Primary Waldenstrom Macroglobulinemia Cells Research Articles

A new role for the SRC family kinase HCK as a driver of SYK activation in MYD88 mutated lymphomas

AbstractThe SRC family kinase (SFK) HCK is transcriptionally upregulated and activated by mutated MYD88 (MYD88Mut), a key adaptor for Toll-receptor signaling. HCK activates BTK, AKT, and ERK in MYD88Mut lymphomas. SYK, a B-cell receptor (BCR) component, is activated in MYD88Mut lymphoma cells. Although the SFK LYN serves as a trigger for SYK activation in MYD88Mut ABC DLBCL cells, LYN activity is muted in MYD88Mut Waldenstrom macroglobulinemia (WM) cells. We therefore investigated a role for HCK in mediating SYK activation. Overexpression of wild-type (WT) (HCKWT) or gatekeeper mutated (HCKThr333Met) HCK in MYD88Mut lymphoma cells triggered SYK activation. Conversely, HCK knockdown reduced p-SYK in MYD88Mut lymphoma cells. Coimmunoprecipitation experiments showed that HCK was complexed with p-SYK in MYD88Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. Rescue experiments in MYD88Mut lymphoma cells expressing HCKThr333Met led to persistent HCK and SYK activation and resistance to the HCK inhibitor A419259. Treatment of primary MYD88Mut WM cells with A419259 reduced p-HCK and p-SYK expression. Taken together, our findings show that SYK is activated by HCK in MYD88Mut B-cell lymphomas cells, broaden the prosurvival signaling generated by aberrant HCK expression in response to MYD88Mut, and help define HCK as an important therapeutic target in MYD88Mut B-cell lymphomas.

VLX1570, a First in Class Dub Inhibitor, Modulates BCR Signaling and CXCR4 Expression and Demonstrates Significant In Vivo Antitumor Activity in a Murine Model of Human Waldenstrom Macroglobulinemia

Background: Aberrant B-cell receptor (BCR) and MYD88 signaling cooperatively drive Waldenstrom macroglobulinemia (WM) tumor cell survival. To sustain BCR-driven growth and maintain optimal cellular homeostasis, WM cells upregulate proteasome function. We have recently described the role of two distinct 19S proteasome-associated deubiquitinating enzymes (DUBs), USP14 and UCHL5, in maintaining WM-cell viability. Pharmacologic inhibition with b-AP15 (commercial compound) results in WM-cell death and accumulation of high molecular weight ubiquitinated protein conjugates; an event that is phenocopied by siRNA knockdown of USP14 and UCHL5. VLX1570 is a lead-optimized clinical grade analog of b-AP15 with enhanced solubility, stability and target residence time. We investigated the effect of VLX1570 on BCR components, WM cell survival proteins and its in vivo activity in a human xenograft model of WM.Materials: VLX1570 was obtained from Vivolux AB and bortezomib from Sellekchem. Human WM cell lines (BCWM.1, MWCL-1 and RPCI-WM1) and their drug-resistant derivatives were used (total n=12). Bone marrow aspirates from WM-patients and from healthy donors were obtained from the Mayo Clinic biospecimens core. PBMCs from healthy donors were used as comparators where needed. NOD/SCID mice were obtained from Taconic Labs and used for profiling the in vivo properties of VLX1570.Results: We first examined for USP14/UCHL5 protein expression by immunohistochemical analysis in primary tumor tissue from WM patients and compared it to healthy donor cells. WM patient-derived cells exhibited greater staining intensity of both DUBs. Preferential binding of VLX1570 to USP14 and UCHL5 in WM cells was probed in a WM cell using an enzyme-based competition assay (HA-Ub-Vs), which demonstrated time-dependent decrease in labeled USP14 and UCHL5 after treatment with VLX1570. In drug-resistant (bortezomib or ibrutinib-resistant) WM cells, VLX1570 induced >50% tumor-specific apoptosis and displayed synergistic activity with ibrutinib (median CI: 0.5). In addition to ER stress markers, we had previously observed that gene expression of BCR-signalosome components were reduced in VLX1570-treated WM cells (NanoString platform) and this was substantiated at the protein level where p-SYK, SYK and BTK decreased in a time-dependent manner. BCR end-effectors were probed in response to VLX1570 and a decrease in nuclear translocation of NFkB (p65) was noted. USP14 has been shown to control the availability of the chemokine receptor CXCR4, which regulates homing of WM cells to bone marrow. After VLX1570 treatment, we noted a significant decrease in CXCR4 surface receptor expression in all WM cell lines tested and primary WM cells. Lastly, to determine the in vivo anti-WM effects of VLX1570, we injected NOD/SCID mice (xenotransplanted with RPCI-WM1-Luc cells) with VLX1570 (I.P. route, 4.4mg/kg every other day) and measured tumor burden along with pharmacodynamic indicators. VLX1570-treated mice (n=7) demonstrated significantly lower tumor volumes by caliper measurement and bioluminescent signal (p=0.01 - 0.003) as well as reduced serum human-IgM, compared to vehicle-treated mice (n=7). Importantly, mice treated with VLX1570 survived longer vs. vehicle-treated mice (median survival, 62 vs. 44 days, respectively, p=0.0008). No unforeseen toxicities were noted and weight of the mice in vehicle vs. VLX1570-treated mice did not significantly change. IHC analysis in mice tumor tissues demonstrated reduced CXCR4, human-IgM, Ki67 and increased cleaved caspase-3 and ubiquitinated (k48-linked) protein level, the latter serving as a more specific indicator of USP14/UCHL5 inhibition.Conclusions: We provide substantial in vitro and in vivo evidence on the anti-WM effects of a novel first-in-class clinical grade USP14/UCHL5 inhibitor, VLX1570. In addition to anticipated induction of ER stress machinery, we describe for the first time VLX1570-mediated modulation of BCR components and WM cell survival proteins as well as demonstrate its robust activity in an aggressive human xenograft model of WM. These data provide the rationale for a clinical trial of single agent VLX1570 in patients with symptomatic WM. DisclosuresMartin:Janssen: Consultancy, Honoraria; Acerta: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bayer: Consultancy. Gullbo:Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

