Waldenstrom Macroglobulinemia Cell Research Articles

Carfilzomib-Dependent Selective Inhibition of the Chymotrypsin-like Activity of the Proteasome Leads to Antitumor Activity in Waldenstrom's Macroglobulinemia

Primary Waldenstrom's Macroglobulinemia (WM) cells present with a significantly higher level of the immunoproteasome compared with the constitutive proteasome. It has been demonstrated that selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive-(c20S) and immuno-(i20S) proteasome represents a valid strategy to induce antineoplastic effect in hematologic tumors. We therefore evaluated carfilzomib, a potent selective, irreversible inhibitor of the CT-L activity of the i20S and c20S in WM cells. We tested the effect of carfilzomib on survival and proliferation of primary WM cells, as well as of other IgM-secreting lymphoma cell lines. Carfilzomib-dependent mechanisms of induced apoptosis in WM cells, and its effect on WM cells in the context of bone marrow (BM) microenvironment have been also evaluated. Moreover, the combinatory effect of carfilzomib and bortezomib has been investigated. In vivo studies have been performed. We demonstrated that carfilzomib targeted the CT-L activity of both i20S and c20S, which led to the induction of toxicity in primary WM cells, as well as in other IgM-secreting lymphoma cells. Importantly, carfilzomib targeted WM cells even in the context of BM milieu. In addition, carfilzomib induced apoptosis through c-jun-N-terminal-kinase activation, caspase cleavage, and initiation of unfolded protein response. Importantly, the combination of carfilzomib and bortezomib synergistically inhibited CT-L activity, as well as caspase-, PARP-cleavage and GRP94 expression. Antitumor activity of carfilzomib has been validated in vivo. These findings suggest that targeting i20S and c20S CT-L activity by carfilzomib represents a valid antitumor strategy in WM and other IgM-secreting lymphomas.

Matrix Metalloproteinase-8 Is Overexpressed in Waldenström's Macroglobulinemia Cells, and Specific Inhibition of this Metalloproteinase Blocks Release of Soluble CD27

Soluble CD27 (sCD27) is produced by Waldenström's macroglobulinemia (WM) cells, with high levels found in WM patients which may facilitate disease expansion. Matrix metalloproteinases (MMP) may facilitate sCD27 release by cleavage of CD27. By gene expression analysis, we observed significantly higher transcription levels of MMP-8 and MMP-9, with 58.5 and 16.7 fold increase in mean transcription levels in WM cells relative to healthy donor peripheral blood B cells (P = .04, and .05, respectively). We developed a model for study of sCD27 release by transfecting BCWM.1 WM cells and BL2126 lymphoblastic B cells, both of which express MMP-8 and MMP-9 with a vector expressing FLAG-tagged CD27 (pFLAG-CD27) which in the presence of phorbol myristate acetate resulted in ≥ 10-fold increase in sCD27 release. MMP inhibitors against MMP-8, but not MMP 2, 3, or 9 blocked release of sCD27. The results suggest that MMP-8 may play a role in the pathogenesis of WM, and that its inhibition may be of therapeutic value in WM.

Key Role of MicroRNAs in Waldenström's Macroglobulinemia Pathogenesis

Epigenetics represent heritable changes in gene expression that are not due to any alteration in the DNA sequence. One of the best-known epigenetic markers is histone acetylation, which has been shown to be deregulated in neoplastic diseases, including B-cell malignancies, such as Waldenström's Macroglobulinemia (WM), a low-grade B-cell lymphoma characterized by the presence of lymphoplasmacytic cells in the bone marrow and a serum monoclonal immunoglobulin M in the circulation. It has been recently demonstrated that microRNAs may be responsible for modulating histone acetylation in WM cells, thus providing the preclinical evidences for using microRNA-based therapeutic strategies in this disease.

Expression of CCL3 by Neoplastic Cells in Patients with Waldenström's Macroglobulinemia: An Immunohistochemical Study in Bone Marrow Biopsies of 67 Patients

C-C motif ligand 3 (CCL3) chemokine plays a crucial role in the inflammation process, cell migration and chemoattraction of monocytes/macrophages, neutrophils and mast cells. CCL3 is overexpressed by malignant cells in B-cell disorders, including chronic lymphocytic leukemia and multiple myeloma. Elevated circulating CCL3 was previously described in Waldenström's macroglobulinemia (WM) but the source of its production was unknown. We performed an immunohistochemical study in bone marrow biopsies of 67 WM patients and found that the whole number of the neoplastic cells express CCL3 in all cases. This finding was constant in newly diagnosed patients with both symptomatic and asymptomatic WM and also in patients with active disease post previous therapies. Our results support, for the first time in the literature, the production of CCL3 by WM cells. They also suggest a possible role of CCL3 in WM biology and reveal CCL3 as a potential target for developing novel drugs against WM.

