Non-canonical Caspase-8 Activation on Autophagosomal Membranes

项目摘要/摘要 这个项目的目标是检验一种假设，即未成熟的自噬小体的积累 膜诱导caspase-8的非规范激活，将细胞保护性自噬转换为细胞凋亡 寻找一种新的抗癌策略。自噬在癌症中是一把双刃剑，因为它可以抑制 致癌或促进癌细胞存活。由于缺乏选择性的自噬通量抑制剂， 很难确定抑制自噬是否是有效的癌症策略。我们和其他人已经证明了这一点 自噬体膜可以作为细胞内死亡诱导信号复合体的平台 (IDISC)激活不依赖于死亡受体信号的caspase-8。从机制上讲，iDISC招募 前caspase-8通过两个臂：1)ATG12-ATG5：FADD：caspase-8；和2)LC3-II： P62：caspase-8。由于ATG12-ATG5在膜关闭和密封时从噬菌体解离 自噬小体运输到溶酶体进行降解，我们假设抑制噬菌体关闭将 稳定iDISC组件以激活caspase-8。事实上，我们的初步数据显示，细胞缺乏 ATG2A/B或VMP1，两个吞噬细胞关闭的调节者，积累未成熟的吞噬细胞，促进 IDISC介导的caspase-8激活。我们认为，阐明噬菌体的分子机制 封闭将导致更多有选择性的自噬抑制靶点，并为癌症提供新的机会 心理治疗。我们将从以下几个具体目标来考察我们的假说：1)为了证明 未成熟噬菌体启动iDISC介导的caspase-8激活及其分子调控特性 2)验证ATG2A/B和VMP1调控噬菌体的假说 通过输送含有ATG9的膜关闭；3)证明受损的吞噬细胞 CLOSE可在体内将自噬转换为IDISC介导的凋亡，以抑制儿童急性髓系白血病 伴有MLL基因重排的白血病(AML)。

Project Summary/Abstract The goal of this project is to test the hypothesis that an accumulation of immature autophagosomal membranes induces the non-canonical activation of caspase-8 to switch cytoprotective autophagy to apoptosis for a novel anti-cancer strategy. Autophagy is a double-edged sword in cancer as it can either suppress oncogenesis or promote cancer cell survival. The lack of selective inhibitors of autophagic flux has made it difficult to determine if inhibition of autophagy is a valid cancer strategy. We, and others, have demonstrated that autophagosomal membranes can serve as platforms for an intracellular death-inducing signaling complex (iDISC) that activates caspase-8 independent of death receptor signaling. Mechanistically, the iDISC recruits pro-caspase-8 to autophagosomal membranes by two arms: 1) ATG12-ATG5: FADD: caspase-8; and 2) LC3-II: p62: caspase-8. As ATG12-ATG5 dissociates from the phagophore upon membrane closure and sealed autophagosomes traffic to lysosomes for degradation, we hypothesize that inhibition of phagophore closure will stabilize iDISC assembly for caspase-8 activation. Indeed, our preliminary data reveal that cells deficient in ATG2A/B or VMP1, two regulators of phagophore closure, accumulate immature phagophores that promote iDISC-mediated caspase-8 activation. We propose that elucidation of the molecular mechanisms of phagophore closure will lead to more selective targets for autophagy inhibition and present novel opportunities for cancer therapy. We will investigate our hypothesis in the following specific aims: 1) to demonstrate that the accumulation of immature phagophores initiates iDISC-mediated caspase-8 activation and characterize molecular regulators of non-canonical caspase-8 activation; 2) to test the hypothesis that ATG2A/B and VMP1 regulate phagophore closure through the delivery of ATG9-containing membranes; 3) to demonstrate that impaired phagophore closure can switch autophagy to iDISC-mediated apoptosis in vivo for the suppression of pediatric acute myeloid leukemia (AML) with MLL (mixed lineage leukemia) gene rearrangements.