Dyer, Martin J.S.

Abstract MALT lymphoma is a genetically unique disorder and five mutually exclusive chromosomal translocations have been identified thus far in this disease: t(11;18), t(14;18), t(1;14), t(1;2) and t(3;14). Despite this genetic heterogeneity, all but one of the translocations affect the NF-κB signaling pathway, which is critical if not essential for antigen receptor mediated B- and T-cell activation and likely enhances MALT lymphoma growth. However, the known translocations are present in only 25% of cases suggesting that additional uncharacterized translocations exist. We used long-distance inverse polymerase chain reaction (LDI-PCR) technique to clone a novel IGH translocation partner in a 60-year-old female with a history of Sjogren’s syndrome and primary MALT lymphoma involving the parotid gland. The breakpoint on chromosome 14 occurred within the IGHSA2 switch region while the breakpoint on chromosome X fell within Xp11.4. The breakpoint on chromosome Xp11.2 fell between two genes, GPR82 and GPR34, both of which code for orphan G-protein coupled receptors (GPCRs). The breakpoint also fell within and disrupted a larger gene called CASK (a membrane-associated guanylate kinase-MAGUK). To determine the prevalence of this translocation in MALT lymphoma we performed interphase FISH studies on 64 MALT lymphomas using a breakapart probe for GPR82. Only the index case had an abnormal split signal pattern. We next designed primers to perform real-time quantitative RT-PCR for the genes located on Xp11 and found that GPR34 RNA was highly expressed, a 49-fold increase, in the patient carrying the t(X;14)(p11.4;q32) translocation compared to a normal splenic B cell control. Expression of GPR82 and CASK RNA was similar between normal B cells and the patient carrying the t(X;14)(p11.4;q32) translocation. These data suggest that the GPR34 gene is dysregulated upon its translocation to the IGHSA2 switch region. We then measured GPR34 RNA expression in a panel of MALT lymphomas (n=12) and found that GPR34 was expressed at levels higher than that seen in normal B cells with an average increase of 11 fold, 9/11 of which had an expression lever greater than 3-fold over normal splenic B cells. In a gastric MALT lymphoma specimen arising in a 67-year-old male, we saw a 64 fold increase in GPR34 expression. Interphase FISH studies performed on this specimen showed an extra intact GPR34 signal but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. GPR34 RNA was also detected in other normal B cell populations and histologic subtypes of NHL but not to the extent seen in MALT lymphoma; values represent the expression of GPR34 normalized to β-actin and a normal B cell control (value of 1.0): t(X;14)(p11.4;q32) specimen, 49.0; MALT lymphoma, 11.0 (n=12); peripheral blood B cells, 0.48 (n=2); normal bone marrow B cells, 0.97 (n=3); follicular lymphoma, 2.47 (n=3); marginal zone B cell lymphoma, 2.47 (n=3); diffuse large cell lymphoma, 0.36, (n=3); mantle cell lymphoma, 2.67 (n=3); and multiple myeloma 0.52 (n=6). The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCR and GPR34 mRNA transcripts are particularly abundant in mast cells while lower levels were detected in other immune cells including B cells. However, little is known about its natural ligand, biologic function, or the signaling cascades activated by its engagement. Because the NF-κB signaling pathway has been shown to be a common downstream target of MALT lymphoma translocations we first examined the impact of GPR34 expression on phosphorylation of Iκ-Bα. Transient expression of a YFP-GPR34 expression plasmid in HeLa cells results in increased phosphorylation of Iκ-Bα compared to the YFP control. Additionally, we observed increased phosphorylation of ERK1/2 in GPR34-expressing cells, however no change in phosphorylation of GSK3β was detected. In summary, these data identify a novel IGHS translocation partner in MALT lymphoma and suggest that dysregulation of GPR34 is commonly found in MALT lymphoma. Overexpression of GPR34 results in activation of the NF-κB and MAP kinase pathways and may be a novel mechanism by which MALT lymphoma occurs.