Hepcidin Is Produced by Lymphoplasmacytic Cells and Is Associated With Anemia in Waldenström's Macroglobulinemia

Waldenström's macroglobulinemia (WM) patients often present with anemia as their primary disease manifestation that may be related to hepcidin, an important regulator of iron homeostasis. We therefore determined hepcidin levels in 53 WM patients, and 20 age-matched healthy patient donors by hepcidin-25 ELISA. Serum hepcidin levels were elevated in WM patients versus healthy patients (P=.04), and correlated with BM disease involvement (P=.004), beta-2-microglobulin levels (P=.029), and inversely with hemoglobin (P=.05). No correlation with serum iron indices was observed, though in patients with high hepcidin levels, increased iron deposition in bone marrow macrophages was observed. Importantly, hepcidin transcripts and protein were produced by primary WM cells. Hepcidin levels correlated with serum IL-6 (P<.001) and C-Reactive Protein (P=.033) levels. The results of this study implicate hepcidin as a contributor to anemia in WM, and suggest that an iron re-utilization defect accompanies hepcidin overproduction leading to its sequestration in WM patients.

Eph-B2/Ephrin-B2 Interaction Plays a Major Role In the Adhesion and Survival of WM Cells In the Context of the Bone Marrow Microenvironment

AbstractAbstract 142 Introduction:Receptor tyrosine kinases (RTKs) are key regulators of the development and progression of many types of cancer. The Eph receptors represent the largest family of RTKs, which are used extensively throughout embryogenesis and development but detected in adult normal tissues. Elevated levels of expression of Eph receptors was found in a wide range of cancers and correlated with aggressive disease and metastasis. This is, triggered by a wide array of cellular responses, including cell boundary formation, motility, adhesion, and repulsion. Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow, indicating homing and adhesion of the malignant cells to specific niches in the bone marrow, which provides a protective environment for the survival and proliferation of these cells. Direct adhesion to endothelial cells and several growth factors and cytokines secreted by endothelial cells in the BM were shown to regulate tumor proliferation. In this study we aimed to characterize the role of RTKs, specifically Eph-B2 receptor, in the interaction of WM cells with the BM microenvironment including cell adhesion, proliferation, and cell-cycle. Methods and Results:We first examined the expression and activation (phosphorylation) of different RTKs in primary WM samples and cell lines using an antibody-based RTK-array. Eph-B2 receptor was activated in all patient samples compared to control, with a 5-fold increase in CD19+ WM cells, and we further confirmed that Eph-B2 was phosphorylated in WM cell lines, BCWM.1, and in IgM secreting cell lines RL, MEC-1. We next examined the activity of Ephrin-B2/Eph-B2 on adhesion and migration of WM cells. Treating starved WM cells with recombinant ephrin-B2 (the ligand of Eph-B2) activated cell-adhesion signaling, including focal adhesion kinase (FAK), Src, p130, paxilin and cofilin, but had no effect on WM cell chemotaxis. Moreover, we found that ephrin-B2 was highly expressed on endothelial cells isolated from the BM of WM patients and on HUVEC cells. Inhibition of ephrin-B2 on the endothelial cells or Eph-B2 on the WM cells with blocking antibodies inhibited the adhesion of the WM cells to endothelial cells, as well as the cytoskeletal signaling in WM cells induced by co-culture with endothelial cells. Consequently, inhibition of the ephrin-B2/Eph-B2 interaction reversed the proliferative and cell cycle effect in WM cell which was induced by co-culture with endothelial cells. This was confirmed using BrdU proliferation assay, flow cytometry for cell cycle, and by immuno-blotting to confirm the effect on proliferative signaling pathways including PI3K kinase and cell cycle related proteins including Cyclin-D, Cyclin-E and p-Rb. Conclusion:This study examines the interaction of Eph-B2 receptor in WM and shows that ephrin- B2/Eph-B2 axis regulates adhesion, activation of downstream signaling of integrin-related molecules, survival and cell cycle progression through the interaction of tumor cells with endothelial cells. This provides the basis for further studies to explore Eph-B2 as a novel therapeutic target in WM. Disclosures:Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

LNA Anti-Microrna-155: a Novel Therapeutic Strategy In Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

AbstractAbstract 4914 Background.We and others have previously demonstrated that microRNA-155 (miR-155) is overexpressed in patients with Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) compared to healthy subjects; indicating that miR-155 may play a crucial role in regulating the pathogenesis of these diseases. We therefore evaluated the role of the novel locked nucleic acid (LNA) antimiR against miR-155 in WM and CLL-derived cell lines. Methods.WM and CLL-derived cell lines (BCWM.1, MEC1) have been treated with 20μM antisense LNA antimiR-155 or scramble probe (Santaris Pharma, Hørsholm, Denmark). Efficiency of delivered LNA oligos into cells was determined by using FAM-labeled LNA, followed by Flow cytometry (FACS). Survival and cell proliferation were assessed by MTT assay and thymidine uptake, respectively. Serum levels of miR-155 from peripheral blood of WM patients or healthy subjects and the level of miR-155-targeted genes were detected by quantitative RT-PCR (qPCR). Results.Efficiency of delivered LNA oligos into both WM and CLL-derived cell lines was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines, as compared to LNA scramble probe used as a control. In contrast, LNA antimiR-155 did not exert any effect on survival of them. We demonstrated increased expression of miR-155-targeted genes, such as CEBPβ, SOCS1, and TP53DINP1 in WM cells upon LNA antimiR-155 treatment. Moreover, we found miR-155 could be detected in peripheral blood serum of WM patients, but not the serum of healthy subjects. Summary.These data provide preclinical evidence for using the novel antisense LNA antimiR-155 in WM and CLL. Moreover, serum levels of miR-155 could potentially be used as a biomarker in patients with WM. Disclosures:No relevant conflicts of interest to declare.

