Multiple Myeloma Disease Progression Research Articles

IDO Pathway Analysis for Identifying Novel Biomarkers in Multiple Myeloma

Background: Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that catalyzes the initial rate-limiting step in Tryptophan (Trp) degradation along the Kynurenine pathway. Trp can be catabolized by tryptophan hydroxylase into serotonin (Ser) or alternatively, by IDO and tryptophan-2,3-dioxygenase (TDO) into Kynurenine (Kyn). A variety of human tumors have been shown to express an active IDO which leads to the induction of immune tolerance and malignant cell survival (Mellor et al, 1999). Elevated IDO activity within tumor microenvironment impairs the function of antigen specific T cells and suppresses the anti-tumor immune response, while in tumor-draining lymph nodes an active IDO inhibits the antigen presentation reducing the population of antigen-specific T cells and stimulates regulatory T cells (Treg) activation (Curti et al. 2009). IDO overexpression has been correlated with poor prognosis in a number of human solid tumors including ovarian, endometrial and colon carcinoma (Okamoto et al. 2005, Ino et al. 2006, Brandacher et al. 2006). In hematological malignancies, such as AML and DLBCL, IDO expression has also shown significant prognostic value (Folgiero et al. 2014, Ninomiya et al. 2011). Currently, our understanding of the role of IDO in multiple myeloma (MM) is limited to a few existing studies correlating the abundance of selected IDO pathway metabolites with the disease (Bonanno et al. 2012, Zdzisinska et al. 2010). We hypothesize that IDO activity correlates with the disease onset and progression in MM patients.Methods: We quantified the levels of Trp, Kyn, and Ser in human serum by mass spectrometry using the Biocrates AbsoluteIDQ p180 kit. Analysis was performed using MetaboAnalyst 3.0 and Graphpad prism software. The serum samples derived from healthy donors and MM patients were obtained commercially.Results: We found significantly reduced levels of Trp (n=13, p<0.001) and elevated levels of Kyn (n=13, p<0.05) in serum samples from MM patients compared to matching (sex and age paired) healthy donor samples, while Ser levels did not differ significantly. Similarly, samples obtained from Newly Diagnosed (ND) MM patients showed a significant reduction in Trp (n=16, p<0.01) and a trend towards increased Kyn. The overall results in Relapsed/Refractory (R/R) MM patients were similar to the ND setting, although we only detected a trend towards Trp reduction, and observed a significant increase in Kyn levels (n=26, p<0.01) when compared to healthy donors. These results were confirmed when analyzing the data using the widely employed Kyn/Trp ratio, a potential marker for assessing IDO activity. A significantly elevated Kyn/Trp ratio was observed in all of the MM malignant populations (paired: n=13, p<0.001; ND: n=16, p<0.01; R/R: n=26, p<0.01) as compared to healthy individuals.Conclusions: Our study suggests that increased activity of IDO, and/or the production of downstream metabolites is one of the features of MM, and could potentially define a valuable set of novel biomarkers. Here we show a consistent pattern of increased Kyn and decreased Trp levels as well as an elevated Kyn/Trp ratio, all of which are hallmarks of high IDO activity in MM subjects when compared to healthy individuals. We did not detect any significant differences in Ser levels between any of the groups which may suggest that the catabolic pathway is not dependent on tryptophan hydroxylase. Additional studies are being initiated to further investigate the role of IDO/Trp pathway in MM pathogenesis as well as better understand its potential prognostic and predictive impact in currently used MM therapies. DisclosuresNo relevant conflicts of interest to declare.

Copper and zinc in stage I multiple myeloma: relation with ceruloplasmin, lipid peroxidation, and superoxide dismutase activity

Background The main aim of this study was to assess the serum levels of copper (Cu), zinc (Zn) with lipid peroxidation, Cu/Zn superoxide dismutase (Cu/Zn SOD) activity, and ceruloplasmin (Cp) in multiple myeloma (MM) patients. Materials and methods The study was conducted in 34 MM patients at stage I. Serum Cu and Zn levels were measured by atomic absorption spectrometry. Also, spectrophotometric assays of malondialdehyde (MDA) levels in addition to Cp and Cu/Zn SOD were quantitated. Results The results showed a significant decrease in the serum Zn levels in patients with MM (p < 0.0001). Also, serum Cu levels were significantly higher (p < 0.0001). However, the serum Cu/Zn ratio was significantly higher in the cancer patients (p < 0.0001). A significant difference was observed in the patients group compared with the control group according to the Cu/Zn SOD activity (p < 0.0001). Moreover, serum levels of Cp and MDA were significantly increased in patients (p < 0.0001, both). Conclusions The elevated levels of serum Cu and MDA with a decrease in Zn and Cu/Zn SOD might explain the increased oxidative stress in MM disease. As the high Cu level was observed in MM patients, therefore, Cu levels should be concentrated in the pathogenesis and progression of MM disease.

