The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5(-/-) differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.

IntroductionMultiple myeloma (MM) is a frequent and still incurable plasma cell malignancy, causing 2% of all cancer deaths. In recent years, treatment of MM has improved remarkably. For example, the proteasome inhibitor (PI) bortezomib (PS-341) proved effective even in the context of heavily pretreated, relapsed, and refractory MM, 1-3 although more than 50% of patients fail to respond to second-line treatment. 4 The molecular bases of different individual responsiveness to bortezomib remain unclear. Age (Ͻ 65 years) and extent of bone marrow plasma cell infiltration (Ͻ 50%) are the conventional factors for successful treatment identified so far. [5][6][7] Identifying the molecular bases underlying PI sensitivity would provide the framework for their improved clinical application.Bortezomib targets the proteasome, a 2.4-MDa multicatalytic protease complex ubiquitously expressed in eukaryotic cells. 1,8 Crucial for degrading proteins involved in cell cycle, angiogenesis, adhesion, cytokine production, and apoptosis, 3,9,10 proteasome inhibition can affect tumor cell growth via direct and indirect mechanisms (eg, by blocking interactions with endothelial and bone cells). 8,11 Proteasomes also dismantle damaged and misfolded/unfolded proteins, which are potentially harmful for the cell. 8 As a result, proteasome impairment causes buildup of polyubiquitinated proteins and eventual cell death. 3 Proteasomes also degrade a significant proportion of newly synthesized proteins in mammalian cells (rapidly degraded polypeptides [RDPs]). 12 Thus, increased protein synthesis or other metabolic unbalances could increase proteasome workload.We recently showed that plasma cell differentiation in vitro, ex vivo, and in vivo entails a dramatic decrease in proteasome expression and activity, correlating with increased sensitivity to PIs. 13,14 Indeed, PIs reduce antibody (Ab) responses in vivo. 14,15 Moreover, inducible expression of orphan Ig-chains sensitizes nonlymphoid tumor cells to PI-induced toxicity. 13 In MM cells (MMCs), the levels of both Ig synthesis and retention correlate with apoptotic sensitivity to PIs, and manipulating Ig synthesis alters sensitivity. 16,17 Altogether, these data suggest that the exquisite sensitivity of certain MMCs to PIs could stem from decreased proteasomal capacity, increased proteasomal workload, or both (ie, an adverse load-versus-capacity ratio).In this study, we exploited MM lines with differential apoptotic sensitivity to PIs to address if proteasome expression and degradative workload vary among different clones, and defined their role in The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 9, 2018. by guest www.bloodjournal.org From determining apoptotic sensitivity to PIs. Moreover, using primary patient-derived MMCs, we revealed ...

After few days of intense immunoglobulin (Ig) secretion, most plasma cells undergo apoptosis, thus ending the humoral immune response. We asked whether intrinsic factors link plasma cell lifespan to Ig secretion. Here we show that in the late phases of plasmacytic differentiation, when antibody production becomes maximal, proteasomal activity decreases. The excessive load for the reduced proteolytic capacity correlates with accumulation of polyubiquitinated proteins, stabilization of endogenous proteasomal substrates (including Xbp1s, IjBa, and Bax), onset of apoptosis, and sensitization to proteasome inhibitors (PI). These events can be reproduced by expressing Ig-l chain in nonlymphoid cells. Our results suggest that a developmental program links plasma cell death to protein production, and help explaining the peculiar sensitivity of normal and malignant plasma cells to PI.

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Multiple 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 de novo 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 load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.