MM Pathogenesis Research Articles

MST1 downregulates TAZ tumor suppressor protein in multiple myeloma and is a potential therapeutic target

We have previously reported that TAZ functions as a tumor suppressor in multiple myeloma. MST1 is a serine-threonine kinase upstream of the Hippo-signaling pathway that functions as a tumor suppressor in many non-haematological malignancies. However, its role in hematological malignancies, including MM is still poorly understood. In this paper, we provide evidence that MST1 expression is higher in MM, and negatively correlates with TAZ expression in both cell lines and patient samples. High MST1 expression was associated with poor clinical outcomes. Genetic or pharmacological inhibition of MST1 leads to increased TAZ expression and cell death. Importantly, MST1 inhibitors sensitizes myeloma cells to frontline antimyeloma agent-lenalidomide and dexamethasone. Taken together, our data reveals a key role for MST1 in MM pathogenesis and provide evidence to explore the therapeutic potential of using MST inhibitors to upregulate TAZ expression in MM to promote response to anticancer agents.

Identification of Metabolic Enzymes ENO1 and KMO As Novel Immunotherapeutic Targets in Myeloma

Introduction. Our previous studies showed an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs are present in increased numbers in MM BM, promote tumor growth and survival, protect tumor from therapy-induced cytotoxicity; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323) . Aberrant pDC function is evidenced from their interactions with MM tumor and T/NK effector cells in the BM milieu (Ray et al, Leukemia 2015, 29:1441-1444) . Direct interruption of pDC-MM and pDC-T cells interactions and their sequelae utilizing novel targeted agents is necessary to further enhance both anti-tumor immunity and cytotoxicity. Based on DNA microarray analysis, we identified several novel targets on MM cells that are upregulated after coculture with pDCs, and focused on metabolic pathway enzymes that are upregulated both in pDC and MM cells. We identified alpha-Enolase (ENO1) and Kynurenine 3-monooxygenase (KMO) as the two most promising therapeutic targets, based on their roles in normal and cancer metabolism. ENO1 is a glycolytic enzyme, and its surface localization is a unique feature of the tumor cells. Moreover, ENO1 promotes tumor metastasis. KMO is one of the two rate-limiting enzymes involved in eukaryotic tryptophan catabolic kynurenine pathway; and importantly, its overexpression has been reported to inhibit functional immune responses in cancer. Here, we utilized our pDC/MM or pDC/T cells co-culture model to show that targeting ENO1 or KMO generates MM-specific cytotoxic T lymphocyte activity.MethodsDNA MicroArray Analysis: MM.1S cells were cocultured with pDCs for 72h, separated using CD138 Microbeads, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM.1S cells cultured in the presence vs absence of pDCs were compared, and heat map was generated (>1.5-fold change in transcript was considered significant, CI > 95%). pDC-induced overexpression of target proteins : MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in target protein expression. CD138+ tumor cells were gated out, and KMO-FITC or ENO1-AlexaFluor488-stained cells were analyzed. C TL activity assays : MM patient BM CD8+ T cells were cocultured with autologous pDCs at 1:10 (pDC:T) ratio in the presence or absence of KMO inhibitor Ro 61-8048 (10 nM) or ENO1 inhibitor ENOblock (0.1-0.2 µM) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T:MM), followed by quantification of viable MM cells by FACS. Statistical parameters were calculated using GraphPad Prism. Pharmacological inhibitors were obtained from Selleck Chemicals.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of both ENO1 and KMO transcripts in MM cells (1.772 and 2.153-fold, respectively, vs MM.1S alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high KMO and surface ENO1 expression. Coculture of MM cells with pDCs further increased ENO1 surface expression levels in MM cells (5-6-fold increase; n=3; p = 0.003), as well as the ENO1-positive MM cell population (3-4-fold versus MM alone; mean ± SD; n=3; p = 0.005). 3) Like ENO1, pDCs also increased both KMO expression on MM cells and KMO-positive MM cells (MFI: 2-3-fold in the presence vs absence of pDCs; p = 0.006). 4) Functional studies showed that treatment of pDC-T cell cocultures with ENO1 antagonists triggers a robust MM-specific CD8+ CTL activity against autologous CD138-positive MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2-fold vs control; p = 0.008). Finally, 6) KMO inhibitor Ro 61-8048 activates patient BM-CD8+T cells in autologous pDCs/T cell cocultures, and triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0-fold vs control; p = 0.009).Conclusions Our study shows that pDC-MM interactions upregulate tumor promoting and immunosuppressive metabolic enzymes in MM. Blockade of ENO1 or KMO enzymes tiggers MM-specific CD8-positive CTL activity. These data therefore identify novel immune-based therapeutic strategies targeting ENO1 and KMO to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

The Role of lncRNAs in the Pathobiology and Clinical Behavior of Multiple Myeloma.

Simple SummaryMultiple myeloma (MM), the second most common hematological neoplasm, is still considered an incurable disease. Long non-coding RNAs (lncRNAs), genes that do not encode proteins, participate in numerous biological processes, but their deregulation, like that of coding genes, can contribute to carcinogenesis. Increasing evidence points to the relevant role of lncRNAs in the development of human tumors, such that they emerge as attractive biomarkers and therapeutic targets for cancer treatment, including MM. Here we review the oncogenic or tumor-suppressor functions of lncRNAs in MM and provide an overview of novel therapeutic approaches based on lncRNAs that will help to improve the management of these patients.MM is a hematological neoplasm that is still considered an incurable disease. Besides established genetic alterations, recent studies have shown that MM pathogenesis is also characterized by epigenetic aberrations, such as the gain of de novo active chromatin marks in promoter and enhancer regions and extensive DNA hypomethylation of intergenic regions, highlighting the relevance of these non-coding genomic regions. A recent study described how long non-coding RNAs (lncRNAs) correspond to 82% of the MM transcriptome and an increasing number of studies have demonstrated the importance of deregulation of lncRNAs in MM. In this review we focus on the deregulated lncRNAs in MM, including their biological or functional mechanisms, their role as biomarkers to improve the prognosis and monitoring of MM patients, and their participation in drug resistance. Furthermore, we also discuss the evidence supporting the role of lncRNAs as therapeutic targets through different novel RNA-based strategies.