Semaphorin-3A inhibits multiple myeloma progression in a mouse model.

Previous studies revealed that progression of multiple myeloma (MM) is associated with downregulation of semaphorin-3A (sema3A) expression in bone marrow endothelial cells. We therefore determined if serum sema3A concentrations are correlated with MM progression and if sema3A can affect MM progression. We find that the concentration of sema3A in sera of MM patients is strongly reduced and that the decrease is correlated with disease progression. A similar depletion is found in patients having acute myeloid leukemia and acute lymphoblastic leukemia but not in cancer forms that do not involve the bone marrow such as in colon cancer. Expression of a modified sema3A [furin-resistant sema3A (FR-sema3A)] stabilized against cleavage by furin-like proprotein convertases in CAG MM cells did not affect their behavior in-vitro. CAG cells injected into the tail vein of severe combined immunodeficient (SCID) mice home to the bone marrow and proliferate, mimicking MM disease progression. Disease progression in mice injected with CAG cells expressing FR-sema3A was inhibited, resulting in prolonged survival and a lower incidence of bone lesions. Histological examination and fluorescence-activated cell sorting analysis revealed that FR-sema3A expression reduced the infiltration of the CAG cells into the bone marrow, reduced bone marrow necrosis and reduced angiogenesis induced by the MM cells in the bone marrow. Our results suggest that measurement of sema3A serum concentrations may be of use for the diagnosis and for the monitoring of malignancies of the bone marrow such as MM. Furthermore, our results suggest that FR-sema3A may perhaps find use as an inhibitor of MM disease progression.

Abstract 454: The role of autophagy in multiple myeloma disease progression

Abstract Multiple Myeloma (MM) is an incurable hematologic neoplasm characterized by accumulation of malignant plasma cells in the bone marrow (BM). MM cells establish close ties with the BM, however at late stages they utilize as yet unknown mechanisms to acquire independence from the microenvironment and survive in extramedullary/nutrient deprived sites. Autophagy, a process responsible for degradation of proteins and organelles under stress conditions, has been suggested as a mechanism whereby tumor cells can withstand metabolic stress. Using two newly acquired human myeloma cell lines (HMCL), TK1 and TK2, from the medullary and extramedullary sites of a MM patient respectively, we investigated the ability of autophagy to confer stress/glutamine deprivation tolerance. Results HMCLs require exogenous L-Glutamine (Gln) for sustained proliferation in vitro. However, when TK1 and TK2 were grown under Gln deprivation conditions (8mM, 4mM, 2mM, 0mM) we observed striking differences in their growth response. The TK2 (peripheral blood) cell line continued proliferating until day 14 (p=0.0046), whereas proliferation of the TK1 cell line significantly decreased in the absence of Gln (p=0.0028 between 8mM and 0mM). Furthermore, the proliferative advantage of TK2 was abrogated at 2mM and 0mM of Gln by the use of an autophagy inhibitor (chloroquine [CQ] (p=0.000235 and p=0.000069 respectively), implicating this process in Gln deprivation tolerance of TK2. Additionally, Gln concentration was inversely correlated with TK1 survival, but not TK2, as measured by annexin-V/PI staining. Cell death in TK1 increased at 24h, from 36.022% at Gln 8mM to 56.2% at Gln 0mM, irrespective of autophagy inhibition. In TK2 cells, Gln deprivation did not cause a significant increase in cell death, whereas inhibition of autophagy increased cell death by two-fold. Accordingly, apoptosis (as measured by PARP cleavage) was induced by the addition of CQ only in TK2 under Gln deprivation. Resistance of TK2 cells to metabolic stress also correlated with up-regulation of the autophagic pathway upon CQ exposure, as demonstrated by a higher LC3BII/I turnover and the presence of autophagic vacuoles in TK2 (but not TK1) cells. In addition to upregulation of autophagy, Gln deprivation also induced the expression of glutamine synthetase in TK2 but not TK1 cells. Chemical inhibition of this enzyme by methionine-sulfoximine (5mM) under Gln deprivation conditions was able to induce significant cell death in TK2 and not TK1 (32.57% and 2.53% increase respectively). These data demonstrate that Gln synthesis is an additional mechanism whereby TK2 cells tolerate Gln starvation. In conclusion we have shown that MM survival outside the BM can be attributed to the ability of cells to overcome “glutamine addiction” due to up-regulation of autophagy and ability to synthesise Gln. Thus, autophagy inhibition is an attractive alternative approach to target late-stage, Gln independent, metastatic cells. Citation Format: Ioanna Savvidou, Sahan Chandrasekara, Tiffany Khong, Andrew Spencer. The role of autophagy in multiple myeloma disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 454.