Abstract Abstract 3927 Poster Board III-863 In previous studies we characterized the 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. As part of a larger MALT gene expression-profiling project, we have now acquired gene expression analysis on the patient carrying the t(X;14)(p11;q32) translocation and have confirmed overexpression of GPR34. We then measured GPR34 mRNA expression in a panel of MALT lymphomas (n=17) and found that GPR34 was expressed at levels higher than that seen in normal B cells (mean, 11.3 fold; median, 5.5; range, 1.4-64 fold). When analyzed separately, 70% (12/17) had an expression level greater than 3-fold over normal B cells. Of note, in a gastric MALT lymphoma specimen, we found a 64 fold increase in GPR34 mRNA expression. FISH studies performed on this specimen showed an extra intact GPR34 signal but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. Elevated expression of GPR34 mRNA was also detected in other histologic types of NHL, but not to the extent seen in MALT lymphoma. Taken together, these data suggest that GPR34 is commonly overexpressed in MALT lymphoma and that deregulation of GPR34 expression can occur independent of a t(X;14)(p11.4;q32) translocation. The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCR and GPR34 mRNA transcripts are abundant in mast cells while lower levels were detected in other immune cells including B cells. Signals from GPR34 have been briefly described and the results to date suggest that overexpression of GPR34 results in an accumulation of inositol phosphates. To further characterize the impact of GPR34 overexpression on cell signaling, HeLa cells were transduced with a retroviral expression plasmid (pBMN-GFP) that expresses GPR34 and GFP. GFP expressing cells were isolated and overexpression of GPR34 mRNA was confirmed by PCR and GPR34 protein expression was detected by flow cytometry. When normalized to the isotype control, pBMN-GPR34 cells expressed 17-fold more GPR34 on their cell surface compared to the pBMN-vector control cells. To determine which signaling pathways were affected by GPR34 overexpression, pBMN-GPR34 or pBMN-vector control cells were transfected with an AP-1, CRE, NF-κB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid. Upon normalization with renilla, pBMN-GPR34 expressing cells had increased luciferase activity (n=3) driven by AP-1 (5.35-fold), CRE (4.7), NF-κB (2.8-fold), and E2F (2.13) when compared to pBMN-vector control cells. ISRE, NFAT, and SRE mediated luciferase expression was similar in the GPR34 and control cells. AP-1 and CRE have been implicated in a large variety of cellular processes, including transformation, and both AP-1 and CRE activity is induced upon activation of MAP kinases. To determine if MAPK activity was also upregulated in GPR34 expressing cells, we analyzed the phosphorylation status of Erk1/2 in pBMN-GPR34 cells by western blot and found that Erk1/2 was constitutively phosphorylated in GRP34 expressing cells (1.8 fold increase) compared to vector control cells. Increased phosphorylation of PKC-α/β was also detected in pBMN-GPR34 cells (3.5 fold increase compared to control cells). To determine the biologic impact of GRP34 overexpression on cell growth, the proliferation rates of pBMN-control and pBMN-GPR34 cells were compared and it was found that proliferation of GPR34 expressing cells was 2.2 times higher than that seen in control cells. Because the MAPK kinase pathway was found to be active in the pBMN-GPR34 cells, we tested the effect of the MEK inhibitor PD98059 on proliferation and saw a dose dependent decrease in proliferation of GPR34 expressing cells. These results suggest that GPR34-mediated proliferation is Erk-dependent. In summary, these data suggest that deregulation of GPR34 is commonly found in MALT lymphoma and that overexpression of GPR34 results in activation of Erk1/2, phosphorylation of PKC, and results is AP-1 and CRE mediated transcription. Additionally, our data suggest that overexpression of GPR34 results in increased cell growth that is MAPK-dependent. Taken together, this studies indicate that overexpression of a GPCR, GPR34, may be a novel mechanism by which MALT, lymphoma, and potentially other subtypes of NHL, develop. Disclosures: No relevant conflicts of interest to declare.