Is absence of CD56 a predictive factor for peripheral blood stem cell mobilization failure in patients with multiple myeloma?

CD56 is believed to play a major role in MM pathogenesis with a 70% to 80% expression rate in malignant plasma cells at the time of diagnosis. Our objective in this study was to investigate the relationship between the characteristics of CD56 expression in bone marrow aspiration material at the time of diagnosis and the success of stem cell mobilization in patients diagnosed with MM. This monocenter study included 94 patients who were diagnosed with MM and had a stem cell mobilization procedure for autologous hematopoietic stem cell transplantation. The primary endpoint of the study was to compare the mobilization success between the groups with and without CD56 expression. The secondary endpoint was to identify other factors affecting mobilization failure outside CD56. At the time of diagnosis, 49 (52.1%) patients had CD56 expression and 45 (47.9%) did not. Mobilization failed in 11 (11.7%) patients. Age, gender, ISS stage and the number of premobilization treatment regimens were not found predictive of mobilization failure. CD56 negativity was 42.2% in the group that had mobilization success and 90.9% in the group that had mobilization failure (P= .001). The fact that CD56 residing on the membrane enables interaction between bone marrow cells and ECM and functions as a signal molecule increases sensitivity to the chemotherapy and G-CSF that are used for mobilization. We found that absence of CD56 can be used as a predictive factor for mobilization failure at the time of diagnosis.

RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy. Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment. To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis. Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment (Figure 4). Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients. Disclosures No relevant conflicts of interest to declare.

Aberrant RHAMM (receptor for hyaluronan-mediated motility) splicing in MM is associated with upregulation of PTBP1/2 (polypyrimidine tract binding protein 1/2): therapeutic implications

Overexpression and/or unbalanced splicing of the RHAMM is linked to various types of hematological malignancies. High RHAMM-V3 variant expression and RHAMM-V3/FL expression ratio induces mitotic instability in MM and correlates with poor survival in MM patients. Thus, understanding the cause of RHAMM splicing alterations can both provide novel insights into MM pathogenesis and identify innovative RNA-based targeted therapy.

Identification of Alpha-Enolase (ENO1) As a Novel Immunometabolic Target in Multiple Myeloma

Introduction. Our previous studies demonstrated an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs interactions with tumor cells and T/NK effector cells in the MM-BM milieu induce immune suppression and MM cell proliferation (Chauhan et al, Cancer Cell 2009, 16:309-323;Ray et al, Leukemia 2015, 29:1441-1444). Here we performed DNA microarray analysis to identify and functionally validate the aberrant moleculer mechanisms mediating pDC-MM interactions with a goal to restore anti-tumor immunity and increase cytotoxicity. Our data shows that pDC-MM interactions trigger significant upregulation of the metabolic enzyme alpha-Enolase (ENO1) in both pDCs and MM cells. ENO1 is known to play a role in metabolic pathways in normal and cancer cells. It functions as a glycolytic enzyme and plasminogen receptor on the surface of tumor cells and MM pDCs. The surface localization of overexpressed ENO1 is a unique feature of tumor cells. We utilized our pDC/MM or pDC/T cells coculture models to show that targeting ENO1 generates MM-specific CTL activity and enhances NK-cell mediated lytic activity. Additionally, we examined whether the combination of ENO1 antagonist with anti-PD-L1 Ab or HDCAi ACY-241 enhances anti-tumor immunity and cytotoxicity in MM.MethodsMicroArray: MM cells were co-cultured with pDCs for 72h, and then separated using CD138 Microbeads; poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared, and heat map was generated ( >1.5-fold change was considered significant, CI > 95%). pDC-induced overexpression of target proteins: MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in ENO1 expression. CTL or NK-lytic activity assays: MM patient BM CD8+ T or NK-cells were cocultured with autologous pDCs (pDC:T/NK; 1:10 ratio) in the presence or absence of ENO1 inhibitor/ENO1i ENOblock (0.1-0.2 µM; Selleck Chemicals) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T/NK:MM), followed by quantification of viable MM cells by FACS. Combination studies were performed using ENO1 antagonists and anti-PD-L1 Ab (5 ug/ml) or ACY-241 (0.1-0.2 uM). Degranulation assay was done to quantify cell surface CD107a. Osteoclast (OCL) function was measured using TRAP staining. Osteoblast (OBL) formation and subsequent bone mineralization were assessed using Alizarin Red staining.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of ENO1 transcript in MM (1.772-fold vs MM alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high surface ENO1 expression, and pDC-MM coculture further increased surface ENO1 expression in MM cells (5-6 fold; n=5; p = 0.003), as well as the ENO1+ MM cell population (3-4 fold vs MM; mean ± SD; n=5; p = 0.005). 3) ENO1 antagonists directly activate pDCs and upregulate functional markers CD80 (1.2-fold) and CD83 (1.3-fold). 4) Treatment of pDC-T cell co-cultures with ENO1i triggers a robust MM-specific CD8+ CTL activity against autologous CD138+ MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2 fold; p = 0.008). 6) In concert with above findings, ENO1 blockade also increased surface CD107a expression on NK and CD8+T cells (CD107a+ NK: 1.4-fold; p = 0.003; CD107a+ CD8+T: 1.3 fold; p= 0.001). 7) Importantly, combination of ENO1i and anti-PD-L1 Ab triggers a more robust allogeneic and autologous MM-specific CD8+ CTL activity than ENO1i alone (%MM lysis: ENO1i: 20%; ENO1i + anti-PD-L1 Ab: 54%; n=7; p = 0.006). Combination of ENOi and ACY-241 also enhances the anti-tumor immunity in MM vs ENOi alone. Finally, 8) ENO1i blocked monocyte-derived osteoclast formation, as well as restored MM patient BM-derived osteoblast formation.Conclusions pDC-MM interactions upregulate immunosuppressive metabolic alpha-enolase enzyme ENO1. Importantly, blockade of ENO1 tiggers MM-specific CD8+ CTL and NK-cell-medaited cytoxicity against MM cells. Our findings therefore identify novel immune-based therapeutic strategies targeting ENO1 to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership.

