磷酸化MARCKS介导E2F1-PUMA轴对多发性骨髓瘤耐药的机制研究

Primary and acquired drug resistance poses formidable challenges in treating Multiple Myeloma (MM) patients. The mechanism of drug resistance is complicated and still not completely clarified. Basing on our preliminary work, we identified and established the molecular characteristics of myeloma resistance, especially phosphorylated form of myristoylated alanine-rich C-kinase substrate (pMARCKS) was overexpressed. pMARCKS promoted cell-cycle progression by inducing p27 ubiquitination degradation and suppressing p27Kip1. This resulted in drug resistance in Myeloma .Additional preliminary experiments provided evidence that p53 upregulated modulator of apoptosis (PUMA) showed extremely high expression after the inhibition of pMARCKS using MPS peptide , however，pMARCKS does not directly activate transcription of the PUMA gene. Moreover，pMARCKS regulated downstream E2F1 which might combine with PUMA via transactivation domain. This suggested the E2F1-PUMA axis play a key role in the drug resistance caused by pMARCKS. Based on our preliminary results, we propose to examine the protein expressions of pMARCKS, E2F1 and PUMA in bone marrow biopsy samples from 200 myeloma patients and summarize the clinical relevance and prognostic value in newly diagnosed and refractory patients. Additionally, the role of pMARCKS through E2F1-PUMA axis in the regulation of drug resistance in myeloma, related molecular mechanisms and the downstream effect pathways/targets will be studied and analyzed in the cell level and animal model. This will provide a theoretical basis to develop small molecule drug targeting pMARCKS.

多发性骨髓瘤（MM）患者出现原发性和获得性耐药是临床治疗中面临的最大难题，其机制一直未能阐明。我们前期工作中建立了MM耐药特征性的分子谱，其中磷酸化MARCKS（pMARCKS）高表达诱导p27泛素化降解，下调p27Kip1加快细胞周期进程导致耐药。进一步试验证实，多肽MPS靶向抑制pMARCKS后，PUMA异常高表达，而pMARCKS不能直接调控PUMA表达，其下游基因E2F1中可能存在PUMA启动子的结合位点，可见E2F1-PUMA轴对于pMARCKS引起的多药耐药至关重要。本研究拟在此基础上，从200例患者骨髓活检样本分析中总结pMARCKS、E2F1、PUMA对初治及难治复发MM的临床相关性及预后价值，同时从细胞水平和动物模型中深入探讨pMARCKS通过E2F1-PUMA轴在MM耐药调控中的作用位点和分子机制，解析下游效应途径及靶点，为靶向pMARCKS药物研发提供理论基础。

多发性骨髓瘤(MM)是一种以骨髓中克隆性浆细胞异常增殖为特征的恶性肿瘤。过去十年在治疗MM方面取得了很大进展，这种疾病仍然无法治愈的。因此，迫切需要寻找新的治疗方案来治疗MM。我们既往研究发现磷酸化肉豆蔻酰化富丙氨酸激酶C底物(pMARCKS)蛋白在耐药MM细胞中高表达，pMARCKS抑制细胞周期蛋白依赖性激酶Skp2/p27促进细胞增殖。MARCKS是如何在MM中被诱导的，它的下游PUMA蛋白表达及作用机制，是困扰我们的问题。.通过本研究，我们发现MM患者及细胞系中miR-34a和MARCKS的表达呈负相关，miR-34a直接与MARCKS 3’-UTR相互作用，负调控MARCKS表达。miR-34a过表达可以抑制MARCKS而诱导细胞死亡，MARCKS过表达可进一步诱导miR-34a的表达。因此，miR-34a通过与MARCKS建立反馈环使细胞产生耐药。.我们既往研究表明，MM细胞中pMARCKS抑制后P53上调凋亡调控因子（PUMA）高表达。在这里，我们证明了通过miR-34a过表达、MARCKS下调或MPS抑制MARCKS，可以诱导PUMA蛋白在耐药MM 细胞株中积聚。PUMA通过释放自噬启动蛋白Beclin1/Vps34复合物，作为自噬的上游调节因子。因此，MARCKS抑制后自噬反应增强可能是PUMA升高的下游效应之一。.自噬是体内清除错误折叠蛋白的主要机制之一，暴露于CQ增强了MARCKS抑制对MM细胞中的细胞毒效应，证明引起自噬增加，促进MM细胞生存，我们在细胞及小鼠体内实验也验证MPS、BTZ和CQ的联合使用对耐药MM细胞株显示出更显著的细胞毒性。.我们发现并验证MARCKS蛋白和表观遗传学的非编码miR34a在耐药MM细胞中的关系，此外还揭示了pMARCKS、PUMA与细胞自噬之间的一种新的关系,为硼替佐米联合自噬抑制剂的治疗提供了理论基础和新的思路。