Supplementary Methods from Targeted Disruption of the S1P2 Sphingosine 1-Phosphate Receptor Gene Leads to Diffuse Large B-Cell Lymphoma Formation

Supplementary Tables 1-3 from Targeted Disruption of the S1P2 Sphingosine 1-Phosphate Receptor Gene Leads to Diffuse Large B-Cell Lymphoma Formation

Supplementary Figure 7 from Targeted Disruption of the S1P2 Sphingosine 1-Phosphate Receptor Gene Leads to Diffuse Large B-Cell Lymphoma Formation

A balance between quiescence and proliferation is critical for proper maintenance of the hematopoietic stem cell (HSC) pool. Although a lot is known about hematopoiesis, molecular mechanisms that control HSC quiescence remain largely unknown. The ubiquitin-editing enzyme A20 functions as a central regulator of inflammation and adaptive immunity. Here, we show that a deficiency of A20 in the hematopoietic system causes anemia, lymphopenia, and postnatal lethality. Lack of A20 in HSCs results in diminished pool size, impaired radioprotection, defective repopulation, and loss of quiescence. A20-deficient HSCs display increased IFN-γ signaling, caused by augmented NF-κB activation. Strikingly, deletion of both IFN-γ and A20 in hematopoietic cells results in partial rescue of the HSC phenotype. We anticipate that our experiments will facilitate the understanding of mechanisms through which A20-mediated inflammatory signals control HSC quiescence and functions.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. These phenotypic subtypes segregate with largely unique genetic lesions, suggesting the involvement of different pathogenetic mechanisms. In this report we show that the BLIMP1/PRDM1 gene is inactivated by multiple mechanisms, including homozygous deletions, truncating or missense mutations, and transcriptional repression by constitutively active BCL6, in ∼53% of ABC-DLBCL. In vivo, conditional deletion of Blimp1 in mouse B cells promotes the development of lymphoproliferative disorders recapitulating critical features of the human ABC-DLBCL. These results demonstrate that BLIMP1 is a bona fide tumor-suppressor gene whose loss contributes to lymphomagenesis by blocking plasma cell differentiation.