Targeting Tryptophan Catabolic Kynurenine Pathway Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Introduction. Plasmacytoid dendritic cells (pDCs) play an integral role in MM pathogenesis (Chauhan et al, Cancer Cell 2009, 16:309-323). pDCs interactions with tumor cells and T/NK effector cells in the MM-BM milieu induce immune suppression and MM cell proliferation (Chauhan et al, Cancer Cell 2009, 16:309-323;Ray et al, Leukemia 2015, 29:1441-1444). A direct interruption of pDC-MM and pDC-T cells interactions with novel agents will enhance cytotoxicity and anti-tumor immunity. In this context, our DNA microarray analysis identified upregulation of tryptophan catabolic kynurenine (Kyn) pathway during pDC-MM interactions. To date, the role of Kyn pathway in MM is unclear, but two rate-limiting enzymes of Kyn pathway, kynurenine 3-monooxygenase (KMO) and indoleamine 2,3-dioxygenase 1 (IDO1), have been implicated in various cancers. Overexpression of these enzymes causes hyper-accumulation of terminal metabolites, thereby triggering immune suppression via disruption of bidirectional signaling axes between antigen-presenting cells and T cells. In our study, we found that KMO and IDO1 are increased in both pDCs and MM cells during pDC-MM interactions. To assess the functional significance of these findings, we used our pDC/MM or pDC/T cells co-culture models, and show that targeting Kyn pathway generates MM-specific CTL activity. Moreover, the combination of KMO/IDO1 inhibitors with anti-PD-L1 Ab enhances anti-tumor immunity and cytotoxicity in MM.MethodsMicroArray: MM cells were cocultured with pDCs for 72h; separated using CD138 Microbeads; poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared ( >1.5-fold change was considered significant, CI > 95%). pDC-induced overexpression of target proteins: MM cells were cocultured with pDCs for 24h; analyzed by flow to detect KMO/IDO1 expression. CTL or NK activity assays: Minimally cytotoxic concentrations of inhibitors were used to assess immune functions. MM-BM CD8+ T or NK- cells were cultured with autologous pDCs (pDC:T/NK; 1:10 ratio) in the presence or absence of KMO (Ro 61-8048: 100 nM) or IDO1 (INCB 024360: 0.1 µM) inhibitors (Selleck Chemicals) for 5 days; drug washed, pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T/NK:MM), followed by quantification of viable MM cells by FACS. Degranulation assay was quantified using cell surface CD107a. Osteoclast and osteoblast formations were assessed from TRAP and Alizarin Red staining, respectively.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of KMO in MM cells (2.153 fold vs MM alone; n=3; CI > 95%). 2) Both pDCs and MM cells expressed KMO and IDO1; pDC-MM coculture further increased KMO expression in MM cells (2-3-fold vs MM; p = 0.006); KMO+ MM cell populations also increased after coculture (~1.2 fold; p <0.05). Importantly, IDO1 expression in MM cells is also enhanced after co-culture; validating the importance of Kyn pathway in the context of pDC-MM interactions (MFI: 1.5-2 fold; p=0.006); IDO1+ MM cell populations increased by ~1.5 fold after coculture (p <0.05). 3) KMOi directly activates pDCs and upregulates functional markers on pDCs (CD80: 1.19 fold; CD83: 1.4 fold; p <0.05). 4) KMOi activates patient BM-CD8+T cells in autologous pDCs/T cell coculture; triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0 fold vs control; p = 0.009). 5) In agreement with above findings, KMOi also increased surface CD107a expression on NK and CD8+T cells (CD56+NK: 1.41 fold; p = 0.004; CD8+T: 1.6 fold; p= 0.001). 6) Importantly, the combination of KMOi or IDO1i and anti-PD-L1 Ab (5 µg/ml) triggers a more robust MM-specific CD8+ CTL activity than single agent (%MM viability: KMOi: 72%; KMOi + anti-PD-L1 Ab: 57%; IDOi: 82%; IDOi + anti-PD-L1 Ab: 70%; n=7; p = 0.01). Finally, 7) Both KMOi and IDO1i blocked monocyte-derived osteoclast formation, as well as restored MM patient BM-derived osteoblast formation.Conclusions pDC-MM interactions upregulate immunosuppressive enzymes in Kyn pathway; and importantly, blockade of Kyn pathway enzymes induces MM-specific CD8+ CTL activity. Our preclinical data therefore provides provides the basis for novel immune-based therapeutic approaches targeting kynurenine (Kyn) pathway enzymes KMO and IDO1 to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Equity Ownership; Oncopep: Equity Ownership; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Takeda Millennium: Consultancy.

Hedgehog Pathway Over Expression Identifies Myeloma Patients With Worse Prognosis

The iperactivation of self-renewal mechanisms, like Hedgehog (HH) pathway, which govern the refuel of MM neoplastic clone, might be critical to the disease recurrence. Most of the current knowledge on MM pathogenesis is mainly based on the CD138+ cells molecular characterization, but recent evidences point up the role of the more immature CD19+ precursor clones.

Exosomes Secreted By Multiple Myeloma Cell Lines (MMCLs) and Bone Marrow Stromal Cells (BMSCs) Reciprocally Modulate Cell Adhesion and Impact on MM Cell Migration and Bortezomib-Induced Apoptosis

