Progression of monoclonal gammopathy of undetermined significance to multiple myeloma is associated with enhanced translational quality control and overall loss of surface antigens.

BACKGROUND: Despite significant advancements in treatment strategies, multiple myeloma remains incurable. Additionally, there is a distinct lack of reliable biomarkers that can guide initial treatment decisions and help determine suitable replacement or adjuvant therapies when relapse ensues due to acquired drug resistance. METHODS: To define specific proteins and pathways involved in the progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM), we have applied super-SILAC quantitative proteomic analysis to CD138 + plasma cells from 9 individuals with MGUS and 37 with MM. RESULTS: Unsupervised hierarchical clustering defined three groups: MGUS, MM, and MM with an MGUS-like proteome profile (ML) that may represent a group that has recently transformed to MM. Statistical analysis identified 866 differentially expressed proteins between MM and MGUS, and 189 between MM and ML, 177 of which were common between MGUS and ML. Progression from MGUS to MM is accompanied by upregulated EIF2 signaling, DNA repair, and proteins involved in translational quality control, whereas integrin- and actin cytoskeletal signaling and cell surface markers are downregulated. CONCLUSION: Compared to the premalignant plasma cells in MGUS, malignant MM cells apparently have mobilized several pathways that collectively contribute to ensure translational fidelity and to avoid proteotoxic stress, especially in the ER. The overall reduced expression of immunoglobulins and surface antigens contribute to this and may additionally mediate evasion from recognition by the immune apparatus. Our analyses identified a range of novel biomarkers with potential prognostic and therapeutic value, which will undergo further evaluation to determine their clinical significance.

IL-32 is induced by activation of toll-like receptors in multiple myeloma cells.

Multiple myeloma (MM) is a hematological cancer characterized by accumulation of malignant plasma cells in the bone marrow. The patients are immune suppressed and suffer from recurrent and chronic infections. Interleukin-32 is a non-conventional, pro-inflammatory cytokine expressed in a subgroup of MM patients with a poor prognosis. IL-32 has also been shown to promote proliferation and survival of the cancer cells. Here we show that activation of toll-like receptors (TLRs) promotes expression of IL-32 in MM cells through NFκB activation. In patient-derived primary MM cells, IL-32 expression is positively associated with expression of TLRs. Furthermore, we found that several TLR genes are upregulated from diagnosis to relapse in individual patients, predominantly TLRs sensing bacterial components. Interestingly, upregulation of these TLRs coincides with an increase in IL-32. Taken together, these results support a role for IL-32 in microbial sensing in MM cells and suggest that infections can induce expression of this pro-tumorigenic cytokine in MM patients.

Mutational analysis and protein profiling predict drug sensitivity in multiple myeloma cell lines.

Introduction: Multiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease. Methods: To identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins. Results: We found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in NRAS/KRAS, and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors. Discussion: Taken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM.

The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells.

BACKGROUND: Multiple myeloma (MM) is an incurable malignancy of plasma cells. The serine protease matriptase is frequently dysregulated in human carcinomas, which facilitates tumor progression and metastatic dissemination. The importance of matriptase in hematological malignancies is yet to be clarified. In this study, we aimed to characterize the role of matriptase in MM. MATERIALS AND METHODS: mRNA expression of matriptase and its inhibitors hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 was studied in primary MM cells from patient samples and human myeloma cell lines (HMCLs). We further investigated the effect of matriptase on migration and proliferation of myeloma cells in vitro. By use of the CoMMpass database, we assessed the clinical relevance of matriptase in MM patients. RESULTS: Matriptase was expressed in 96% of patient samples and all HMCLs tested. Overexpression of matriptase in vitro reduced proliferation, and significantly decreased cytokine-induced migration. Conversely, matriptase knockdown significantly enhanced migration. Mechanistically, overexpression of matriptase inhibited activation of Src kinase. CONCLUSIONS: Our findings may suggest a novel role of matriptase as a tumor suppressor in MM pathogenesis.

Clonal evolution after treatment pressure in multiple myeloma: heterogenous genomic aberrations and transcriptomic convergence.

