t(X;14)(p11;q32) in MALT lymphoma involving GPR34 reveals a role for GPR34 in tumor cell growth

Abstract

Genetic 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.

Figure 1

Cloning of the of the t(X;14)(p11;q32) translocation breakpoint in MALT lymphoma. (A) Sequence of the t(X;14)(p11;q32) breakpoint. DNA sequence from Xp11.4 is shown in gray and IGHA2 in black. (B) Chromosomal location of the breakpoints on chromosome 14 and X and the structure of der. Idiogram Album: copyright 1994 David Adler. (C) Interphase FISH analysis of the t(X;14) index case. In the left panel the Xp11.4 BAP probe shows a 1R2G1F pattern, indicative of splitting of Xp11.4 as well as an extra copy of the distal (green) Xp arm. In the middle panel the IGH BAP probe shows a 2R1G1F pattern, consistent with the presence of 2 translocations, the first occurring within the typical IGH breakpoint region (indicated by 1 red and 1 green signal) and the second occurring proximally to the typical IGH breakpoint (indicated by 1 red and 1 fusion signal). In the right panel the t(X;14)(p11.4;q32) dual fusion probe shows a 1R1G3F signal pattern. (D) Array CGH analysis of tumor cells from the t(X;14) patient, showing an extra copy of the distal portion of chromosome X up to the position of the Xp11.4 BAP FISH probe. (E) Predicted gene organization of der.

Figure 2

GPR34 is deregulated by the t(X;14) translocation and is elevated in lymphoma tissue. (A) qPCR for GPR34, GPR82 and CASK from mRNA isolated from the t(X;14) specimen or CD19⁺ B cells from peripheral blood (PBL; n = 2), bone marrow (BM; n = 2), or spleen (Spl; n = 3) from healthy donors. Relative concentrations of GPR34 (GPR34/GAPDH) are expressed in copies/μL. (B) qPCR for GPR34 in MALT (n = 35), NMZBCL (n = 21), SMZBCL (n = 33), LPL (n = 23), DLBCL (n = 9), FL (n = 10), and MCL (n = 10) lymphomas, and normal resting (n = 11) or activated (n = 6) CD19⁺ B cells from peripheral blood or bone marrow (n = 5), resting and activated CD3⁺ T cells (n = 6) and CD14⁺ monocytes (n = 6). Analyses shown are the average of 2 independent experiments for each sample. *Compared with normal resting B cells, MALT (P = .03), LPL (P = .0006), NMZBCL (P = .0007), and SMZBCL (P = .001) had significantly higher GPR34 expression. (C) GPR34 gene expression analysis in lymphoma and brain tumor tissue; MALT (n = 24), NMZBCL (n = 24), SMZBCL (n = 28), LPL (n = 24), ABC DLBCL (n = 74), GCB DLBCL (n = 71), PBMCL (n = 31), normal LN (n = 8), naive B cells (n = 2), memory B cells (n = 2), glioma (n = 21), brain cell lines (n = 12), and normal brain (n = 3). (D) GPR34 expression on CD19⁺ tumor cells from MALT or SMZBCL biopsies, normal blood lymphocytes, and JeKo-1 lymphoma B cells. Expression of GPR34 is shown in the open histogram, isotype control in the gray histogram.

Figure 3

Overexpression of GPR34 drives ERK1/2 activation and ERK1/2-mediated gene transcription in HeLa and lymphoma B cells. (A) HeLa cells expressing vector control, WT or DRY were analyzed for GPR34 expression by flow cytometry. (B) HeLa cells were transiently transfected with a YFP vector control or a YFP-tagged GPR34 and were visualized by confocal microscopy. (C) HeLa vector control, WT, or DRY cells were analyzed for total and phosphorylated forms of ERK1/2 with and without PMA activation by Western blot. (D) Average fold increase in ERK1/2 phosphorylation. Data are normalized to total ERK1/2. *Compared with GPR34-DRY, GPR34 WT cells had a significant increase in ERK1/2 phosphorylation (P = .05, n = 5). (E) HeLa vector control or WT cells were transiently transfected with reporter gene plasmids that have the firefly luciferase gene under the control of a specific cis-acting; pNFAT-luc, pISRE-luc, pSRE-luc, pE2F-luc, pNFKB-luc, pCRE-luc, or pAP1-luc. Data represent an average fold increase in luciferase activity over vector control (n = 3). (F) Activation of the pAP1 and pCRE reporters in vector control, WT, and DRY cells in the absence or presence of increasing doses (25-100μM) of the MEK inhibitor PD98059 (n = 2). *Compared with the nil control, WT cells have a significant increase in pAP1 (P = .04) and pCRE (P = .002) activity. (G) OCI-Ly19 vector control or WT cells were analyzed for total and phosphorylated forms of ERK1/2, PKC, and CREB Western blot.

Figure 4

GPR34 overexpression enhances cell proliferation of HeLa and lymphoma B cells. (A) Proliferation of HeLa vector control, WT, or DRY cells alone or in the in the presence of 25 to 100μM PD98059 was analyzed. *Compared with the vector control, WT cells have a significant increase in cell proliferation (P < .0001). (B) Tumor colony-forming assays were performed on HeLa vector control, WT, and DRY cells. A media control with no cells was included as a negative control. Photomicrograph of tumor colony in inset, scale bar, 100μM. *Compared with the vector control (P = .05) or DRY (P = .01), WT cells have a significant increase in colony formation (n = 3). (C) Proliferation of OCI-Ly19 vector control or WT cells alone or in the presence of 50 to 100μM PD98059 was analyzed (n = 3, representative experiment shown). *Compared with the vector control, WT cells have a significant increase in cell proliferation (P = .0002).

Figure 5

High expression of GPR34 distinguishes a unique molecular subset of MZLs and correlates with GPR82 and SOX11 expression. (A) GEP data from NMZBL tumors (n = 34) was used to classify GPR34 high and low-expressing tumors. High is defined as those tumors that express GPR34 at levels greater than 2 standard deviations above the mean of the normal controls. (B) Heatmap of GPR34 high and low expressing NMZBCL tumors. (C) Table of 10 most up-regulated and down-regulated genes in GPR34 high tumors. (D) Correlation of GPR34 expression with GPR82 and SOX11 in MZL (n = 83).