Introduction: In MM pathogenesis, the interaction of plasma cells and the bone marrow microenvironment plays a crucial role. Exosomes are secreted by nearly all cell types, including cancer cells, and have been implicated in intercellular communication, drug resistance and tumor progression. We therefore purified, characterized and studied the influence of exosomes secreted by MMCLs and BMSCs on cell adhesion, bortezomib-induced apoptosis and cell migration.Methods: Exosomes were purified from the supernatants of MMCLs (IM-9, MM.1S, U266, RPMI8226) and M2-10B4 BMSCs after 72h of culture with RPMI1640+10% exosome-depleted FBS by differential centrifugation. Purity and quality of exosomes was confirmed by electron microscopy and western blot for CD63. Exosomal protein concentration per 5x106 cells was quantified by Bradford. CD44 and ICAM1 were evaluated by flow cytometry. Apoptosis was assessed via annexin V/7-AAD-staining. Chemotaxis to M2-10B4 exosomes (100-500μg/ml) was studied using 24-well chemotaxis chambers. Migrated cells were counted by flow cytometry.Results: Exosomes from IM-9 cells were identified by electron microscopy, thereby showing a characteristic saucer-like morphology, ranging in diameter between 30 and 120nm. Both MM cell and M2-10B4 exosomes expressed CD63. Mean exosomal protein concentration was 1.4μg/μl for M2-10B4 and varied for MMCLs between 1.2 and 1.7μg/μl. M2-10B4 cells expressed higher levels of both adhesion molecules CD44 and ICAM-1 when incubated with exosomes from MMCLs compared to basal conditions, however, not reaching statistical significance. Conversely, M2-10B4 exosomes induced a significant increase in CD44 expression on IM-9 cells (p=0.03), reflecting an increased adhesion to components of the extracellular matrix that might contribute to cell-adhesion mediated drug resistance. IM-9 apoptotic cells after 24h with 10nM bortezomib decreased from 72% to 61% in the presence of M2-10B4 exosomes and from 74% to 62% with IM-9 exosomes, suggesting a lower sensitivity to bortezomib. Migration of IM-9 cells slightly increased with 200μg/ml M2-10B4 exosomes and decreased with higher exosome concentrations.Conclusions: Our findings underline the role of M2-10B4 exosomes on adhesion and migration of one representative MMCL (IM-9), and the impact of M2-10B4 and IM-9 exosomes on bortezomib cytotoxicity in this MMCL, suggesting their involvement in a paracrine/autocrine loop modulating treatment response and MM cell survival. DisclosuresNo relevant conflicts of interest to declare.

Abnormalities Of FAM46C, AHCYL1, CDC14A and CDKN2C Genes Located At Chromosome 1p Detected By QM-FISH Identifies Deletion Of 1p32.3 Covered CDKN2C Is An Independent Adverse Prognostic Marker In Multiple Myeloma

SNP arrays and FISH showed Chromosome 1 appeared as a critical region in MM pathogenesis and was associated with adverse survival, such as: 1q gain and 1p deletion. Recent years, some studies found deletions of some genes in the common minimum deletion regions on 1p were related to adverse prognosis, such as FAM46C at 1p12, CDC14A at 1p21.2, CDKN2C at 1p32.3. However, these studies were limited to one or two regions and lacked the comparison among the effects of different sites on prognosis. The characteristics of chromosome 1p abnormalities are not clear and no uniform consensus has been obtained that which locus is the best prognostic factor suitable for clinical routine detection. In addition, there's lack of data on the impact of 1p abnormalities on homogeneous group of patients. We investigated the abnormalities of FAM46C, AHCYL1, CDC14A, CDKN2C genes located at chromosome 1p12, 1p13.3, 1p21.2 and 1p32.3 by quantitative multicolor-fluorescence in situ hybridization (QM-FISH) using bacterial artificial chromosome (BAC) clones in 230 previously untreated myeloma patients. FAM46C, AHCYL1, CDC14A, CDKN2C genes were labeled with Green-dUTP (green), promoFluor-555-aadUTP (orange), PF590-dUTP (Red) and PF415-dUTP (Blue) and used as a FISH probe. The results demonstrated the deletion rates of 1p12, 1p13.3, 1p21.2 and 1p32.3 were 13.0%, 18.7%, 20.8% and 9.06% respectively. The deletion rate of 1p was 24.2%. Amplifications of 1p with 3.04%, 2.60%, 3.77% and 4.15% respectively in 1p12, 1p13.3, 1p21.2 and 1p32.3 were detected in some patients. These amplifications were significant lower than those of deletions (P=0.000, 0.000, 0.000, 0.021). Interestingly, we found chromosome 1p abnormalities were complex, various in forms. Among 57 patients with del(1p), 26.3% of patients presented with one locus deletion, 22.8% with two loci loss, 35.1% with three loci loss, 15.8% with four loci loss. Most (73.7%) of del (1p) were large size of deletion (≥ two regions). Del (1p) was positively correlated with high LDH (≥220U/L) (P=0.026), del (13q14) (P=0.023), and high percentage of plasma cells in bone Marrow (≥ 50%) (P=0.001). 108 patients were homogeneously treated with thalidomide-based chemotherapy (TAD/MPT). Survival analysis showed the median progression-free survival (PFS) of patients with and without del (1p) were 13.0 vs.26 months (P=0.002), median overall survival (OS) were 15.5 vs. 39.5 months (P=0.000). In addition, we found patients with sole del (1p) involving 1p12 or/and 1p13 had no worse prognosis. Patients with del (1p32.3) had shorter PFS (9.0 vs. 23.0, P=0.001) and OS (9.0 vs. 39.0, P=0.000). In multivariate analyses, 1p32.3 deletion appeared as an independent negative prognostic factor regarding to complex karyotype, LDH≥220U/L and del (17p13) in thalidomide-based chemotherapy group. Hazard ratio showed that the progression risk and death risk in patients with 1p32.3 were increased 5.64 (P=0.031) and 8.314 times respectively (P=0.011). Conclusionour datas show that chromosome 1p abnormalities are complex, various in forms, mainly with large size of deletion, rarely amplification. 1p deletions are negative prognostic factors for PFS and OS in MM patients receiving thalidomide-based chemotherapy. 1p32.3 deletion is the most important 1p deletion and is an independent poor factor. Disclosures:No relevant conflicts of interest to declare.

