Multiple myeloma (MM) originates from malignant plasma cells, leading to multiple destructive lytic bone lesions that occur in more than 80% of MM patients. MicroRNAs have been reported to be involved in development of bone lesions in MM. However, the circulating microRNA as diagnostic and prognostic biomarkers for bone lesions has not been elucidated yet. In this study, we identified differentially expressed miRNAs that are potentially involved in myeloma-related bone disease in serum of MM patients. MiR-214 and miR-135b was shown to be increased in serum of MM patients with bone lesions. Serum level of miR-214 and miR-135b was highly correlated with the severity of lytic bone lesions and demonstrated as a diagnostic tool for identifying bone diseases based on results of a receiver operating characteristic analysis (ROC). In addition, patients with high levels of serum miR-214 had a dismal survival with significantly shortened progression free survival (PFS) and overall survival (OS). Interestingly, bisphosphonates treatment significantly extended PFS and OS in patients with higher level of miR-214 comparing to patients without bisphosphonates treatment. Taken together, our findings revealed the significance of circulating miR-214 and miR-135b levels in detection of bone disease and in prediction of prognosis of patients with multiple myeloma, suggesting its potential clinical applications. The result of this study also set the foundation for searching more circulating miRNA as biomarker for tumor bone lesions.

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Multiple myeloma (MM) is an incurable B-cell malignancy. The proteasome inhibitor bortezomib (BTZ) is a frontline MM drug; however, intrinsic or acquired resistance to BTZ remains a clinical hurdle. As BTZ induces oxidative stress in MM cells, we queried if altered redox homeostasis promotes BTZ resistance. In primary human MM samples, increased gene expression of copper-zinc superoxide dismutase (CuZnSOD or SOD1) correlated with cancer progression, high-risk disease, and adverse overall and event-free survival outcomes. As an in vitro model, human MM cell lines (MM.1S, 8226, U266) and the BTZ-resistant (BR) lines (MM.1SBR, 8226BR) were utilized to determine the role of antioxidants in intrinsic or acquired BTZ-resistance. An up-regulation of CuZnSOD, glutathione peroxidase-1 (GPx-1), and glutathione (GSH) were associated with BTZ resistance and attenuated prooxidant production by BTZ. Enforced overexpression of SOD1 induced BTZ resistance and pharmacological inhibition of CuZnSOD with disulfiram (DSF) augmented BTZ cytotoxicity in both BTZ-sensitive and BTZ-resistant cell lines. Our data validates CuZnSOD as a novel therapeutic target in MM. We propose DSF as an adjuvant to BTZ in MM that is expected to overcome intrinsic and acquired BTZ resistance as well as augment BTZ cytotoxicity.

Therapeutic advances have markedly prolonged overall survival in multiple myeloma (MM) but the disease currently remains incurable. In a panel of MM cell lines (MM.1S, OPM-2, H929, and U266), using CD138 immunophenotyping, side population staining, and stem cell-related gene expression, we demonstrate the presence of stem-like tumor cells. Hypoxic culture conditions further increased CD138low stem-like cells with upregulated expression of OCT4 and NANOG. Compared to MM cells, these stem-like cells maintained lower steady-state pro-oxidant levels with increased uptake of the fluorescent deoxyglucose analog. In primary human MM samples, increased glycolytic gene expression correlated with poorer overall and event-free survival outcomes. Notably, stem-like cells showed increased mitochondrial mass, rhodamine 123 accumulation, and orthodox mitochondrial configuration while more condensed mitochondria were noted in the CD138high cells. Glycolytic inhibitor 2-deoxyglucose (2-DG) induced ER stress as detected by qPCR (BiP, ATF4) and immunoblotting (BiP, CHOP) and increased dihydroethidium probe oxidation both CD138low and CD138high cells. Treatment with a mitochondrial-targeting agent decyl-triphenylphosphonium (10-TPP) increased intracellular steady-state pro-oxidant levels in stem-like and mature MM cells. Furthermore, 10-TPP mediated increases in mitochondrial oxidant production were suppressed by ectopic expression of manganese superoxide dismutase. Relative to 2-DG or 10-TPP alone, 2-DG plus 10-TPP combination showed increased caspase 3 activation in MM cells with minimal toxicity to the normal hematopoietic progenitor cells. Notably, treatment with polyethylene glycol conjugated catalase significantly reduced 2-DG and/or 10-TPP-induced apoptosis of MM cells. Also, the combination of 2-DG with 10-TPP decreased clonogenic survival of MM cells. Taken together, this study provides a novel strategy of metabolic oxidative stress-induced cytotoxicity of MM cells via 2-DG and 10-TPP combination therapy.