Involvement of Lipogenic Pathway In 5-Azacytidine-Induced Cytotoxicity In Waldenstrom's Macroglobulinemia

AbstractAbstract 29915-azacitidine and 5-aza-2‘-deoxycytidine are demethylation agents used for the treatment of myelodysplastic syndrome (MDS). Despite a putative role for the reactivation of epigenetically silenced tumor-suppressor genes, the mechanism of action for these agents remains to be clarified. Recent studies suggest that 5-azacitidine may also act at the post-transcriptional level by insertion into RNA transcripts. As part of these efforts, we investigated the therapeutic potential of these agents in Waldenstrom's Macroglobulinemia (WM), and characterized molecular changes associated with treatment. We observed that 5-azacitidine was more potent than 5-aza-2‘-deoxycytidine in inducing dose-dependent cytotoxicity against BCWM.1 WM cells and primary WM lymphoplasmacytic cells. 5-azacitidine-induced cell cycle arrest at G1 was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21 (Cip1/WAF1) through a p53-independent pathway. Activation of caspase-3, 7, 8, and 9, and PARP was also observed indicating involvement of both mitochondrial and death receptor pathways for 5-azacitidine-induced apoptosis. The BH3 domain proteins Bim and Puma were increased in WM cells treated with 5-azacitidine, and siRNA-mediated knockdown of Bim and Puma provided moderate protection from 5-azacitidine-induced apoptosis. In contrast, PI3K/AKT/mTOR and NF- B activities were not significantly altered by 5-azacitidine. To further characterize the molecular changes with 5-azacitidine, we profiled mRNA expression in the WM cells associated with 5-azacitidine treatment. The most strikingly down-regulated genes by 5-azacitidine include FASN and SCD1, which encode enzymes essential for lipogenesis. Importantly, by quantitative RT-PCR, FASN mRNA expression levels were significantly higher in WM patients versus healthy donors (p=0.001). WM patients who expressed higher levels of FASN mRNA showed greater bone marrow involvement (p=0.003). Importantly, siRNA-mediated knockdown of FASN or SCD1 induced significant growth arrest and apoptosis in WM cells. siRNA-mediated knockdown of the sterol regulatory element binding protein 1 (SREBP-1), a transcriptional activator for FASN and SCD1 was associated with reduced transcription of FASN and SCD1, along with increased cytotoxicity in WM cells. Inhibition of FASN activity by Cerulenin also induced significant apoptosis, and the combination of 5-azacitidine and Cerulenin resulted in a synergistic induction of apoptosis in WM cells. In conclusion, the results of this study support an important role for the lipogenic genes in the 5-azacitidine-induced cytotoxicity in WM cells, and highlight a novel mechanistic pathway for this agent, as well as novel targets for drug therapy in WM. Disclosures:Off Label Use: Role of 5-azacytidine in Waldenstrom's macroglobulinemia.

MLN4924, a Novel Investigational NEDD8 Activating Enzyme Inhibitor, Exhibits Preclinical Activity In Multiple Myeloma and Waldenström's Macroglobulinemia through Mechanism Distinct From Existing Proteasome Inhibitors

AbstractAbstract 2988 Background:Multiple myeloma (MM) and Waldenström Macroglobulinemia (WM) have both shown clinical responses to Bortezomib therapy which blocks the elimination of ubiquitin tagged regulatory proteins by the proteasome. The NEDD8 activating enzyme (NAE)-inhibitor MLN4924 is a novel agent which demonstrates selective inhibition of the proteins for degradation in the ubiquitin pathway and may offer benefits to MM and WM patients through the more targeted approach. Methods:A panel of human MM and WM cell lines were tested for their in vitro response to MLN4924 using MTT colorimetric survival assays. MM and WM cell lines tested exhibited dose and time dependent decrease of their viability upon exposure to MLN4924 (IC50=25-150 nM). In addition, miRNA and gene expression studies in response to MLN4924 were compared to treatment of the same cells with bortezomib. In vivo safety studies were performed in mice and animal efficacy studies are ongoing in both MM and WM engrafted mice. Results:A panel of MM and WM cells were treated with MLN4924 for 72hrs and compared to the colon carcinoma line HCT116 and normal cell lines HS-5 (stroma) and THLE-3 (hepatocytes). In addition, a longitudinal assessment of viability of MM1S (MM) and BCWM1 (WM) cells during a 72hr incubation with MLN4924 (500nM) showed commitment to death <48hrs. This result, coupled with the observation that normal donor peripheral blood mononuclear cells (PBMCs) and HS-5 stromal cells were less sensitive (IC50 >1000 nM) than the MM or WM cell lines tested, suggest that this compound exhibits a rapid, tumor-selective effect at clinically relevant conditions. We also evaluated primary MM (CD138+) and WM (CD19+) patient bone marrow cells and observed sub-μ M activity by MLN4924. In addition, we tested a series of combinations of MLN4924 with dexamethasone, doxorubicin and bortezomib in both MM1S and BCWM1 cells lines and observed additive activity or greater with MLN4924. Gene expression profiling revealed distinct signatures, in MM1S and BCWM1 lines, as well as distinct patterns of gene expression changes which were induced by MLN4924 vs. bortezomib. For instance, while bortezomib potently induces a compensatory upregulation of transcripts for ubiquitin/proteasome and heat shock protein genes which, in MM1S or BCWM1 cells, were not observed in response to MLN4924 treatment. Additional studies with the proteasome inhibitor MLN9708 revealed similar patterns of expression as bortezomib. These results indicate that MLN4924 does not induce pronounced proteotoxic stress in MM or WM cells, highlighting the distinct effect of MLN4924 on the ubiquitin/proteasome pathway compared to inhibitors which target the 20S proteasome subunit. Longitudinal miRNA profiling revealed a distinct pattern of miRNA expression in MLN4924-treated vs. bortezomib-treated MM and WM cells. Lastly, animal safety studies showed that MLN4924 was tolerated at doses up to 60mg/kg 2x daily for 1 week. Efficacy studies in MM and WM are ongoing. Conclusions:MLN4924 induces cell killing at sub-μ M concentrations for both MM and WM cells with higher sensitivity of tumor cells compared to normal tissues, exhibits selective gene expression and miRNA regulation and can be safely administered to mice. These studies provide the framework for the clinical investigation of MLN4924 in MM and WM. Disclosures:McMillin:Axios Biosciences: Equity Ownership. Smith:Millennium: Employment. Birner:Millennium: Employment. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Promoter-Wide Transcriptional Deregulation In Waldenstrom Macroglobulinemia