The Comprehensive Genomic Characterization Of All Commercially and Non-Commercially Available Multiple Myeloma Cell Lines

Human myeloma cell lines (HMCL) provide both a discovery and validation platform to improve our understanding of the molecular pathogenesis of multiple myeloma. We have completed a project to characterize the underlying genetics of all commonly used HMCL with a primary goal of identifying appropriate model systems for findings from large scale patient studies like the multiple myeloma genomics initiative (MMGI). We first purchased all 37 commercially available HMCL from DSMZ, JCRB, ECACC, and ATCC. An additional, 32 non-commercial cell lines were obtained from Diane Jelinek and Leif Bergsagel. Subsequently each HMCL was thawed and cultured under strict parameters, which yielded cells for analysis, by comparative genomic hybridization, whole exome sequencing (Agilent SureSelect V4+UTR) and mRNA sequencing. The combination of these two assays provides a detailed map of the genetic complexity underlying this deadly disease.For variant discovery, alignment was done using BWA followed by realignment, quality recalibration and duplicate removal. High quality calls were identified using Mutect by requiring a minor allele frequency of 0.25 and a minimum depth of coverage of 10x. This identified a median of 81999 mutations per sample with 25th and 75thpercentile values of 76621 and 87440 respectively. To identify likely somatic mutations, we filtered out variants found in dbSNP, 1000 genomes project and the NHLBI Exome Sequencing Project when any of the SNPs within each database had a global minor allele frequency of greater than 0.1%. In addition, we included only those variants in gene space defined by RefSeq or GENCODE and targeted by Agilent exome capture V4+UTR. After these filtering steps a median of 1389 putative somatic mutations remained. To identify key events responsible for mutagenesis, we extracted non-synonymous variants in genes, which show substantially expression in RNASeq(FPKM>5). This identified a median of 371 and 135 variants per sample respectively. Overall, across all the 66 samples, there were at total of 9074 non-synonymous mutations in 4453 genes, which are significantly expressed.To identify potential oncogenes we focused on mutations that occurred at the same position in the genome or altered the same amino acid in expressed genes. This identified 63 genes including KRAS(n=16) and NRAS(n=11) as expected but also BRD9, CCNE1 and MAP2K3. To identify putative tumor suppressors, we looked for genes with homozygous mutations or genes with more than one mutation in the same sample. This identified 112 genes including TP53, TRAF3, FAM46C and DIS3 as expected along with LTB and KDM6B. Independently, to attempt to identify genes involved in MM pathogenesis, genes with mutations having a SIFT score of less than 0.05 which occurred in at least 5%(n>3) of the cell-lines were identified. This identified 185 genes of which 25 are in our putative oncogene list and 37 are in the tumor suppressor list.These study results provide the myeloma community with the unique resource of a fully characterized series of models that can be used for laboratory-based tests. To increase the utility of the dataset to the community all these data are freely available at www.keatslab.org or by request. Disclosures:No relevant conflicts of interest to declare.

1115PD - A Phase 2 Randomized Study of Ramucirumab (IMC 1121B; RAM) with or Without Dacarbazine (DTIC) in Patients (PTS) with Metastatic Melanoma (MM) (CP12-0604/NCT00533702)

ABSTRACT Background Vascular endothelial growth factor (VEGF)-mediated angiogenesis contributes to MM pathogenesis. RAM is a fully human IgG1 recombinant monoclonal antibody that inhibits VEGF receptor-2 (VEGFR-2) ligand binding and signaling. We investigated RAM alone and in combination with DTIC in chemotherapy-naive MM pts. Methods Eligible pts had stage IV cutaneous MM, ECOG PS 0-1, adequate hematologic, hepatic, and renal function. Therapy (Rx) in Arm A: RAM (10 mg/kg) + DTIC (1000 mg/m2); Arm B: RAM (10 mg/kg). Rx was every (q) 3 weeks (wk); tumor assessments were q6 wk. The primary endpoint was progression-free survival (PFS); other endpoints were safety, response, overall survival (OS). Results 106 pts were randomized; 102 received study Rx. Arm A (n = 52) median (medn) age was 63, 71% male, 37% had elevated LDH, 23% stage M1c. Arm B (n = 50) medn age was 62, 76% male, 38% had elevated LDH, 36% stage M1c. Medn PFS was 2.6 months (m) Arm A and 1.7m Arm B; 6m PFS rates were 31% and 18%; 12m PFS rates were 24% and 16% on Arms A and B, respectively. There were 9 (17%) partial responses (PR) & 19 (37%) with stable disease (SD); 2 (4%) PR & 21(42%) SD on Arms A and B, respectively. Medn OS was 8.7m Arm A; 11m Arm B. Nonhematologic adverse events (AEs) considered at least possibly related to RAM included fatigue (50%; 4% Grade [G] ≥3), hypertension ([HTN] 21%; 15% G ≥3), infusion-related reactions ([IRR] 8%; 0 G ≥3), proteinuria ([PU] 8%; 4% G ≥ 3), headache ([HA] 10%; 2% G ≥3) on Arm A; fatigue (30%; 2% G ≥3), HTN (22%; 14% G ≥3), IRR (14%; 6% G ≥3), PU (12%; 2% G ≥3), HA (20%; 0 G ≥3) on Arm B. IRR incidence was reduced following a recommendation for premedication after 37 pts received initial Rx. Hematologic AEs were neutropenia ([NP] 33%; 19% G ≥3), thrombocytopenia ([TP] 39%; 15% G ≥3), and anaemia ([A]14%; 4% G ≥3) on Arm A; NP (0%), TP (6%; 0 G ≥3) and A (2%; 0 G ≥3) on Arm B. Conclusions RAM alone or in combination with DTIC was associated with acceptable incidence of AEs in MM. Clinical activity was modest on both arms. Although the study was not powered for definitive comparison between treatment arms, PFS appeared greater in Arm A. Biomarker analysis is ongoing. Disclosure R. Carvajal: Cosulting, Novartis - money paid to me Consulting, Morphotek - money paid to me Grants, NIH/NCI - money paid to me Grants, FDA - money paid to me Grants, ASCO - money paid to me. J. Thompson: ImClone provided funding to the University of Washington to support this research. K. Lewis: Research Funding - ImClone. J. Wolchok: Consultant - Bristol Myers Squibb; Merck; GlaxoSmithKline Research support - Bristol Myers Squibb and GlaxoSmithKline. P. Rojas: I am an employee of Lilly and own Lilly stock. J. Schwartz: I am an employee of Lilly and own Lilly stock. All other authors have declared no conflicts of interest.

