Abstract
Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined1,2. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells3,4. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor’s Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt’s lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.
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Acknowledgements
We thank S. Coughlin for Gna13f/f and Arhgef1−/− mice and R. Proia for S1pr2−/− mice. We thank X. Geng and G. Doitsch for assistance with processing of human tonsil, A. Reboldi for discussion, and T. Arnon and O. Bannard for reading the manuscript. J.R.M. is supported by a Fellow Award from the Leukemia & Lymphoma Society and by National Institutes of Health (NIH) institutional training grants (T32 DK007636 and T32 CA1285835); R.S. is supported by the Dr Mildred Scheel Stiftung für Krebsforschung (Deutsche Krebshilfe). N.V. was supported by NIH grant GM097261 for the modelling work. J.G.C. is an Investigator of the Howard Hughes Medical Institute. The human lymphoma samples were studied under the auspices of the Lymphoma/Leukemia Molecular Profiling Project. The work was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and NIH grant AI45073.
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Authors and Affiliations
Contributions
J.R.M. designed and performed experiments, interpreted the results and wrote the manuscript. R.S. performed sequencing of cell lines and primary samples, and analysed data. J.A.G. designed experimental procedures used in the manuscript. W.X. analysed sequence data. A.B.L and N.V. performed computer modelling of S1PR2. S.E.B. performed western blots of S1PR2. J.A. performed mouse genotyping and cared for the mouse colony. Y.X. performed quantitative PCR. A.R., G.O., R.D.G., L.M.R., E.C., E.S.J., J.D., E.B.S., R.M.B., R.R.T., J.R.C., D.D.W. and W.C.C. supplied lymphoma patient samples or lines, and reviewed pathological and clinical data. L.M.S. coordinated human sequence analysis, analysed data and supervised research. J.G.C. designed experiments, supervised research and wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Lymphoma-associated mutations result in loss of expression and function of S1PR2.
a–c, Surface expression of Flag (a) quantitative PCR of human S1PR2 (b) or Thy1.1 reporter expression (c) in mouse WEHI231 B lymphoma cells transduced as described in Fig. 1b. Shown in a are histograms of transduced cells (Thy1.1+) in blue and untransduced cells (Thy1.1−) in grey. Five of eight S1PR2 mutations showed loss of protein expression despite strong transcript and reporter expression. Loss of expression in these five mutants was probably a result of degradation of improperly folded proteins in the endoplasmic reticulum. d, Representative FACS plots of transwell migration of WEHI231 cells transduced with vector, WT or R147C mutant S1PR2 to the indicated stimuli or the input sample. Numbers indicate percentage of cells positive for the Thy1.1 reporter. e, WEHI231 cells stimulated as in Fig. 1d were analysed for phosphorylation of Akt (pAkt S473) by western blot or by intracellular FACS. Data in a and c are representative of four independent experiments. Data in b are from one experiment. Data in d and e are representative of three independent experiments.
Extended Data Figure 2 Frequency of mutations in GNA13, S1PR2 and P2RY8 in aggressive lymphoma.
a, b, Summary of overall mutation frequencies (a) and allelic frequencies (b) of non-synonymous coding mutations in S1PR2, GNA13 and P2RY8 in GCB-DLBCL, Burkitt’s lymphoma or ABC-DLBCL cases shown in Supplementary Table 2. Unmutated indicates no coding region mutations in the genes shown. Since the sequencing was performed on genomic DNA, the data may underestimate the frequency of biallelic cases as some disruptive mutations may occur in non-coding regulatory elements.
Extended Data Figure 3 S1PR2 heterozygosity confers a survival advantage to GC B cells and R147C S1PR2 fails to function.
a, b, Flow cytometry of follicular and GC B cells from mesenteric lymph node and Peyer’s patches of mixed bone-marrow chimaeras generated as in Fig. 1e. Gating strategy for follicular B cells and GCB in mesenteric lymph node is shown in a and percentages of CD45.2+ cells in follicular and GC B cells from Peyer’s patches are shown in b. Data in b are pooled from four independent experiments. c, d, Gating strategy of Thy1.1 reporter expression in follicular and GC B cells from Peyer’s patches (c) or fold change in Thy1.1+ cells in GC relative to follicular B cells of mesenteric lymph node (d) of retrovirally transduced bone-marrow chimaeras as described in Fig. 1f. Data in d are pooled from three independent experiments. *P < 0.05, ***P < 0.001, unpaired two-tailed Student’s t-test. There was increased variability in mesenteric lymph node relative to Peyer’s patches when WT S1PR2 was transduced into S1PR2+/− bone marrow. Nine of 17 animals reconstituted with S1PR2+/− bone marrow transduced with WT S1PR2 showed a reduction in expression of Thy1.1 in mesenteric lymph node GC relative to follicular B cells, whereas in six of eight animals reconstituted with R147C S1PR2 there was increased reporter expression. e, The hydrogen bond formed between Y141 in ICL2 and D130 on transmembrane helix 3 (TM3) has been observed only in the active state of β2-adrenergic receptor (shown in pink) and not in the inactive state (shown in cyan). f, Population distribution of the conformational states showing the predicted hydrogen bond network between R147 (TM4), Y140 (ICL2) and E129 (TM3) of the WT (solid lines) and R147C mutant (dashed lines) of S1PR2. g, The network of predicted hydrogen bonds mediated by Y140 on ICL2. The hydrogen bond network tightens the interactions between transmembrane helices TM3 and TM4. We hypothesize that this network stabilizes the putative active state conformation of S1PR2. Such a network is broken in the R147C mutant and hence this mutant does not activate the G protein.