AbstractAbstract 3620 Introduction:Waldenstrom macroglobulinemia (WM) is a rare indolent non-Hodgkin lymphoma, characterized by bone marrow infiltration of clonal lymphoplasmacytic cells. Despite recent advances in understanding the pathogenesis of this disease, the molecular basis of WM etiology has not been clearly defined. We therefore performed genome-wide analysis of RNA polymerase II (pol II) binding sites and gene expression profiling in primary WM cells in order to comprehensively define the aberrant transcriptional regulation and related genes in WM. Methods: Primary CD19+ bone marrow derived WM cells and normal primary bone marrow were used. Genomic DNA was extracted using genome isolation kit (QIAGEN) after cross linking. All the DNA samples were sent for Chip assay and human promoter 1.0R array (Genepathway Inc.) which comprised of over 4.6 million probes tiled through over 25.500 human promoter regions. Each promoter region covers approximately 7.6kb upstream through 2.45kb downstream of the transcription start sites. For over 1,300 cancer associated genes, coverage of promoter regions was expanded to additional genomic content; for selected genes total coverage spans from 10kb upstream through 2.45kb downstream of transcription start sites. The published gene expression datasets (GDS2643) which included 10 CD19+ B cell from bone marrow of 10 WM patients and 8 normal controls was analyzed by d-chip software and normalized to normal control. The motif analysis was performed using Cistrome online tools from the Dana Farber Cancer Institute. The gene sets enrichment analysis (GSEA) was performed using GSEA online software from Broad institute. Results:A total of 13,546 high-confidence pol II sites were identified in WM samples and share a small percentage of overlap (11.5%) with the binding sites identified in normal controls. Combining the expression microarray data of WM patient samples and normal controls, we demonstrated a significant correlation between high levels of gene expression and enriched promoter binding of pol II. Notably, we also observed that the WM-unique pol II binding sites are localized in the promoters of 5,556 genes which are involved in important signaling pathways, such as Jak/STAT and MAPK pathways by applying gene set enrichment analysis (GSEA). Interestingly, we found that STAT, FOXO and IRF family binding sites motifs were enriched in the pol II-bound promoter region of IL-6 which plays a crucial role in cell proliferation and survival of WM cells. Moreover, the CpG island associated c-fos promoter was enriched for Pol II binding as compared to the normal control. Conclusion:The presence of increased Pol II binding and the identification of transcription factor motifs in the promoters of key oncogenes may lead to a better understanding of WM. Our findings suggest that altered transcriptional regulation may play an important role in the pathogenesis of WM. In addition, this study will provide novel insights into the molecular mechanism of WM etiology, and may lead to discovery of novel diagnostic molecular biomarkers and therapeutic targets for WM. Disclosures:Leleu:Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