A Novel Murine Model of Human Disseminated Multiple Myeloma with A Subcutaneous Human Bone Chip from an Adult Donor Confers Proliferative Advantage and Is Suitable for the In Vivo Evaluation of Novel Drugs

AbstractAbstract 4077Several murine models resembling multiple myeloma have been developed in the last years: the subcutaneous plasmocytoma model and the disseminated model, based on the iv or intracardiac injection of MM cells, have been widely used to test the efficacy of novel agents. Nevertheless, it is well known the crucial role that the interaction between MM plasma cells and the bone marrow microenvironment plays in MM pathogenesis as well as in the proliferation of MM cells and drug resistance; however, unfortunately, most murine models do not recapitulate these interactions. In order to overcome this caveat, other models such as the SCIDhu or the SCIDrab have been developed. However, in these models, the induced microenvironmental milleu is derived from a fetus and a rabbit respectively, which can be relatively different from that found in an adult patient with MM. We have developed a novel model of a human disseminated MM with the presence of human adult microenvironment.For this purpose, CB17-SCID mice, which lack B and T lymphocytes, were anesthetized and a human bone chip obtained from a healthy adult patient undergoing a hip replacement due to a non-malignant fracture, was subcutaneously implanted in the back of the mice. In the same day 3×106 MM1S-Luc cells were iv injected through the tail vein. Two groups were also included as controls in the study, one with MM1S-Luc cells without the bone chip, and the other with only the human bone chip (without MM cells). Luciferase activity was measured three times per week using a Xenogen-IVIS 50 system after ip injection of 0,15 mg/Kg luciferin. Mice were sacrificed when they developed signs associated with advanced disease, such as body weight loss or back limb paralysis.Although the group of mice receiving MM1S-Luc without bone, did develop a disseminated myeloma, this was much more precocious in the group of mice with a human bone subcutaneous implant. In fact, in mice with human bone and MM1S-Luc cells, the luciferase activity was detectable a median of 9 days after the injection of the cells whereas it was necessary 16 days for obtaining the same effect in mice without the bone chip. This is pointing out that the bone chip in these mice induces a favourable environment for myeloma growth and could be appropriate model to study the interactions of MM cells with human stroma. The increase of tumour growth rate also correlated with a decrease in survival. Median survival (CI 95%) of 64 (47-80) days for mice without bone vs 48 (45-50) days for mice with human bone (Log Rank=0.004). Regarding the sites of infiltration, luminescence images showed a disseminated involvement in all skeletal regions, and this was confirmed by histopathology, as there was a general infiltration of spine, femora, tibia and cranium of these mice. By contrast we did not find infiltration of extra-skeletal regions such as heart, lung or spleen in any mice and only unclear infiltrates were observed in the kidney and liver of some mice.In order to validate the suitability of this model for testing the in vivo antimyeloma activity of novel drugs in the presence of a human microenvironment, 6 mice bearing this novel model were randomized to receive: bortezomib (0.5 mg/Kg ip five days per week) + dexamethasone (0.5 mg/Kg 2 days per week) or vehicle control. A second group of the same number of mice with MM1S-Luc but without the bone chip were randomized in the same way and used as control. Bortezomib + dex significantly decreased tumour growth in both groups of mice without any clear differences in activity between both models, suggesting that this combination overcomes the influence of the microenvironment.In summary we have developed a murine model of MM that may allows us to 1) gain insights into the role of the BM microenvironment interactions in MM pathogenesis. 2) Evaluate the in vivo activity of different anti-MM agents in the presence of human microenvironment. 3) Potentially, this model could also contribute to analyze the effect of different agents in the myeloma bone disease. Disclosures:No relevant conflicts of interest to declare.

Abnormally High Levels of E- and N-Cadherin Expression Add to Altered Regulation of b-Catenin In Multiple Myeloma

AbstractAbstract 2982Gene expression profiling (GEP) of normal and malignant plasma cells and B-cells, revealed that MM is uniquely characterized by elevated expression of E- or N-cadherin. Classical cadherins are integral plasma membrane proteins, that together with a- and b-catenin form calcium-dependent adherent junctions. Homotypic interaction of N-cadherin+ hematopoietic stem cells and N-cadherin+ bone lining cells in the endosteal niche regulates HSC function. Adherent junctions contribute to the regulation of Wnt/b-catenin signaling by modulating the balance between membrane-bound and free cytosolic b-catenin, the latter of which is required for TCF transcriptional activity. Overexpression of DKK1 and suppression of Wnt/b-catenin osteoblasts causes a loss in self-renewal of HSC and only stromal cells with active nuclear b-catenin can support hematopoiesis in-vitro. On the other hand, disruption of adherent junctions and release of b-catenin contributes to epithelial-to-mesenchymal transition and solid tumor metastases. We, and others, have demonstrated that Wnt/β-catenin signaling is active in MM. However, emerging evidence suggests that loss of Wnt/b-catenin activity, rather than its activation, is central to MM pathogenesis. Nearly 90% of primary MM cells express and secrete DKK1 and/or SFRP3 or SFRP2, potent inhibitors of Wnt/b-catenin signaling. Moreover, loss-of-function mutations of APC or axin genes or gain-of-function mutations in the β-catenin gene common in colon cancer have not been found in MM. We therefore hypothesized that elevated expression of N/E-cadherin in MM cells contributes to the abnormally increase of b-catenin in MM. We first assessed the steady-state levels of β-catenin protein in MMCL with immunoblotting analysis. β-Catenin protein was expressed in all tested MMCL, with variable levels in individual lines. Interestingly, relative levels of β-catenin protein were comparable to N-cadherin expression in all eight tested myeloma cell lines. CD138-enriched plasma cells from the BM of 72 patients newly diagnosed MM revealed β-catenin protein levels are highly variable. After normalization of β-catenin with β-tubulin levels we segregated cases into three groups: 39% had low, 23% moderate, and 38% high levels of β-catenin. Analysis of correlation of b-catenin protein levels with U133Plus microarray data revealed there are striking positive correlations between N- or E-cadherin mRNA levels with levels of b-catenin protein. Importantly, b-catenin levels were not correlated with known Wnt/b-catenin target genes. To evaluate the role of N-cadherin in regulating β-catenin signaling in MM, we used a lentiviral expression system to express wild-type N-cadherin (NCadW/MMS1) or empty vector (EV/MMS1) in MMS1 cells. Significant increases in total and free b-catenin correlated with N-cadherin protein expression. These results indicate that N-cadherin protein modulates b-catenin levels MM cells. Results of experiments to determine whether N-cadherin-mediated regulation of b-catenin translates into altered TCF/b-catenin transcriptional activity in MM cells will be reported. Disclosures:Shaughnessy:Myeloma Health LLC: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Genzyme: Patents & Royalties; Millennium: Honoraria; Celgene: Honoraria; OrthoBiotech: Honoraria; Array BioPharma: Honoraria.

