Elsawa, Sherine F.

Waldenström macroglobulinemia (WM) is a B cell disorder with a highly variable clinical outcome, where some patients remain asymptomatic, while others have significant symptoms and require therapeutic intervention. Clinical symptoms include infiltration of lymphoplasmacytic cells into the bone marrow, production of a monoclonal IgM protein, anemia, lymphadenopathy, and serum hyperviscosity. Despite the introduction of multiple chemotherapeutic regimens over the past several decades, WM remains an incurable disease. 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester derivative (CDDO-Me) and imidazolide derivative (CDDO-Im) are synthetic triterpenoids derived from oleanolic acid. These compounds have been shown to induce apoptosis of several tumor cell types including breast cancer, lung cancer, ovarian cancer, melanoma, osteosarcoma, leukemia, and multiple myeloma cells. The goal of this study was to evaluate the potential role of synthetic triterpenoids in WM. Preliminary studies on malignant B cells indicated that CDDO-Im induced the greatest amount of cell death and we therefore used this derivative of CDDO for our studies. CD19+ CD138+ cells from bone marrow biopsy specimens obtained from WM patients were isolated by positive selection and were treated with varying concentrations of CDDO-Im (62.5 nM to 750 nM ) and cell viability was determined after 24 hours (n=3). Compared to the nil control 47% of the malignant cells remained viable at a CDDO-Im concentration of 62.5 nM and only 11% remained viable at 125 nM CDDO-Im. To determine if CDDO-Im had specific toxic effects on non-malignant cells, we cultured CD19- CD138- cells from WM patient bone marrows with CDDO-Im and found that non-malignant cells were less sensitive to the drug, 80% being viable at 62.5 nM and 65% being viable at 125 nM. Similarly, we found that normal peripheral blood B cells and CD19+ CD138+ bone marrow B cells from healthy donors were less sensitive to CDDO-Im. Compared to the nil control 93% of the CD19+ CD138+ bone marrow B cells and 70% of the peripheral blood B cells remained viable at a CDDO-Im concentration of 62.5 nM and 95% and 68% remained viable at 125 nM CDDO-Im respectively. We next examined the effect of CDDO-Im on WM cell proliferation and found that CDDO-Im inhibited cell proliferation in a dose-dependent manner. Similar to the viability assays, there was a differential effect of CDDO-Im on malignant and non-malignant cells. Compared to the nil control, at 125 nM, there was a complete inhibition of malignant cell growth, while approximately 40% of the non-malignant cells remained proliferative. To determine the mechanism of cell death, CD19+ CD138+ cells were cultured in the presence or absence of various doses of CDDO-Im for 6 hours and cell lysates were examined for cleavage of PARP. There was evidence of PARP cleavage in a dose-dependent manner, suggesting that CDDO-Im induced malignant cell death occurs through a caspase-dependent mechanism. In summary, the synthetic triterpenoid CDDO-Im decreased the viability of WM B cells in a dose-dependent manner, and CDDO-Im had a greater effect on the viability of the malignant cells compared to non-malignant cells from the same WM patients. CDDO-Im also inhibited malignant cell growth in a dose-dependent manner and the mechanism of CDDO-Im mediated cell death appears to be a caspase-mediated event. Overall, our data indicate that CDDO-Im may have potential efficacy in WM patients.

