Targeted disruption of the S1P2 sphingosine 1-phosphate receptor gene leads to diffuse large B-cell lymphoma formation

Abstract

S1P(2) sphingosine 1-phosphate receptor signaling can regulate proliferation, survival, morphology, and migration in many cell types in vitro. Here, we report that S1P(2)(-/-) mice develop clonal B-cell lymphomas with age, such that approximately half of the animals display this neoplasm by 1.5 to 2 years of age. Histologic, immunophenotypic, and molecular analyses revealed a uniform tumor phenotype with features of germinal center (GC)-derived diffuse large B-cell lymphoma (DLBCL). Tumor formation was preceded by increases in GC B cells and CD69(+) T cells, as well as an increased formation of spontaneous GCs, suggesting that S1P(2) loss may promote lymphomagenesis in part by disrupting GC B-cells homeostasis. With the sole exception of rare lung tumors, the effect of S1P(2) gene disruption is remarkably restricted to DLBCL. In humans, 28 of 106 (26%) DLBCL samples were found to harbor multiple somatic mutations in the 5' sequences of the S1P(2) gene. Mutations displayed features resembling those generated by the IgV-associated somatic hypermutation mechanism, but were not detected at significant levels in normal GC B cells, indicating a tumor-associated aberrant function. Collectively, our data suggest that S1P(2) signaling may play a critical role in suppressing DLBCL formation in vivo. The high incidence of DLBCL in S1P(2)(-/-) mice, its onset at old age, and the relative lack of other neoplasms identify these mice as a novel, and potentially valuable, model for this highly prevalent and aggressive human malignancy.

Figure 1. Histopathology of DLBCL in S1P₂^−/− mice

Representative DLBCL, featuring H&E staining (note the large tumor cells in the Wright-Giemsa stained touch preparation in the inset). These tumor cells display positive BCL6 (dark blue), B220 (purple-grey) and Peanut Agglutinin (PNA; reddish-brown) staining and negative IRF4 (dark blue) and CD138 (dark blue) staining. The lymph node is totally replaced by diffuse large cell lymphoma proliferation, containing lymphocytes and inflammatory cells (BCL6, B220 and PNA negative, IRF4 and CD138 positive). The PNA stained section also received a light blue nuclear counterstain. Scale bars: bottom panels: 10μm; top panels: 1μm.

Figure 2. DLBCL immunophenotype in S1P₂^−/− mice

A) Surface immunophenotype by FCM of a representative DLBCL from a S1P₂^−/− mouse and a histologically normal spleen from a littermate control S1P₂^+/+ mouse. Note the low/absent surface Ig, the loss of CD23 and the absence of CD138 in the DLBCL sample. B) AID expression in representative DLBCL from S1P₂^−/− mice, as compared to purified normal GC B cells from two S1P₂^−/− and one S1P₂^+/+ mice. Mouse lung tissue was used as a negative control for the AID RT-PCR.

Figure 3. Clonal rearrangements of the IgH locus and evidence of somatic hypermutation in DLBCL from S1P₂^−/− mice

A) Southern blot analysis of EcoRI digested DNA from representative S1P₂^−/− DLBCLs, and control tail DNA, hybridized with a murine JH region probe. The germline immunoglobulin heavy chain EcoRI fragment is ~6.5 Kb (arrow). Clonal rearrangements of the IgH gene in the tumors analyzed are indicated by asterisks. The predominant germline band observed in some tumors is most likely due to the presence of infiltrating normal cells (see text). B) Mutation analysis of clonally rearranged IgV genes in lymphomas from representative S1P₂^−/− animals. Note that, even though Southern blot analysis in tumors #246 and #297 suggests the presence of two distinct clones, only one rearranged allele could be successfully amplified, possibly due to the usage of a less common V gene family member not recognized by the primers used, or to the presence of mutations in the primer binding site. C) Nucleotide sequence alignment of the rearranged IgV gene from S1P₂^−/− DLBCL #245 (as obtained by direct sequencing) with the germline VH7183 and JH region (GenBank accession # X53774). Dashes indicate sequence identities, while the indicated nucleotides correspond to mutations. Note that, since direct sequencing only detects clonally represented events, the observed mutations can be unequivocally attributed to the malignant clone.

Figure 4. Morphometric analysis of splenic GC formation in S1P₂^−/− mice

A) Unimmunized mice; total area of BCL6+ aggregates in each size range (measured in pixels [1,000 pixels = 1 Kpx]) normalized for spleen section area. The inset presents the percentage of splenic B220+ PNA+ GC cells in old, unimmunized control and knockout mice measured by flow cytometry. The difference is significant (p<0.0015). B) Unimmunized mice; number of BCL6+ aggregates per spleen section area, calculated as described in A. The numbers presented are per average spleen section. *ANOVA analyses indicated that both the size and number of >2 Kpx aggregates are significantly (p<0.05) increased in S1P₂^−/− spleens. C) Number and area of the GC aggregates in SRBC immunized mice. Note the expected increase in larger aggregates for immunized control and knockout mice compared to values for unimmunized mice in A and B. N= 7 young control; 3 young S1P₂^−/−; 4 old control; 3 old S1P₂^−/−. All values are presented as mean ± SEM.

Figure 5. Mutational analysis of the S1P₂ gene in human DLBCL

Top panel: S1P₂ genomic locus with untranslated (empty boxes) and translated (filled boxes) exons; arrow indicates the transcriptional start site per GeneBank accession No. NM_004230. The region amplified for analysis is expanded in the bottom panel and aligned to sequences of mutated DLBCL cases (one line = 2 alleles), where each small segment represents a 25 bp interval and position +1 corresponds to the first nucleotide of the reference mRNA. Ovals, single basepair substitutions; brackets, deletions; triangles, insertions.