The pathways regulating formation of the germinal center (GC) dark zone (DZ) and light zone (LZ) are unknown. In this study we show that FOXO1 transcription factor expression was restricted to the GC ...DZ and was required for DZ formation, since its absence in mice led to the loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B cells displayed normal somatic hypermutation but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involved the trans-activation of the chemokine receptor CXCR4, and cooperation with the BCL6 transcription factor in the trans-repression of genes involved in immune activation, DNA repair, and plasma cell differentiation. These results also have implications for the role of FOXO1 in lymphomagenesis because they suggest that constitutive FOXO1 activity might be required for the oncogenic activity of deregulated BCL6 expression.
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•FOXO1 expression in the germinal center is restricted to dark zone B cells•FOXO1-null mouse germinal centers lack dark zones and lose architectural polarity•FOXO1 deletion impairs affinity maturation and IgG1 class switch recombination•FOXO1 instructs the dark zone gene program directly and by licensing BCL6 activity
The factors that control germinal center polarity and cyclic reentry are unknown. Dalla-Favera and colleagues demonstrate that the transcription factor FOXO1 instructs a gene program that is required for germinal center dark zone development. Mouse germinal centers devoid of FOXO1 expression fail to support affinity maturation and class switch recombination.
Chronic lymphocytic leukemia (CLL) is a malignancy of B cells of unknown etiology. Deletions of the chromosomal region 13q14 are commonly associated with CLL, with monoclonal B cell lymphocytosis ...(MBL), which occasionally precedes CLL, and with aggressive lymphoma, suggesting that this region contains a tumor-suppressor gene. Here, we demonstrate that deletion in mice of the 13q14-minimal deleted region (MDR), which encodes the
DLEU2/miR-15a/16-1 cluster, causes development of indolent B cell-autonomous, clonal lymphoproliferative disorders, recapitulating the spectrum of CLL-associated phenotypes observed in humans. miR-15a/16-1-deletion accelerates the proliferation of both human and mouse B cells by modulating the expression of genes controlling cell-cycle progression. These results define the role of 13q14 deletions in the pathogenesis of CLL.
► Deletion of chromosomal region 13q14 in mice causes chronic lymphocytic leukemia ► 13q14
−/− mice recapitulate the spectrum of lymphoproliferative diseases in humans ► The 13q14 region controls expansion of the B cell compartment ► The microRNA cluster miR-15a/16-1 functions as a tumor suppressor
Inactivating mutations of the CREBBP acetyltransferase are highly frequent in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), the two most common germinal center (GC)-derived ...cancers. However, the role of CREBBP inactivation in lymphomagenesis remains unclear. Here, we show that CREBBP regulates enhancer/super-enhancer networks with central roles in GC/post-GC cell fate decisions, including genes involved in signal transduction by the B-cell receptor and CD40 receptor, transcriptional control of GC and plasma cell development, and antigen presentation. Consistently,
-deficient B cells exhibit enhanced response to mitogenic stimuli and perturbed plasma cell differentiation. Although GC-specific loss of
was insufficient to initiate malignant transformation, compound
-haploinsufficient/BCL2-transgenic mice, mimicking the genetics of FL and DLBCL, develop clonal lymphomas recapitulating the features of the human diseases. These findings establish
as a haploinsufficient tumor-suppressor gene in GC B cells and provide insights into the mechanisms by which its loss contributes to lymphomagenesis.
Loss-of-function mutations of
are common and early lesions in FL and DLBCL, suggesting a prominent role in lymphoma initiation. Our studies identify the cellular program by which reduced CREBBP dosage facilitates malignant transformation, and have direct implications for targeted lymphoma therapy based on drugs affecting CREBBP-mediated chromatin acetylation.
.
B cells producing high-affinity antibodies are destined to differentiate into memory B cells and plasma cells, but the mechanisms leading to those differentiation pathways are mostly unknown. Here we ...report that the transcription factor IRF4 is required for the generation of plasma cells. Transgenic mice with conditional deletion of Irf4 in germinal center B cells lacked post-germinal center plasma cells and were unable to differentiate memory B cells into plasma cells. Plasma cell differentiation required IRF4 as well as the transcriptional repressor Blimp-1, which both acted 'upstream' of the transcription factor XBP-1. In addition, IRF4-deficient B cells had impaired expression of activation-induced deaminase and lacked class-switch recombination, suggesting an independent function for IRF4 in this process. These results identify IRF4 as a crucial transcriptional 'switch' in the generation of functionally competent plasma cells.