IL-21 in the Bone Marrow Microenvironment Contributes to IgM Secretion and Proliferation of Malignant Cells in Waldenstrom's Macroglobulinemia

Abstract 770 Background:Waldenstrom's macroglobulinemia (WM) is a B-cell lymphoma characterized by high serum monoclonal IgM and infiltration of lymphoplasmacytic cells into the bone marrow. As with many hematologic malignancies, cytokines within the tumor microenvironment play an important role in supporting the growth and survival of malignant WM cells. IL-21 is a pleiotropic cytokine involved in the differentiation of B cells into plasma cells. During malignancy, IL-21 has demonstrated diverse effects promoting the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in WM. Our objective here was to assess the expression of IL-21 and its receptor in WM cells and to examine whether IL-21 contributes to the biology of WM. Results:When compared to normal bone marrows, immunohistochemistry revealed significant IL-21 staining in the bone marrow of patients with WM (n=5). To determine whether WM cells are susceptible to IL-21 in the microenvironment, expression of the IL-21 receptor (IL-21R) was assessed via PCR in CD19+CD138+ cells isolated by positive selection from patients with WM (n=8) and in the newly characterized WM cell line, MWCL-1. Nearly all (7/8) CD19+CD138+ WM cells expressed IL-21R, as did MWCL-1 cells. Using flow cytometry we detected expression of IL-21R protein on the surface of WM cells as well. The contribution of microenvironmental IL-21 to the biology of WM tumors was then examined. In MWCL-1 cells, IL-21 (100 ng/mL) increased proliferation by 37% (p=0.005) over untreated controls as determined by thymidine incorporation at 72 hr, and in primary WM cells, proliferation increased by nearly 50% (p=0.003). Interestingly, the immortalized B cell line, IM-9, responded to IL-21 with a significant decrease in proliferation, consistent with previous data indicating differential effects of IL-21 depending on the pathological status of the B-cell in question. IL-21 also significantly induced (p<0.0005) IgM secretion in WM as measured by ELISA (MWCL-1 5,956 +/− 393 ng/mL vs. 10,013 +/− 730 ng/mL; CD19+138+ WM 504 +/− 33 ng/mL vs. 811 +/− 32.5 ng/mL). Annexin/PI staining was used to assess viability, but no apoptotic effects were associated with IL-21 in WM. To better understand the mechanisms through which IL-21 increases cellular proliferation and IgM secretion in WM, we characterized STAT activation in response to this cytokine. In MWCL-1 cells, IL-21 significantly increased the phosphorylation of both STAT1 and STAT3, and to a lesser extent, STAT5. Treatment with a STAT3 inhibitor completely abolished the effects of IL-21 on cellular proliferation and IgM secretion suggesting IL-21 mediates its biologic activity through a STAT3-dependent mechanism. The expression of transcription factors involved in B-cell differentiation was also measured in MWCL-1 cells treated with IL-21. Both BLIMP-1 and Bcl-6 levels significantly increased upon addition of IL-21, whereas PAX5 was significantly decreased. IL-21 had no effect on the expression of XBP-1, which is involved in regulating Ig secretion, suggesting that the increase in IgM secretion in MWCL-1 cells may occur secondary to the increase in proliferation, as opposed to an actual increase in the production of IgM. Lastly, IL-21 significantly enhanced IL-10 secretion from MWCL-1 cells (669 +/− 152 pg/mL vs. 1,948 +/− 279 pg/mL, p=0.0002). While the interplay between IL-10 and IL-21 in WM remains to be examined, IL-10 is known to be involved in normal B-cell development and may have synergistic effects with IL-21 in malignant WM cells. Overall our data indicate that IL-21 in the bone marrow microenvironment significantly affects the biology of WM tumor cells through a STAT3-dependent mechanism. Disclosures:No relevant conflicts of interest to declare.