MicroRNAs 15a/16-1 function as tumor suppressor genes in multiple myeloma

Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. Here, we functionally validate the role of the microRNAs-15a/16-1 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using "sponge" lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Importantly, miR-16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load and host angiogenesis. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes including FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. We validated designated genes showing binding sites within the conserved 3'-UTR and also within the mRNA coding region as direct miR-16 targets, thus indicating that the miRNAs may have many more targets than anticipated by conventional prediction methods. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.

MicroRNA Expression Profiling in Multiple Myeloma: Correlation with Genetic Abnormalities

MicroRNAs (miRNAs) are small non-coding RNA that regulate gene expression by inducing RNA degradation or translational inhibition of their mRNA targets. Recently, deregulation of miRNAs has been associated with cancer development. MiRNA expression profiling has been analysed in different hematological malignancies, but the information about miRNA expression in MM is limited. We investigated the expression levels of 368 miRNAs using a quantitative PCR-method (“TaqMan low density arrays-microRNA assay”, Applied Biosystems) in 49 patients with newly diagnosed MM. Moreover, 4 MM cell lines (MM1S, OPM2, U266 and RPMI) and 4 samples of normal human plasma cells (PC) were also included in the study. In all the bone marrow samples a CD138 positive PC isolation was performed. Relative quantification of miRNA expression was calculated with the 2−ΔΔCt method. The data were normalized respect to RNU48 and relative to a calibrator sample (average of normal human PC samples). Hierarchical clustering based on the average-linkage method with the centered correlation metric was used for unsupervised analysis. Differentially expressed miRNAs were identified using “Significant Analysis of Microarrays” (SAM) algorithm, the t test and the nonparametric Wilcoxon rank sum test. Primary myeloma cells displayed a distinctive miRNA expression pattern compared to normal PC, characterized by the downregulation of the miRNAs differentially expressed. Unsupervised analysis of the data revealed that the MM samples segregated mainly into two clusters: one contained 12 out the 14 MM cases with t(4;14), 20 out of the 30 MM cases with RB deletion, and the 3 samples with t(14;16) which were tightly clustered. The other cluster contained 5 out of the 7 MM samples without genetic abnormalities (IGH translocation, RB and P53 deletions and gain on 1q were ruled out by FISH analysis). MM samples with t(11;14) and those with gains on 1q were dispersed along the dendrogram. The comparative analysis of the miRNA expression profile between MM with t(4;14) and the remaining MM samples showed a set of 5 up-regulated miRNA (let-7c, miR-125a, miR-125b, miR-135a, miR-99a) in the t(4;14) group. Although MM samples with t(11;14) were not clustered together, the supervised analysis identified two upregulated miRNAs (miRNA-345 and miRNA-193a) compared to those MM cases without this translocation. None of the differentially expressed miRNA was located in the chromosomal regions involved in the specific translocations. Upon comparing the miRNA expression in cases with or withouth RB deletion it was found that the three most differentially expressed miRNA were miR-145, miRNA-378 and let-7b (downregulated in MM with RB deletion). There was no correlation between the expression level of miRNAs located on chromosome 13 and the RB status by FISH. In the same way, the miRNAs differentially expressed in MM with 1q gains were not located in 1q. In summary, these results indicate that miRNA expression is deregulated in myeloma cells, and what is more important, the miRNA pattern of expression in MM seems to be associated with specific genetic abnormalities, which indicate that they may play a relevant role in MM pathogenesis.

Over Expression of YY1 and RKIP in Multiple Myeloma (Cell Lines and MM Tissues).

There is a need to identify underlying molecular mechanisms and gene products (targets for intervention and biomarkers) involved in MM chemo- or immuno-resistance. Recent findings from our laboratory have reported high expression of the transcription factor Yin Yang 1 (YY1) in several tumor types and have demonstrated its fundamental role in tumor cell chemo/immuno-resistance (Gordon, S., et al., Oncogene 25:1125, 2006). In contrast, low levels of the Raf kinase inhibitor protein (RKIP) has been shown to play a pivotal role in the negative regulation of cell survival signaling pathways, and has been considered a metastasis tumor suppressor. The overall objective of our studies is to examine whether MM cell lines and tissues derived from MM patients present deregulated expression patterns of RKIP and YY1, and to demonstrate the roles of YY1 and RKIP in MM pathogenesis and response to various therapies. In the present study, the myeloma cell lines RPMI-8226 and MM-1S were screened for RKIP and YY1 expression at the protein and m-RNA levels. In addition, fresh tissues from MM patients were also examined for RKIP and YY1 expression by IHC and their patterns compared with those observed in normal bone marrow cells. The findings demonstrate that both MM cell lines express remarkably higher levels of RKIP protein and transcripts compared to other tumor cell lines examined (lymphomas and prostate cancer), as assessed by western, RT-PCR and IHC. There was significantly higher expression of RKIP in MM patient's samples compared to normal bone marrow cells. YY1 protein and m-RNA levels were detected in the MM cell lines; however, they were lower compared to Ramos and PC-3 cells. YY1 expression in MM tissues was significantly elevated compared to normal bone marrow cells. RKIP was basically present in the cytoplasm while YY1 was mainly accumulated in the nucleus. In contrast, normal bone marrow cells presented primarily cytoplasmic YY1 and RKIP expression. The YY1 expressing population was the CD38+/CD138− cell subset, which has been reported to be more susceptible to apoptosis (Mitsiadis, S.C., et al., Blood 98:795, 2001). Overall, the present findings support the potential involvement of two new gene products, such as YY1 and RKIP, in MM pathogenesis and their implication in regulating MM resistance to conventional therapeutics. Moreover, these gene products suggest their potential use as new therapeutic targets and/or biomarkers in multiple myeloma. Future studies with large cohorts of tissues will elucidate the importance of the above findings and will give new insights in the unexpected RKIP over expression and activity in multiple myeloma.