SOCS1 and SOCS3 Genes Are Not Methylated In Waldenstrom Macroglobulinemia

AbstractAbstract 2481 Background.The suppressor of cytokine signalling (SOCS) 1 and 3 are inhibitors of interleukin 6 (IL6) signalling pathway and demonstrated tumour-suppressor activity. The methylation of SOCS family, resulting in transcriptional silencing, has been suggested to play an important role in development of several hematological disorders, including multiple myeloma. Waldenstrom's macroglobulinemia (WM) is a lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM immunoglobulin. Gene expression profiling has demonstrated an up-regulation in IL6 signalling in WM. IL6 also plays a critical role in cell proliferation and survival of WM cells and in the interaction between the tumour cells and their bone marrow microenvironment. We hypothesized that aberrant SOCS1 and SOCS3 expression may play a role in the pathogenesis of MW. Our study aimed to explore genetic and epigenetic aberrations of SOCS1 and SOCS3 genes. Methods.32 patients (pts) diagnosed with WM were included. Diagnosis was done following guidelines, and pts were not treated at time of BM collection. Approval of this protocol was obtained from the CHRU of Lille and was in accordance with the declaration of Helsinki. We studied RL and MEC1, cell lines that secrete IgM, and BCWM1 developed from a pt with WM. DNA and RNA were extracted following negative CD19 B cells selection. The levels of mRNA of SOCS1 and SOCS3 were evaluated by real-time PCR. Samples were examined at steady state and following PMA stimulation. The CpG island methylation status of SOCS1 and SOCS3 genes in the promoter region was investigated by methylation-specific (MSP) PCR. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both loss of heterozygosity (LOH) and copy number alteration (CNA). Paired DNA samples (tumor cells / normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. Results.SOCS1 and SOCS3 genes were expressed in BCWM1, RL and MEC1 cell lines but the baseline expression in cell lines was heterogeneous. PMA induced SOCS1 and SOCS3 expression in all cell lines. No methylation of SOCS1 gene was observed in these cell lines. In contrast, partial methylation of SOCS3 promoter gene was observed in RL and BCMW1. SOCS1 and SOCS3 genes were constitutively expressed at heterogeneous levels in WM pts. The expression varied for SOCS1 from 0.31 to 43 and for SOCS3 from 1.35 to 73. SOCS1 and SOCS3 expressions were enhanced by PMA treatment (n=3 pts). In order to explore genetic aberration on SOCS1 and SOCS3 genes in WM patients, SNP arrays were performed in 17 WM pts. No deletion of SOCS 1 and 3 genes were observed. In one case (5.8%), SNP array karyotyping identified an acquired uniparental disomy (UPD), a LOH without variation of copy number changes, at the SOCS3 locus (17q25). Gain of SOCS1 gene (16p13) was observed in another pt with high mRNA SOCS1 expression. Gene methylation in the promoter was studied in 25 WM pts. No methylation of CpG islands in the SOCS1 and SOCS3 gene promoters was detected in the WM pts. Conclusion.SOCS1 and SOCS3 genes are expressed in WM cells. Copy number aberration, UPD and epigenetic inactivation of SOCS1 and SOCS3 genes seem to be rare events in WM. Other mechanisms might explain for the heterogeneous expression levels of SOCS1 and SOCS3 genes that may contribute for IL6 signalling deregulation in WM. Disclosures:Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Interaction Between Heat Shock Protein Inhibitors and Isoprenoid Biosynthetic Pathway Inhibitors In Multiple Myeloma Cells

AbstractAbstract 2895Multiple myeloma (MM) is characterized by the production of substantial quantities of monoclonal protein (MP) by malignant plasma cells. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of MM cells via inhibition of Rab geranylgeranylation, leading to disruption of MP trafficking and induction of the unfolded protein response (UPR) pathway. Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) have also been shown to induce components of the UPR and apoptosis in MM cells and are currently under clinical investigation. Given the sensitivity of MM cells to stressors which activate the UPR, we hypothesized that combining agents that disrupt protein folding (HSP90 inhibitors) with agents that disrupt protein trafficking (IBP inhibitors) would yield enhanced cytotoxicity via their effects on MP levels and the UPR. We therefore evaluated the effects of combining IPB and HSP90 inhibitors in MM, Waldenstrom macroglobulinemia (WM), and IgM-secreting cells. Studies were performed in 2 lambda light chain-secreting human MM cell lines (RPMI-8226, U266), a WM cell line (WM-WSU), and the IgM-secreting RL lymphoblastoid cell line. IBP inhibitors included lovastatin (Lov) (HMG-CoA reductase inhibitor), digeranyl bisphosphonate (DGBP) (geranylgeranyl synthase inhibitor), and 3-PEHPC (geranylgeranyl transferase II inhibitor), while 17-AAG and 17-DMAG were used as HSP90 inhibitors. The combination of Lov and 17-AAG resulted in enhanced cytotoxicity in all tested cell lines at both 24 & 48 h as determined by MTT assays. Isobologram analysis revealed a synergistic interaction for the RPMI-8226, RL, and WM cells and an additive interaction for the U266 cells. Pre-treatment with Lov for 24 h prior to the addition of 17-AAG resulted in a synergistic interaction in all tested cell lines. The enhanced cytotoxicity was prevented by co-incubation with either mevalonate or geranylgeranyl pyrophosphate. Pre-treatment with DGBP yielded a synergistic interaction between DGBP and 17-AAG in the RPMI-8226 cell line. The addition of 3-PEHPC to either 17-AAG or 17-DMAG resulted in a synergistic interaction in the RPMI-8226 cells. Treatment with 17-AAG and 17-DMAG led to a concentration-dependent decrease in secreted lambda light chain (RPMI-8226, U266) or IgM (RL) levels after 24 h as measured with ELISA. As we have previously demonstrated, Lov decreased secreted MP levels. The combination of Lov and the HSP90 inhibitors resulted in a further decrease in secreted MP. Interestingly, 17-AAG and 17-DMAG decreased intracellular lambda light chain levels in the RPMI-8226 & U266 lines while increasing intracellular IgM levels in the RL line. The increase in l light chain induced by Lov in the MM lines was partially prevented by co-incubation with either of the HSP90 inhibitors. The increase in HSP70 levels induced by HSP90 inhibition was not significantly altered by co-incubation with Lov; however, the accumulation of unmodified Rap1a (a marker for inhibition of geranylgeranylation) induced by Lov was diminished in the presence of the HSP90 inhibitors. Pre-treatment with Lov prior to the addition of 17-AAG resulted in enhanced apoptosis, as determined by PARP and calnexin cleavage, in the MM cells while significant changes were not observed following 24 h concurrent treatment. As a measure of induction of the UPR, levels of phosphorylated eIF2a (p-eIF2a) were assessed. While no significant changes were noted following 24 h concurrent treatment, pre-treatment with Lov did yield an increase in p-eIF2a levels compared with 17-AAG alone. In conclusion, these studies are the first to demonstrate synergistic cytotoxic interactions between select IBP inhibitors and HSP90 inhibitors in MM cells and to evaluate the effects of these agents on MP levels. That the effects of these agents on components of the UPR and apoptosis are most evident following pre-incubation with Lov suggests that inhibition of prenylation is required for the interaction. Further studies will be required to determine whether the observed decrease in intracellular and secreted light chain levels induced by the HSP90 inhibitors is a consequence of increased protein degradation or decreased protein synthesis. These studies form the basis for further pre-clinical and clinical investigation into the use of this novel combination in MM and related MP-secreting disorders. Disclosures:No relevant conflicts of interest to declare.