We investigated genomic and transcriptomic changes in paired tumor samples of 29 in-house multiple myeloma (MM) patients and 28 patients from the MMRF CoMMpass study before and after treatment. A change in clonal composition was found in 46/57 (82%) of patients, and single-nucleotide variants (SNVs) increased from median 67 to 86. The highest increase in prevalence of genetic aberrations was found in RAS genes (60% to 72%), amp1q21 (18% to 35%), and TP53 (9% to 18%). The SBS-MM1 mutation signature was detected both in patients receiving high and low dose melphalan. A total of 2589 genes were differentially expressed between early and late samples (FDR < 0.05). Gene set enrichment analysis (GSEA) showed increased expression of E2F, MYC, and glycolysis pathways and a decreased expression in TNF-NFkB and TGFbeta pathways in late compared to early stage. Single sample GSEA (ssGSEA) scores of differentially expressed pathways revealed that these changes were most evident in end-stage disease. Increased expression of several potentially targetable genes was found at late disease stages, including cancer-testis antigens, XPO1 and ABC transporters. Our study demonstrates a transcriptomic convergence of pathways supporting increased proliferation and metabolism during disease progression in MM.

Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells.

Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo. High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism.

TAK1-inhibitors are cytotoxic for multiple myeloma cells alone and in combination with melphalan.

Multiple myeloma (MM) is an incurable cancer caused by malignant transformation of plasma cells. Transforming growth factor-ß activated kinase 1 (MAP3K7, TAK1) is a major regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. Both NF-κB and MAPK control expression of genes with vital roles for drug resistance in MM. TAK1 is an attractive drug target as it switches these survival pathways to cell death. Our analysis showed that patients with high MAP3K7 expression in the tumor had shorter overall and progression free survival. The TAK1-inhibitors NG25 and 5Z-7-oxozeaenol (5Z-7) were cytotoxic to MM cell lines and patient cells. NG25 reduced expression of MYC and E2F controlled genes, involved in tumor cell growth, cell cycle progression and drug resilience. TAK1 can be activated by genotoxic stress. NG25 and 5Z-7 induced both synergistic and additive cytotoxicity in combination with the alkylating agent melphalan. Melphalan activated TAK1, NF-κB, and the MAPKs p38 and c-Jun N-terminal kinase (JNK), as well as a transcriptional UV-response. This was blocked by NG25, and instead apoptosis was activated. MM induce elevated bone-degradation resulting in myeloma bone disease (MBD), which is the main cause of disability and morbidity in MM patients. NG25 and 5Z-7 reduced differentiation and viability of human bone degrading osteoclasts, suggesting that TAK1-inhibition can have a double beneficial effect for patients. In sum, TAK1 is a promising drug target for MM treatment.

Bystander Memory T Cells and IMiD/Checkpoint Therapy in Multiple Myeloma: A Dangerous Tango?

In this review article we discuss the role of the memory T cells in multiple myeloma (MM) and how they may influence immune responses in patients that received immunomodulating drugs and check point therapy.

Phosphatase of regenerating liver-3 regulates cancer cell metabolism in multiple myeloma.

Cancer cells often depend on microenvironment signals from molecules such as cytokines for proliferation and metabolic adaptations. PRL-3, a cytokine-induced oncogenic phosphatase, is highly expressed in multiple myeloma cells and associated with poor outcome in this cancer. We studied whether PRL-3 influences metabolism. Cells transduced to express PRL-3 had higher aerobic glycolytic rate, oxidative phosphorylation, and ATP production than the control cells. PRL-3 promoted glucose uptake and lactate excretion, enhanced the levels of proteins regulating glycolysis and enzymes in the serine/glycine synthesis pathway, a side branch of glycolysis. Moreover, mRNAs for these proteins correlated with PRL-3 expression in primary patient myeloma cells. Glycine decarboxylase (GLDC) was the most significantly induced metabolism gene. Forced GLDC downregulation partly counteracted PRL-3-induced aerobic glycolysis, indicating GLDC involvement in a PRL-3-driven Warburg effect. AMPK, HIF-1α, and c-Myc, important metabolic regulators in cancer cells, were not mediators of PRL-3's metabolic effects. A phosphatase-dead PRL-3 mutant, C104S, promoted many of the metabolic changes induced by wild-type PRL-3, arguing that important metabolic effects of PRL-3 are independent of its phosphatase activity. Through this study, PRL-3 emerges as one of the key mediators of metabolic adaptations in multiple myeloma.

Monoclonal immunoglobulins promote bone loss in multiple myeloma.

Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.

Conversion of ATP to adenosine by CD39 and CD73 in multiple myeloma can be successfully targeted together with adenosine receptor A2A blockade.