Abstract Immunoglobulin (Ig) secretion by terminally differentiated B-cells is important in the immune response to pathogens. Its over production is a defining characteristic of many B-cell malignancies, including Waldenström Macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Thus, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for these diseases. Here, we define a novel pathway involving the oncogenic transcription factor, GLI2, modulating IgM secretion via regulation of IL-6 receptor alpha subunit (IL6Rα) expression in WM cells. We have previously identified components of the Hedgehog (HH)-GLI pathway as modulators of Ig secretion. Pharmacological (GANT61) and genetic (shRNA) inhibition of GLI proteins resulted in no significant effect on cell proliferation or viability, while there was a significant reduction in IgM secretion (60%) in BCWM.1 (p<0.0001), (55%) in MWCL-1 (p<0.0001) and (50%) in RPCI-WM1 (p<0.0001) cells. Interestingly, this occurred independent of HH signaling as manipulation of HH had no effect on IgM secretion, cell growth or viability. Screening for a mechanism identified IL6Rα as a downstream direct target of GLI activity. GANT61 treatment significantly reduces IL6Rα promoter activity in BCWM.1 (p=0.0002), MWCL-1 (P=0.0003) and RPCI-WM1 (p<0.0001) cells. GLI2 knockdown using two independent shGLI2 constructs resulted in reduced IL6Rα mRNA expression (p<0.0001) determined by qPCR, and IL6Rα protein expression examined by FACS analysis. Similarly, GLI2 knockdown diminished IL6Rα promoter activity in BCWM.1 (p=0.0218), MWCL-1 (p=0.0008) and RPCI-WM1 (p=0.0002) cells. Using bioinformatics analysis, we identified three candidate GLI2 binding sites in the IL6Rα promoter region. Using chromatin immunoprecipitation assay (ChIP) followed by qPCR, we confirmed that GLI2 directly binds to the IL6Rα promoter region (-1294 / -1065). Finally, we were able to rescue the reduction in IgM secretion in GLI2 knockdown group by overexpressing IL6Rα (p=0.0142). Taken together, these data reveal a novel mechanism where GLI2 modulates IgM secretion in WM through the regulation of IL6Rα expression. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Abstract Waldenstrom’s macroglobulinemia (WM) is a serious and frequently fatal disorder characterized by the production of a monoclonal IgM protein, a lymphoplasmacytic infiltrate in the bone marrow, and associated symptoms including anemia, lymphadenopathy and hyperviscosity. Many of the mechanisms leading to this disease are not yet known. It is clear, however, that there is dysregulation of the balance between cell proliferation and programmed cell death. BLyS (B-lymphocyte stimulator) is a TNF family member expressed by monocytes, neutrophils, macrophages, and dendritic cells. BLyS has been shown to be critical for maintenance of normal B cell development and homeostasis, and has been found to stimulate lymphocyte growth. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B-cells. Studies of the effects of BLyS on B cell physiology have shown that it also regulates immunoglobulin secretion. In previous work, we have shown that malignant B cells from patients with WM are able to bind soluble BLyS and variably express the BLyS receptors, BAFF-R, TACI and BCMA. We also found expression of BLyS in bone marrow specimens by immunohistochemistry and elevated serum BLyS levels in patients with WM. The goal of this study was to determine the functional role of BLyS-receptor ligand system in Waldenstrom’s macroglobulinemia and its relevance to the increased immunoglobulin production seen in this disease. Using cells from WM patients, we first examined the ability of BLyS to increase the secretion of IgM by malignant B cells. BLyS, alone or in combination with cytokines that induce plasmacytic differentiation and immunoglobulin production (IL-2, IL-6, IL-10 and IL-12), was found to increase IgM secretion by malignant B cells. Mean baseline IgM levels significantly increased in cells treated with BLyS (p=0.03), cytokines (p=0.0002) and a combination of BLyS and cytokines (p<0.0001). We then determined the effect of BLyS on the survival of malignant B cells using Annexin-V/PI staining. Compared to cells cultured in media alone, BLyS was found to increase viability of malignant B cells from WM patients. Cell viability was normalized relative to the media-alone control and the median relative viability increased significantly compared to controls (median increase 41.2%; range 8 – 46%). Next, we examined the ability of BLyS to modulate cell proliferation using thymidine incorporation. Using WM patient samples, BLyS was found to significantly enhance the proliferation of malignant B cells (p=0.0002). Furthermore, addition of anti-Ig antibody further enhanced the ability of BLyS to promote the proliferation of malignant B cells (p<0.0001). In summary, we have demonstrated that BLyS enhances IgM secretion by malignant B cells from patients with Waldenstrom’s macroglobulinemia. We have also demonstrated the ability of BLyS to enhance the survival and proliferation of malignant B cells. Strategies to inhibit BLyS may potentially have therapeutic efficacy in Waldenstrom’s macroglobulinemia.

