The HMG-CoA reductase inhibitor simvastatin overcomes cell adhesion-mediated drug resistance in multiple myeloma by geranylgeranylation of Rho protein and activation of Rho kinase

Primary drug resistance is a major problem in multiple myeloma, an incurable disease of the bone marrow. Cell adhesion-mediated drug resistance (CAM-DR) causes strong primary resistance. By coculturing multiple myeloma cells with bone marrow stromal cells (BMSCs), we observed a CAM-DR of about 50% to melphalan, treosulfan, doxorubicin, dexamethasone, and bortezomib, which was not reversed by secreted soluble factors. Targeting the adhesion molecules lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) by monoclonal antibodies or by the LFA-1 inhibitor LFA703 reduced CAM-DR significantly. Only statins such as simvastatin and lovastatin, however, were able to completely restore chemosensitivity. All these effects were not mediated by deadhesion or reduced secretion of interleukin 6. Targeting geranylgeranyl transferase (GGTase) and Rho kinase by specific inhibitors (GGTI-298 and Y-27632), but not inhibition of farnesyl transferase (FTase) by FTI-277, showed similar reduction of CAM-DR. Addition of geranylgeranyl pyrophosphate (GG-PP), but not of farnesyl pyrophosphate (F-PP), was able to inhibit simvastatin-induced CAM-DR reversal. Our data suggest that the 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA)/GG-PP/Rho/Rho-kinase pathway mediates CAM-DR and that targeting this pathway may improve the efficacy of antimyeloma therapies by reduction of CAM-DR.     

2-Methoxyestradiol overcomes drug resistance in multiple myeloma cells

2-Methoxyestradiol (2ME2) an estrogen derivative, induces growth arrest and apoptosis in leukemic cells and is also antiangiogenic. In this study, we demonstrate that 2ME2 inhibits growth and induces apoptosis in multiple myeloma (MM) cell lines and patient cells. Significantly, 2ME2 also inhibits growth and induces apoptosis in MM cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40 and Dox-6), and dexamethasone (MM.1R). In contrast to its effects on MM cells, 2ME2 does not reduce the survival of normal peripheral blood lymphocytes. Moreover, 2ME2 enhances Dex-induced apoptosis, and its effect is not blocked by interleukin-6 (IL-6). We next examined the effect of 2ME2 on MM cells in the bone marrow (BM) milieu. 2ME2 decreases survival of BM stromal cells (BMSCs), as well as secretion of vascular endothelial growth factor (VEGF), and IL-6 triggered by the adhesion of MM cells to BMSCs. We show that apoptosis induced by 2ME2 is mediated by the release of mitochondrial cytochrome-c (cyto-c) and Smac, followed by the activation of caspases-8, -9, and -3. Finally, 2ME2 inhibits MM cell growth, prolongs survival, and decreases angiogenesis in a murine model. These studies, therefore, demonstrate that 2ME2 mediates anti-MM activity directly on MM cells and in the BM microenvironment. They provide a framework for the use of 2ME2, either alone or in combination with Dex, to overcome drug resistance and to improve outcome in MM.     

HSP70 inhibition reverses cell adhesion mediated and acquired drug resistance in multiple myeloma

Heat shock proteins (HSPs) are a super family of highly conserved molecular chaperone proteins, which are induced in response to stress. HSP70 has been demonstrated to inhibit apoptosis induced by a number of chemotherapeutic agents. Previous investigations have suggested the development of drug resistance in multiple myeloma (MM) cells after adhesion to stroma. This study used MM cell lines and primary plasma cells to determine if HSP70 had a role in development of chemo resistance. Adhesion of MM cells to either bone marrow stromal cells or fibronectin (FN) enhanced HSP70 expression. Inhibition of the HSP70 expression decreased 8226 cell adhesion to stroma or FN and induced more apoptosis in FN-adhered 8226 cells than in suspension cultures at 24 h. Further, HSP70 inhibitors enhanced melphalan-induced apoptosis and reversed melphalan-induced cell adhesion-mediated drug resistance (CAM-DR) phenotype. In addition, compared to parental cells, KNK-437, a heat shock factor inhibitor caused more apoptosis in melphalan-resistant 8226/LR5 cells and sensitized them to melphalan. Primary CD138 positive cells showed high expression of HSPA4 mRNA, and KNK-437 caused apoptosis in these cells. In conclusion, our data suggest inhibition of HSP70, reduced adhesion and caused apoptosis of both acquired and de novo drug resistant MM cells.     

