蟾毒灵靶向AHA1/HSP90通路抑制多发性骨髓瘤增殖及蛋白酶体抑制剂类药物耐受的作用和机制研究

多发性骨髓瘤（MM）为第二大血液恶性肿瘤，蛋白酶体抑制剂类（PI，如硼替佐米）一线药物的耐药复发是影响其治愈的主要原因之一。本课题前期以具有抗MM活性的蟾毒灵为探针，运用蛋白芯片、MM基因文库高通量筛选发现AHA1蛋白为蟾毒灵潜在靶标；热泳动试验证明蟾毒灵与该蛋白相互结合；AHA1表达升高与MM不良预后、硼替佐米耐药复发显著相关，过表达AHA1的MM细胞增殖、PI耐药性显著升高；免疫沉淀及蛋白组学研究发现AHA1/HSP90通路调控CDK6、PSMD2等增殖耐药相关的功能蛋白。本课题计划：运用基因编辑、模式动物等技术，体内外验证AHA1作为MM增殖耐药靶标的可行性；采用点突变、分子相互作用、离体磷酸化、泛素化等手段，阐明蟾毒灵靶向AHA1的具体位点及作用机制；使用原代细胞、PDX等MM模型，体内外检测AHA1靶向抑制剂联合PI对MM的治疗作用。本研究拟为MM临床治疗提供新靶标与先导化合物。

Multiple myeloma (MM) is the second most hematological malignancy, which is incurable mainly due to the relapse and drug-resistance to the first-class drugs, proteasome inhibitors (PI) like Bortezomib, in the MM patients. In this study, using Bufalin，an actively anti-MM compound, as probe, we have discovered AHA1 as a potential target of Bufalin through screening protein chip and MM gene database. Further microscale thermophoresis assay (MST) verified the binding between Bufalin and AHA1 protein. Intriguingly, increased AHA1 expression led to poor survival and Bortezomib resistance in MM patients, while overexpression of AHA1 in MM cell lines induced cellular proliferation and PIs-resistance. Besides, co-immunoprecipitation combined with mass spectrum assay discovered that AHA1 mediated CDK6 and PSMD2 in the AHA1/HSP90 signaling pathway, which were functional in cellular proliferation and drug-resistance. Our project aim to: 1) Verify if AHA1 is feasible as an therapeutic marker for MM proliferation and drug-resistance using technics like gene editing, animal models et al; 2) Elucidate the precise targeting site of Bufalin on AHA1 protein and its related AHA1/HSP90 signaling pathway by site mutation, molecular interaction, phosphorylation and ubiquitinaiton ex vivo assay et al. 3) Detect the therapeutic effect of AHA1 inhibitor combining with PIs on MM using primary MM cells and animal model. Summarily, this projects aims to provide novel therapeutic target and active compounds for MM.