Abstract The interaction between tumor cells and their surrounding microenvironment is essential for the growth and persistence of cancer cells within the host. Cytokines play a key role in mediating this crosstalk between malignant cells and the tumor microenvironment. Although this interaction is clearly established, many of the molecular mechanisms mediating these signaling events remain elusive. In previous work, we have shown that the cytokine CCL5 can regulate the expression and activity of the transcription factor GLI2 in stromal cells. GLI2 is an effector of hedgehog (HH) signaling; however, our studies reveal a role for GLI2 independent of HH. GLI2, in turn, increases the expression and secretion of IL-6 in the tumor microenvironment, highlighting a novel role for GLI2 in directly modulating cytokine genes. We therefore screened for additional cytokines regulated by this axis in HS-5 bone marrow stromal cells using expression arrays. We found that GLI2 can modulate the expression of several cytokines including CD40 ligand (CD40L), CCL2, CCL7 and CXCL11. CD40L is an immunomodulatory and proinflammatory molecule that has been shown to play a role in multiple hematologic malignancies including chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and acute myeloid leukemia (AML) pathogenesis. We first characterized the regulation of CD40L, because of the importance of CD40L in malignant cell biology. Gene expression results were validated by RT-PCR in two additional bone marrow stromal cell lines Saka and L88 where overexpression of GLI2 resulted in increased CD40L expression. Furthermore, we detected increased levels of soluble CD40L (sCD40L) in L88 and Saka cells overexpressing GLI2. Conversely, knockdown of GLI2 resulted in reduced expression and secretion of this cytokine. Biochemical mapping of the domains critical for this GLI2 regulatory mechanism reveals that N-terminal transcriptional domain is dispensable, as a mutant form of GLI2 lacking this domain (delta N-GLI2) increases CD40L gene expression by RT-PCR, protein expression by western blot and sCD40L secretion by ELISA, beyond that of the full-length GLI2 levels. Bioinformatics analysis of the CD40L promoter indicates the presence of 3 candidate binding sites for GLI proteins, suggesting the modulation of CD40L by GLI2 is mediated by direct interaction with its promoter. Ongoing experiments are aimed at determining the mechanism by which GLI2 regulates CD40L expression (either by direct binding to the CD40L promoter or through another indirect pathway). Together, our data identify a novel mechanism controlling CD40L in stromal cells in the bone marrow microenvironment. Because of the importance of CD40L in malignant cell biology, understanding the mechanism of its regulation is of great importance for the development of therapies aimed at targeting the malignant bone marrow tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1691 Poster Board I-717 Waldenström macroglobulinemia (WM) is a B-cell malignancy that is characterized by the production of a monoclonal IgM protein and a lymphoplasmacytic infiltrate in the bone marrow. The aberrant production of the monoclonal IgM can result in serum hyperviscosity that can cause significant morbidity in patients with this disease. In previous work, we have shown that IL-6 significantly upregulates IgM secretion by WM cells and that IL-6 secretion is regulated by CCL5 (Rantes). We have also shown that IL-6 mediated IgM secretion in WM requires phosphorylation of Stat1 and Stat3. Because IL-6 induced signaling involves the Jak/Stat pathway, we tested whether the use of a Jak/Stat inhibitor, TG101348, would result in down regulation of CCL5, IL-6 and IgM production and inhibit cell proliferation and viability in WM. First, we determined whether TG101348 could inhibit the production of CCL5 because other Jak inhibitors have been shown to inhibit cytokine production. Using the BCWM.1 cell line as well CD19+ malignant cells from bone marrow specimens from WM patients, we measured CCL5 by ELISA in the culture supernatant 24 hours after treatment with increasing concentrations of the inhibitor. We found that CCL5 secretion was decreased by 50% at a concentration of TG101348 of 250nM and was completely inhibited at 2μM. Next, we measured IL-6 production after treatment with TG101348. We had previously shown that stromal cells are the primary source of IL-6 and therefore used the stromal cell line HS-5 to measure IL-6 by ELISA after treatment with the inhibitor. Our previous work had also shown that IL-6 secretion was mediated by GLI (a member of the Hedgehog pathway) rather than the Jak/Stat pathway. Interestingly, we found that IL-6 secretion was inhibited in a dose dependent fashion but required higher doses for complete suppression (8μM). We then measured IgM production by malignant B-cells 24 hours after treatment with TG101348. Our previous work had shown that IL-6 mediated IgM secretion was dependent on the Jak/Stat pathway. We found that IgM production was inhibited by 50% at 500nM and completely suppressed at 2μM. Finally, we measured the effect of TG101348 on cell proliferation and survival. Using the BCWM.1 cell line, we found that cell proliferation as determined by tritiated thymidine uptake was inhibited in a dose dependent fashion with 50% inhibition at 1μM. Inhibition of cell viability as measured by Annexin V/propidium iodide staining, however, required higher concentrations and cell viability was inhibited with an IC50 of 8μM. These data confirm the role of Jak/Stat signaling in the CCL5-IL-6-IgM axis in WM. We found that TG101348 generally suppressed the signaling and growth of WM cells but that pathways that were known to be Jak/Stat dependent required significantly lower doses to be completely inhibited. These data provide a strong rationale for the use of inhibitors of this pathway, such as TG101348, in the treatment of patients with WM. Disclosures No relevant conflicts of interest to declare.

Abstract Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.

Key PointsThe NF-κB subunits p52 and RelB increase IRF4 promoter activity and expression in PTCL cells. A positive feedback loop involving CD30, NF-κB, and IRF4 drives PTCL cell proliferation and can be blocked by NF-κB inhibitors.

