Enrico Milan

Plasma cells require autophagy for sustainable immunoglobulin production

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.

Pivotal Advance: Protein synthesis modulates responsiveness of differentiating and malignant plasma cells to proteasome inhibitors

A previously unsuspected, considerable proportion of newly synthesized polypeptides are hydrolyzed rapidly by proteasomes, possibly competing with endogenous substrates and altering proteostasis. In view of the anti‐cancer effects of PIs, we set out to achieve a quantitative assessment of proteasome workload in cells hallmarked by different PI sensitivity, namely, a panel of MM cells, and in a dynamic model of plasma cell differentiation, a process that confers exquisite PI sensitivity. Our results suggest that protein synthesis is a key determinant of proteasomal proteolytic burden and PI sensitivity. In different MM cells and in differentiating plasma cells, the average proteolytic work accomplished per proteasome ranges over different orders of magnitude, an unexpected degree of variability, with increased workload invariably associated to increased PI sensitivity. The unfavorable load‐versus‐capacity balance found in highly PI‐sensitive MM lines is accounted for by a decreased total number of immunoproteasomes/cell coupled to enhanced generation of RDPs. Moreover, indicative of cause‐effect relationships, attenuating general protein synthesis by the otherwise toxic agent CHX reduces PI sensitivity in activated B and in MM cells. Our data support the view that in plasma cells protein synthesis contributes to determine PI sensitivity by saturating the proteasomal degradative capacity. Quantitating protein synthesis and proteasome workload may thus prove crucial to design novel negative proteostasis regulators against cancer.

A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells.

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.

Combined Inhibition of p97 and the Proteasome Causes Lethal Disruption of the Secretory Apparatus in Multiple Myeloma Cells

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

The amyloidogenic light chain is a stressor that sensitizes plasma cells to proteasome inhibitor toxicity

Systemic light chain (AL) amyloidosis is caused by the clonal production of an unstable immunoglobulin light chain (LC), which affects organ function systemically. Although pathogenic LCs have been characterized biochemically, little is known about the biology of amyloidogenic plasma cells (PCs). Intrigued by the unique response rates of AL amyloidosis patients to the first-in-class proteasome inhibitor (PI) bortezomib, we purified and investigated patient-derived AL PCs, in comparison with primary multiple myeloma (MM) PCs, the prototypical PI-responsive cells. Functional, biochemical, and morphological characterization revealed an unprecedented intrinsic sensitivity of AL PCs to PIs, even higher than that of MM PCs, associated with distinctive organellar features and expression patterns indicative of cellular stress. These consisted of expanded endoplasmic reticulum (ER), perinuclear mitochondria, and a higher abundance of stress-related transcripts, and were consistent with reduced autophagic control of organelle homeostasis. To test whether PI sensitivity stems from AL LC production, we engineered PC lines that can be induced to express amyloidogenic and nonamyloidogenic LCs, and found that AL LC expression alters cell growth and proteostasis and confers PI sensitivity. Our study discloses amyloidogenic LC production as an intrinsic PC stressor, and identifies stress-responsive pathways as novel potential therapeutic targets. Moreover, we contribute a cellular disease model to dissect the biology of AL PCs.

Autophagy in Plasma Cell Ontogeny and Malignancy

Autophagy is a highly conserved pathway that recycles cytosolic material and organelles via lysosomal degradation. Once simplistically viewed as a non-selective survival strategy in dire straits, autophagy has emerged as a tightly regulated process ensuring organelle function, proteome plasticity, cell differentiation and tissue homeostasis, with key roles in physiology and disease. Selective target recognition, mediated by specific adapter proteins, enables autophagy to orchestrate highly specialized functions in innate and adaptive immunity. Among them, the shaping of plasma cells for sustainable antibody production through a negative control on their differentiation program. Moreover, memory B cells and long-lived plasma cells require autophagy to exist. Further, the plasma cell malignancy, multiple myeloma deploys abundant autophagy, essential for homeostasis, survival and drug resistance.

MHC class II transactivator is an in vivo regulator of osteoclast differentiation and bone homeostasis co-opted from adaptive immunity.

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co‐activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte‐osteoclast lineage. Both CIITA‐overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro‐computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone‐forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA‐overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c‐fms and receptor activator of NF‐κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA‐overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re‐expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo.

The Autophagic Process Occurs in Human Bone Metastasis and Implicates Molecular Mechanisms Differently Affected by Rab5a in the Early and Late Stages

Autophagy favours metastatic growth through fuelling energy and nutrients and resistance to anoikis, typical of disseminated-tumour cells. The autophagic process, mediated by a unique organelle, the autophagosome, which fuses with lysosomes, is divided into three steps. Several stages, especially early omegasome formation and isolation-membrane initiation, remain controversial; molecular mechanisms involve the small-GTPase Rab5a, which regulates vesicle traffic for autophagosome formation. We examined Rab5a involvement in the function of key members of ubiquitin-conjugation systems, Atg7 and LC3-lipidated, interacting with the scaffold-protein p62. Immunohistochemistry of Rab5a was performed in human specimens of bone metastasis and pair-matched breast carcinoma; the autophagic-molecular mechanisms affected by Rab5a were evaluated in human 1833 bone metastatic cells, derived from breast-carcinoma MDA-MB231 cells. To clarify the role of Rab5a, 1833 cells were transfected transiently with Rab5a-dominant negative, and/or stably with the short-hairpin RNA Atg7, were exposed to two inhibitors of autolysosome function, and LC3II and p62 expression was measured. We showed basal autophagy in bone-metastatic cells and the pivotal role of Rab5a together with Beclin 1 between the early stages, elongation of isolation membrane/closed autophagosome mediated by Atg7, and the late-degradative stages. This regulatory network might occur in bone-metastasis and in high-grade dysplastic lesions, preceding invasive-breast carcinoma and conferring phenotypic characteristics for dissemination.

Autophagy mediates epithelial cancer chemoresistance by reducing p62/SQSTM1 accumulation

To cope with intrinsic and environmental stress, cancer cells rely on adaptive pathways more than non-transformed counterparts. Such non-oncogene addiction offers new therapeutic targets and strategies to overcome chemoresistance. In an attempt to study the role of adaptive pathways in acquired drug resistance in carcinoma cells, we devised a model of in vitro conditioning to three standard chemotherapeutic agents, cisplatin, 5-fluorouracil, and docetaxel, from the epithelial cancer cell line, HEp-2, and investigated the mechanisms underlying reduced drug sensitivity. We found that triple-resistant cells suffered from higher levels of oxidative stress, and showed heightened anti-stress responses, including the antioxidant Nrf2 pathway and autophagy, a conserved pleiotropic homeostatic strategy, mediating the clearance of aggregates marked by the adapter p62/SQSTM1. As a result, re-administration of chemotherapeutic agents failed to induce further accumulation of reactive oxygen species and p62. Moreover, autophagy proved responsible for chemoresistance through the avoidance of p62 accumulation into toxic protein aggregates. Indeed, p62 ablation was sufficient to confer resistance in parental cells, and genetic and pharmacological autophagic inhibition restored drug sensitivity in resistant cells in a p62-dependent manner. Finally, exogenous expression of mutant p62 lacking the ubiquitin- and LC3-binding domains, required for autophagic engulfment, increased chemosensitivity in TDR HEp-2 cells. Altogether, these findings offer a cellular system to investigate the bases of acquired chemoresistance of epithelial cancers and encourage challenging the prognostic and antineoplastic therapeutic potential of p62 toxicity.