Ziesmer, Steve

Abstract 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.

Abstract The objective of this study was to analyze molecular changes that occur at the protein level in WM and IgM-MGUS in order to identify proteins dysregulated at disease progression and design new therapies. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between WM, IgM-MGUS and normal lymphoplasmacytic cells as control. The antibody array is a novel technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto a glass slide spotted with 512 different monoclonal antibodies specific for human proteins. CD 19+ and CD138+ purified lymphoplasmacytic cells were obtained from cryopreserved bone marrow samples of 5 newly diagnosed patients with WM and 5 patients with IgM-MGUS. Control plasma cells were obtained from 3 pooled CD19+ and CD138+ purified normal donor bone marrow cells. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software and changes of protein expression ≥2 fold in 60% of the samples as compared to normal control were identified in both WM and MGUS groups. There were 20 proteins upregulated in WM and 21 proteins in MGUS as compared to normal control cells These included proteins from the small GTPases such as the Ras, Rho and Ran families (Rab 4, MONA, RCC1, and CDC42GAP), Kinases/signaling proteins (ROK alpha, p62 dok, JAK, 14-3-3e), cyclin dependent kinase (CDK2), fatty acid synthase (HDAC3), NFkB pathway (IKKalpha, IKBe), and histone deacetylase. The WM and IgM MGUS differed in only 3 proteins using the 2-fold threshold. The WM samples showed overexpression of the heat shock protein Hsp90, the Ras family protein CDC25C, and the chemotaxis protein p43/EMAPII as compared to the MGUS samples. This is the first proteomic study of WM and MGUS patients. It identifies novel proteins dysregulated in WM that may serve in future clinical trials as targets for therapy such as Hsp90, NFkB, HDAC and fatty acid synthase. In addition, it identifies 3 proteins that identify disease progression from MGUS to WM, which could be useful for monitoring MGUS patients and designing targeted therapies to prevent progression to WM.

Abstract Abstract 1570 B-cell non-Hodgkin lymphoma encompasses a heterogeneous group of B-lymphocyte-derived malignancies that are characterized by chromosomal translocations involving the immunoglobulin (IG) gene loci and specific histological subtypes of disease are associated with a different spectrum of IG translocations. Marginal zone derived B cell lymphomas encompass three distinct entities: extranodal marginal zone B lymphoma (MZL) of mucosa associated lymphoid tissue (MALT), nodal MZL (NMZL), and splenic MZL (SMZL). MALT lymphoma is genetically unique and four mutually exclusive chromosomal translocations have been identified in this disease thus far. However, the known translocations are only present in a subset of cases suggesting that additional uncharacterized translocations or other genetic events may exist that contribute to disease development. In previous studies we characterized the novel t(X;14)(p11.4;q32) translocation in a patient with MALT lymphoma and found that GPR34, an orphan G-protein coupled receptor (GPCR), was highly expressed due to its juxtaposition to the IGHSA2 switch region. We then measured GPR34 mRNA expression in tissue biopsies from a panel of MALT, NMZL, SMZL, lymphoplasmacytic (LPL), diffuse large B cell (DLBCL), follicular (FL), and mantle cell (MCL) lymphomas. Expression of GPR34 was detected in all tissues examined, but was significantly increased in MALT (37 fold), LPL (23 fold), NMZL (18 fold), and SMZL (21 fold) compared to normal CD19+ B cells. GPR34 mRNA expression was also analyzed by gene expression profiling (GEP) of a panel of lymphomas or control lymphoid tissue and we confirmed elevated expression of GPR34 in the t(X;14) case and detected elevated GPR34 mRNA expression in MALT, LPL, NMZL, SMZL, and ABC DLBCL. When we grouped specimens by high and low expression of GPR34, a clear molecular subtype of SMZL could be identified and suggests that GPR34 high expressing tumors have a unique GEP. To confirm the qPCR and GEP analysis of GPR34 expression, we analyzed surface expression of GPR34 by flow cytometry of normal and malignant tissues and found that expression of GPR34 was detected on all MALT and SMZL B cells with an average DMFI of 2.08 (DMFI range 1.26–3.18, n=12) and on the JeKo-1 lymphoma B cell line (DMFI= 4.75). Analysis of CD19+ cells from normal controls (n=3) revealed a low level of GPR34 on (average DMFI =1.12). Little is known about the function of GPR34 and to further characterize the impact of GPR34 overexpression on cell signaling, OCI-LY19 lymphoma B cells and HeLa cells were transduced with GPR34 or a vector control. To determine if MAPK activity, a pathway regulated by GPCRs, was upregulated, the phosphorylation status of ERK1/2 in GPR34 or control cells was analyzed and we found that ERK1/2 was constitutively phosphorylated at significantly higher levels in GRP34 expressing cells. To further explore which signaling pathways were affected by GPR34 overexpression, GPR34 or vector control cells were transfected with an AP-1, CRE, NF-kB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid and GPR34 expressing cells had significantly increased luciferase activity driven by AP-1 (5.35-fold), CRE (4.7-fold), NF-kB (2.6-fold), and E2F (2.1) when compared to vector control cells. To determine the biologic impact of GRP34 overexpression, the proliferation rates of vector control and GPR34 cells were compared and we found that proliferation of GPR34 WT cells was significantly higher than that seen in the control. We next tested the effect of the MEK inhibitor on proliferation and saw a dose dependent decrease in proliferation of GPR34 expressing cells. We then tested the ability of GPR34 cells to generate colony formation in a soft-agar colony formation assay. After 3 weeks of culture, the GPR34 cells had formed significantly more colonies than the vector control suggesting that overexpression of GPR34 can result in increased anchorage-independent cell growth. Taken together, our results identify a novel translocation in MALT lymphoma, emphasize a novel role for GPR34 in regulation of gene expression and tumor cell growth, and suggest that MEK inhibitors may be useful in a subset of translocation–harboring or GRP34 high-expressing tumors. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Experiments were designed to characterize endothelin receptors in arteries after chronic increases in blood flow. A fistula was created between the femoral artery and vein in one hindlimb of dogs; contralateral blood vessels were sham operated. Sham- and fistula-operated arteries were removed 6 wk postoperatively. Some arteries were prepared for measurement of isometric force or for isolation of membrane proteins. Other arteries were used for histological staining with an endothelin-B (ETB) receptor antibody. In arteries suspended for the measurement of isometric force, endothelin-1 produced concentration-dependent increases in tension that were significantly greater in fistula- than in sham-operated arteries without endothelium. The ETB-receptor-selective peptide sarafotoxin S6c produced concentration-dependent increases in tension only in fistula-operated arteries. In receptor-binding studies of membrane proteins, Scatchard analysis of saturation binding with 125I-labeled endothelin-1 (125I-endothelin-1) indicated that the total number of receptors was greater in fistula-operated arteries; affinity was threefold less in fistula- than in sham-operated arteries. Competitive displacement of 125I-endothelin-1 by endothelin-3 was significant for a two-site model in membranes prepared from sham-and fistula-operated arteries. Competitive inhibition of 125I-endothelin-1 binding by sarafotoxin S6c was significant for a one-site binding model in all arteries. Sarafotoxin S6c binding sites were elevated significantly in fistula-operated arteries. Immunohistochemical staining for the ETB receptor was significantly greater in both the endothelium and smooth muscle of fistula- than in sham-operated arteries. These results suggest that chronic increases in blood flow upregulate endothelin receptors, including ETB receptors in arterial smooth muscle.