Deletion of chromosomal region 13q14 represents the most common genetic aberration in B-cell chronic lymphocytic leukemia (CLL). 13q14 deletions are commonly large and heterogeneous in size and ...affect multiple genes. We recently found that targeted deletion in mice of the 0.11 megabase (mb)–long minimal deleted region (MDR) encompassing the DLEU2/miR-15a/16-1 cluster recapitulates the spectrum of CLL-associated lymphoproliferations in humans, including CLL, CD5+ monoclonal B-cell lymphocytosis, and CD5− non-Hodgkin lymphomas. In the present study, we demonstrate that additional deletion of the 0.69-mb large genomic region telomeric to the MDR called the common deleted region (CDR) changed the spectrum of lymphoproliferations developing in CDR- versus MDR-deleted mice in that the number of CLL among B-cell lymphoproliferations was significantly elevated in the former. In addition, CDR-deleted mice seemed to succumb to their disease faster than MDR-deleted mice. Comparing HCDR3 regions of CD5+ lymphoproliferations derived from this and published CLL mouse models, 44% (29 of 66) of junctions could be assigned to 8 sets of highly similar HCDR3 regions, demonstrating that CLL developing in mice frequently expresses almost identical, stereotypic Ag receptors. These results suggest that the size of 13q14 deletions influences the phenotype of the developing lymphoproliferations and potentially the severity of disease, suggesting a tumor-suppressor function for genetic elements in addition to DLEU2/miR-15a/16-1.
Diffuse large B cell lymphomas (DLBCL) derive from germinal center (GC) B cells and display chromosomal alterations deregulating the expression of BCL6, a transcriptional repressor required for GC ...formation. To investigate the role of BCL6 in DLBCL pathogenesis, we have engineered mice that express BCL6 constitutively in B cells by mimicking a chromosomal translocation found in human DLBCL. These mice display increased GC formation and perturbed post-GC differentiation characterized by a decreased number of post-isotype switch plasma cells. Subsequently, these mice develop a lymphoproliferative syndrome that culminates with the development of lymphomas displaying features typical of human DLBCL. These results define the oncogenic role of BCL6 in the pathogenesis of DLBCL and provide a faithful mouse model of this common disease.
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.
► Disruption of
BLIMP1 by inactivating mutations and biallelic deletions in ABC-DLBCL ► Transcriptional silencing of
BLIMP1 in ABC-DLBCL cases carrying
BCL6 translocations ► B cell conditional
Blimp1 knockout mice model the human ABC-DLBCL
Heterozygous inactivating mutations of the KMT2D methyltransferase and the CREBBP acetyltransferase are among the most common genetic alterations in B cell lymphoma and co-occur in 40 to 60% of ...follicular lymphoma (FL) and 30% of EZB/C3 diffuse large B cell lymphoma (DLBCL) cases, suggesting they may be coselected. Here, we show that combined germinal center (GC)-specific haploinsufficiency of
and
synergizes in vivo to promote the expansion of abnormally polarized GCs, a common preneoplastic event. These enzymes form a biochemical complex on select enhancers/superenhancers that are critical for the delivery of immune signals in the GC light zone and are only corrupted upon dual
/
loss, both in mouse GC B cells and in human DLBCL. Moreover, CREBBP directly acetylates KMT2D in GC-derived B cells, and, consistently, its inactivation by FL/DLBCL-associated mutations abrogates its ability to catalyze KMT2D acetylation. Genetic and pharmacologic loss of CREBBP and the consequent decrease in KMT2D acetylation lead to reduced levels of H3K4me1, supporting a role for this posttranslational modification in modulating KMT2D activity. Our data identify a direct biochemical and functional interaction between CREBBP and KMT2D in the GC, with implications for their role as tumor suppressors in FL/DLBCL and for the development of precision medicine approaches targeting enhancer defects induced by their combined loss.