LNA Anti-MicroRNA-155: A Novel Therapeutic Strategy in Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

Abstract 2728 Background.We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods.WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results.The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn’t exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary.A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures:Roccaro:Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

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.

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.

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.

Selective Inhibition of the Chymotrypsin-Like Activity of the Immunoproteasome and Constitutive Proteasome Represents a Valid Anti-Tumor Strategy in Waldenstrom Macroglobulinemia.

Abstract 4911 IntroductionProteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom macroglobulinemia (WM), where single-agent Bortezomib has been tested in phase 2 clinical trials, achieving 40% to 80% responses. Nevertheless, a significant fraction of patients relapse, or develop significant neuropathy. Therefore preclinical evaluation of new proteasome inhibitor is needed in order to improve patient outcome. We tested PR-047, a new orally bioavailable analog of carfilzomib which selectively inhibits the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome, in WM. MethodsWM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining/flow cytometry analysis and DNA fragmentation, respectively. NF-kB activity has been evaluated on nuclear proteins using a DNA-binding ELISA-based assay. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. ResultsPrimary bone-marrow derived WM cells are characterized by higher expression of the immunopreoteasome as compared to the constitutive proteasome. PR-047 inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, leading to induction of cytotoxicity in primary WM cells; as well as to programmed cell death in a caspase-dependent and -independent manner, as shown by activation of c-jun-N-terminal kinase; inhibition of NF-kB; and initiation of the unfolded protein response. PR-047 induced cytotoxicity and inhibited DNA synthesis in primary WM cells (IC50: 50-80nM), as well as in IgM secreting low grade lymphoma cells (IC50: 30-50nM). Importantly, PR-047 exerted cytotoxicity in WM cells, even in the context of bone marrow milieu, by inhibiting IL-6- and IGF1-BMSC secretion and BMSCs-induced phosphorylation of Akt and ERK in WM cells. Moreover, combination of PR-047 and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced caspases-, PARP-cleavage; NF-kB inhibition; and synergy in inhibiting the chymotrypsin- and caspase-like activities of the immunoproteasome and constitutive proteasome. ConclusionThese preclinical findings demonstrate that PR-047 targets WM cells, due to its anti-CT-L activity of both immunoproteasome and constitutive proteasome, providing the framework for testing PR-047 in this disease. DisclosuresAujay:Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Primary Waldesntrom Macroglobulinemia Cells Harbor Constitutive Activation of Akt, mTOR, Rictor and Raptor: Rational for Testing a Dual Inhibitor of the PI3K/Akt and mTOR Pathways in This Disease.

