V-ATPase与溶酶体生物合成交互调控在白血病耐药中的作用及其机制

Multi-drug resistance is a primary hindrance in curative leukemia therapy. The vacuolar-type H+-ATPase (V-ATPase) mediates lysosome acidification and contributes to lysosomal sequestration of hydrophobic weak base chemotherapeutics, therefore reducing their anti-cancer efficacy. While the intrinsic number of lysosome per cell is limited, upon accumulation in the lysosome, hydrophobic weak base compounds induce lysosomal alkalization which disrupts lysosomal activity. Therefore, to maintain a sustainable cellular function, there is a burning need for the cancer cells to upregulate lysosome biogenesis in response to chemotherapeutics, the mechanism by which remains elusive. Our previous studies have shown inhibition of V-ATPase activity can induce caspase-dependent leukemic cell death. Furthermore, a weak base chemotherapeutic agent, as well as a V-ATPase inhibitor triggered lysosome biogenesis, as shown by marked increase in lysosome number, size , and expression of lysosomal-associated membrane protein 1(LAMP1), indicating an upregulation of lysosome biogenesis via a feedback mechanism. Recently, lysosome biogenesis was found to be regulated by transcription factor EB (TFEB), a master regulator of genes in the Coordinated Lysosomal Expression and Regulation (CLEAR) network. The activation of TFEB requires its translocation from cytoplasm to nucleus, which can be negatively regulated by the mammalian target of rapamycin complex 1(mTORC1) via phosphorylation. We have postulated that weak base hydrophobic weak base chemotherapeutics may function through V-ATPase, by interfering with V-ATPase dependent activation of mTORC1, trigger the transcription of TFEB-CLEAR network genes, lysosome biogenesis and the expression of V-ATPase. This forms a feedback regulatory mechanism, in which through upregulation of V-ATPase expression and producing more lysosomes, further enhances lysosomal entrapment of weak base chemotherapeutics and ultimately leads to chemoresistance. To test this hypothesis, we will make use of drug -sensitive and -resistant cell lines and observe the changes in mTORC1 activation, TFEB subcellular localization and downstream CLEAR gene express as well as lysosome function parameters without or in the presence of a weak base chemotherapeutic agent. While utilizing the morphology and molecular biology methods, we sought to analyze the cross-talk between V-ATPase and lysosome biogenesis, and the mechanism by which they leads to multi-drug resistance in leukemia and provide evidence that targeting lysosome drug sequestration and lysosome as a strategy to overcome this chemoresistance.

白血病多药耐药是临床治疗的瓶颈。现认为，细胞通过V-ATPase调控溶酶体酸化，使弱碱性化疗药富集于溶酶体中，降低抗癌疗效。细胞固有溶酶体数量有限，需调控溶酶体生物合成等维持溶酶体功能，但机制尚未阐明。我们发现：抑制V-ATPase诱导白血病细胞凋亡；弱碱性药与V-ATPase抑制剂增加溶酶体数量、大小及LAMP1表达，表明反馈性上调了溶酶体生物合成。研究表明TFEB调控溶酶体生物合成，且其活性受mTORC1调控。我们推测：V-ATPase基于溶酶体状态，通过mTORC1调控TFEB-CLEAR基因激活，反馈性上调溶酶体生物合成，使药物被溶酶体隔离，降低抗癌疗效，导致溶酶体依赖的耐药。本项目拟通过观察化疗药物作用下mTORC1激活、TFEB定位及下游CLEAR基因表达、溶酶体功能等改变，揭示V-ATPase与溶酶体生物合成交互调控白血病多药耐药的产生机制，为克服肿瘤耐药提供新线索。