Abstract Abstract 148 Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Numerous genetic alterations segregate with ABC-DLBCL, namely translocations of the BCL6 proto-oncogene, BLIMP1 inactivation and constitutive NF-κB activation. We recently reported that A20, a negative regulator of NF-κB signaling, is biallelically inactivated by mutations and deletions in one-third of ABC-DLBCL (Compagno et al, Nature, 2009), indicating a tumor suppressor role in this disease. Notably, A20 inactivation is commonly associated with chromosomal translocations deregulating BCL6 (n=11/20 DLBCL cases). Furthermore, the two genes are linked in the same pathway, where NF-κB induced activation of IRF4 leads to BCL6 down-regulation (Saito et al. Cancer Cell 2007) and consequent release of the BCL6 target BLIMP1, a master regulator of plasma cell differentiation. These observations suggest that A20 inactivation and BCL6 translocations cooperate in DLBCL pathogenesis. In order to examine the individual and combined contribution of these two lesions in vivo, we have generated an A20 conditional knockout allele in which a loxP-flanked exon 3 of the A20 gene can be deleted upon Cre-mediated recombination. The resulting mice were crossed with both a Cγ1-Cre deletor strain, which expresses the Cre recombinase in germinal centre (GC) B cells, and the lymphoma-prone Iμ HABCL6 mouse model (Cattoretti et al., Cancer Cell, 2005), which mimics a BCL6 translocation to the immunoglobulin heavy chain locus. When analyzed at 3 months of age, GC B-cell conditional heterozygous (A20Cγ1HET) and homozygous (A20Cγ1KO) A20 knockout mice showed a significant increase in the B220dimCD138+ plasma cell population (0.6% and 0.5%, respectively, versus 0.3% for wild-type littermates) and a corresponding 2-fold increase in IgG1 serum immunoglobulin levels after immunization with sheep red blood cells. Furthermore, A20 knockout splenic B cells had increased proliferative capacity and survival after stimulation ex vivo with lipopolysaccharides, B-cell receptor cross-linking or CD40 activation, consistent with enhanced NF-κB activity in these cells. Interestingly, the increase in plasma cells was not observed in compound Iμ HABCL6/ A20Cγ1HET and Iμ HABCL6/ A20Cγ1KO animals, presumably due to the known role of BCL6 in blocking plasma cell differentiation (Tunyaplin et al., J. of Immunol., 2004). In contrast, these animals displayed a marked increase in the B220+PNAhi GC B cell compartment, as compared to both A20 knockout and Iμ HABCL6 mice (2.5% and 3.9% respectively, versus 1.9% for wild-type littermates). Overall, these findings document that A20 acts as a negative regulator of B cell proliferation and survival as well as of plasma cell differentiation in vivo, and support a model by which loss of A20 synergizes with BCL6 deregulation to promote the expansion of GC B cells while preventing terminal differentiation. Long-term follow-up of these cohorts will provide critical information on the role of A20 as a tumor suppressor gene in vivo and on its cooperative activity with BCL6 deregulation in the pathogenesis of DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 445 Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma, is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. We have previously reported that the BLIMP1 gene, a master regulator of plasma cell differentiation normally expressed in a subset of germinal center (GC) B cells and in all plasma cells, is inactivated by truncating mutations in a fraction of ABC-DLBCL, but not in GCB-DLBCL (Pasqualucci et al, J Exp Med 2006). In addition, most ABC-DLBCL lack expression of the BLIMP1 protein despite the presence of IRF4, another key regulator of plasma cell differentiation known to be invariably co-expressed with BLIMP1 in normal B cells, thereby suggesting that additional genetic or epigenetic mechanisms may inactivate BLIMP1 in these tumors. Here we report the characterization of the full spectrum of genetic lesions affecting the BLIMP1 locus in DLBCL, as determined by genome-wide copy number analysis (Affymetrix SNP 6.0 array), fluorescence in situ hybridization, and direct sequencing of the entire BLIMP1 coding region in 158 primary biopsies, classified by gene expression profile analysis (51 ABC-DLBCL; 62 GCB-DLBCL; 11 unclassified) and/or by immunohistochemistry (24 non-GC and 10 GC-DLBCL). This analysis uncovered a total of 22 mutations, distributed in 21 cases and segregating with an activated DLBCL phenotype (13/51 ABC-, 3/11 unclassified and 5/24 non-GC-DLBCL, vs 0/71 GCB/GC-DLBCL). The vast majority of the mutations were represented by frameshift insertions/deletions (n=10), splice site mutations (n=7) and nonsense mutations (n=1) leading to severely truncated polypeptides that lack critical functional domains and have therefore lost their activity. Interestingly, in the remaining three cases, 4 missense mutations introduced amino acid changes that were documented to severely impair BLIMP1 function by either causing protein instability (n=3) or abrogating its ability to bind chromatin and repress its known target genes CIITA and ID3 (n=1). When transduced into the GCB-DLBCL cell line BJAB, the wild type, but not three of the BLIMP1 missense mutant constructs induced cell cycle arrest. Copy number analysis confirmed deletion of the second allele in 9/12 mutated ABC-DLBCL, and identified three additional cases harbouring biallelic loss of the gene, including a focal homozygous deletion of 274Kb, which encompasses the BLIMP1 gene but not the two proximal genes ATG5 and PREP. Thus, 31% (n=16/51) of ABC-DLBCL have inactivation of BLIMP1 due to mutations or biallelic deletions. Moreover, immunohistochemical analysis revealed the lack of Blimp1 protein expression in 90% (n=27/30) of IRF4+ ABC-DLBCL carrying normal BLIMP1 loci. Notably, ten of these samples (30%) were found to harbour chromosomal translocations affecting BCL6, a master regulator of the GC and a direct transcriptional repressor of BLIMP1, suggesting that deregulated BCL6 expression was responsible for the lack of BLIMP1 expression in these cases. With the exception of two cases, BCL6 translocations and BLIMP1 structural alterations were mutually exclusive. Collectively, these data identify a novel mechanism by which missense mutations of BLIMP1 can impair its function in human DLBCL, and demonstrate that the IRF4-BCL6-BLIMP1 pathway is inactivated by structural alterations in over half of ABC-DLBCLs, strongly suggesting that BLIMP1 inactivation and BCL6 translocations may represent alternative mechanisms contributing to the pathogenesis of this disease by blocking terminal B cell differentiation.Disclosures: No relevant conflicts of interest to declare.

The full set of microRNAs (miRNAs) in the human genome is not known. Because presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, many tissue-specific miRNAs remain unrevealed. To understand the role of miRNAs in B cell function and lymphomagenesis, we generated short-RNA libraries from normal human B cells at different stages of development (naive, germinal center, memory) and from a Burkitt lymphoma cell line. A combination of cloning and computational analysis identified 178 miRNAs (miRNome) expressed in normal and/or transformed B cell libraries. Most notably, the B cell miRNome included 75 miRNAs which to our knowledge have not been previously reported and of which 66 have been validated by RNA blot and/or RT-PCR analyses. Numerous miRNAs were expressed in a stage- or transformation-specific fashion in B cells, suggesting specific functional or pathologic roles. These results provide a resource for studying the role of miRNAs in B cell development, immune function, and lymphomagenesis.

