Inhibition of Cytochrome c-induced Caspase Activation

描述（由申请人提供）：化疗药物和其他细胞损伤剂诱导的凋亡细胞死亡通常涉及细胞色素c从线粒体膜间隙释放到细胞质中，在那里它诱导Apaf-1的寡聚化形成“凋亡体”并激活原aspase 9。然后激活的半胱天冬酶9激活刽子手半胱天冬酶3和7来拆除注定要死亡的细胞。功能受损或细胞凋亡形成是许多癌症的标志，包括卵巢癌、黑色素瘤、肺癌和其他癌症。我们已经证明，在表达活化酪氨酸激酶（Bcr-Abl、Tel-PDGFR2和活化FLT3）的白血病中，凋亡细胞的形成是有缺陷的。这一缺陷可追溯到在白血病细胞中Hsp90的β亚型与Apaf-1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）磷酸化状态的差异可以解释白血病细胞和未转化细胞之间Apaf-1结合和细胞色素c诱导的caspase激活的差异。此外，模拟白血病形式的非磷酸化Hsp902变体的表达赋予未转化细胞对细胞色素c诱导的凋亡的抗性，并促进bcr - abl转化的骨髓源造血干细胞对前线治疗伊马替尼的耐药性。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。本研究的目的是阐明Hsp902磷酸化在白血病细胞中是如何失调的，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作用，评估Hsp902在患者样本中的磷酸化状态。这项工作的长期目标是充分了解白血病细胞凋亡抵抗的分子基础，并最终将这些知识转化为白血病的治疗药物。

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.