A Plastic SQSTM1/p62-Dependent Autophagic Reserve Maintains Proteostasis and Determines Proteasome Inhibitor Susceptibility in Multiple Myeloma Cells

ABSTRACTMultiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 <i>de novo</i> expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome <i>load-versus-capacity</i> model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.

摘要：多发性骨髓瘤（multiple myeloma, MM）是典型的对蛋白酶体抑制剂（proteasome inhibitor, PI）敏感的癌症，但众多患者仍对其治疗无应答。增强其敏感性的极具潜力的靶点之一是（巨）自噬（macroautophagy），近期研究发现（巨）自噬对骨髓浆细胞——多发性骨髓瘤的正常同源细胞——的存活至关重要。本研究整合蛋白质组学与假说驱动策略，鉴定出自噬货物受体与衔接蛋白（autophagic cargo receptor and adapter protein）SQSTM1/p62是自噬储备的核心组分：该组分不仅可协同蛋白酶体维持蛋白质稳态（proteostasis），还能介导对蛋白酶体抑制剂的可塑性适应性应答，且可精准反映固有蛋白酶体抑制剂敏感性。慢病毒工程实验证实，SQSTM1对多发性骨髓瘤细胞的存活不可或缺，且可赋予细胞针对蛋白酶体抑制剂的特异性保护作用。在基础状态下，依赖SQSTM1的自噬可通过持续降解大量泛素化蛋白，减轻蛋白酶体的降解负荷。事实上，抑制或激活该自噬通路可分别显著增敏或拮抗蛋白酶体抑制剂诱导的蛋白质聚集与细胞死亡。此外，在蛋白酶体应激状态下，骨髓瘤细胞可选择性上调SQSTM1的从头表达，并重塑其庞大的内源性相互作用组（interactome），使SQSTM1脱离信号通路结合伴侣，以最大化其与泛素化蛋白的结合。以未降解的SQSTM1阳性聚集体在细胞内的蓄积为指征的自噬储备饱和状态，可特异性区分患者来源的、固有对蛋白酶体抑制剂敏感的骨髓瘤细胞与原发耐药者。此类聚集体与内质网（endoplasmic reticulum）的蓄积相关，而比较蛋白质组学（comparative proteomics）鉴定出内质网是多发性骨髓瘤细胞中自噬靶向的主要细胞区室。综上，本研究将自噬纳入我们此前建立的蛋白酶体负荷-容量模型（proteasome load-versus-capacity model），并揭示SQSTM1聚集体可作为蛋白质稳态缺陷的精准标志物，为多发性骨髓瘤的预后评估与治疗策略开发提供了全新框架。