Beclin-1过表达诱导的自噬对Bcr-Abl的抑制作用及其分子机制研究

Although the process of autophagy was initially described as a survival strategy in times of cellular stress, it may, however, also lead to programmed cell death, presumably by an excessive activation of this self-degrading system. Recently, it was reported that Bcr-Abl suppressed autophagy through ATF5-mediated regulation of mTOR transcription. In contrast, it has been demonstrated that treatment with arsenic trixide resulted in p62/SQSTM1-mediated localization of Bcr-Abl oncoprotein to the autolysosomes and subsequent degradation mediated by the protease cathepsin B, suggesting that autophagic degradation of Bcr-Abl is critical for the antileukemic effects of arsenic. We recently found that chronic myeloid leukemia (CML) cells including K562, and 32D with T315I mutation treated with oncolytic adenovirus expressing Beclin-1 showed inhibition of Bcr-Abl mRNA and degradation of the oncoprotein. In addition, autophagic cell death was also demonstrated in the cells treated with the virus. Our data suggest that autophagy-induced by overexpression of Beclin-1 contributes to the degradation of Bcr-Abl. However, the mechanisms of action on Bcr-Abl remain elusive. In this study, we use genetic deletion of Bcr-Abl in 32D cells to address whether Beclin-1 bind with allosteric regions, SH2- or SH3-kinase in the Bcr-Abl. Furthermore, we will study that whether autophagy-induced by overexpression of Beclin-1 inhibits mRNA of Bcr-Abl by detecting the promoter activity of the oncogene and its effects on tranlation of gene. In addition, we will observe that the mechanisms of degradation of Bcr-Abl protein via ubiquitination or autophagolysosome pathway. Finally, we will study the effects of autophagy-induced by overexpression of Beclin-1 on Bcr-Abl inhibition and cell killing in CD34+CD38- leukemic stem cells obtained from the CML patients with blast crisis. Taken together, our study will provide a new apporach for the treatment of kinase inhibitor-resistant CML patients.

自噬性死亡是抗肿瘤治疗的新途径。新近研究表明，自噬和白血病融合基因Bcr-ABL、PML/RARα有密切关系。一些研究发现融合基因能促进自噬发生，而另一些研究则发现自噬能使融合蛋白降解。我们在前期研究中应用携带Beclin-1基因的嵌合型溶瘤病毒感染慢性髓细胞白血病（CML）细胞，发现其能诱导自噬性死亡，增强溶瘤病毒抗白血病作用。重要的是，Beclin-1过表达能导致Bcr-Abl mRNA、蛋白水平和磷酸化Bcr-Abl下调。但其分子机制尚不清楚。本研究拟应用表达Beclin-1的非增殖嵌合型腺病毒感染CML细胞，从基因转录、mRNA翻译和蛋白降解三个层面研究Beclin-1诱导自噬导致Bcr-Abl降解的分子机制；探明Beclin-1蛋白能否作用于Bcr-Abl异构区；进一步研究Beclin-1过表达诱导的自噬能否使得CML白血病干细胞的Bcr-Abl降解，并显著杀伤白血病干细胞。

新近研究表明，自噬诱导的细胞死亡是杀伤肿瘤细胞特别是耐细胞凋亡肿瘤细胞的新途径。一些研究表明，自噬和白血病融合基因Bcr-ABL、PML/RARα有密切关系。据报道白血病融合基因能促进自噬发生，而另一些研究则发现自噬能使白血病融合基因蛋白降解。我们在前期研究中应用携带Beclin-1 基因的嵌合型溶瘤病毒感染慢性髓细胞白血病（CML）细胞，发现其能诱导CML细胞发生自噬性死亡，从而增强了溶瘤病毒抗白血病作用。重要的是，Beclin-1 过表达能导致Bcr-Abl mRNA、蛋白水平和磷酸化Bcr-Abl下调。但其分子机制尚不清楚。本研究拟应用表达Beclin-1 的非增殖嵌合型腺病毒感染CML细胞，从基因转录、mRNA 翻译和蛋白降解三个层面研究Beclin-1诱导自噬导致Bcr-Abl 降解的分子机制。通过本项目探明了Beclin-1 蛋白与Bcr-Abl蛋白具有相互作用；进一步研究证明了Beclin-1 过表达诱导的自噬能否使得CML细胞的Bcr-Abl 降解，并显著杀伤白血病细胞。在开辟治疗耐药特别是BCR-ABL突变的CML提供了理论和实验基础。