Epigenetic silencing of MIR203 in multiple myeloma

Epigenetic inactivation of tumour suppressor microRNAs has been implicated in carcinogenesis. We studied the promoter methylation of MIR203 in eight normal marrow controls, eight multiple myeloma (MM) cell lines, 20 monoclonal gammopathy of undetermined significance (MGUS), 123 diagnostic MM and 19 relapsed MM samples by methylation-specific polymerase chain reaction. Promoter of MIR203 was unmethylated in normal controls but homozygously methylated in 25% MM cell lines. Treatment with 5-Aza-2'-deoxycytidine led to promoter demethylation and MIR203 re-expression. Cyclic AMP responsive element binding protein 1 (CREB1) mRNA was predicted as a MIR203 direct target. Luciferase activity was reduced in constructs carrying wild-type CREB1 3'UTR upon MIR203 expression but not in those carrying mutant CREB1 3'UTR. Moreover, restoration of MIR203 led to downregulation of CREB1 protein and inhibition of myeloma cell proliferation. In primary samples, MIR203 methylation occurred in 25·0% MGUS, 23·6% diagnostic MM, and 21·1% relapsed MM samples. In conclusion, MIR203 methylation is disease-specific with reversible gene silencing in MM. MIR203 is a tumour suppressor microRNA inhibiting cellular proliferation by targeting CREB1 mRNA in MM. Comparable occurrence of MIR203 methylation in MGUS and MM at diagnosis or relapse suggested that MIR203 methylation may be an early event in myelomagenesis instead of being acquired during disease progression.     

Environmental-mediated drug resistance: a target for multiple myeloma therapy

Multiple myeloma is an incurable malignancy of mature clonal B cells. The refractory nature of this disease has long been attributed to the acquisition of drug resistance. Traditionally, mechanisms of drug resistance have been defined by genetic, acquired changes in the expression or function of specific genes products. However, over the past 10 years a large body of evidence has emerged demonstrating that in addition to mechanisms of drug resistance intrinsic to the cancer cell, there exist dynamic, de novo mechanisms coordinated by the tumor microenvironment resulting in a environmental-mediated drug resistance (EM-DR). Within this review we will provide an overview of some of these mechanisms of drug resistance and how they contribute to minimal residual disease and subsequent treatment failure. By understanding mechanisms of EM-DR, therapeutic targets can be identified and interventions designed to reduce minimal residual disease and improve clinical outcomes.     

ADP-ribosylation factor 1 (ARF1) takes part in cell proliferation and cell adhesion-mediated drug resistance (CAM-DR)

Cell adhesion-mediated drug resistance (CAM-DR) remains the primary obstacle in human multiple myeloma (MM) therapy. In this study, we aimed at investigating the expression and biologic function of ARF1 in MM. We determined that ARF1 expression was positively correlated with cell proliferation and knockdown of ARF1 contributed to CAM-DR. The enhancement in the adhesion of MM cells to fibronectin (FN) or the bone marrow stroma cell line HS-5 cells translated to an increased CAM-DR phenotype. Importantly, we showed that this CAM-DR phenotype was correlated with the phosphorylation of Akt and ERK in MM cells. Moreover, we sought to determine whether ARF1 could interact with p27 in RPMI8226 cells. Knockdown of ARF1 also significantly decreased pT157-p27 protein expression in RPMI8226 cells. Our research shows ARF1 may reverse CAM-DR by regulating phosphorylation of p27 at T157 in MM. Taken together, our data shed new light on the molecular mechanism of CAM-DR in MM, and targeting ARF1 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in MM.     