Multiple Myeloma Treatment in Real-world Clinical Practice: Results of a Prospective, Multinational, Noninterventional Study

BackgroundThe present prospective, multinational, noninterventional study aimed to document and describe real-world treatment regimens and disease progression in multiple myeloma (MM) patients. Patients and MethodsAdult patients initiating any new MM therapy from October 2010 to October 2012 were eligible. A multistage patient/site recruitment model was applied to minimize the selection bias; enrollment was stratified by country, region, and practice type. The patient medical and disease features, treatment history, and remission status were recorded at baseline, and prospective data on treatment, efficacy, and safety were collected electronically every 3 months. ResultsA total of 2358 patients were enrolled. Of these patients, 775 and 1583 did and did not undergo stem cell transplantation (SCT) at any time during treatment, respectively. Of the patients in the SCT and non-SCT groups, 49%, 21%, 14%, and 15% and 57%, 20%, 12% and 10% were enrolled at treatment line 1, 2, 3, and ≥ 4, respectively. In the SCT and non-SCT groups, 45% and 54% of the patients had received bortezomib-based therapy without thalidomide/lenalidomide, 12% and 18% had received thalidomide/lenalidomide-based therapy without bortezomib, and 30% and 4% had received bortezomib plus thalidomide/lenalidomide-based therapy as frontline treatment, respectively. The corresponding proportions of SCT and non-SCT patients in lines 2, 3, and ≥ 4 were 45% and 37%, 30% and 37%, and 12% and 3%, 33% and 27%, 35% and 32%, and 8% and 2%, and 27% and 27%, 27% and 23%, and 6% and 4%, respectively. In the SCT and non-SCT patients, the overall response rate was 86% to 97% and 64% to 85% in line 1, 74% to 78% and 59% to 68% in line 2, 55% to 83% and 48% to 60% in line 3, and 49% to 65% and 36% and 45% in line 4, respectively, for regimens that included bortezomib and/or thalidomide/lenalidomide. ConclusionThe results of our prospective study have revealed great diversity in the treatment regimens used to manage MM in real-life practice. This diversity was linked to factors such as novel agent accessibility and evolving treatment recommendations. Our results provide insight into associated clinical benefits.

Multiple myeloma: update on pathophysiology and management

Proteasome inhibitors and immunomodulatory drugs have substantially improved the clinical outcomes in patients with multiple myeloma (MM) since 2000. In 2015, the new monoclonal antibodies, daratumumab and elotuzumab, were approved for treating relapsed and/or refractory MM (RRMM). Furthermore, venetoclax, a selective BCL-2 inhibitor, and chimeric antigen receptor (CAR) T-cell therapy that work against B-cell maturation antigen (BCMA) have reportedly shown great efficacy in phase 1 studies. The efficacy of venetoclax has been observed in RRMM with t (11;14) and higher BCL-2/BCL-XL expression. BCMA CAR-T therapies have caused considerable remissions in highly refractory MM. These suggest that personalized medicine and therapy aiming at cure are becoming reality in the near future. MM is a genetically complex and heterogeneous disease that develops via a multistep transformation process. Recent next-generation sequencing (NGS) studies have revealed the molecular landscape, providing insights into the biology, including the intraclonal heterogeneity and disease progression in MM. In this review, we discuss the current knowledge regarding MM genomics reported by NGS studies as well as the recent progress in MM therapy. The agents and treatment reviewed here include elotuzumab, daratumumab, ixazomib, venetoclax, and BCMA CAR-T therapies.

Abstract A176: Quantitative parabolic optimization platform for the design of epigenetic-based drug combinations against bortezomib-resistant multiple myeloma

Abstract Proteasome inhibitor-based drug combinations have improved multiple myeloma median survival beyond 5 years. Multiple myeloma, however, remains incurable as patients inevitably relapse and become resistant to proteasome inhibitors, such as bortezomib. Thus, there is a need to identify improved drug combinations that may serve as later lines of treatment against bortezomib-resistant multiple myeloma. Epigenetic inhibitors have been explored in treating advanced multiple myeloma; however, clinical trials have shown mixed results. There is increasing evidence from these clinical trials that epigenetic-based therapies may see greater benefit as part of drug combinations rather than as monotherapies. Rationally identifying optimal epigenetic-based drug combinations against aggressive cancers, such as bortezomib-resistant multiple myeloma, however, remains a challenge. Utilizing a quantitative parabolic optimization platform (QPOP), optimal drug combinations against bortezomib-resistant multiple myeloma were identified. QPOP analysis uses quantifiable phenotypic outputs to identify significant effects amongst a set of drugs using a minimal number of experiments through parabolic response surface mapping. By comparing therapeutic response of bortezomib-resistant multiple myeloma cells to bortezomib-sensitive multiple myeloma cells as well as normal control cells, a series of drug combinations were identified from an initial pool of 129 FDA approved oncology drugs. QPOP does not rely on molecular mechanism modeling but rather uses experimental data to rationally identify optimal drug combinations. As a result, targetable molecular mechanisms of cancer progression can be identified without any previous assumptions or bias. Thus, QPOP revealed that drug resistance in multiple myeloma may involve specific epigenetic changes as identified by top-ranked epigenetic inhibitor containing drug combinations. While it is known that DNA hypermethylation accumulates during multiple myeloma progression, reversal of DNA hypermethylation by DNA methyltransferase inhibitors as monotherapies had no significant therapeutic effect in vitro or in vivo. DNA hypermethylation can be targeted, however, with the appropriate drug combinations to effectively treat in vitro and in vivo models of bortezomib-resistant multiple myeloma, as well as ex vivo primary multiple myeloma cells from a wide range of patients. QPOP analysis also revealed that optimal drug combinations for other classes of epigenetic inhibitors, such as HDAC inhibitors, change from naïve multiple myeloma to drug-resistant multiple myeloma. Thus, QPOP-based drug combination optimization not only revealed effective epigenetic-based drug combinations but also the molecular mechanisms by which epigenetic modifiers affect multiple myeloma disease progression. This work highlights the need for rational identification of different epigenetic-based drug combinations throughout the progression of multiple myeloma. Beyond multiple myeloma, this platform can be used to identify optimal drug combinations for other cancers as well as provide insight into therapeutically targetable molecular mechanisms that drive cancer progression in various cancer subtypes. Citation Format: Edward Kai-Hua Chow. Quantitative parabolic optimization platform for the design of epigenetic-based drug combinations against bortezomib-resistant multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A176.