Abstract Abstract 446 The PRDM1/ BLIMP1 gene encodes a zinc finger transcriptional repressor that is expressed in a subset of germinal center (GC) B cells and in all plasma cells, and is required for terminal B cell differentiation. The BLIMP1 locus is biallelically inactivated by structural alterations in approximately one third of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) (Pasqualucci et al, J Exp Med 2006). Moreover, the expression of the Blimp1 protein is absent in up to 80% of ABC-DLBCL due to alternative genetic and epigenetic mechanisms. These findings suggest that BLIMP1 may function as a tumor suppressor gene whose loss may contribute to the pathogenesis of this lymphoma type by blocking terminal B cell differentiation. To investigate the role of BLIMP1 inactivation in lymphomagenesis in vivo, we tested whether conditional deletion of the Blimp1 gene in mouse B cells can promote the growth of lymphomas recapitulating the features of ABC-DLBCL. Toward this end, a mouse model carrying a loxP-flanked exon 5 of the Blimp1 gene that can be deleted by Cre-mediated recombination (Ohinata et al, Nature 2005) was crossed with a CD19-Cre deletor strain, expressing the Cre recombinase in all B cells. The resulting mice were monitored for tumor development and survival. Consistent with previous observations in a similar model (Shapiro-Shelef et al, Immunity 2003), Blimp1 conditional knockout (Blimp1CD19KO) mice showed a severe impairment in the generation of CD138+ plasma cells and had decreased serum immunoglobulin levels of all isotypes, together with a two-fold increase in the number of PNAhiCD95+ GC B cells. Over time, significantly reduced survival was observed in the Blimp1CD19KO cohort, with only 27% of the animals being alive at 15 months of age (LogRank p value<0.0001). Macroscopic and flow cytometric analysis of the lymphoid compartments revealed the presence of splenomegaly in 32/38 (84%) Blimp1CD19KO, as compared to 1/25 (4%) age-matched wildtype (WT) littermates, and a significant increase in IgM+IgD-CD21+CD23lo splenic B cells, indicative of marginal zone B cell expansion. In addition, 79% (n=30/38) of Blimp1CD19KO mice showed markedly hyperplastic bronchus-associated lymphoid tissue (BALT). Notably, between 10 and 16 months of age 34% (13/38) of these animals developed clonal lymphoproliferative disorders with a mature B cell phenotype (B220+Pax5+) and histologic features of DLBCL (n=6) or less aggressive lymphoid proliferations (LPD: n=6; marginal zone lymphoma: n=1), in contrast with 1/27 heterozygous and 0/25 WT animals. Sequencing analysis of the rearranged immunoglobulin variable region genes in lymphoma biopsies revealed the presence of somatic mutations in 6/8 samples investigated, demonstrating their origin from a GC-experienced B cell. Moreover, immunohistochemical staining for Bcl6 and Irf4 documented a late-GC “activated” B cell phenotype (Bcl6-Irf4+) in all tumors tested (n=4), consistent with the expansion of cells that had been committed to plasma cell differentiation. These data demonstrate that Blimp1 is a bona-fide tumor suppressor gene whose B-cell specific inactivation in vivo promotes the development of lymphomas sharing features of the human ABC-DLBCL. Disclosures: No relevant conflicts of interest to declare.

AbstractS1P2 sphingosine 1-phosphate receptor signaling can regulate proliferation, survival, morphology, and migration in many cell types in vitro. Here, we report that S1P2−/− 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 S1P2 loss may promote lymphomagenesis in part by disrupting GC B-cells homeostasis. With the sole exception of rare lung tumors, the effect of S1P2 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 S1P2 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 S1P2 signaling may play a critical role in suppressing DLBCL formation in vivo. The high incidence of DLBCL in S1P2−/− 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. [Cancer Res 2009;69(22):8686–92]

(1988). Science and friendship: The Societe Philomathique de Paris, 1788–1835. History and Technology: Vol. 5, French Institutions from the Revolution to the Restoration, pp. 179-192.

Bioinformatics Analysis of short-RNA libraries The bioinformatics microRNA (miRNA) analysis pipeline includes (a) identification of short-RNAs from each library, (b) identification of exact and partial matches of the short-RNA sequences to the human genome, (c) testing each short-RNA genomic region for compatibility with hairpin secondary structures, (d) clustering genomic regions to predict mature miRNAs, (e) annotating and filtering short-RNAs and miRNAs candidates, (f) estimation of predicted miRNA frequencies in the libraries, (g) clustering short-RNAs that do not support miRNAs candidates.