Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). IntroductionA cardinal clinical feature of multiple myeloma (MM) is the presence of osteolytic bone lesions. Myeloma cells disrupt the delicate balance between bone formation and bone resorption. 1,2 Various clinical observations 3 and experimental studies 4,5 have linked the level of MM bone disease with disease burden. Increased osteoclastic activity and its molecular basis have long been considered a primary pathogenic event in MM bone disease. However, a molecular basis for the well recognized lack of osteoblast (OB) function, specifically DKK-1, in the MM bone disease has only recently been described. 6,7 Canonical Wnt pathway plays an important role in controlling proliferation, differentiation, and survival of OB. [8][9][10][11] Previous studies have reported high expression levels of the canonical Wnt inhibitor DKK1 and osteolytic bone lesions in various tumor types including breast, 12,13 neuroblastoma, 14 esophageal, and lung cancer, 15 and conversely enhanced OB activity and osteoblastic bone lesions associated with decreased DKK1 levels in prostate and colon cancers. [16][17][18] In MM, high serum DKK1 levels were correlated with focal bone lesions. 19 The DKK1 produced by MM cells can inhibit the differentiation of OB precursor cells 19 and bone formation in vitro 20 through a DKK1-mediated attenuation of Wnt3a-induced stabilization of ␤-catenin. 21 These findings confirm DKK1 as an important regulator of bone formation in the bone microenvironment. The importance of DKK1 secretion in diseases associated with bone destruction is reinforced by a recent study showing that DKK1 mediates the bone destructive effects of rheumatoid arthritis and that a neutralizing antibody to DKK1 could inhibit the bone destructive process in that disease. 22 There is also emerging evidence that the cellular bone compartment affects MM cell growth and progression. This is supported by the observation that osteoclasts can support long-term survival and proliferation of primary MM cells, 23,24 and OB may impede MM cell growth. 7,25 Thus, targeting these cellular elements may also favorably affect disease control. Therefore, we have evaluated DKK1 as a therapeutic target in MM in the context of the bone marrow (BM) microenvironment, analyzing the effect of a human DKK1 neutralizing antibody (BHQ880). We show that this clinically applicable antibody increases OB function and number and also has anti-MM effect when evaluated in the presence of the BM milieu. Methods ReagentsBHQ880 is a phage-derived DKK1 neutralizing human immunoglobulin G1 (IgG1) antibody (provided by Novartis, Cambridge, MA). BHQ880 has a high affinity for and can neutralize both human DKK1 and murine DKK1. IgG1 isotype antibody was used as control. CellsBone marrow mononuclear cells (BMMNCs) and primary MM cells were isolated using Ficoll-Hypaque density gradient sedimentation from BM Submitted November 25, 2008; accepted April 20, 2009. Prepublished online ...

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Monoclonal gammopathy of undetermined significance (MGUS) is