Hypoxia promotes IL-32 expression in myeloma cells, and high expression is associated with poor survival and bone loss.

Multiple myeloma (MM) is a hematologic cancer characterized by expansion of malignant plasma cells in the bone marrow. Most patients develop an osteolytic bone disease, largely caused by increased osteoclastogenesis. The myeloma bone marrow is hypoxic, and hypoxia may contribute to MM disease progression, including bone loss. Here we identified interleukin-32 (IL-32) as a novel inflammatory cytokine expressed by a subset of primary MM cells and MM cell lines. We found that high IL-32 gene expression in plasma cells correlated with inferior survival in MM and that IL-32 gene expression was higher in patients with bone disease compared with those without. IL-32 was secreted from MM cells in extracellular vesicles (EVs), and those EVs, as well as recombinant human IL-32, promoted osteoclast differentiation both in vitro and in vivo. The osteoclast-promoting activity of the EVs was IL-32 dependent. Hypoxia increased plasma-cell IL-32 messenger RNA and protein levels in a hypoxia-inducible factor 1α-dependent manner, and high expression of IL-32 was associated with a hypoxic signature in patient samples, suggesting that hypoxia may promote expression of IL-32 in MM cells. Taken together, our results indicate that targeting IL-32 might be beneficial in the treatment of MM bone disease in a subset of patients.

Alteration of mitochondrial biogenesis promotes disease progression in multiple myeloma.

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.

Monoclonal IgG in MGUS and multiple myeloma targets infectious pathogens.

Subsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1-specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher β2-microglobulin and inflammation/infection-linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients.

Cutting edge genomics reveal new insights into tumour development, disease progression and therapeutic impacts in multiple myeloma.

Multiple Myeloma (MM) is a haematological malignancy characterised by the clonal expansion of plasma cells (PCs) within the bone marrow. Despite advances in therapy, MM remains a largely incurable disease with a median survival of 6years. In almost all cases, the development of MM is preceded by the benign PC condition Monoclonal Gammopathy of Undetermined Significance (MGUS). Recent studies show that the transformation of MGUS to MM is associated with complex genetic changes. Understanding how these changes contribute to evolution will present targets for clinical intervention. We discuss three models of MM evolution; the linear, the expansionist and the intraclonal heterogeneity models. Of particular interest is the intraclonal heterogeneity model. Here, distinct populations of MM PCs carry differing combinations of genetic mutations. Acquisition of additional mutations can contribute to subclonal lineages where "driver" mutations may influence selective pressure and dominance, and "passenger" mutations are neutral in their effects. Furthermore, studies show that clinical intervention introduces additional selective pressure on tumour cells and can influence subclone survival, leading to therapy resistance. This review discusses how Next Generation Sequencing approaches are revealing critical insights into the genetics of MM development, disease progression and treatment. MM disease progression will illuminate possible mechanisms underlying the tumour.