Abstract 3843Poster Board III-779 BackgroundThe PI3K/Akt and mTOR pathways play a pivotal role in the initiation and progression of malignancies, enhancing cell survival by stimulating cell proliferation and inhibiting apoptosis. Therefore, it is critical to examine therapeutic agents that explicitly target this pathway, specifically in tumors that harbor activation of the PI3K/Akt pathway, such as Waldenstrom macroglobulinemia (WM). MethodsPrimary-CD19+ bone marrow-derived WM cells, -bone marrow stromal cells, WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL), and primary normal CD19+ peripheral blood-derived (CD19+ PB)cells were used. Gene-expression and microRNA profiling have been performed on primary WM cells, as compared to CD19+ PB cells. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining, and Apo2.7/flow cytometry analysis, respectively. Cell signaling and apoptotic pathways were delineated by Western Blot and immunofluorescence analysis. In vivo homing has been assessed by in vivo flow cytometry. ResultsPrimary bone-marrow derived WM cells are characterized by lower expression of PTEN gene and protein; higher expression of pospho(p)-Akt, p-mTOR, rictor and raptor, as compared to their normal cellular counterpart (CD19+ PB cells). We also observed that microRNA-542-3p and -494 are more highly expressed in primary WM cells as compared to normal CD19+ PB cells (P<.01); and they both target PTEN, as predicted using TargetScan, PicTar, and miRanda algorithms, suggesting their role in inhibiting PTEN expression. We next assessed the effect of the dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 (Novartis, Basel, Switzerland). This agent induced cytotoxicity and inhibited DNA synthesis (IC50 20-25nM) in BCWM.1 at 48 hours. Similar effects were demonstrated in all IgM secreting cell lines and in primary CD19+ WM cells (IC50 20-50nM). No cytotoxicity was observed on CD19+ PB cells, indicating selective toxicity of the compound on the malignant lymphoplasmacytic clone. NVP-BEZ235 inhibited p-Akt and p-mTOR, as well as the downstream Akt- targeted proteins GSK3a/b, p-S6R and p-p70S6, in a dose-dependent manner. Akt and mTOR in vitro kinase activity was also inhibited by NVP-BEZ235 treatment. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation in WM cells. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the forkhead box transcription factors. Finally, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment evidenced by significant inhibition of migration, adhesion in vitro and homing in vivo. ConclusionThese studies therefore show that dual targeting of the PI3K/mTOR pathway represents a promising therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade such as WM. Disclosures:Ghobrial:Millennium : Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Involvement of Fatty Acid Synthase in Azacytidine-Induced Cytotoxicity in Waldenstrom's Macroglobulinemia.

Abstract 3731Poster Board III-667Azacytidine is a potent demethylation agent and has been used for the treatment of myelodysplastic syndrome. Despite a role in reactivating epigenetically silenced tumor-suppressor genes, the mechanisms of action for azacytidine are not well understood. The relationships between the reactivation of tumor-suppressor genes and the significance for clinical responses also remain to be established. Azacytidine can incorporate into both RNA and DNA strands and may therefore act through multiple pathways. We therefore investigated the therapeutic potential of azacytidine in Waldenstrom's Macroglobulinemia (WM), and characterized certain molecular changes associated with azacytidine. We found that azacytidine exhibited significant dose-dependent cytotoxicity against WM cell lines, and primary WM cells. Treatment of BCWM.1 WM cells with 2 uM of azacytidine rapidly induced cell cycle arrest at G1, and also induced apoptosis at 48 hours. Cleavage of caspase 3, 7, 8, 9 and PARP-1 suggested an involvement of mitochondrial and death receptor pathways in azacytidine-induced apoptosis. The BH3 proteins Puma and Bim, and the pro-apoptotic protein Bax were up-regulated while the anti-apoptotic proteins Bcl-2 and Bcl-xl remained unchanged after treatment with azacytidine. In addition, azacytidine did not induce Akt and NFκB pathways. To further elucidate molecular changes associated with azacytidine treatment, we performed genome-wide microarray expression in BCWM.1 WM cells treated with azacytidine. We observed that the gene encoding fatty acid synthase (FASN) was among the most down-regulated of targets. FASN has been linked to tumor cell proliferation, and its expression was observed by us to be upregulated by microarray studies in WM patients, and further validated as a highly over-expressed target by quantitative RT-PCR analysis in WM patients versus healthy donors. Moreover, by these studies we show that knockdown of FASN by small interfering RNA resulted in growth arrest, and also induced apoptosis in BCWM.1 WM cells. Cerulenin, a specific inhibitor of FASN derived from a natural product, induced strong cytotoxicity against BCWM.1 and primary WM cells at 20 uM in 24 hours. In addition, a synergistic induction of apoptosis in BCWM.1 WM cells was observed in the presence of both azacytidine and cerulenin. The results of this study support an important role for FASN in azacytidine-induced cytotoxicity in WM cells, and highlight a novel mechanistic pathway for this agent, as well as a novel target for drug therapy in WM. A clinical study investigating the activity of azacytidine in WM is planned. Disclosures:No relevant conflicts of interest to declare.

