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）是对蛋白酶体抑制剂（proteasome inhibitor, PI）敏感的典型恶性肿瘤，但诸多患者仍会出现耐药。增强其敏感性的极具潜力的靶点之一是（大）自噬（(macro)autophagy），近期研究发现自噬对骨髓浆细胞——MM的正常同源细胞——的存活至关重要。本研究通过整合蛋白质组学与假说驱动研究策略，鉴定出自噬货物受体（autophagic cargo receptor）与衔接蛋白SQSTM1/p62是自噬储备的核心组分：其不仅可与蛋白酶体协同维持蛋白质稳态（proteostasis），还能介导细胞对PI的可塑性适应性应答，并可精准反映肿瘤细胞固有的PI敏感性。慢病毒（lentiviral）工程化实验证实，SQSTM1对MM细胞的存活不可或缺，且可提供特异性的PI耐药保护。在基础生理状态下，依赖SQSTM1的自噬会持续降解大量泛素化蛋白，从而减轻蛋白酶体的降解负荷。事实上，抑制或激活该自噬通路，可分别显著增强或减弱PI诱导的蛋白聚集与细胞死亡。此外，在蛋白酶体应激状态下，骨髓瘤细胞会选择性上调SQSTM1的从头（de novo）表达，并重塑其庞大的内源相互作用组，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。当此类自噬储备达到饱和时，会表现为细胞内积累未被降解的SQSTM1阳性聚集体，该特征可精准区分患者来源的、固有PI敏感型与固有耐药型骨髓瘤。这类聚集体与内质网（endoplasmic reticulum）的积累呈正相关，而比较蛋白质组学研究证实，内质网是MM中自噬靶向的主要细胞亚区室。综上，本研究将自噬通路纳入此前建立的「蛋白酶体负荷-容量」模型，同时证实SQSTM1聚集体可作为蛋白质稳态缺陷的精准标志物，为MM的预后评估与治疗策略开发提供了全新的研究框架。