Microarray Analysis of the Peripheral Monocytes From Waldenstrom's Macroglobulinemia Patients Reveals a Distinct Gene Expression Profile

AbstractAbstract 2010 Background:Waldenstrom's macroglobulinemia (WM) is a B-cell malignancy characterized by the accumulation, predominantly in the bone marrow, of clonally related IgM-secreting lymphoplasmacytic cells. Macrophage derived inflammatory factors are elevated in WM suggesting a possible contribution by monocytes to the growth and survival of WM cells. Patients and Method:Monocytes (CD14+) from the peripheral blood of 8 untreated WM patients, and 6 healthy donors (HDs) were isolated by immunomagnetic bead sorting. Gene expression profiling was then performed using Human Genome U133 Plus 2.0 chips, and data obtained from microarray was analyzed by dChip software with fold change >=2 and p<=0.05 as cut off points. Validation was performed by real time PCR. Results:Using Panther classification, 284 transcripts were identified as significantly different in monocytes derived from WM patients versus HDs. The resulted transcripts are involved in many critical signaling pathways, such as Toll-like immune receptor pathway (i.e. TLR1, TLR4, TLR8, TICAM); inflammatory response (i.e. CD40, PTAFR, FPR2), integrin binding (i.e. RAC2, ILK), chemokinesis (i.e. CCR2, CX3CR1), apoptosis (i.e. TNFSF10), p53 signaling (i.e. GADD45A, 1433F) and G-protein coupled receptors (i.e. CX3CR1). Fifteen of 21 genes were validated by real-time PCR, and were over-expressed in peripheral blood monocytes from WM patients in comparison to healthy age matched donors:GeneMean Fold ChangeMann-Whitney TestCD401.6440570.02857IL2RG1.9627680.02857CKLF1.9839210.1143TLR82.5739330.02857CD323.0265440.02857CD643.1445550.02857UBE2D13.195680.02857FCGR2C3.8325840.02857TLR13.8882540.02857CX3CR14.3014270.05714TNFSF105.3654510.02857TLR45.9844860.02857TICAM7.3245130.02857CLU15.106270.02857CCR217.157380.02857 Conclusion:Peripheral monocytes from WM patients demonstrate a distinct gene expression profile characterized by up-regulation of genes affecting Toll-like innate immunity, inflammation, and apoptosis. These studies define a distinct microenvironmental signature, and provide a framework for the exploration of novel targets for prognosis and therapy in WM. Disclosures:No relevant conflicts of interest to declare.

Aberrant Expression of Regulatory miRNAs and Transcripts for IRS-PI3K Growth and Survival Signaling In Waldenstrom's Macroglobulinemia

AbstractAbstract 1912 Background:Waldenstrom's Macroglobulinemia (WM) is a rare low grade non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells in (LPC) the bone marrow, an elevated serum IgM, and frequently accompanied with hyperviscosity syndrome. The insulin receptor substrates (IRS) are important mediators of the insulin like receptor family and PI3K signaling leading to PKC and AKT activation. Methods:TaqMan low density arrays were used to evaluate the relative levels of 667 miRNAs in 11 WM patient and 5 age matched healthy donor (HD) CD19+ bone marrow cells. The results of this screen were validated using individual stem loop RT-PCR assays. Additional mRNA targets were identified using an existing gene expression profiling (GEP) data set of 22 WM patients and 8 HD using the Affymetrix U133 plus 2 platform. GEP findings were validated using an independent cohort of 18 WM patients and 7 HD. Results:Aberrant miRNAs identified were miR-21 (+3.27 fold p=0.035), miR-29c (+3.17 fold; p=0.003), miR-155 (+5.53 fold; p=0.082), miR-9* (-3.94 fold; p=0.001), miR-27b (-4.94 fold; p=0.001), miR-126 (-21.52 fold; p=0.006), miR-126* (-25.55 fold; p=0.039), miR-145 (-34.27 fold; p<0.001), miR-223 (-24.25 fold; p=0.041), and miR-886-5p (-3.01 fold; p=0.004). Importantly, 5 of these 10 miRNAs targeted members of the IRS-PI3K signaling pathway, a pathway important for growth and survival of WM cells: miR-29c (PIK3R1); R-155 (SHIP1); miR-21 (PTEN); miR-145 (IRS1); miR-126 (IRS1 and PIK3R2), and predict for increased protein levels for PIK3R2 and IRS1 with lower protein levels for PIK3R1, PTEN and SHIP1. Moreover, GEP and confirmatory RT-PCR revealed down-regulation of the IRS-PI3K pathway members IRS2 (-2.0 fold; p=0.004) and PIK3R1 (-2.0 fold; p=0.04). Conclusions:The results of this demonstrate aberrant expression of regulatory miRNAs and transcripts for IRS-PI3K growth and survival signaling in WM, and provide support for the development of IRS/PI3K targeted therapeutics in WM. Disclosures:No relevant conflicts of interest to declare.