11β-Hydroxysteroid dehydrogenases type 2 (11β-HSD2), a key regulator for pre-receptor metabolism of glucocorticoids (GCs) by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion) through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action.

The G protein-coupled receptor 109 A (GPR109A) is robustly expressed in osteoclastic precursor macrophages. Previous studies suggested that GPR109A mediates effects of diet-derived phenolic acids such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on promoting bone formation. However, the role of GPR109A in metabolic bone homeostasis and osteoclast differentiation has not been investigated. Using densitometric, bone histologic and molecular signaling analytic methods, we uncovered that bone mass and strength were significantly higher in tibia and spine of standard rodent diet weaned 4-week-old and 6-month-old GPR109A gene deletion (GPR109A-/-) mice, compared to their wild type controls. Osteoclast numbers in bone and in ex vivo bone marrow cell cultures were significantly decreased in GPR109A-/- mice compared to wild type controls. In accordance with these data, CTX-1 in bone marrow plasma and gene expression of bone resorption markers (TNFα, TRAP, Cathepsin K) were significantly decreased in GPR109A-/- mice, while on the other hand, P1NP was increased in serum from both male and female GPR109A-/- mice compared to their respective controls. GPR109A deletion led to suppressed Wnt/β-catenin signaling in osteoclast precursors to inhibit osteoclast differentiation and activity. Indeed, HA and 3-3-PPA substantially inhibited RANKL-induced GPR109A expression and Wnt/β-catenin signaling in osteoclast precursors and osteoclast differentiation. Resultantly, HA significantly inhibited bone resorption and increased bone mass in wild type mice, but had no additional effects on bone in GPR109A-/- mice compared with their respective untreated control mice. These results suggest an important role for GPR109A during osteoclast differentiation and bone resorption mediating effects of HA and 3-3-PPA on inhibiting bone resorption during skeletal development.

Metabolic alterations are important cancer-associated features that allow cancer cell transformation and survival under stress conditions. Multiple myeloma (MM) plasma cells show increased glycolysis and oxidative phosphorylation (OXPHOS), which are characteristics associated with recurrent genetic aberrations that drive the proliferation and survival of MM cells. The protein kinase B/AKT acts as a central node in cellular metabolism and is constitutively active in MM cells. Despite the known role of AKT in modulating cellular metabolism, little is known about the downstream factors of AKT that control the metabolic adaptability of MM cells. Here, we demonstrate that negative regulation of the forkhead box O (FOXO) transcription factors (TFs) by AKT is crucial to prevent the metabolic shutdown in MM cells, thus contributing to their metabolic adaptability. Our results demonstrate that the expression of several key metabolic genes involved in glycolysis, the tricarboxylic acid (TCA) cycle, and OXPHOS are repressed by FOXO TFs. Moreover, the FOXO-dependent repression of glycolysis- and TCA-associated genes correlates with a favorable prognosis in a large cohort of patients with MM. Our data suggest that repression of FOXO by AKT is essential to sustain glycolysis and the TCA cycle activity in MM cells and, as such, predicts patient survival.

The total therapy (TT) IIIB phase 2 study incorporated bortezomib into tandem melphalan-based hematopoietic stem cell transplantation with dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide, and etoposide for induction/consolidation and bortezomib, lenalidomide, and dexamethasone (VRD) for maintenance in patients with newly diagnosed multiple myeloma (MM). This updated analysis presents a 15.4-year median follow-up. Of 177 patients, 21% patients had gene expression profile (GEP)-defined high-risk MM. 15-year progression free survival (PFS) was 27.9%. Median PFS was better in GEP-defined low-risk patients at 7.8 years and in International Staging System stage 1 patients at 8.7 years. Overall, median OS was 9.1 years, and 15-year overall survival (OS) was 35.9%. GEP-defined low-risk patients' median OS was 11.2 years, and that of GEP-defined high-risk patients was 2.8 years. There was no difference in OS between TT IIIB and TT IIIA. This study includes the longest follow-up of patients treated with maintenance VRD reported to date. In patients with GEP-defined low-risk, nearly half and one-third of patients without ongoing treatment showed no signs of progression at 10 and 15 years, respectively. One-third of patients survived more than 15 years, but 3 years of VRD maintenance did not improve outcomes for patients with GEP-defined high-risk MM. The study was registered on www.clinicaltrials.gov as #NCT00572169.