The IgM Flare Following Rituximab and IVIG Administration in Waldenstrom's Macroglobulinemia Is Related to IL-6 Production by Bystander Immune Cells, Possibly through Stimulation of the Fcgriia Receptor.

Abstract 761 Introduction:Rituximab is an important treatment option in Waldenstrom's Macroglobulinemia (WM). We and others have previously reported a paradoxical flare in serum IgM levels following rituximab administration which can occur within hours of its administration, and can affect 40-50% of WM patients leading often to symptomatic hyperviscosity, and or aggravation of other IgM mediated morbidities. We have observed a similar flare phenomenon in 3 WM patients receiving IVIG therapy, suggesting that the IgM flare may result from Fcg receptor signaling. We therefore sought to clarify the mechanism for the IgM flare, in hopes that knowledge of its mechanistic attributes might facilitate development of pre-emptive treatments. Patients and methods:CD19+ sorted BM lymphoplasmacytic cells (LPC) from WM patients were incubated in the presence or absence of rituximab or IVIG for 24-48 hours, alone and in the presence of co-cultured monocytes. IgM levels in supernatant were then determined at 24-48 hours by ELISA. Sera and genomic DNA from 68 WM patients treated with rituximab, of whom 27 (40%) experienced an IgM flare, were also evaluated. Serum IgM and cytokine levels were determined for these patients before and following rituximab. Polymorphisms in FcgRIIA-27, -131, FcgRIIB-187, FcgRIIIA-48, -158, IL-6 Promoter -174, -6331, IL-6R-358, and IL6ST(gp130)-148 were also determined for all 68 WM patients, including both patients who did and did not demonstrate an IgM flare. Results:We first observed that the direct incubation of WM LPC with either rituximab or IVIG did not induce significant IgM release. We therefore sought to delineate if other bystander immune cells contributed to the IgM flare phenomenon either directly or by cytokine release. We observed that rituximab and IVIG could stimulate both healthy donor and WM patient monocytes to produce IL-6, which was assessed by both RT-PCR and ELISA. IL-6 transcription was increased as early as 3 hours, and was associated with an increase in IL-6 secretion. Since monocytes express various Fcg receptors, we next sought to clarify which receptor was potentially responsible for the induction of IL-6 release. We therefore stimulated monocytes with activating F(ab')2 fragments directed at FcgRI, FcgRIIA, FcgRIIB, and FcgRIIIA. Only treatment with an FcgRIIA F(ab')2 activating fragment resulted in robust IL-6 transcription and secretion by monocytes. Since IL-6 plays an important role in stimulating IgM secretion by WM cells, we next examined IL-6 levels in patients who received rituximab and compared their levels over the course of therapy in patients with and without an IgM flare. A spike in IL-6 levels was observed in those patients who experienced an IgM flare, and correlated with the time course of the IgM flare. Given these findings, we co-cultured primary BM WM cells as well as BCWM.1 cells in the presence or absence of monocytes using a Tran swell co-culture system, and observed that overnight incubation with rituximab or IVIG resulted in increased IgM production. This effect could be blocked by simultaneous incubation with an IL-6 blocking antibody. Similar results were obtained by culturing WM LPC with supernatants obtained from rituximab or IVIG stimulated monocytes incubated with and without an IL-6 blocking antibody. Lastly, the frequency of polymorphisms for all 68 WM patients for FcgRIIA-27, -131, FcgRIIB-187, FcgRIIIA-48, -158, IL-6 Promoter -174, -6331, IL-6R-358, and IL6ST(gp130)-148 did not defer from published data bases, and were similar between rituximab treated patients who either experienced or not the IgM flare. Conclusion:Taken together, the above data suggest that the IgM flare observed in WM patients following rituximab and IVIG administration is likely triggered by IL-6 in response to stimulation of bystander immune cells including monocytes, possibly through FcgRIIA binding. Efforts aimed at blocking IL-6 release should be explored as a pre-emptive means of preventing the IgM flare in WM patients being considered for rituximab and IVIG therapy. Disclosures:No relevant conflicts of interest to declare.