Pleiotropic Role of GSK3 Kinase in Myeloma Cell Biology.

GSK3 is a cellular serine-threonine kinase discovered because of its involvement in insulin, growth factor and Wnt signalling, downstream of which it is inhibited through the action of the PI3K/AKT cell survival pathway or through other Wnt signalling-dependent mechanisms. GSK3, therefore, has been included in the group of “tumor suppressors”, as it can antagonize cell proliferation triggered by these cascades. Recent findings, however, have challenged this notion in that GSK3β has been found central for cell survival and NF-κB signalling. Since growth factor, Wnt and cytokine-dependent signalling pathways have been implicated in MM pathogenesis, we decided to investigate the role of GSK3 in multiple myeloma cell biology. GSK3 kinase activity was found slightly higher in malignant plasma cells as compared to normal resting B-lymphocytes and normal in vitro generated plasmablasts. GSK3 enzymatic activity was hampered by stimulation of MM cells with IL-6 and IGF-I but, remarkably, not with TNFα. IL-6 and IGF-I driven MM cell proliferation was significantly increased by GSK3 blockade as it was MM cell survival upon serum starvation or contact with bone marrow stromal cells (BMSC). At molecular level, IL-6-dependent STAT3 phosphorylation was unaffected by GSK3 inhibition, however, ERK1, 2 phosphorylation was increased. Importantly, NF-κB activation and transcriptional activity downstream from TNFα were only slightly affected when GSK3 function was inhibited in MM cells. However, when GSK3 inhibitors were added to MM cell cultured with BMSC, IL-6 secretion in the medium was reduced and the expression of NF-κB-dependent antiapoptotic genes was altered. Lastly, the addition of GSK3 inhibitors in MM cells-BMSC cultures led to an increased expression of Wnt/β-catenin-dependent genes both in MM and in BMSC cells. Our data indicate a different involvement of GSK3 downstream from growth factors or TNFα-induced signalling pathways in MM cells; the observed effects of GSK3 inhibition on the Wnt-signalling pathway indicate that, whereas they would be desirable in the BMSC compartment (i.e. antagonisms of Wnt-inhibitors released in the MM bone marrow, such as Dikkopf-1 (DKK1) or secreted Frizzled-Related Protein (sFRP)-2 and favouring of the osteoblast maturation) on the other hand they could lead, together with the loss of a brake downstream from growth factor signals, to uncontrolled and enhanced MM cell proliferation; thus, the inhibition of this kinase for therapeutic purposes in MM is likely to be accompanied by dangerous and unwanted side effects that may promote the progression of this disease.

Immunoglobulin (Ig) Repertoire in Multiple Myeloma: High Frequency of Recurrent Aminoacid Substitutions in the FR2 and CDR2.

Background. Characterization of the heavy chain (IgH) repertoire and assessment of rate and nature of somatic mutations (SM) provided important clues to understand the pathogenesis of CLL and other lymphoid tumors. The IgH repertoire of MM has been studied less extensively. This aspect has been analyzed in a large population of Italian MM patients. The results have been compared to published repertoires from normal subjects (Brezinschek et al J Immunol 1995) and to our bank of more than 200 CLL.Methods. MM-specific Ig heavy chain rearrangements were sequenced as described elsewhere (Ladetto et al BBMT 2000) starting from 220 patients. The analysis was successful in 162 with a close-to-expected success rate. IgH sequences were analyzed using the Basic local alignment search toll (BLAST), (http://www.ncbi.nlm.nih.gov/igblast) to identify V, D and J regions and to establish the rate of somatic mutations and their silent to replacing (S/R) ratio. Finally the aminoacid sequence derived from the IgH rearrangement was analyzed to identify recurrent aminoacid substitutions. The clinical features of our patients are: age median 55 years (34–73), male sex 53%, Stage III MM 61%, IgG MM 53%, IgA MM 24%, light chain MM 17%.Results. Most VH families were not over- or under-represented compared to normal controls. The only family strongly over-represented in MM compared to both CLL and normal repertoires was the VH 3–33 (22% of patients) (p<0.0001). In addition, some families were under-represented (VH 3–49,VH 4–59, VH4–31) compared to normal subjects (p<0.05). Three D families were under-representred (DH 2–2, DH 5–5 DH 6–13) and two over-represented (DH 4–17, DH 5–24) (p<0.05). JH usage was not skewed in MM. The median rate of VH SM in MM was 5.3% (0.5%–18.8%). When the position and type of aminoacid replacements were evaluated a striking number of recurrent substitutions were noticed. In three families (VH 3–21, 3–23 and 3–33) recurrent mutations occurring in more than 40% of cases were noticed. In family 3–21, 6 of 10 patients (60%) had an identical triple aminoacid substitution in CDR2 at position 5 (S to D), 6 (S to G) and 8 (Y to N). In the VH 3–23 a S to D substitution at position 5 and a S to N substitution at position 8 of the CDR2 occurred in 11 (44%) and 10 (40%) of 25 patients, without apparent association or mutual exclusion. Finally, in the over-represented VH 3–33 family an A to S mutation involving the FR2 (position 14) occurred in 25 of 36 patients (69.4%) and a W to S mutation in the CDR2 at position 3 occurred in 24 patients (66.7%). The two mutations co-existed in all but three cases. These mutations were much less common in VH-mutated sequences of CLL (median rate: 20% range 9–36%) and normal repertoires (median 0%, range 0–28%) despite a comparable overall rate of SM. These differences reached statistical significance for all but one mutation. Moreover the recurrent triple and double mutation pattern of VH3–21 and VH 3–33 were observed only occasionally in non-MM populations.Conclusions. This extensive analysis on Ig repertoire in MM suggest that malignant plasmacells derive from lymphocytes that underwent specific pathways of antigen selection which might be relevant for MM pathogenesis. Additional studies are required to address epidemiological aspects (VH3–33 over-representation was not seen in previous smaller studies from other countries) and to identify potential responsible antigens.