In most myeloma patients, even after several rounds of intensive therapy, drug resistant tumor cells survive and proliferate aggressively leading to relapse. In the present study, gene expression profiling of tumor cells isolated from myeloma patients after sequential rounds of chemotherapy, revealed for the first time that heparanase, a potent promoter of myeloma growth and progression, was elevated in myeloma cells that survived therapy. Based on this clinical data, we hypothesized that heparanase was involved in myeloma resistance to drug therapy. In several survival and viability assays, elevated heparanase expression promoted resistance of myeloma tumor cells to chemotherapy. Mechanistically, this enhanced survival was due to heparanase-mediated ERK signaling. Importantly, use of the heparanase inhibitor Roneparstat in combination with chemotherapy clearly diminished the growth of disseminated myeloma tumors in vivo. Moreover, use of Roneparstat either during or after chemotherapy diminished regrowth of myeloma tumors in vivo following therapy. These results provide compelling evidence that heparanase is a promising, novel target for overcoming myeloma resistance to therapy and that targeting heparanase has the potential to prevent relapse in myeloma and possibly other cancers.

This study evaluates the role of scavenger receptor class A member 3 (SCARA3) in multiple myeloma (MM). SCARA3 expression was induced upon treatment with oxidative stressors (ionizing radiation and chemotherapeutic drugs). An epigenetic inactivation of SCARA3 was noted in MM.1S myeloma cells. Myeloma cell killing by dexamethasone and bortezomib was inhibited by up-regulation of SCARA3 while SCARA3 knockdown sensitized myeloma cells to the drugs. Clinical samples showed an inverse correlation between SCARA3 gene expression, myeloma progression, and favorable clinical prognosis. In MM, SCARA3 protects against oxidative stress-induced cell killing and can serve as predictor of MM progression and therapeutic response.

Natural killer (NK) cell-based treatments are promising therapies for multiple myeloma (MM). Carfilzomib (CFZ), is a second-generation proteasome inhibitor, used to treat relapsed and refractory MM. In this study, we determined that CFZ treatment enhanced the sensitivity of MM cells to NK cell-mediated lysis. Here, we report that CFZ decreased the expression of human leukocyte antigen (HLA) class I in a time- and dose-dependent manner. CFZ also down-regulated the expression of newly formed HLA class I on MM cells. Treatment of MM with CFZ enhanced NK cell degranulation and significantly sensitized patients' MM cells to NK cell-mediated lysis. Furthermore, the enhancement of NK cell-mediated lysis was linked with the decreased expression of HLA class I. Our findings show a novel activity of CFZ as an immunomodulating agent and suggest a possible approach to therapeutically augment NK cell function in MM patients.

High-dose chemotherapies to treat multiple myeloma (MM) can be life-threatening due to toxicities to normal cells and there is a need to target only tumor cells and/or lower standard drug dosage without losing efficacy. We show that pharmacologically-dosed ascorbic acid (PAA), in the presence of iron, leads to the formation of highly reactive oxygen species (ROS) resulting in cell death. PAA selectively kills CD138+ MM tumor cells derived from MM and smoldering MM (SMM) but not from monoclonal gammopathy undetermined significance (MGUS) patients. PAA alone or in combination with melphalan inhibits tumor formation in MM xenograft mice. This study shows PAA efficacy on primary cancer cells and cell lines in vitro and in vivo.

Aerobic glycolysis, a hallmark of cancer, is characterized by increased metabolism of glucose and production of lactate in normaxia. Recently, pyruvate kinase M2 (PKM2) has been identified as a key player for regulating aerobic glycolysis and promoting tumor cell proliferation and survival.

Colorectal cancer (CRC) has a high mortality rate, and therapeutic approaches to treat these cancers are varied and depend on the metabolic state of the tumour. Profiles of CRC tumours have identified several biomarkers, including microRNAs. microRNA-210 (miR-210) levels are directly correlated with CRC survival. miR-210 expression is higher in metastatic colon cancer cells versus non-metastatic and normal colon epithelium. Therefore, efficient methods to inhibit miR-210 expression in CRC may provide new advances in treatments.

Oncogenic overexpression of integrin-β7 (ITGB7) in cases of high-risk multiple myeloma (MM) was reported to promote enhanced interactions between neoplastic plasma-B cells and stromal cells to develop cell-adhesion mediated drug resistance.