Carfilzomib Exerts Anti-Neoplastic Activity in Waldenstrom Macroglobulinemia.

Abstract 4916 IntroductionProteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom's macroglobulinemia (WM), where single-agent Bortezomib has been successfully tested in phase 2 clinical trials. Nevertheless, a significant fraction of patients relapse, or develop significant toxicity due to high toxicity in non-transformed cells. Therefore preclinical evaluation of new proteasome inhibitor with a more selective inhibition of neoplastic cells is needed in order to increase efficacy and improve patient outcome. We tested Carfilzomib, a tetrapeptide epoxyketone selective inhibitor of the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in WM. MethodsWM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity and DNA synthesis were measured by thymidine uptake and MTT, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. ResultsPrimary CD19 bone-marrow derived WM cells express higher level of the immunopreoteasome as compared to the constitutive proteasome. Carfilzomib inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, in a dose-dependent manner; leading to inhibition of proliferation (IC50: 5nM; 48h) and induction of cytotoxicity (IC50: 7.5nM; 48h) in WM cells. Carfilzomib mediated apoptosis in WM by increasing PARP-, caspase-9- and -3-cleavage; as well as by inducing activation of c-jun-N-terminal kinase and ER-stress in a dose-dependent manner. Moreover, combination of Carfilzomib and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced PARP-, caspase-9- and -3-cleavage; and synergy in inhibiting the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome. ConclusionTaken together, these findings provide the pre-clinical rational for testing Carfilzomib in Waldenstrom Macroglobulinemia. DisclosuresAujay:Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Regulation of the New CXCR7 Receptor in Plasma Cell Dyscrasias.

Background: Multiple Myeloma (MM) and Waldenstrom Macroglobulinemia (WM) are characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of tumor cells at that site. The receptor for SDF-1, CXCR4 has already been implicated by us and others in the migration and homing of tumor cells to the bone marrow. Recently, a new receptor for the chemokine SDF-1 has been described, namely CXCR7. To date, it has been implicated in the migration of embryonic cells during neurogenesis in zebrafish. It has been reported to be expressed in cancer cells but its function remains unknown. We have previously shown that disruption of the CXCR4/SDF-1 axis interferes with homing of MM cells to the BM. To better understand the homing and migration of MM and WM tumor cells, we sought to study the expression and function of CXCR7.Methods: MM (U266, MM1.S, RPMI, OPM2, OPM1, KMS12BM) and WM (BCWM.1 and WSU-WM) cell lines were used. CD19+ and CD138+ cells were extracted from patient bone marrow samples by microbeads selection after informed consent. To determine the expression of CXCR7 on MM and WM cell lines as well as primary samples, we used flow cytometry and RT-PCR analysis. The migration of tumor cells towards SDF-1 was studied using the transwell boyden chamber migration assay. The adhesion of tumor cells to fibronectin using a fluorometric assay. The CXCR7 inhibitor CCX-754 (ChemoCentryx Inc., Mountain View, CA) was used.Results: CXCR7 was expressed in all cell lines tested except WSU-WM by flow cytometry and RT-PCR. Interestingly, U266 cell line did not express surface CXCR4 and expressed CXCR7 and therefore was used in further experiments to determine the differential role of CXCR7 in SDF-1 related function. Primary WM (n=14) and MM (n=7) cells expressed low intensity CXCR7 by flow cytometry. Use of the above cells in experiments utilizing small molecule antagonists of both CXCR4 and CXCR7 suggested that inhibiting the binding of SDF-1 to one receptor could impact the signaling functions of the other. Experiments are ongoing to clarify the nature of the interaction between these two SDF receptors. Furthermore, adhesion of U266 cells to fibronectin was increased in presence of SDF-1, and inhibited in the presence of CCX-7754. Further analysis to examine CXCR7 and CXCR4 antagonism in adhesion are ongoing.Conclusion: we showed for the first time CXCR7 expression on MM and WM tumor cells. Our results lead us to conclude that through binding of a shared ligand, CXCR7 and CXCR4 may modulate the biological activity of the other.