IntroductionT regulatory (T reg ) cells play an important role in the maintenance of self-tolerance, control of auto-immunity, and regulation of T-cell homeostasis, and they modulate overall immune responses against infectious agents and tumor cells. 1 Natural T reg cells develop during normal T-cell maturation in the thymus and represent 5% to 10% of the CD4 ϩ cell compartment in the peripheral blood. 2 These cells express CD4 and CD25 surface antigens as well as CTLA-4, GITR, CD103, CD62L, CD69, CD134, CD71, CD54, and CD45RA. 3 The suppressive activity of T reg cells is associated with the overexpression of FOXP3, a member of the forkhead/winged helix family, which acts as a transcriptional repressor. 4 T reg cells suppress CD25 Ϫ CD4 ϩ T-cell proliferation on the basis of cell-cell contact and suppress immune responses by secreting immunosuppressive cytokines such as IL-10 and TGF-␤. 5 A significant impairment of T-cell function is observed in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Although phenotypic and functional aberrations in CD4 and CD8 cells have been described in MM and MGUS, 6-9 the biologic basis for these abnormalities remains unclear. Because T reg cells play an important role in modulating normal immune responses, the abnormal T reg -cell activity in myeloma patients could contribute to immune dysfunction in MM and could provide a new target to enhance immune responses. Therefore, in this study we evaluated natural T reg -cell number and function in patients with MGUS and MM and compared them with those of healthy donors. Study design Phenotypic characterizationCD4 ϩ CD25 ϩ T reg -cell numbers were analyzed by flow cytometric analyses in peripheral-blood mononuclear cells (PBMCs) collected from healthy donors, patients with MGUS, and patients with newly diagnosed MM. Approval for these studies was obtained from the institutional review board of the Dana-Farber Cancer Institute and the Veterans Administration (VA) Boston Healthcare System. Informed consent was provided according to the Declaration of Helsinki. Measurement of FOXP3 expressionAs FOXP3 is specifically expressed by T reg cells and is required for their suppressive activity, we analyzed the proportion of PBMCs expressing intracellular FOXP3 using anti-FOXP3 antibody (eBiosciences, San Diego, CA) using dual-color flow cytometry and multiphoton microscopy. Level of protein expression was quantitated by Western blotting and by real-time reverse transcription-polymerase chain reaction (RT-PCR) using previously described methods. 10 Suppressive activity of T regulatory cellsTo evaluate the function of T reg cells, PBMCs were first depleted of CD25 ϩ T cells (which contain T reg cells) by positive selection using anti-CD25-coated microbeads (Miltenyi Biotech, Auburn, CA), according to the manufacturer's instructions. 11 PBMCs depleted of CD25 ϩ cells and control PBMCs containing CD25 ϩ cells were stimulated with anti-CD3 antibody for 3 days, and proliferation was measu...

BCMA targeting chimeric antigen receptor (CAR) T cell therapy has shown deep and durable responses in multiple myeloma. However, relapse following therapy is frequently observed, and mechanisms of resistance remain ill-defined. Here, we perform single cell genomic characterization of longitudinal samples from a patient who relapsed after initial CAR T cell treatment with lack of response to retreatment. We report selection, following initial CAR T cell infusion, of a clone with biallelic loss of BCMA acquired by deletion of one allele and a mutation that creates an early stop codon on the second allele. This loss leads to lack of CAR T cell proliferation following the second infusion and is reflected by lack of soluble BCMA in patient serum. Our analysis suggests the need for careful detection of BCMA gene alterations in multiple myeloma cells from relapse following CAR T cell therapy.

Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, is an antioxidant with chemopreventive and chemotherapeutic actions. Based on its ability to modulate growth factor-mediated cell proliferation, we evaluated its efficacy in multiple myeloma (MM).

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumoconjugating enzyme Ube2I, the Sumoligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumoconjugating enzyme in mammalian cells and cells transfected with a dominantnegative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal- IntroductionMultiple myeloma (MM) is a neoplasia hallmarked by the clonal expansion of malignant plasma cells (PCs) and the accumulation of a monoclonal immunoglobulin (Ig) or Ig fragment detectable in the serum and/or urine. [1][2][3] MM is the second most commonly diagnosed hematologic malignancy in the Western world, accounts for nearly 20% of all hematologic malignancies, and despite conventional treatment or high-dose therapy with stem cell transplantation is generally considered incurable. 4,5 Recent advances in mechanistic understanding and treatment modalities have extended median survival to exceed 6 years, and 10% of MM patients survive beyond 10 years. [6][7][8] Although high-dose therapy and novel agents that include thalidomide, its analog lenalidomide, and proteasome inhibitors have significantly improved prognosis, patient survival remains highly variable and cannot be accurately predicted with current models in part because the cellular pathways that determine patient response to treatment remain unidentified.In eukaryotes, a highly conserved multienzyme system is used in a sequential process to covalently attach the polypeptide ubiquitin to proteins targeted for degradation. 9,10 Ubiquitination is an essential process that maintains cellular homeostasis through dynamic switches in protein functional states. Ubiquitin-protein conjugates are then degraded in by the ATP-dependent 26S proteasome complex. 11,12 Deregulation of ubiquitination in tumor models has resulted in malignant transformation and tumor progression. 13 In myeloma, proteasomal-dependent catabolis...

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