Summary Multiple Myeloma (MM) remains incurable despite novel therapies, suggesting the need for further identification of factors mediating tumorigenesis and drug resistance. Using both in vitro and in vivo MM xenograft models, we show that plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) microenvironment both mediate immune deficiency characteristic of MM and promote MM cell growth, survival, and drug resistance. Microarray, cell signaling, cytokine profile and immunohistochemical analysis delineate the mechanisms mediating these sequelae. Although pDCs are resistant to novel therapies, targeting Toll-like Receptors with CpG ODNs both restores pDC immune function and abrogates pDC-induced MM cell growth. Our study therefore validates targeting pDC-MM interactions as a therapeutic strategy to overcome drug resistance in MM.

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 ...

Purpose PD1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated anti-tumor immune responses. Here we assessed the impact of single and dual blockade of PD1/PD-L1, alone or in combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma (MM) cell growth in the BM milieu. Experimental Design Surface expression of PD1 on immune effector cells, and PD-L1 expression on CD138+MM cells and myeloid derived suppressor cells (MDSC) were determined on tumor cells from newly diagnosed (ND)-MM and relapsed/refractory (RR)-MM BM versus healthy donor (HD). We defined the impact of single and dual blockade of PD1/PD-L1, alone and with lenalidomide, on autologous anti-MM immune response and tumor cell growth. Results Both ND and RR patient MM cells have increased PD-L1 mRNA and surface expression compared to HD. There is also a significant increase in PD1 expression on effector cells in MM. Importantly, PD1/PD-L1-blockade abrogates BM-stroma cell (BMSC)-induced MM growth, and combined blockade of PD1/PD-L1 with lenalidomide further inhibits BMSC-induced tumor growth. These effects are associated with induction of intracellular expression of IFNγ and Granzyme-B in effector cells. Importantly, PD-L1 expression in MM is higher on MDSC than on antigen presenting cells, and PD1/PD-L1-blockade inhibits MDSC-mediated MM growth. Finally, lenalidomide with PD1/PD-L1-blockade inhibits MDSC-mediated immune suppression. Conclusion Our data therefore demonstrates that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in MM, and that PD1/PD-L1-blockade induces anti-MM immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy.

Over the past 4 decades, basic research has provided crucial information regarding the cellular and molecular biology of cancer. In particular, the relevance of cancer microenvironment (including both cellular and noncellular elements) and the concept of clonal evolution and heterogeneity have emerged as important in cancer pathogenesis, immunologic escape, and resistance to therapy. Multiple myeloma (MM), a cancer of terminally differentiated plasma cells, is emblematic of the impact of cancer microenvironment and the role of clonal evolution. Although genetic and epigenetic aberrations occur in MM and evolve over time under the pressure of exogenous stimuli, they are also largely present in premalignant plasma cell dyscrasia such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), suggesting that genetic mutations alone are necessary, but not sufficient, for myeloma transformation. The role of bone marrow microenvironment in mediating survival, proliferation, and resistance to therapy in myeloma is well established; and although an appealing speculation, its role in fostering the evolution of MGUS or SMM into MM is yet to be proven. In this review, we discuss MM pathogenesis with a particular emphasis on the role of bone marrow microenvironment. (Blood. 2015;125(20):3049-3058) Characteristics of the myeloma cancer clone Ontogenesis of myelomaMultiple myeloma (MM) represents the far end of the spectrum of B cell-derived neoplasms. It is the neoplastic counterpart of terminally differentiated, immunoglobulin-producing, long-lived plasma cells (PCs). Long-lived PCs are a subset of PCs characterized by long-term (months to years) survival within the bone marrow (BM) and thought to be key for immunologic memory.1,2 Based on the sequencing of the immunoglobulin heavy chain (IgH) variable region of MM cells (MMCs), the first oncogenic events in MM appear to occur in the germinal center, likely during the processes of isotype class switching and somatic hypermutation, which are, by nature, mutation prone. 3 The observation that patients with premalignant PC dyscrasia monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM) also carry these initial mutations suggests that they are necessary, but not sufficient, in MM pathogenesis. Late oncogenic events are thought to occur in the BM, after the founder cancer clone is completely differentiated into a long-lived PC (Figure 1). 4 There is ongoing debate regarding the identity of the MM stem cells. Different groups have shown that both CD138 1 and CD192 cells are capable of tumorigenesis in mouse models.However, CD138 1 tends to lose self-renewing potential after a few cycles of serial transplantation, whereas the putative B-cell stem clone was never proved to be clonally related to its putative CD138 1 progeny.Overall, modification in the cytokine composition of the media used to maintain CD138 1 ex vivo successfully overcame the first issue, suggesting that the MM stem cell may be CD1381 . 5Ev...

Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/ additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P ‫؍‬ .007) and overall survival was significantly increased (P < . IntroductionMultiple myeloma (MM) is a B-cell disease characterized by accumulation of malignant plasma cells in the bone marrow (BM), bone lesions, and immunodeficiency. Genetic analysis shows that approximately 55% to 60% of MM patients have a hyperdiploid karyotype, which confers a better prognosis than nonhyperdiploid disease. 1 The most frequent chromosomal abnormalities observed in nonhyperdiploid MM are translocations between immunoglobulin heavy chain gene located on chromosome 14q32 and an oncogene chromosome 11q13 (CYCLIN D1), 4p16.3 (FGFR3 and MMSET), 6p21 (CYCLIN D3), 16q23 (MAF), or 20q11 (MAFB); or less frequently, the immunoglobulin light chain locus (2p12, or 22q11). 2 During cell proliferation, activation of each cell-cycle phase is dependent on the progress and completion of the previous one. Regulation of the cell cycle involves detecting and repairing genetic damage, as well as controlling various checkpoints to prevent uncontrolled cell division. MM cells are further influenced by the BM microenvironment because adhesion of MM cells to extracellular-matrix proteins supports cell adhesion-mediated drug resistance. In addition, binding of MM cells to BM accessory cells induces secretion of cytokines, which further promote growth, survival, and migration of tumor cells, as well as resistance to conventional chemotherapy. 2,3 Aberrant or overexpression of D-type cyclins is ubiquitous in MM, 4,5 and Aurora kinases regulate cell-cycle checkpoints 6 and cell cycle-regulatory molecules, including cyclins and cyclindependent kinases. [7][8][9] Aurora serine/threonine kinases localize in the centrosome and play a crucial role in cell division by regulating chromatid segregation in mitotic cells 10 ; moreover, defective chromatid segregation causes genetic instability, leading to tumorigenesis. 11 They were first identified in Xenopus Eg2, yeast Ipl1, and Drosophila aurora. The human genome expresses 3 members of the mitotic Aurora kinase family: Aurora-A serine/threonine kinases, Aurora-B serine/threonine kinases, and Aurora-C s...

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.

Smoldering multiple myeloma (SMM) carries a 50% risk of progression to multiple myeloma (MM) or related malignancy within the first 5 years following diagnosis. The goal of this study was to determine if high levels of circulating plasma cells (PCs) are predictive of SMM transformation within the first 2–3 years from diagnosis. Ninety-one patients diagnosed with SMM at Mayo Clinic from January 1994 through January 2007 who had testing for circulating PCs using an immunofluorescent assay and adequate follow up to ascertain disease progression, were studied. High level of circulating PCs was defined as absolute peripheral blood PCs >5000 ×106/L and/or > 5% cytoplasmic immunoglobulin (Ig) positive PCs per 100 peripheral blood mononuclear cells. Patients with high circulating PCs (14 of 91 patients, 15%) were significantly more likely to progress to active disease within 2 years compared with patients without high circulating PCs, 71% versus 25%, respectively, P=0.001. Corresponding rates for progression within 3 years were 86% versus 35%, respectively, P<0.001. Overall survival (OS) after both SMM diagnosis and MM diagnosis was also significantly different. High levels of circulating PCs identify SMM patients with an elevated risk of progression within the first 2 to 3 years following diagnosis.

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.