Abstract Background/Aim: Glioma-associated oncogene 1 (GLI1) is one of the most important transcription factors in the hedgehog (Hh) signaling pathway and has been shown to be involved in embryonic development and tumor progression. The epithelial-to-mesenchymal transition (EMT) is an important cellular program which contributes to invasion and metastasis of cancer. We have previously shown that GLI1 may promote HCC metastasis via EMT. Here, we demonstrate that GLI1 upregulates SNAI1 expression, induces EMT, and promotes HCC recurrence in vivo and in vitro. Methods: Gli1 mRNA was assessed in 139 HCCs by microarray and its correlation with HCC recurrence and SNAI1 mRNA expression was analyzed. In vitro, the mRNA and protein expression of GLI1 was measured in Gli1-negative Huh7 cells transfected with GU1 and GU1-expressing SNU398 cells transfected with antisense oligonucleotides targeting GU1 (GLI1 ASO). GLI1-dependent transcriptional activity was assessed with GU reporter luciferase assay and electrophoretic mobility shift assay (EMSA). The interaction between Gli1, SNAI1 and EMT was assessed by real-time PCR, Western immunoblotting and immunofluorescence. Effects of GLI1 on proliferation, viability, migration, and invasion of tumor cells were also assessed. Results: GLI1 mRNA was detected in 74 HCC tumors and found to be upregulated in 43 (58.1 %) HCCs. Increased Gli1 mRNA in HCCs was associated with tumor recurrence after resection. There was a positive correlation between GLI1 and SNAI1 mRNA expression. In HCC cells, GLI1 overexpression increased Gli1-dependent transcriptional activity, proliferation, viability, migration, and invasion of tumor cells. GU1 also increased SNAI1 expression and induced the EMT. Opposite effects were observed in GU1-knockdown models. Conclusion: Together, these findings identify a novel mechanism mediating HCC recurrence that includes GLI1-mediated activation of EMT via upregulation of SNAI1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B20.

AbstractAlthough proinflammatory and chemotactic cytokines can profoundly affect the tumor microenvironment, and many of them have been shown to have therapeutic efficacy in preclinical models, the role of these molecules in Waldenström macroglobulinemia (WM) remains poorly understood. In this study, simultaneous analysis of WM patient sera and bone marrow biopsies identified a set of dysregulated cytokines including CCL5, G-CSF, and soluble IL-2 receptor, that were significantly elevated in WM patients whereas IL-8 and EGF levels were significantly lower in these patients compared with healthy controls. Interestingly, CCL5 levels positively correlated with features of disease aggressiveness such as elevated IgM levels and bone marrow involvement. Functional analysis of tumor microenvironment revealed a functional correlation between CCL5 levels and IL-6 levels, a proinflammatory cytokine with an important role in normal and malignant B-cell biology. Furthermore, CCL5 stimulated IL-6 secretion in WM stromal cells resulting in increased IgM secretion by WM malignant cells via the JAK/STAT signaling pathway. Thus, together these results define a novel signaling network in the WM tumor microenvironment controlling IgM secretion and suggest CCL5 as a potential target for the treatment of this disease.

Abstract Crosstalk between signaling pathways is crucial to the initiation, progression and maintenance of the transformed phenotype. Therefore, characterization of these signaling interactions is fundamental for the understanding of the complex and varied network of events that leads to the development and spread of tumors. The Hedgehog (HH)-GLI pathway, an important carcinogenic cascade, is required for the growth and survival of tumors of diverse origin. Recent reports, mainly from animal models, suggest that key signaling interactions are required by HH-GLI signaling to modulate cancer-associated cellular functions. However, the molecular mechanisms underlying this phenomenon in cancer and non-cancer cells remain elusive. Our preliminary data show a crosstalk between the HH-GLI axis and TGF-beta pathway, a well-characterized tumor suppressor/oncogenic signaling pathway. Luciferase reporter and expression assays in combination with genetic and pharmacological manipulations of the TGFbeta pathway show that this cascade is able to activate GLI transcriptional activity, even in the absence of an active HH signaling. The TGFbeta pathway is required to maintain active GLI transcriptional complexes at GLI target genes. Further analysis of this molecular interaction suggests that TGFbeta pathway-mediated activation of GLI transcription factors requires an intact SMAD4, thus suggesting a potential mechanism mediating this functional interaction. The knowledge derived from this study provides both mechanistic insight into the contribution of this HH-GLI/TGF-beta pathway interaction to carcinogenesis and could serve as a foundation for development of new therapeutic approaches for tumors with active HH-GLI and TGF cascades. 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 3917.