BACKGROUND: PD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its immunosuppressive receptor should be considered. CD39 and CD73 convert extracellular ATP to adenosine, which inhibits T-cell effector functions via the adenosine receptor A2A (A2AR). We set out to investigate whether blocking the adenosine pathway could be a therapy for MM. METHODS: Expression of CD39 and CD73 on BM cells from patients and T-cell proliferation were determined by flow cytometry and adenosine production by Liquid chromatograpy-mass spectrometry (HPCL/MS). ENTPD1 (CD39) mRNA expression was determined on myeloma cells from patients enrolled in the publicly available CoMMpass study. Transplantable 5T33MM myeloma cells were used to determine the effect of inhibiting CD39, CD73 and A2AR in mice in vivo. RESULTS: Elevated level of adenosine was found in BM plasma of MM patients. Myeloma cells from patients expressed CD39, and high gene expression indicated reduced survival. CD73 was found on leukocytes and stromal cells in the BM. A CD39 inhibitor, POM-1, and an anti-CD73 antibody inhibited adenosine production and reduced T-cell suppression in vitro in coculture of myeloma and stromal cells. Blocking the adenosine pathway in vivo with a combination of Sodium polyoxotungstate (POM-1), anti-CD73, and the A2AR antagonist AZD4635 activated immune cells, increased interferon gamma production, and reduced the tumor load in a murine model of MM. CONCLUSIONS: Our data suggest that the adenosine pathway can be successfully targeted in MM and blocking this pathway could be an alternative to PD1/PDL1 inhibition for MM and other hematological cancers. Inhibitors of the adenosine pathway are available. Some are in clinical trials and they could thus reach MM patients fairly rapidly.

Stability and uniqueness of clonal immunoglobulin CDR3 sequences for MRD tracking in multiple myeloma.

Minimal residual disease (MRD) tracking, by next generation sequencing of immunoglobulin sequences, is moving towards clinical implementation in multiple myeloma. However, there is only sparse information available to address whether clonal sequences remain stable for tracking over time, and to what extent light chain sequences are sufficiently unique for tracking. Here, we analyzed immunoglobulin repertoires from 905 plasma cell myeloma and healthy control samples, focusing on the third complementarity determining region (CDR3). Clonal heavy and/or light chain expression was identified in all patients at baseline, with one or more subclones related to the main clone in 3.2%. In 45 patients with 101 sequential samples, the dominant clonal CDR3 sequences remained identical over time, despite differential clonal evolution by whole exome sequencing in 49% of patients. The low frequency of subclonal CDR3 variants, and absence of evolution over time in active multiple myeloma, indicates that tumor cells at this stage are not under selective pressure to undergo antibody affinity maturation. Next, we establish somatic hypermutation and non-templated insertions as the most important determinants of light chain clonal uniqueness, identifying a potentially trackable sequence in the majority of patients. Taken together, we show that dominant clonal sequences identified at baseline are reliable biomarkers for long-term tracking of the malignant clone, including both IGH and the majority of light chain clones.

Correction: MYC amplifications in myeloma cell lines: correlation with MYC-inhibitor efficacy.

[This corrects the article DOI: 10.18632/oncotarget.4245.].

BMPR2 inhibits activin and BMP signaling via wild-type ALK2.

TGF-ß/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-ß superfamily member, inhibits growth of multiple myeloma cells, but the mechanism for this is unknown. We therefore aimed to clarify how activins affect myeloma cell survival. Activin A activates the transcription factors SMAD2/3 through the ALK4 type 1 receptor, but may also activate SMAD1/5/8 through mutated variants of the type 1 receptor ALK2 (also known as ACVR1). We demonstrate that activin A and B activate SMAD1/5/8 in myeloma cells through endogenous wild-type ALK2. Knockdown of the type 2 receptor BMPR2 strongly potentiated activin A- and activin B-induced activation of SMAD1/5/8 and subsequent cell death. Furthermore, activity of BMP6, BMP7 or BMP9, which may also signal via ALK2, was potentiated by knockdown of BMPR2. Similar results were seen in HepG2 liver carcinoma cells. We propose that BMPR2 inhibits ALK2-mediated signaling by preventing ALK2 from oligomerizing with the type 2 receptors ACVR2A and ACVR2B, which are necessary for activation of ALK2 by activins and several BMPs. In conclusion, BMPR2 could be explored as a possible target for therapy in patients with multiple myeloma.This article has an associated First Person interview with the first author of the paper.