Serum Soluble SLAMF7 is Correlated With Disease Progression in Multiple Myeloma and May Affect Anti-SLAMF7 Antibody Therapy

Serum Soluble SLAMF7 is Correlated With Disease Progression in Multiple Myeloma and May Affect Anti-SLAMF7 Antibody Therapy

Development of a New BCMAxCD3 Duobody® Antibody for Multiple Myeloma

B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predominantly expressed on terminally differentiated B-cells. BCMA signals through P38/NF-κB pathway upon binding to its ligands; a proliferation inducing ligand (APRIL) and B-cell activator of the TNF family (BAFF) and promote anti-apoptotic gene expression. BCMA expression is elevated in plasma blasts, plasma cells from spleen and bone marrow and correlates with disease progression in multiple myeloma (MM). BCMA expression in premalignant MM settings such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) also gives an opportunity for early cancer interception. To target cancer cells expressing BCMA, we developed a BCMAxCD3 bispecific antibody using the Genmab DuoBody® technology (Ab-957) to recruit T cells to BCMA-expressing MM cells so that T cells could be activated and induced to kill BCMA+ cancer cells. This antibody can induce cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells: EC50=0.15nM, MM1.R cells: EC50=0.06nM, RPMI8226 cells: EC50=0.45nM) with a concomitant T cell activation (H929 cells: EC50=0.21nM, MM1.R cells: EC50=0.1nM, RPMI8226 cells: EC50=0.28nM). In contrast, this antibody was unable to kill BCMA- cancer cell line (MV4-11), demonstrating the specificity of the cytotoxicity. Ab-957 also inhibited tumor development or growth in two BCMA+ MM murine xenograft models inoculated with human T cells. Furthermore, this antibody could deplete BCMA+ cells in bone marrow samples from MM patient’s in an ex-vivo assay with an average EC50 value of 2.5 nM. Lastly, Ab-957 is well-tolerated in cynomolgus monkey and is being developed for Phase I clinical trial in patients with multiple myeloma. DisclosuresPillarisetti:Janssen: Employment. Baldwin:Janssen: Employment. Babich:Janssen: Employment. Majewski:Janssen: Employment. Barone:Janssen: Employment. Li:Janssen: Employment. Zhang:Janssen: Employment. Chin:Janssen: Employment. Luistro:Janssen: Employment. Mendonça:Janssen: Employment. Nanjunda:Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Bellew:Janssen: Employment. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.

The Pathophysiologic Role of JunB in Multiple Myeloma Pathogenesis: Focus on Bone Marrow Angiogenesis

Angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and is correlated with adverse prognosis. The increase in vascular density within the bone marrow (BM) microenvironment is triggered by oncogene-mediated expression and secretion of pro-angiogenic growth factors and cytokines, most prominently including VEGF. Members of the AP-1 family of transcription factors (TFs) have emerged as actively pursued therapeutic targets over the past years. Our previous studies demonstrated a critical role for the AP-1 family member JunB in MM cell proliferation, survival and drug resistance. Whether JunB also contributes to MM BM angiogenesis is currently unknown. We first sought to identify correlative expression patterns of JUNB and angiogenic factors using the Oncomine software. Indeed, similar to JUNB our data identified significant induction of VEGF, VEGFB, IGF-1 and PlGF, progressing from normal plasma cells to cells from patients with monoclonal gammopathy of undetermined significance (MGUS) and MM in two independent gene expression profiling data sets. In contrast, no correlation was observed between expression of JUNB and ANGPT1, ANGPT2, SDF-1 and FGF4. Besides BM stromal cells (BMSCs) also osteoblasts (OBs) upregulate JunB protein levels in MM: stromal cell co-cultures. This effect is, at least in part, mediated by the humoral milieu, IL-6 in particular. Whether BMSC- and OB- mediated production and secretion of angiogenic factors is mediated via JunB in MM cells was investigated next. Our data show that doxycyclin- induced inhibition of BMSC: OB- mediated JunB upregulation in TetR-shJUNB/ MM.1S cells abrogated production and secretion of VEGF, VEGFB, IGF-1 and PlGF as evidenced by qPCR and ELISA assay. Similar effects were observed in MM: stromal cell co-cultures using the IL-6R inhibitor tocilizumab. Consequently, supernatant of both doxycycline- treated BMSC: Tet-shJUNB/ MM.1S and OB: Tet-shJUNB/ MM.1S co-cultures as well as tocilizumab significantly inhibited angiogenesis, as evidenced by matrigel- based tube formation as well as wound healing assays. Conversely, tamoxifen- induced JunB activity in JunB-ER/MM cells triggered the expression and secretion of angiogenic factors and angiogenesis. Importantly, the functional role of JunB on BM angiogenesis was also verified in vivo using a MM xenograft mouse model. To this purpose, immunodeficient NSG mice were inoculated subcutaneously with Tet-SCR/ MM.1S or Tet-shJUNB/ MM.1S together with BMSCs into the left and right flanks of mice, respectively, and fed with doxycycline in their drinking water for 5 weeks. Treatment with doxycycline inhibited JunB protein levels in Tet-shJUNB/ MM.1S, but not in Tet-SCR/ MM.1S and induced a significant reduction in growth and angiogenesis in tumors formed by Tet-shJUNB/ MM.1S versus control cells, as evidenced by Ki-67 and anti-CD31 staining. In summary, our findings demonstrate for the first time a role for JunB in MM bone marrow angiogenesis, thereby strongly supporting that this TF is a promising new therapeutic target in MM. DisclosuresHose:Takeda: Other: Travel grant; Sanofi: Research Funding; EngMab: Research Funding. Goldschmidt:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Podar:Novartis: Research Funding; Eutropics: Research Funding.