Many cancers, including multiple myeloma (MM), retain more cytosolic iron to promote tumor cell growth and drug resistance. Higher cytosolic iron promotes oxidative damage due to its interaction with reactive oxygen species generated by mitochondria. The variation of mitochondrial biogenesis in different stages of MM disease was evaluated using gene expression profiles in a large clinical dataset. Sixteen of 18mitochondrial biogenesis related gene sets, including mitochondrial biogenesis signature and oxidative phosphorylation, were increased in myeloma cells compared with normal plasma cells and high expression was associated with an inferior patient outcome. Relapsed and drug resistant myeloma samples had higher expression of mitochondrial biogenesis signatures than newly diagnosed patient samples. The expression of mitochondrial biogenesis genes was regulated by the cellular iron content, which showed a synergistic effect in patient outcome in MM. Pharmacological ascorbic acid induced myeloma cell death by inhibition of mitochondria oxidative phosphorylation in an in vivo model. Here, we identify that dysregulated mitochondrial biogenesis and iron homeostasis play a major role in myeloma progression and patient outcome and that pharmacological ascorbic acid, through cellular iron content and mitochondrial oxidative species, should be considered as a novel treatment in myeloma including drug-resistant and relapsed patients.

Anti-multiple myeloma B cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies represent a promising treatment strategy with high response rates in myeloma. However, durable cures following anti-BCMA CAR-T cell treatment of myeloma are rare. One potential reason is that a small subset of minimal residual myeloma cells seeds relapse. Residual myeloma cells following BCMA-CAR-T-mediated treatment show less-differentiated features and express stem-like genes, including CD24. CD24-positive myeloma cells represent a large fraction of residual myeloma cells after BCMA-CAR-T therapy. In this work, we develop CD24-CAR-T cells and test their ability to eliminate myeloma cells. We find that CD24-CAR-T cells block the CD24-Siglec-10 pathway, thereby enhancing macrophage phagocytic clearance of myeloma cells. Additionally, CD24-CAR-T cells polarize macrophages to a M1-like phenotype. A dual-targeted BCMA-CD24-CAR-T exhibits improved efficacy compared to monospecific BCMA-CAR-T-cell therapy. This work presents an immunotherapeutic approach that targets myeloma cells and promotes tumor cell clearance by macrophages.

Following up on previous work demonstrating the involvement of the transcription factor forkhead box M1 (FOXM1) in the biology and outcome of a high-risk subset of newly diagnosed multiple myeloma (nMM), this study evaluated whether FOXM1 gene expression may be further upregulated upon tumor recurrence in patients with relapsed multiple myeloma (rMM). Also assessed was the hypothesis that increased levels of FOXM1 diminish the sensitivity of myeloma cells to commonly used myeloma drugs, such as the proteasome inhibitor bortezomib (Bz) and the DNA intercalator doxorubicin (Dox).

Here we demonstrate the crucial role of CKS1B in multiple myeloma (MM) progression and define CKS1B-mediated SKP2/p27(Kip1)-independent down-stream signaling pathways. Forced-expression of CKS1B in MM cells increased cell multidrug-resistance. CKS1B activates STAT3 and MEK/ERK pathways. In contrast, SKP2 knockdown or p27(Kip1) over-expression resulted in activation of the STAT3 and MEK/ERK pathways. Further investigations showed that BCL2 is a downstream target of MEK/ERK signaling. Stimulation of STAT3 and MEK/ERK signaling pathways partially abrogated CKS1B knockdown induced MM cell death and growth inhibition. Targeting STAT3 and MEK/ERK signaling pathways by specific inhibitors induced significant MM cell death and growth inhibition in CKS1B-overexpressing MM cells and their combinations resulted in synergy. Thus, our findings provide a rationale for targeting STAT3 and MEK/ERK/BCL2 signaling in aggressive CKS1B-overexpressing MM.

Despite the development of several new agents for multiple myeloma (MM) therapy over the last decade, drug resistance continues to be a significant problem. Patients with relapsed/refractory disease have high mortality rates and desperately need new precision approaches that directly target specific molecular features that are prevalent in the refractory setting. Reolysin is a proprietary formulation of reovirus for cancer therapy that has demonstrated efficacy in multiple clinical trials. Its selective effects against solid tumors have been largely attributed to RAS-mediated control of reovirus replication. However, the mechanisms regulating its preferential anti-neoplastic effects in MM and other hematological malignancies have not been rigorously studied. Here we report that the reovirus receptor, junctional adhesion molecule-A (JAM-A) is highly expressed in primary cells from patients with MM and the majority of MM cell lines compared to normal controls. A series of experiments demonstrated that JAM-A expression, rather than RAS, was required for Reolysin-induced cell death in MM models. Notably, analysis of paired primary MM specimens revealed that JAM-A expression was significantly increased at relapse compared to diagnosis. Two different models of acquired resistance to bortezomib also displayed both higher JAM-A expression and elevated sensitivity to Reolysin compared to parental cells, suggesting that Reolysin may be an effective agent for patients with relapsed/refractory disease due to their high JAM-A levels. Taken together, these findings support further investigation of Reolysin for the treatment of patients with relapsed/refractory MM and of JAM-A as a predictive biomarker for sensitivity to Reolysin-induced cell death.