Analysis of Chemokine and Adhesion Markers in Waldenstrom Macroglobulinemia.

Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), indicating continuous entry and egression of malignant cells in and out of the BM. The process of homing and trafficking involves chemokines, cytokines, and adhesion molecules. In this study, we sought to determine the level of chemokines, cytokines and adhesion molecules in the BM and peripheral blood (PB) of patients with WM.Method: We compared BM supernatant to serum samples from 10 patients with WM, including 5 matched samples from the same patients, and PB serum and BM supernatant from 2 normal healthy controls. The Luminex® xMAP® Multiplex (Upstate, VA) allows detection of human cytokines, chemokines, and growth factors using the Beadlite® products (Upstate, VA). We focused on chemokines that regulate migration: Eotaxin, GRO, IP10, MIP1a, MIP1b, MCP1, MCP3, MDC/CCL22, and RANTES; cytokines that regulate proliferation: Interleukin (IL)-2, -4, -6, -8, -10, and -15; and adhesion molecules: ICAM, VCAM. The means fluorescence intensity (MFI) was measured for all molecules. Chemokine and adhesion receptor expression on primary WM cells and WM cell line BCWM.1 was assessed using flow cytometry. Adhesion was measured using the in vitro adhesion assay (EMD Biosciences, CA), and the anti-VLA-4 antibody (10–100ug/ml, BD Pharmingen, CA).Results: We first compared the BM supernatant of patients with WM to healthy donors. Adhesion molecules ICAM (mean MFI 5624 in WM vs 720 in control) and VCAM (mean MFI 9096 in WM vs 2135 in control) were significantly higher in the WM supernatant, p=0.03 and p=0.05 respectively. In addition, the chemokines IP-10 (Mean MFI 8186 in WM vs 1647 in control), MDC/CCL22 (mean MFI 13015 in WM vs 4221 in control), and GRO (mean MFI 7463 in WM vs 1337 in control), that regulate migration, were significantly upregulated in WM BM supernatant. We found higher expression of MIP1a in the PB of WM patients compared to patient BM supernatant (mean MFI 5715 in PB vs 1484 in BM), p=0.02. Similarly, RANTES and IL-10 were significantly higher in patients' PB sera. There was no statistically significant difference between the PB sera of patients and healthy controls. The expression of CXCR3 (receptor of IP-10) and CCR4 (receptor of MDC/CCL22) were highly expressed on BCWM.1 (mean 90% expression). We then determined the expression of adhesion receptors on BM CD19+ cells from patient samples and BCWM.1. The WM cells and cell line expressed very high levels of surface adhesion receptors VLA4 and LFA1 (mean expression 95%). Anti-VLA-4 antibody 10ug/ml completely abrogated adhesion to fibronectin, 6% compared to control.Conclusion: Adhesion molecules VCAM and ICAM and their receptors VLA-4 and LFA-1 were significantly elevated in the bone marrow of WM patients, indicating activation of adhesion receptors and localizing WM cells in the BM. The anti-VLA-4 antibody completely abrogated adhesion in vitro. In addition, the chemokines IP-10, MDC/CCL22 and GRO were upregulated in the BM of patients, which induce homing of cells into the BM. This study, therefore, systematically defines chemokines, cytokines and adhesion markers in WM, providing the preclinical framework for inhibition of adhesion molecules in clinical protocols to improve patient outcome in WM. * HN and XL are co-first authors.