Inhibition of Cytochrome c-induced Caspase Activation

描述（由申请人提供）：由化疗剂和其他细胞损伤剂诱导的凋亡性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，在细胞质中它诱导Apaf-1的寡聚化以形成“线粒体”并激活半胱氨酸天冬氨酸蛋白酶原9。然后，活性半胱天冬酶9激活刽子手半胱天冬酶3和7，以拆除注定的细胞。 受损的功能或寡聚体的形成是许多癌症的标志，包括卵巢癌、黑色素瘤、肺癌等。我们已经证明，在表达活化酪氨酸激酶（Bcr-Abl、Tel-PDGFR 2和活化FLT 3）的白血病中，溶酶体形成是有缺陷的。这种缺陷被追溯到紧密抑制性结合的β亚型的Hsp 90 Apaf-1在白血病细胞，但不是正常细胞。具体而言，在两个位点（S226/S255）的Hsp 902的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的Apaf-1结合和细胞色素c诱导的caspase激活的差异。此外，模拟白血病形式的不可磷酸化的Hsp 902变体的表达在未转化的细胞中赋予对细胞色素c诱导的细胞凋亡的抗性，并在体外促进Bcr-Abl转化的骨髓来源的造血干细胞中对一线治疗性伊马替尼的抗性。这些发现指出调节Hsp 90的激酶/磷酸酶是潜在的有吸引力的治疗靶点。该提案的目的是阐明Hsp 902磷酸化在白血病细胞中是如何被错误调节的，以确定Hsp 902磷酸化不足如何影响Apaf-1和其他Hsp 902靶点，分析Hsp 902磷酸化不足在动物中的影响，并评估患者样本中Hsp 902磷酸化状态。这项工作的长期目标是充分了解白血病中的多体耐药的分子基础，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.