Abstract 468
Deletion of chromosomal region 13q14 represents the most common genetic aberration in chronic lymphocytic leukemia (CLL) and occurs with less frequency in other lymphoid malignancies. ...13q14 deletions are commonly large and heterogeneous in size, affecting multiple genes. We recently demonstrated that the targeted deletion in mice of miR-15a/16-1 alone causes lymphoproliferations and recapitulates the spectrum of CLL-associated phenotypes in humans. However, both the penetrance and severity of the disease increased markedly upon deletion of the 110 kilobase (kb)-long minimal deleted region (MDR) that encompasses the DLEU2/miR-15a/16-1 cluster (Klein et al., Cancer Cell, 2010). Thus, we investigate here the extent to which the additional deletion of genetic material encoded in the 690 kb large genomic region telomeric of the MDR, including the DLEU7 and RNASEH2B genes, affects the phenotype and spectrum of lymphoproliferations and disease course. To this end, we generated a conditional allele that allows in vivo deletion of this 800 kb large region termed common deleted region (CDR) specifically in B cells. B-cell conditional deletion was necessary since homozygous CDR germ-line deletion led to embryonic lethality.
We demonstrate the technical feasibility of mimicking a large chromosomal deletion (800 kb) occurring in human cancer in vivo and in a cell type-specific fashion, thus establishing an animal model for this genomic aberration. We found that despite a similar time-point of disease onset (∼12 months) and disease penetrance (∼40%), the spectrum of lymphoproliferations developing in CDR-deleted mice differed from those found in MDR-deleted mice in that the fraction of CLL among the various types of B-lymphoproliferations (including CLL, CD5+ monoclonal B-cell lymphocytosis (MBL) and CD5– non-Hodgkin lymphomas (NHL)) was significantly elevated. Specifically, 19/20 mice with homozygous deletion of the CDR developed lymphoproliferations that represented CLL (95% CLL; 5% MBL) vs. 12/22 of the corresponding MDR-deleted mice (55% CLL; 27% MBL; 18% NHL). Since we previously observed that MDR+/– mice compared to wild-type mice showed a trend towards developing clonal lymphoproliferations, we established and monitored cohorts of CDR+/– mice and wild-type littermates. 25% of CDR+/– mice developed CD5+ lymphoproliferations vs. 8% of control mice. Determination of event-free survival demonstrated that CDR+/– mice succumb to their disease earlier than wild-type littermates (P=0.0002). Compared to MDR+/– mice, the CDR+/– mice showed a trend towards a more aggressive disease course (70% of CDR+/– vs. 45% of MDR+/– mice have died at 20 months). In summary, these results suggest that the additional loss of genetic elements located telomeric of the MDR impacts the phenotype of the lymphoproliferations and potentially the severity of disease.
Notably, lymphoproliferations derived from 4 independent CLL mouse models, i.e. TCL1-tg (Yan et al., PNAS, 2006) as well as miR-15a/16-1, MDR (Klein et al., Cancer Cell, 2010) and CDR-deleted mice, expressed antibody genes with nearly identical, stereotypic antigen binding regions. Specifically, comparing all HCDR3 regions derived from MBL or CLL cases of our own (n=46) and the TCL1-tg (n=20) studies, we observed that 44% (29/66) of the junctions can be assigned to 8 sets of highly similar HCDR3 regions among the sequence collection, 5 of which have previously been defined by Yan et al. These findings provide a rationale for defining clusters of stereotypic antigen receptors in CLL-prone mice. They also suggest that genetic aberrations disrupting the control of cell growth and survival and chronic antigen-stimulation co-operate in the clonal expansion of CLL tumor cells.
In summary, published and the present results suggest that whereas deletion of the miR-15a/16-1 cluster is the critical mechanism in the pathogenesis of CLL with 13q14 aberrations, the additional loss of genetic elements encoded in the 13q14 tumor suppressor locus can significantly influence the penetrance of the phenotype, the spectrum of the lymphoproliferations, and the severity of the disease course. Since 13q14 deletions in humans are usually heterogeneous, these findings provide a clear rationale for determining the pathogenic role of the various genetic elements affected by deletions of the 13q14 locus.
No relevant conflicts of interest to declare.
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.
No relevant conflicts of interest to declare.
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