Epigenetics in Waldenström‘s Macroglobulinemia

Waldenström's macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by the presence of lymphoplasmacytic cells in the bone marrow and a serum monoclonal immunoglobulin, known as IgM, in the circulation. Although indolent, it remains incurable with a median overall survival of 5-6 years, and most patients succumb to disease progression. Cytogenetic and molecular studies on gene-expression analysis at the mRNA level have demonstrated minimal changes in WM cells. Therefore, multilevel characterization of this disease at the genetic and epigenetic level is required to improve our understanding of the underlying molecular changes that lead to the initiation and progression of this disease. In this study it has been demonstrated that WM patients present with a specific miRNA signature. Among deregulated miRNAs, miR-155 and miR-9* play a pivotal role in the pathogenesis of this disease.

Selective inhibition of chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in Waldenström macroglobulinemia

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor kappaB (NF-kappaB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)-derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

Resveratrol and simvastatin decrease IgM secretion in Waldenström macroglobulinemia: One-year follow-up.

e18556 Background: Waldenström macroglobulinemia (WM) is a distinct B-cell lymphoproliferative disorder characterized by lympho plasmacytic bone marrow infiltration along with an immunoglobulin M (IgM) monoclonal gammopathy.Therapy is postponed for asymptomatic patients, and progressive anemia is the most common indication for initiation of treatment. Published data show that resveratrol and simvastatin have significant antitumor activity in WM cells line. Data is still lacking on the use of these two drugs in vivo. Methods: 6 patients (3 males and 3 females), median age 41.5 yr (range, 42–73) and asymptomatic WM (IgM-MGUS or smouldering WM(SWM), were treated with resveratrol 40 mg/die and simvastatin 20 mg/die for at least 180 days. Immunoglobulin(Ig)-M monoclonal gammopathy of undetermined significance (IgM-MGUS) was defined as a serum IgM monoclonal protein < 3 g/dL,bone marrow lymphoplasmacytic infiltration < 10%, and no evidence of constitutional symptoms, anemia, hyperviscosity, lymphadenopathy, or hepatosplenomegaly related to the lymphoplasmacytic process. Smoldering Waldenström macroglobulinemia was defined as serum IgM monoclonal protein level ≥3 g/dL and/or bone marrow lymphoplasmacytic infiltration ≥10% and no evidence of end-organ damages.At enrollment patients characteristic were: Hb level median, 12.3 g/dL, serum beta(2)-microglobulin level median 2.4 mg/L, and IgM peaks median, 2.2 g/dL.5 patients were classified as IgM-MGUS and only one patient as SWM because of bone marrow lymphoplasmacytic infiltration > 10%. All patients took regularly the drugs and there have been no adverse events.CK and LDH serum levels were kept in the normal range. Results: In all IgM-MGUS patients a reduction between 30 and 50% of the IgM peak was observed after 6 months of therapy and is still maintained at 14 months of follow-up.In SWM patient the reduction was about 25% and is maintained over time. Conclusions: Our data demonstrate clearly that the association between resveratrol with simvastatin decreases IgM secretion both in IgM-MGUS and in smouldering patients not having any adverse effects. No significant financial relationships to disclose.

Activity of the CD20-directed monoclonal antibody GA101 relative to rituximab in Waldenstrom's macroglobulinemia (WM), and applicability to patients expressing FcγRIIIA-158 F/F.