Extended Data Figure 4 Aged Gα13-deficient mice develop GC-derived lymphoma.
a, Quantitative PCR analysis of Gna12 and Gna13 transcript abundance in follicular and GC B cells relative to the control gene Ptprc. b, Flow cytometry of follicular and GC B cells from Peyer’s patches of mixed bone-marrow chimaeras as described in Fig. 1g. c, PCR analysis of VHJ558–DJH, Vκ–Jκ and Vλ–Jλ rearrangements from indicated tissues of Gna13 KO animals. The space in the gel image marks the position of lanes that were not relevant to this experiment and were removed for clarity. This PCR analysis was done using bulk rather than sorted GC B cells from tumours and thus probably under-reports the number of animals with clonal outgrowths. Samples scored as having clonal outgrowths (and thus probably harbouring tumours) were numbers 307, 377, 418, 1310 and 443. In the case of number 307, the splenic nodule and enlarged Peyer’s patches showed enrichment of the same VHJ558 clonal bands observed in the mesenteric lymph node. d, Gross appearance of small intestine of Gna13 KO number 307 mouse. Box denotes enlarged Peyer’s patches analysed by PCR in c; arrows denote two uninvolved Peyer’s patches. Scale bar, 1 cm. e, Immunohistochemical analysis of splenic nodule from number 307 (see Fig. 1l) for GC marker GL7 (blue) and naive B-cell marker IgD (brown). Scale bar, 500 μm. f, Control or enlarged Gna13 KO mesenteric lymph nodes were stained for the GC B-cell markers GL7 and Bcl6, the plasma cell markers CD138 and IRF4, and the follicular B-cell marker IgD. Scale bar, 200 μm in all samples in f.
Extended Data Figure 5 Defective regulation of pAkt and cell migration in human GCB DLBCL cell lines harbouring mutations in the S1PR2 signalling pathway.
a, Frequency of non-synonymous coding mutations in S1PR2, GNA13 and ARHGEF1 in GCB-DLBCL lines, and the fraction that were mono- or biallelic, summarized from Supplementary Table 3. Unmutated indicates no coding region mutations in the genes shown. b, c, Intracellular FACS (b) or western blot (c) for pAkt in human GCB-DLBCL cell lines that are WT or mutant for S1PR2, GNA13 or ARHGEF1 as indicated and which were stimulated with CXCL12 (100 ng ml−1) in the presence or absence of S1P (10 nM) for 5 min. pAkt staining of cells treated with wortmannin (200 nM) for 5 min is shown in grey as a staining control for each cell line. d, Transwell migration of GNA13 WT (Ly7, Ly8, NUDUL1) or mutant (DOHH2) cell lines to CXCL12 (100 ng ml−1) in the presence or absence of S1P (10 nM). e, f, Intracellular FACS for pAkt of the GNA13 mutant cell lines Karpas422 (d) or DOHH (e) transduced with retrovirus expressing the reporter alone (vector) or GNA13 in the presence or absence of S1P (10 nM) or wortmannin (200 nM; staining control). g, Intracellular FACS for pAkt in the ARHGEF1 mutant cell line Ly19 transduced with retrovirus expressing reporter alone (vector) or ARHGEF1 that were treated as in b or with the PI3K inhibitor GS-1101 (2 μM; staining control). h, Quantitative PCR analysis of S1PR2 transcript abundance in human GCB-DLBCL cell lines relative to GAPDH. i, Intracellular FACS for pAkt in NUDUL1 cells transduced with retrovirus expressing reporter alone (vector), S1PR2, GNA13 or ARHGEF1, treated as in d. Data in b and d are representative of at least three independent experiments. Pooled data from at least three independent experiments are shown in b, e, f, g and i. Data in b are one experiment representative of two. **P < 0.01, paired two-tailed Student’s t-test.