Abstract Cytokines within the tumor microenvironment play an important role in supporting the growth and survival of B-cell malignancies. One such cytokine, IL-21, promotes 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 Waldenstrom macroglobulinemia (WM), a B-cell lymphoma characterized by elevated serum IgM and a lymphoplasmacytic bone marrow infiltrate. We report here on the presence of IL-21 in the bone marrow of patients with WM and have identified activated T cells as the source of this cytokine. We readily detected the IL-21 receptor on malignant WM B cells and show that IL-21 significantly increases both IgM secretion and cellular proliferation of these cells with no effect on viability. IL-21 rapidly induces phosphorylation of STAT3 in WM cells, and treatment of the WM cell line MWCL-1 with a STAT3 inhibitor abolished the IL-21–mediated increases in cellular proliferation and IgM secretion. IL-21 also increased the expression of known STAT3 targets involved in B-cell differentiation, including BLIMP-1, XBP-1, IL-6, and IL-10. 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.

AbstractCytokines are deregulated in cancers and can contribute to tumor growth. In patients with diffuse large-cell lymphoma (DLBCL), we observed higher levels of JAK/STAT pathway-related serum cytokines (ie, IL-6, IL-10, epidermal growth factor, and IL-2) compared with controls. Of these, only IL-10 activated the JAK2 pathway in lymphoma cells in vitro. Patients with high serum IL-10 had shorter event-free survival (EFS) than patients with low levels (P > .01) and high IL-10 was correlated with high lactase dehydrogenase (P = .0085) and higher International Prognostic Index scores (P = .01). To explore the mechanism by which IL-10 may contribute to an inferior EFS, we investigated the effect of IL-10 on the JAK2 pathway and found that the IL-10/IL-10 receptor complex up-regulated JAK2 signaling. Neutralizing Ab to IL-10 inhibited constitutive and IL-10–induced JAK2/STAT3 phosphorylation. JAK2 inhibition dephosphorylated JAK2 and STAT3 and caused an inhibitory effect on phospho-JAK2–positive DLBCL cells; there was a minimal effect on phospho-JAK2–negative cells. Apoptosis induced by JAK2 inhibition was dependent on inhibition of autocrine IL-10 and c-myc expression and independent of Bcl-2 family expression. These results provide the rationale for testing JAK2 inhibitors in DLBCL patients, and indicate that serum IL-10 may be a biomarker to identify patients more likely to respond to JAK2-targeted therapy.