The Abnormal Repression of Mir-375 Expression By Overlapping Epigenetic Dysregulations in Myelomas

[Introduction] Multiple myeloma (MM) remains mostly incurable despite major advances in treatment strategies. The complex interplay among various cell-intrinsic and -extrinsic molecular mechanisms confers inter-patients diversity and intraclonal heterogeneity in MM. However, as the universal and relevant therapeutic target molecule against MM, we have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and its major downstream substrate RSK2 were universally active in MM (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014), while we also identified that the repression of miR-375 was found to be the universal underlying mechanism for the overexpression/activation of PDPK1 in MM (Tatekawa S, ASH meeting 2015). In this study, we further extended our study to assess the clinical relevance of miR-375 repression and the molecular mechanisms for the miR-375 repression in MM for the future clinical translation of miR-375/PDPK1/RSK2 signaling axis in the diagnosis and treatment development for MM.[Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 113 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells; N=10, MGUS; N=30, newly diagnosed MM (NDMM); N=34, relapsed/refractory (RRMM); N=39). The level of miR-375 expression was calculated with 2-ΔΔCt methods. Human U6 snRNA was examined as the reference and cDNA of RPMI8226 was used as a calibrator sample. The methylation status of miR-375 upstream regions including promoter site were analyzed by methylation-specific PCR (MSP) and bisulfite sequence. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine the histone modification status in miR-375 upstream regions. The circulating plasma RNA samples were converted to cDNA libraries followed by sequencing using the multiplex small RNA library primer set. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent.[Result] When compared to normal plasma cells, the miR-375 expression was significantly decreased in CD138 positive plasma cells from MGUS (p<0.05), NDMM and RRMM (both p<0.01), and tended to be decreased in HMCL cells (p=0.085). As the causative of miR-375 repression, we focused upon epigenetic deregulation for miR-375 gene. The bisulfite sequence analyses disclosed that CGI-1 and CGI-2, which were CpG-rich regions within 3Kbp from pre-miR-375 gene, were hypermethylated in all three patient-derived myeloma cells of each stage patients examined (MGUS, NDMM and RRMM) and both two HMCLs examined. The abnormal hypermethylation status of CGI-1 and CGI-2 in MM were further conformed with larger number of genomic DNA samples by MSP, i.e., the CGI-1 region and promoter sites (CGI-2) of miR-375 gene were hypermethylated in all 8 HMCLs examined and in 58 patient-derived myeloma cells. Furthermore, the treatment with Trichostatin A, a histone deacetylase inhibitor, upregulated acetylated histones in several fragments including the promoter site of pre-miR375 in HMCLs. Finally, the treatment with SGI-110, a hypomethylating agent, and/or Trichostatin A, markedly increase miR-375 expression in HMCLs, suggesting that the overlapping epigenetic deregulations, such as abnormal DNA hypermethylation or histone deacetylation, are involved in the silencing of miR-375 in MM. In contrast, the expression levels of circulating plasma miR-375 were not significantly different between myeloma patients and healthy donors, indicating that the abnormally repressed miR-375 is the event specific to myeloma cells, but not in normal tissues in MM patients.[Conclusion and Discussion] This study revealed that the expression level of miR-375 decreased specifically in clonal plasma cells from MGUS to RRMM patients, and the repression of miR-375 was caused by the overlapping epigenetic dysregulations. The abnormal repression of miR-375 which is the major causative for the constitutive hyperactivation of the PDPK1/RSK2 signaling axis in MM is likely to be involved in the myelomagenesis in MGUS as well as the disease progression in MM, and the epigenetic deregulations may be a novel therapeutic target for MM. DisclosuresIida:Celgene: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding. Kuroda:Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Astra Zeneca: Research Funding; Janssen: Honoraria.