Anti-Cancer Potential of Homemade Fresh Garlic Extract Is Related to Increased Endoplasmic Reticulum Stress.

The use of garlic and garlic-based extracts has been linked to decreased incidence of cancer in epidemiological studies. Here we examine the molecular and cellular activities of a simple homemade ethanol-based garlic extract (GE). We show that GE inhibits growth of several different cancer cells in vitro, as well as cancer growth in vivo in a syngeneic orthotopic breast cancer model. Multiple myeloma cells were found to be especially sensitive to GE. The GE was fractionated using solid-phase extractions, and we identified allicin in one GE fraction; however, growth inhibitory activities were found in several additional fractions. These activities were lost during freeze or vacuum drying, suggesting that the main anti-cancer compounds in GE are volatile. The anti-cancer activity was stable for more than six months in −20 °C. We found that GE enhanced the activities of chemotherapeutics, as well as MAPK and PI3K inhibitors. Furthermore, GE affected hundreds of proteins involved in cellular signalling, including changes in vital cell signalling cascades regulating proliferation, apoptosis, and the cellular redox balance. Our data indicate that the reduced proliferation of the cancer cells treated by GE is at least partly mediated by increased endoplasmic reticulum (ER) stress.

Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™.

Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan.

Monitoring multiple myeloma by quantification of recurrent mutations in serum.

Circulating tumor DNA is a promising biomarker to monitor tumor load and genome alterations. We explored the presence of circulating tumor DNA in multiple myeloma patients and its relation to disease activity during long-term follow-up. We used digital droplet polymerase chain reaction analysis to monitor recurrent mutations, mainly in mitogen activated protein kinase pathway genes NRAS, KRAS and BRAF Mutations were identified by next-generation sequencing or polymerase chain reaction analysis of bone marrow plasma cells, and their presence analyzed in 251 archived serum samples obtained from 20 patients during a period of up to 7 years. In 17 of 18 patients, mutations identified in bone marrow during active disease were also found in a time-matched serum sample. The concentration of mutated alleles in serum correlated with the fraction in bone marrow plasma cells (r=0.507, n=34, P<0.002). There was a striking covariation between circulating mutation levels and M protein in ten out of 11 patients with sequential samples. When relapse evaluation by circulating tumor DNA and M protein could be directly compared, the circulating tumor DNA showed relapse earlier in two patients (3 and 9 months), later in one patient (4 months) and in three patients there was no difference. In three patients with transformation to aggressive disease, the concentrations of mutations in serum increased up to 400 times, an increase that was not seen for the M protein. In conclusion, circulating tumor DNA in myeloma is a multi-faceted biomarker reflecting mutated cells, total tumor mass and transformation to a more aggressive disease. Its properties are both similar and complementary to M protein.

Src Family Kinases Are Regulated in Multiple Myeloma Cells by Phosphatase of Regenerating Liver-3.

Phosphatase of regenerating liver-3 (PTP4A3/PRL-3) is a dual-specificity phosphatase that is upregulated in various types of cancers and is related to poor prognosis and aggressive tumor behavior. The expression level of PRL-3 is elevated in response to several antiapoptotic cytokines, including IL6, in cancer cells from patients with multiple myeloma (MM) and can promote survival and migration. Here, it is demonstrated that PRL-3 activates Src kinase in the IL6-dependent MM cell line INA-6. Inhibition of PRL-3 by a small-molecule inhibitor of PRL-3 or by shRNA resulted in inactivation of Src. In addition to activation of Src, PRL-3 also activated the Src family kinase (SFK) members LYN and HCK in INA-6 cells. Forced expression of catalytically inactive mutant PRL-3 decreased the activation of these three SFK members while the total level of HCK and FYN remained elevated. Inhibitors of Src increased sensitivity of cells overexpressing PRL-3 to the PRL-3 inhibitor through joint downregulation of both PRL-3 and Mcl-1. In conclusion, PRL-3 protected MM cells against apoptosis by dysregulating both the total levels and the activation levels of specific SFK members that are important for IL6 signal transduction in MM cells. Eventually, this led to increased levels of Mcl-1. IMPLICATIONS: This study suggests PRL-3 and SFKs are key mediators of the IL6-driven signaling events and points to both PRL-3 and SFK members as potential targets for treatment of MM. Mol Cancer Res; 15(1); 69-77. ©2016 AACR.