First clinical experience with simvastatin to overcome drug resistance in refractory multiple myeloma

In vitro statins overcome cell adhesion-mediated drug resistance at non-toxic concentrations that are achievable in humans by standard dose simvastatin. A pilot phase-II trial was initiated to determine feasibility and antimyeloma efficacy. In six myeloma patients refractory to two cycles of bortezomib or bendamustine simvastatin was concomitantly administered during further two cycles. The therapy was well tolerated without grade 3/4 toxicity. Intrapatient (cycles I/II vs. III/IV) and interpatient comparison (vs. ten patients without simvastatin) showed reduction of drug resistance by inhibition of HMG-CoA-reductase. In summary, this is the first phase II experience to study antimyeloma activity of statins in humans.     

Epigenetic mechanisms of liver tumor resistance to immunotherapy

Hepatocellular carcinoma(HCC) is the most common primary liver tumor, which stands fourth in rank of cancer-related deaths worldwide. The incidence of HCC is constantly increasing in correlation with the epidemic in diabetes and obesity, arguing for an urgent need for new treatments for this lethal cancer refractory to conventional treatments. HCC is the paradigm of inflammation-associated cancer, since more than 80% of HCC emerge consecutively to cirrhosis associated with a vast remodeling of liver microenvironment. In the recent decade, immunomodulatory drugs have been developed and have given impressive results in melanoma and later in several other cancers. In the present review, we will discuss the recent advancements concerning the use of immunotherapies in HCC, in particular those targeting immune checkpoints, used alone or in combination with other anticancers agents. We will address why these drugs demonstrate unsatisfactory results in a high proportion of liver cancers and the mechanisms of resistance developed by HCC to evade immune response with a focus on the epigeneticrelated mechanisms.     

Immunomodulatory drug CC-5013 overcomes drug resistance and is well tolerated in patients with relapsed multiple myeloma

Thalidomide (Thal) can overcome drug resistance in multiple myeloma (MM) but is associated with somnolence, constipation, and neuropathy. In previous in vitro studies, we have shown that the potent immunomodulatory derivative of thalidomide (IMiD) CC-5013 induces apoptosis or growth arrest even in resistant MM cell lines and patient cells, decreases binding of MM cells to bone marrow stromal cells (BMSCs), inhibits the production in the BM milieu of cytokines (interleukin-6 [IL-6], vascular endothelial growth factor [VEGF], tumor necrosis factor-alpha [TNF-alpha]) mediating growth and survival of MM cells, blocks angiogenesis, and stimulates host anti-MM natural killer (NK) cell immunity. Moreover, CC-5013 also inhibits tumor growth, decreases angiogenesis, and prolongs host survival in a human plasmacytoma mouse model. In the present study, we carried out a phase 1 CC-5013 dose-escalation (5 mg/d, 10 mg/d, 25 mg/d, and 50 mg/d) study in 27 patients (median age 57 years; range, 40-71 years) with relapsed and refractory relapsed MM. They received a median of 3 prior regimens (range, 2-6 regimens), including autologous stem cell transplantation and Thal in 15 and 16 patients, respectively. In 24 evaluable patients, no dose-limiting toxicity (DLT) was observed in patients treated at any dose level within the first 28 days; however, grade 3 myelosuppression developed after day 28 in all 13 patients treated with 50 mg/d CC-5013. In 12 patients, dose reduction to 25 mg/d was well tolerated and therefore considered the maximal tolerated dose (MTD). Importantly, no significant somnolence, constipation, or neuropathy has been seen in any cohort. Best responses of at least 25% reduction in paraprotein occurred in 17 (71%) of 24 patients (90% confidence interval [CI], 52%-85%), including 11 (46%) patients who had received prior Thal. Stable disease (less than 25% reduction in paraprotein) was observed in an additional 2 (8%) patients. Therefore, 17 (71%) of 24 patients (90% CI, 52%-85%) demonstrated benefit from treatment. Our study therefore provides the basis for the evaluation of CC-5013, either alone or in combination, to treat patients with MM at earlier stages of disease.     