8112 Background: Rituximab alone and in combination is an important mainstay of therapy in WM. Rituximab interacts with Fcγ receptors whose binding is largely influenced by polymorphisms in FcγRIIIA-158. Polymorphisms in FcγRIIIA-158 predict overall response to rituximab alone, as well as attainment of VGPR or CR with rituximab combination therapy in WM. Patients expressing V/V or V/F at FcγRIIIA-158 show improved outcomes versus F/F. We therefore have sought novel antibodies which can improve outcomes for patients expressing FcγRIIIA-158-F/F. GA101 is a novel humanized anti-CD20 antibody with a glyco-engineered Fc receptor domain that exhibits increased FcγRIIIA binding. Methods: We examined ADCC activity for GA101 versus rituximab using 1) healthy donor derived NK cells which were genotyped for polymorphic expression at FcγRIIIA-158 against BCWM.1 cells; as well as autologous NK cells which were genotyped against WM patient LPCs. Results: These studies showed a 2.88, 1.88, and 1.22 fold increase in ADCC activity by healthy donor NK cells expressing FcγRIIIA-158 F/F, V/F, and V/V, respectively, against BCWM.1 WM cells for GA101 versus rituximab. Moreover, ADCC activity was enhanced by 1.98, 1.37 and 1.38 fold for autologous NK cells expressing FcγRIIIA-158 F/F, V/F, and V/V, respectively against WM LPCs. In addition, we also observed direct cytotoxicity against BCWM.1 and primary WM patient LPCs by GA101; in comparison, little or no direct apoptotic activity was observed by rituximab. Western blot analysis showed no activation of caspases 3, 6, 7, 9 in GA101 treated BCWM.1 cells, though nuclear translocation of apoptosis inducing factor (AIF) was observed by immunofluorescence microscopy. Conclusions: GA101 is associated with enhanced ADCC activity relative to rituximab by NK cells, particularly those expressing FcγRIIIA-158 F/F. In addition, GA101 initiates direct cell death in WM LPCs through a caspase independent manner. These studies provide the framework for the investigation of GA101 in WM, and suggest potential benefit for those patients who express FcγRIIIA-158 F/F. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Biogen Idec, Celgene, Genetic Technologies, Millennium Biogen Idec, Celgene, Millennium Celgene, Millennium

Abstract 2060: microRNA-dependent modulation of histone acetylation in Waldenstrom's Macroglobulinemia

Abstract Introduction. Epigenetic regulation of gene-expression, including histone acetylation, is commonly deregulated in many malignancies leading to aberrant transcription, but microRNA (miRNA)-dependent modulation of histone acetylation in Waldenstrom's Macroglobulinemia (WM) has not been evaluated yet. Methods. miRNA and gene-expression profiling have been performed on bone-marrow-derived CD19+ WM cells, compared to their normal cellular counterparts. Purified cRNA was hybridized to HG-U133Plus2.0 GeneChip (Affimetrix). A liquid phase Luminex microbead miRNA profiling (Luminex, Austin, TX) has been used for miRNA studies. To further define those miRNAs differentially expressed between groups (patients vs normal), the data were filtered on significance of differences using ANOVA test, (P &amp;lt; 0.01). Microbead-miRNA profiling data were validated data by stem-loop qRT-PCR. To identify specific predicted miRNA-targeted mRNAs, TargetScan, PicTar, and miRanda algorithms were used. Functional studies were performed on precursor-miRNA-9* and anti-miRNA-206-transfected WM cells, as compared to either scramble probe-transfected or non-transfected cells. HDAC activity was determined by using Colorimetric HDAC Activity Assay Kit. Effect on signaling cascades have been evaluated by western blot and immunofluorescence. DNA proliferation, cytotoxicity, cell cycle, and apoptosis were assessed by thymidine incorporation, MTT, PI, and Apo2.7 staining, respectively. Results. WM cells present with a specific miRNA signature characterized by increased expression of miRNA-206 and decreased expression of miRNA-9* (ANOVA;P&amp;lt; 0.01). Predicted targets for miRNA-206 and −9* include histone-deacetylases (HDAC4; HDAC5) and -acetyltransferases (Myst3). We first demonstrated that primary WM cells are characterized by unbalanced expression of HDACs and HATs at gene level with higher level of HDACs and lower level of HATs, responsible for decreased acetylated-histone-H3 and -H4, at protein level and increased HDAC activity. miRNA-206 and −9* played a functional role in regulating histone-acetylation and HDAC activity in WM cells, based on their ability to target HDACs and HATs; leading to induction of toxicity in precursor-miRNA-9* or anti-miRNA-206-transfected cells, as shown by reduced proliferation rate, cell cycle arrest, induction of apoptosis, supported by PARP-, caspase-8- and −9-cleavage. In addition, miRNA-9*-induced autophagy in WM cells by modulating Rab7 and LC3B. Conclusion. These findings confirm that histone-modifying genes and HDAC activity are de-regulated in WM cells; this may be partially driven by the aberrant expression of miRNA-206 and −9* in the tumor clone; and provide the basis for the development of miRNA-based targeted therapies in WM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2060.

Dual targeting of the PI3K/Akt/mTOR pathway as an antitumor strategy in Waldenstrom macroglobulinemia

AbstractWe have previously shown clinical activity of a mammalian target of rapamycin (mTOR) complex 1 inhibitor in Waldenstrom macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR in WM, and mechanisms of overcoming resistance to therapy. We first demonstrated that primary WM cells show constitutive activation of the PI3K/Akt pathway, supported by decreased expression of phosphate and tensin homolog tumor suppressor gene (PTEN) at the gene and protein levels, together with constitutive activation of Akt and mTOR. We illustrated that dual targeting of the PI3K/mTOR pathway by the novel inhibitor NVP-BEZ235 showed higher cytotoxicity on WM cells compared with inhibition of the PI3K or mTOR pathways alone. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the Forkhead box transcription factors. In addition, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment, leading to significant inhibition of migration, adhesion in vitro, and homing in vivo. These studies therefore show that dual targeting of the PI3K/mTOR pathway is a better modality of targeted therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade, such as WM.