic imatinib in Bcr-Abl-transformed bone marrow-derived hematopoietic stem cells in vitro. These findings point to the kinases/phosphatases regulating Hsp90 as potentially attractive therapeutic targets. The aims of this proposal are to elucidate how Hsp902 phosphorylation is misregulated in leukemic cells, to determine how Hsp902 hypophosphorylation impacts Apaf-1 and other Hsp902 targets, to analyze the effects of Hsp902 hypophosphorylation in animals and to evaluate Hsp902 phosphorylation status in patient samples. The long term goal of this work is to fully understand the molecular basis for apoptosome resistance in leukemia and, ultimately, to translate this knowledge into therapeutic agents for leukemias. PUBLIC HEALTH RELEVANCE: This proposal addresses the molecular pathways linking activated tyrosine kinases to the suppression of programmed cell death (apoptosis) in leukemias. Understanding these pathways may have broad implications for the treatment of leukemias and other cancers expressing activated tyrosine kinases. The goal of this work is to fully understand these signaling pathways in order to design better therapeutic agents for the treatment of leukemias.