Extended Data Figure 6 Loss of GC B-cell confinement in the absence of Gα13 or Arhgef1.
a, Additional examples of mesenteric lymph node sections from Gna13 WT or KO mice stained for GC B cells (GL7, blue) and naive B cells (IgD, brown). In the absence of Gα13, the GC border is indistinct and IgD-positive follicular B cells are interspersed with GL7-positive GC B cells throughout the central region of the follicle. The disruption of mesenteric lymph node GC architecture caused by Gα13 deficiency appears more severe than observed in S1pr2-deficient mice3. b, Mixed B-cell transfer showing exclusion of Gna13-deficient GC B cells from the interior of otherwise WT GCs. Gna13 WT or KO CD45.2+ B cells were mixed with WT CD45.1+ B cells and transferred into MD4 Ig-transgenic CD45.1+ recipients that were then immunized with SRBCs intraperitoneally, and splenic tissue was analysed by immunohistochemistry and FACS after 8 days for CD45.2+ B cells. This transfer approach allows efficient participation of transferred polyclonal B cells in the GC as the Ig-transgenic recipient B cells are hen-egg lysozyme specific and do not respond to SRBCs. Note that CD45.2+ WT B cells are distributed uniformly through the GL7+ GCs (upper panels) whereas the CD45.2+ Gna13 KO B cells are located at the perimeter of the GC or in the surrounding follicle (lower panels). In each case, two example images are shown and the GL7 and CD45.2 stains are of adjacent sections. c, Additional sections of mesenteric lymph nodes from Arhgef1 WT or KO mice, stained for GL7 and IgD. Scale bar, 200 μm in a–c. Data in b are one experiment representative of two.
Extended Data Figure 7 Augmented GC B-cell survival is not sufficient to promote dissemination of GC B cells.
a, b, Transduced GC B-cell frequency among total cells in mesenteric lymph node (a) and lymph (b) of mice reconstituted with bone marrow transduced with B-cell-restricted control (vector, n = 5) or myr-Akt (n = 5) expressing retrovirus. c, Immunohistochemical analysis of mesenteric lymph node sections from mice in a, stained for GL7 and IgD. Scale bar, 100 μm. d, e, BCL2-tg or Gna13 KO GC B-cell frequency among total cells in mesenteric lymph node (d) and lymph (e) of BCL2-tg:Gna13 KO mixed chimaeras (n = 8). f, Immunohistochemical analysis of mesenteric lymph nodes from BCL2-tg Gna13 WT or BCL2-tg Gna13 KO mice. Scale bar in low-magnification images (left) is 200 μm and in high-magnification images (right) is 100 μm. Data in a, b, d and e are pooled from two independent experiments. Data in c and f are representative of at least three mice of each type.
Extended Data Figure 8 Human P2RY8 suppresses GC B-cell growth and promotes B-cell confinement to the GC in mice.
a, b, P2RY8 mutations arising in GCB-DLBCL and Burkitt’s lymphoma disrupt receptor expression. Flag-tagged versions of six point mutants and the WT receptor were expressed in WEH231 B cells and surface expression examined by Flag flow cytometry (a). The transduction efficiency of each construct was confirmed to be similar based on IRES-Thy1.1 reporter expression (b). c, d, Fold change in Thy1.1 reporter+ GC relative to follicular B cells from mesenteric lymph node of chimaeras described in Fig. 4d, e. e–g, Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (e), Gpr183+/− (f) or Gna13 WT or KO (g) B cells transduced as in Fig. 4f, g and assessed 24 h after cell transfer. Data in a and b are representative of three independent experiments. Data in e and g are additional examples of the experiments shown in Fig. 4f, g, respectively. Data in f are representative of four independent experiments. Scale bar, 200 μm in e–g. *P < 0.05, **P < 0.01, unpaired two-tailed Student’s t-test.
Extended Data Figure 9 P2RY8-dependent suppression of GC B-cell survival and promotion of B-cell confinement to the GC niche is receptor specific.
a, Transwell migration of WEHI231 cells transduced with retrovirus encoding the control Gα13-coupled receptor, Tbxa2r, towards CXCL12 (100 ng ml−1) in the presence or absence of the thromboxane A2 analogue, U-46619. b, c, Fold change in frequency of Thy1.1 reporter+ GC relative to follicular B cells of Peyer’s patches (b) or mesenteric lymph node (c) from bone marrow chimaeras reconstituted with S1pr2 KO bone marrow transduced with empty vector (control) or Tbxa2r. d, Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Gpr183+/− B cells transduced with empty vector, Tbxa2r or P2RY8, and assessed 24 h after cell transfer. Scale bar, 200 μm. Data in a and d are one experiment representative of two. Data in b and c are from one experiment (n = 4 in each group). **P < 0.01, unpaired two-tailed Student’s t-test.
Extended Data Figure 10 Model relating disruptions in S1PR2/P2RY8–Gα13–ARHGEF1 migration- and Akt-inhibitory pathway to increases in GC B-cell survival, dispersal in the follicle, egress into circulation and dissemination to bone marrow.
a, Summary of signalling pathway. b, Schematic diagram showing GC-containing lymph node follicle, with connection to efferent lymphatic, blood and bone marrow. Suggested distribution of S1P and of putative P2RY8 ligand within lymph node is shown by dots. Comparative migration and survival behaviour of GC B cells with loss (S1PR2, P2RY8, GNA13, ARHGEF1) or gain (BCL2) of function mutations is summarized.
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Muppidi, J., Schmitz, R., Green, J. et al. Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma. Nature 516, 254–258 (2014). https://doi.org/10.1038/nature13765
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DOI: https://doi.org/10.1038/nature13765
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