CXCL12 and CXCR7 are relevant targets to reverse cell adhesion‐mediated drug resistance in multiple myeloma

Cell adhesion‐mediated drug resistance (CAM‐DR) by the bone marrow (BM) is fundamental to multiple myeloma (MM) propagation and survival. Targeting BM protection to increase the efficacy of current anti‐myeloma treatment has not been extensively pursued. To extend the understanding of CAM‐DR, we hypothesized that the cytotoxic effects of novel anti‐myeloma agents may be abrogated by the presence of BM stroma cells (BMSCs) and restored by addition of the CXCL12 antagonist NOX‐A12 or the CXCR4 inhibitor plerixafor. Following this hypothesis, we evaluated different anti‐myeloma agents alone, with BMSCs and when combined with plerixafor or NOX‐A12. We verified CXCR4, CD49d (also termed ITGA4) and CD44 as essential mediators of BM adhesion on MM cells. Additionally, we show that CXCR7, the second receptor of stromal‐derived‐factor‐1 (CXCL12), is highly expressed in active MM. Co‐culture proved that co‐treatment with plerixafor or NOX‐A12, the latter inhibiting CXCR4 and CXCR7, functionally interfered with MM chemotaxis to the BM. This led to the resensitization of MM cells to the anti‐myeloma agents vorinostat and pomalidomide and both proteasome inhibitors bortezomib and carfilzomib. Within a multicentre phase I/II study, NOX‐A12 was tested in combination with bortezomib‐dexamethasone, underlining the feasibility of NOX‐A12 as an active add‐on agent to antagonize myeloma CAM‐DR.     

Proline promotes proliferation and drug resistance of multiple myeloma by downregulation of proline dehydrogenase

Amino acids in the bone marrow microenvironment (BMME) are a critical factor for multiple myeloma (MM) progression. Here, we have determined that proline is elevated in BMME of MM patients and links to poor prognosis in MM. Moreover, exogenous proline regulates MM cell proliferation and drug resistance. Elevated proline in BMME is due to bone collagen degradation and abnormal expression of the key enzyme of proline catabolism, proline dehydrogenase (PRODH). PRODH is downregulated in MM patients, mainly as a result of promoter hypermethylation with high expression of DNMT3b. Thus, overexpression of PRODH suppresses cell proliferation and drug resistance of MM and exhibits therapeutic potential for treatment of MM. Altogether, we identify proline as a key metabolic regulator of MM, unveil PRODH governing MM progression and provide a promising therapeutic strategy for MM treatment.     

beta-lapachone,a novel plant product,overcomes drug resistance in human multiple myeloma cells

OBJECTIVE: To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). MATERIALS AND METHODS: Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. CONCLUSION: These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.     

Plasma cell karyotype in multiple myeloma

Karyotypic abnormalities were studied in multiple myeloma and were correlated with clinical features. Among 115 evaluable patients, 46% had an abnormal karyotype. Trisomy 3, 5, 9, and 15 and monosomy 13 and 16 were the most common clonal abnormalities. Translocations described previously in other B cell malignancies occurred in nine patients, including four with t(8;14)(q24;q32) translocations. The association of all t(8;14) abnormalities with IgA protein type suggested a pathogenetic relationship between a specific karyotypic abnormality and myeloma protein type. Hypodiploidy occurred mainly in patients with only Bence Jones protein, was associated with resistance to therapy, and justified the early consideration of investigational therapies.     

Honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis

Honokiol (HNK) is an active component purified from magnolia, a plant used in traditional Chinese and Japanese medicine. Here we show that HNK significantly induces cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM. Neither coculture with bone marrow stromal cells nor cytokines (interleukin-6 and insulin-like growth factor-1) protect against HNK-induced cytotoxicity. Although activation of caspases 3, 7, 8, and 9 is triggered by HNK, the pan-caspase inhibitor z-VAD-fmk does not abrogate HNK-induced apoptosis. Importantly, release of an executioner of caspase-independent apoptosis, apoptosis-inducing factor (AIF), from mitochondria is induced by HNK treatment. HNK induces apoptosis in the SU-DHL4 cell line, which has low levels of caspase 3 and 8 associated with resistance to both conventional and novel drugs. These results suggest that HNK induces apoptosis via both caspase-dependent and -independent pathways. Furthermore, HNK enhances MM cell cytotoxicity and apoptosis induced by bortezomib. In addition to its direct cytotoxicity to MM cells, HNK also represses tube formation by endothelial cells, suggesting that HNK inhibits neovascurization in the bone marrow microenvironment. Taken together, our results provide the preclinical rationale for clinical protocols of HNK to improve patient outcome in MM.