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 1999 Recurrent chromosomal translocations involving the IGH locus are characteristic of B-cell non-Hodgkin lymphoma (NHL) and MALT lymphoma in particular has been pathologically characterized by specific IGH translocations. However, these translocations are present in only a subset of cases, suggesting that additional uncharacterized translocations might exist. 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. Although the t(X;14)(p11;q32) translocation was only identified in one patient, we wanted to determine its expression across a spectrum or normal and malignant lymphoma tissue, as other mechanisms may contribute to overexpression of genes. We measured GPR34 mRNA expression by quantitative PCR in tissue biopsies from a panel of lymphomas. As controls, we also measured GPR34 mRNA expression in normal resting or activated CD19+ B cells, resting and activated CD3+ T cells, and CD14+ monocytes. Expression of GPR34 was significantly increased in MALT (37-fold, n=35), LPL (23-fold, n=23), nodal marginal zone B cell lymphoma (18-fold, n=21), and splenic marginal zone B cell lymphoma (21 fold, n=33) compared to resting CD19+ B cells (n=11). In a gastric MALT lymphoma specimen we detected GPR34 expression at levels similar to the t(X;14)(p11;q32) patient and interphase FISH studies showed an extra intact GPR34 signal, but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCRs and signaling studies suggest that overexpression of GPR34 results in an accumulation of inositol phosphates and MAP-kinase activation. To further characterize the impact of GPR34 overexpression on cell signaling, HeLa cells were transduced with WT GPR34, a signaling deficient GPR34 (GPR34 DRY), or a vector control. To determine if MAPK activity was upregulated, the phosphorylation status of ERK1/2 in GPR34 WT, GPR34 DRY, and vector control cells was analyzed and we found that ERK1/2 was constitutively phosphorylated at higher levels (1.6-fold) in GRP34 WT cells compared to GRR34 DRY or vector control cells. To further explore which signaling pathways were affected by GPR34 overexpression, GPR34 WT or vector control cells were transfected with an AP-1, CRE, NF-kB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid. GPR34 expressing cells had 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. GPR34 DRY was unable to activate these pathways. AP-1 and CRE have been implicated in a large variety of cellular processes, including transformation, and both AP-1 and CRE activity is induced upon activation of MAP kinases. In untreated cells, both AP-1 and CRE activity were significantly higher in GPR34 WT cells compared to GPR34 DRY cells and both AP-1 and CRE luciferase activity was inhibited by the MEK1 inhibitor PD98059, suggesting that activation of these pathways is MAP-kinase dependent. To determine the biologic impact of GRP34 overexpression, the proliferation rates of vector control, GPR34 WT, and GPR34 DRY cells were compared and we found that proliferation of GPR34 WT cells was significantly higher (5.6-fold) than that seen in GPR34 DRY cells. We next tested the effect of the MEK inhibitor on proliferation and saw a dose dependent decrease in proliferation of GPR34 WT expressing cells. These results suggest that GPR34-mediated proliferation is dependent on MAPK. The presence of elevated GPR34 expression in tumors and the increased proliferation mediated by GPR34 suggests that it may have oncogenic potential. We therefore tested the ability of GPR34 WT cells to generate tumors in a tumor colony-forming assay. Only the GRP34 WT cells had the ability to form tumors in soft agar suggesting that overexpression of GPR34 can result in tumor formation. Taken together, our results identify a novel translocation in MALT lymphoma, emphasize a novel role for GPR34 in tumor cell growth, and suggest that MEK inhibitors may be useful in a subset of translocation-harboring or GRP34 high-expressing tumors. Disclosures: Fonseca: Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

AbstractGenetic aberrations, including trisomies 3 and 18, and well-defined IGH translocations, have been described in marginal zone lymphomas (MZLs); however, these known genetic events are present in only a subset of cases. Here, we report the cloning of an IGH translocation partner on chromosome X, t(X;14)(p11.4;q32) that deregulates expression of an poorly characterized orphan G-protein–coupled receptor, GPR34. Elevated GPR34 gene expression was detected independent of the translocation in multiple subtypes of non-Hodgkin lymphoma and distinguished a unique molecular subtype of MZL. Increased expression of GPR34 was also detected in tissue from brain tumors and surface expression of GPR34 was detected on human MZL tumor cells and normal immune cells. Overexpression of GPR34 in lymphoma and HeLa cells resulted in phosphorylation of ERK, PKC, and CREB; induced CRE, AP1, and NF-κB–mediated gene transcription; and increased cell proliferation. In summary, these results are the first to identify a role for a GPR34 in lymphoma cell growth, provide insight into GPR34-mediated signaling, identify a genetically unique subset of MZLs that express high levels of GPR34, and suggest that MEK inhibitors may be useful for treatment of GPR34-expressing tumors.

Abstract We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10−16). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.

Abstract Chronic lymphocytic leukemia (CLL) and other B-cell lymphoproliferative disorders display familial aggregation. To identify a susceptibility gene for CLL, we assembled families from the major European (ICLLC) and American (GEC) consortia to conduct a genome-wide linkage analysis of 101 new CLL pedigrees using a high-density single nucleotide polymorphism (SNP) array and combined the results with data from our previously reported analysis of 105 families. Here, we report on the combined analysis of the 206 families. Multipoint linkage analyses were undertaken using both nonparametric (model-free) and parametric (model-based) methods. After the removal of high linkage disequilibrium SNPs, we obtained a maximum nonparametric linkage (NPL) score of 3.02 (P = .001) on chromosome 2q21.2. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under a common recessive model of disease susceptibility (HLOD = 3.11; P = 7.7 × 10−5), which was significant at the genome-wide level. In addition, 2 other chromosomal positions, 6p22.1 (corresponding to the major histocompatibility locus) and 18q21.1, displayed HLOD scores higher than 2.1 (P < .002). None of the regions coincided with areas of common chromosomal abnormalities frequently observed in CLL. These findings provide direct evidence for Mendelian predisposition to CLL and evidence for the location of disease loci.