靶向SQSTM1/p62介导的选择性自噬及其在多发性骨髓瘤治疗和耐药中的意义

In daily clinical practice, drug resistance remains a major challenge for treatment of cancer, including multiple myeloma (MM), primarily due to lack of effective approaches to manage patients who are refractory or have become relapsed and resistant to current standard therapy (e.g., the first-line proteasome inhibitors such as bortezomib). Autophagy is a well conserved cytoprotective “self-cleaning” response to diverse stresses. While autophagy is considered as a “double-edged" sword in a context-specific manner in tumorigenesis and treatment, emerging evidence supports its crucial pro-oncogenic roles in advanced tumors (e.g., promoting stress adaption, maintaining cellular metabolism, preventing energy crisis and cell death, suppressing immune responses, and mediating cross-talk with host microenvironment). Moreover, autophagy is also involved in virtually all aspects of tumor metastasis (e.g., cell migration and invasion, resistance to anoikis, epithelial-to-mesenchymal transition, dormancy, and escape from immune surveillance). Thus, there is no doubt that autophagy represents one of the most attractive therapeutic targets in cancer as well as many other life-threatening diseases. However, despite the tremendous advances in understanding the machinery governing autophagy, it is still in debate how to target autophagy in cancer treatment. In the recent studies involving MM, we have identified the key autophagic receptor SQSTM1/p62, also a multi-functional oncoprotein, as a novel target for an entirely new autophagy-targeted therapy. We found that p62 down-regulation led to "inefficient" autophagy due to cargo-loading failure, resulting in accumulation of the BH3-only protein NBK/Bik that in turn triggered activation of the apoptotic signaling cascade. Based on these findings, we here propose three specific aims to further validate and develop this potential strategy as follows: (1) to expand and validate the strategy targeting p62 in MM cells and their drug-resistant counterparts; (2) to understand the molecular basis for the functions of p62 as a therapeutic target, including autophagic receptor (focusing on aggrephagy and proteaphagy induced by proteasome inhibition) and oncogenic signaling hub; (3) to discover small molecular inhibitors that target p62 as well as validate their anti-tumor activity and mechanism of action in vitro and in vivo in MM models. We participate that these studies would provide a basis for future development of this first-in-class autophagy-targeted therapy.

耐药是当前肿瘤（包括多发性骨髓瘤，MM）治疗中亟待解决的最大难题。在肿瘤治疗中，自噬能拮抗凋亡，导致耐药，是重要靶点之一。尽管自噬基础研究已十分深入，但其靶向治疗的研究一直处于“瓶颈”。在前期MM靶向治疗研究中，我们发现自噬受体SQSTM1/p62是一个理想的分子靶点，据此提出一种自噬靶向新策略：通过干预选择性自噬过程的关键环节 - 货物识别/装载，导致“无效”自噬，触发凋亡。本课题拟以MM为疾病模型中，确证SQSTM1/p62作为自噬靶向治疗的新分子靶点；基于p62分子结构，阐明靶向p62自噬受体和促瘤信号枢纽的分子基础；通过干预p62基因转录/重新合成和靶向抑制p62自噬受体功能两种途径，发现先导小分子化合物，体内、外研究验证其提高抗MM一线药物（侧重蛋白酶体抑制剂）疗效及克服耐药的功能，阐明其靶向自噬的分子作用机制。以期为干预自噬的精准靶向治疗奠定基础。

复发耐药是当前多发性骨髓瘤（MM）临床治疗的最大挑战之一，迄今仍缺乏有效的干预策略和措施。虽然自噬的基础研究已非常深入（获2016年诺贝尔生理学或医学奖），其在疾病（特别是肿瘤）发生、发展和治疗中的潜在价值，不言而喻，但迄今尚未见其向临床的成功转化，而在肿瘤治疗中如何靶向自噬吗，仍不明确。本研究基于我们前期提出并初步验证的科学假设（即精准靶向SQSTM1/p62的功能，将保护性自噬转变为杀伤性凋亡，以克服耐药），以MM为疾病模型，探索精准靶向自噬的治疗策略。主要完成了以下三部分研究内容：1）SQSTM1/p62通过蛋白酶体自噬介导硼替佐米（Btz）耐药的机制；2）选择性诱导MM细胞铁死亡克服硼替佐米（Btz）耐药；3）SQSTM1/p62及其翻译后修饰在MM细胞铁自噬中的作用。重要结果包括：1）转录组学分析揭示了蛋白酶体自噬相关蛋白酶体亚基的表达及其与MM预后负相关；2）验证了蛋白酶体抑制剂（如Btz）能诱导蛋白酶体自噬；3）PSMD4的K40位点泛素化是蛋白酶体自噬的必要条件；4）SQSTM1/p62磷酸化（S349）是其泛素化的前提，并直接调节蛋白酶体自噬；5）SQSTM1/p62泛素化（K435）促进底物PSMD4与LC3结合，从而介导蛋白酶体自噬；6）蛋白酶体自噬底物PSMD4参与Btz耐药，selinexor通过下调PSMD4，与Btz发挥协同作用，为其联合Btz治疗复发难治MM提供了新的理论依据；7）不同铁死亡诱导剂选择性诱导MM细胞铁死亡；8）铁代谢通过脂质过氧化介导MM细胞铁死亡；9）MM细胞铁死亡伴GPX4亚型转换，并向线粒体转位；10）巨噬细胞通过摄取铁离子保护MM细胞免招铁死亡；11）铁死亡诱导剂与Btz具有显著协同作用，并克服Btz耐药；12）铁死亡诱导剂能同时诱导铁自噬；13）抑制铁自噬显著提高MM细胞对铁死亡的敏感性；14）MM细胞铁死亡伴SQSTM1/p62的磷酸化和泛素化；15）SASTM1/p62磷酸化或泛素化位点突变，抑制铁自噬，导致其细胞保护功能的丧失。因此，本项目研究系统阐明了SQSTM1/p62在蛋白酶体自噬及铁死亡/铁自噬中的功能及其作用机制，为精准靶向自噬克服MM耐药提供了新的思路和靶点。