Characterization of an In Vitro model of MGUS Progression to Multiple Myeloma

Introduction: Multiple Myeloma (MM) is an incurable cancer characterized by a pre-malignant clonal phase of disease called monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS do not develop overt MM and the biology underlying this potential transformation is unclear. Investigations to prevent the development of MM from MGUS are limited by the relative infrequency of MGUS progression. Unfortunately, MGUS cells have historically proven difficult to grow in vitro because of slow rates of proliferation and difficulty in sustaining cell cultures. We present evidence of an in vitro model that generates MM-like plasma cells from patients diagnosed with only MGUS. We further present gene expression patterns of primary patient cells versus the induced MM cells to provide guidance as to important initiating events within our model.Methods: We collected a CD38+ cell fraction and a mononuclear (CD38-) fraction from 4 patients with MGUS using a Miltenyl Biotec column Separator. The CD38- fraction was grown in RPMI with 10% FBS and 1% sodium pyruvate with or without a polyglycolic acid/ poly L-lactic acid 90/10 (PLGA) copolymer scaffold to create 3D culture conditions. The mononuclear layer from healthy donors and the MM cell line, U266, were grown as controls. We then analyzed the initial CD38+ fraction, the initial CD38- fraction, and the CD38- fraction grown in media or 3D co-culture by flow cytometry for expression of kappa, lambda, CD38, CD138, CD45, CD19, and CD56. Gene expression analysis was performed using RNA-sequencing data from the CD38+, CD38-, cultured CD38-, and control cells. Expression of the top 100 ranked differentially expressed genes, which demonstrated the largest variation, were further analyzed using the nCounter® Analysis System (NanoString Technologies).Results: The CD38- fraction from MGUS patients grew into an adherent layer of elongated cells, consistent with bone marrow stromal cells. After several months, the stromal cells were noted to change shape and new, round cells were observed budding off from the stromal layer. Over time, the stromal layer disappeared and the round plasmacytoid cells remained. Characterization of these round cells revealed them to be plasma cells by IHC and flow cytometry. When comparing these in vitro generated cells to the initial CD38+ fraction removed from patients, the new cells showed the re-emergence of CD38 and CD138, increased expression of CD56 and CD19, and decreased expression of CD45. Gene expression analysis revealed 3 distinct populations of cells. The initial CD38- fraction separated with the healthy mononuclear layer. The initial CD38+ fraction clustered independently while the grown plasma cells clustered with the U266 cells. Analysis of the differential gene expression patterns revealed differences in the expression of immunoglobulin genes, as well as alterations in expression of extracellular matrix and cell adhesion markers including PAI-1, MMP2, COL1A2, and GREM1; and alterations in expression of mitochondrial genes.Conclusion: To our knowledge, this is the first in vitro simulation of disease progression from MGUS to MM. Our model induced the growth of plasma cells with an aggressive phenotype as assayed by flow cytometry. The gene expression profile further demonstrates gene expression patterns from our induced plasma cells consistent with MM versus MGUS. The alterations in extracellular matrix proteins as seen in our induced plasma cells are consistent with an epithelial to mesenchymal type transition implicated in disease progression, metastasis, and bone lesions. Additionally, the alterations in mitochondrial gene expression have been implicated in early disease progression in colon cancer and MM. These findings provide further evidence that our model simulates disease transformation and the expression data suggest possible pathways that may be important in myeloma disease progression that can be further evaluated in vivo. DisclosuresNo relevant conflicts of interest to declare.

The Pathophysiologic Role of JunB in Multiple Myeloma Pathogenesis: Focus on Angiogenesis

Angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and is correlated with adverse prognosis. The increase in vascular density is triggered by oncogene-mediated expression of pro-angiogenic growth factors and cytokines e.g. VEGF. A critical role of JunB, a member of the AP-1 family of transcription factors, has been proposed in VEGF regulation and tumor angiogenesis. This study aims to clarify the pathophysiologic role of JunB in multiple myeloma angiogenesis.

Signaling Interplay between Bone Marrow Adipose Tissue and Multiple Myeloma cells.

In the year 2000, Hanahan and Weinberg (1) defined the six Hallmarks of Cancer as: self-sufficiency in growth signals, evasion of apoptosis, insensitivity to antigrowth mechanisms, tissue invasion and metastasis, limitless replicative potential, and sustained angiogenesis. Eleven years later, two new Hallmarks were added to the list (avoiding immune destruction and reprograming energy metabolism) and two new tumor characteristics (tumor-promoting inflammation and genome instability and mutation) (2). In multiple myeloma (MM), a destructive cancer of the plasma cell that grows predominantly in the bone marrow (BM), it is clear that all these hallmarks and characteristics are in play, contributing to tumor initiation, drug resistance, disease progression, and relapse. Bone marrow adipose tissue (BMAT) is a newly recognized contributor to MM oncogenesis and disease progression, potentially affecting MM cell metabolism, immune action, inflammation, and influences on angiogenesis. In this review, we discuss the confirmed and hypothetical contributions of BMAT to MM development and disease progression. BMAT has been understudied due to technical challenges and a previous lack of appreciation for the endocrine function of this tissue. In this review, we define the dynamic, responsive, metabolically active BM adipocyte. We then describe how BMAT influences MM in terms of: lipids/metabolism, hypoxia/angiogenesis, paracrine or endocrine signaling, and bone disease. We then discuss the connection between BMAT and systemic inflammation and potential treatments to inhibit the feedback loops between BM adipocytes and MM cells that support MM progression. We aim for researchers to use this review to guide and help prioritize their experiments to develop better treatments or a cure for cancers, such as MM, that associate with and may depend on BMAT.

Bortezomib-induced pro-inflammatory macrophages as a potential factor limiting anti-tumour efficacy.

Multiple myeloma (MM) is a chronic progressive malignancy of plasma cells. Although treatment with the novel proteasome inhibitor, bortezomib, significantly improves patient survival, some patients fail to respond due to the development of de novo resistance. We have previously shown that cytotoxic drugs can induce pro-tumorigenic host-mediated effects which contribute to tumour re-growth and metastasis, and thus limit anti-tumour efficacy. However, such effects and their impact on tumour cell aggressiveness have not been investigated using cytostatic agents such as bortezomib. Here we show that plasma from bortezomib-treated mice significantly increases migration, viability and proliferation of MM cells in vitro, compared to plasma from vehicle treated mice. In vivo, bortezomib induces the mobilization of pro-angiogenic bone marrow cells. Furthermore, mice treated with bortezomib and subsequently were used as recipients for an injection of MM cells succumb to MM earlier than mice treated with the vehicle. We show that bortezomib promotes pro-inflammatory macrophages which account for MM cell aggressiveness, an effect which is partially mediated by interleukin-16. Accordingly, co-inoculation of MM cells with pro-inflammatory macrophages from bortezomib-treated mice accelerates MM disease progression. Taken together, our results suggest that, in addition to the known effective anti-tumour activity of bortezomib, host-driven pro-tumorigenic effects generated in response to treatment can promote MM aggressiveness, and thus may contribute to the overall limited efficacy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Autocrine and Paracrine Regulatory Mechanisms of Growth Arrest-Specific Gene 6 Contribute to Disease Progression of Multiple Myeloma

Abstract Multiple myeloma (MM) is a lethal hematologic neoplasm characterized by proliferation of malignant plasma cells in part due to abnormal surrounding microenvironment, but the mechanism of disease progression remains largely unknown in MM. Thus, we screened molecules differentially expressed in bone marrow (BM) cells of MM patients using microarray analysis, which revealed significant upregulation of Gas6 (growth arrest-specific gene 6), a vitamin K-dependent protein with a structural homology with protein S. Since upregulation of Gas6 and its tyrosine kinase receptors have been reported to associate with various malignancies, we hypothesized that Gas6 contributed to the pathogenesis of MM. Gas6 was overexpressed in the BM of MM patients and in various MM cell lines. We found that the serum levels of Gas6 protein were markedly increased in 18 symptomatic MM patients compared to 16 healthy volunteers using human Gas6 ELISA. High levels of soluble Gas6 were detected in the supernatants of in vitro cultured MM cell lines RPMI-8226 and AMO-1 using Gas6 ELIZA. Analysis of apoptosis by flow-cytometry (FCM) with annexin V showed that exogenous Gas6 (100-200 ng/mL) inhibited the apoptosis of MM cells. MTT cell proliferation assay showed that exogenous Gas6 induced cell proliferation of MM cells with increased extracellular signal regulated kinase (ERK) phosphorylation. These results suggest that the presence of an autocrine loop of Gas6 and its receptor with ERK signaling enhanced the proliferative and anti-apoptotic effects on MM cells. Bone marrow stromal cell (BMSC) line HS-5 was found to secrete high levels of soluble Gas6 into culture supernatants using Gas6 ELIZA. Reduction of MM cell apoptosis by HS-5 cell-conditioned medium (CM) was suppressed by Gas6 neutralizing antibody (20 microg/mL) using FCM with annexin V. HS-5 cell-CM induced a marked increase in cell proliferation of MM cells, which was completely inhibited by Gas6 neutralizing antibody (0.2-20 microg/mL) using MTT cell proliferation assay. Our results showed a critical role of Gas6 in the pathology of apoptosis inhibition and cell proliferation of MM cells through paracrine mechanisms. Interleukin (IL)-6 is identified as a major growth and anti-apoptotic factor of MM cells through autocrine mechanisms. Conversely, BMSCs provide efficient support for MM cell survival by paracrine IL-6 stimulation. As determined by western blotting and real-time PCR, HS-5 cell-CM induced Gas6 upregulation, which was suppressed by IL-6 neutralizing antibody (1-10 microg/mL in a dose-dependent manner in MM cells. Meanwhile, HS-5 cell-CM induced the increased IL-6 expression, which was reversed by Gas6 neutralizing antibody in MM cells. Our findings indicate that Gas6 possessed both autocrine and paracrine functions similar to IL-6 between MM cells and BMSCs. Intercellular cell adhesion molecule-1 (ICAM-1) enhanced adhesion of MM cells to BMSCs and subsequently resulted in MM disease progression. Our immnoblotts showed that HS-5 cell-CM induced ICAM-1upregulation, and the ICAM-1 upregulation was reversed by Gas6 neutralization antibody, indicating that Gas6 stimulation increased ICAM-1 synthesis and subsequently accelerated the proliferation of MM cells. The present study offers new insights into autocrine and paracrine actions of Gas6 in concert with IL-6 between MM cells and BMSCs on apoptosis inhibition and cell proliferation of MM cells. We suggest that Gas6-related signaling pathways may be an attractive therapeutic target for the treatment of MM. Disclosures No relevant conflicts of interest to declare.