E2F4 Plays a Critical Role in Early B-Cell Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 318-318
Author(s):  
Clayton Smith ◽  
Michelle Glozak ◽  
Maura Gasparetto ◽  
Rachel Rempel ◽  
Jos Domens ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
Immunoglobulin Gene ◽  
B Cell Differentiation ◽  
Cycle Progression

Abstract The E2Fs are important mediators of cell cycle control, DNA synthesis and apoptosis in many cell types. Recently E2F4 has been shown to play a role in hematopoietic cell growth and development (Rempel et al. Mol Cell, 6 p293, 2000). Here we report the effects of loss of E2F4 specifically on B-cell development. E2F4−/− mice have a partial block in early B-cell development prior to immunoglobulin gene rearrangement. The block is intrinsic to B-cell progenitors rather than secondary to micro-environmental effects since it occurs following transplant of E2F4−/− marrow into wild type recipients. Increases in apoptosis and abnormalities in cell cycle progression were found in B220+CD43+ B-cells of E2F4−/− mice indicating that E2F4 plays an important role in these processes in early B-cells. Expression of a variety of genes important in B-cell development including E2A, RAG, IL-7, EBF and Pax-5 were decreased in early E2F4−/− B-cells. In contrast, Id1 and Id2, regulators of a variety of genes critical to B-cell development, were relatively over-expressed in early E2F4−/− B-cells while Id3 was relatively under-expressed in these cells. E2F binding sites were identified in the Id2 and Id3 promoters and E2F4 was found to directly bind to these promoters in splenic B-cells. These findings suggest that E2F4 may also regulate early B-cell development by directly and indirectly modulating expression of the genes critical to B-cell differentiation. Together, these observations indicate that E2F4 is a critical mediator of early B-cell development via its effects on multiple pathways including those involved with apoptosis, cell cycle progression and differentiation. These findings also suggest that the E2Fs may serve to link cell survival and proliferation pathways to differentiation pathways in early B-cells and perhaps other cells aswell.

Detection of Genomic Lesions in Childhood Precursor-B Cell ALL in Diagnosis and Relapse Samples Using High Resolution Genomic Profiling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 995-995
Author(s):  
Roland P. Kuiper ◽  
Frank N. van Leeuwen ◽  
Suzanne T.M. Keijzers-Vloet ◽  
Simon V. van Reijmersdal ◽  
Jayne Y. Hehir-Kwa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
High Resolution ◽  
B Cell ◽  
Cell Cycle Progression ◽  
Clinical Decision Making ◽  
Lymphoblastic Leukemia ◽  
Cell Development ◽  
B Cell Development ◽  
Genomic Profiling ◽  
Cycle Progression

Abstract Due to advances in therapeutic regimens developed during the last two decades, the majority of children with acute lymphoblastic leukemia (ALL) respond well to therapy. However, in approximately 25% of the patients relapses occur. Chomosome aneuploidies and recurrent chromosomal translocations are of considerable prognostic importance, and are routinely used in the course of clinical decision making. Current technological developments in molecular cytogenetic techniques have revealed that genetic lesions driving tumorigenesis frequently occur at the submicroscopic level and, consequently, escape standard cytogenetic observations. Therefore, we have previously performed high resolution genomic profiling of precursor-B-cell ALL samples obtained at diagnosis, using 250k NspI SNP-based oligoarrays from Affymetrix (Kuiper et al., 2007). By doing so, we detected multiple de novo genetic lesions, some of which were subtle and affected single genes. Many of these lesions involved recurrent (partially) overlapping deletions and duplications, encompassing various established leukemia-associated genes, such as ETV6, RUNX1, and MLL. Importantly, the most frequently affected genes were those controlling G1/S cell cycle progression (e.g. CDKN2A, CDKN1B, and RB1), followed by genes associated with B-cell development. The latter group included the B-lineage transcription factors PAX5, EBF, E2-2, and IKZF1 (Ikaros), as well as genes with other established roles in B-cell development, i.e., RAG1 and RAG2, FYN, PBEF1, or CBP/PAG. Here we have selected 34 additional precursor-B cell ALL cases that suffered from relapses 6 months to 9 years after diagnosis. Lesions affecting genes involved in G1/S cell cycle progression and B-cell development were observed with similar frequencies in the diagnosis and relapse samples as compared to our previous cohort of patients with unknown therapy response. However, additional (secondary) lesions were observed in the relapse samples in nearly all patients analyzed, indicating that these relapse samples are genomically distinct. In addition, several cases were encountered in which the diagnosis and relapse samples carried alternative lesions affecting the same gene(s), including CDKN2A and PAX5, suggesting that inactivation of these genes were secondary but essential events required to develop a full blown leukemia.

scholarly journals CtIP is essential for early B cell proliferation and development in mice

2019 ◽  
Vol 216 (7) ◽  
pp. 1648-1663 ◽  
Author(s):  
Xiangyu Liu ◽  
Xiaobin S. Wang ◽  
Brian J. Lee ◽  
Foon K. Wu-Baer ◽  
Xiaohui Lin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Essential Gene ◽  
Nuclease Activity ◽  
Cell Development ◽  
Nuclear Body ◽  
B Cell Development ◽  
Immunoglobulin Gene ◽  
End Resection

B cell development requires efficient proliferation and successful assembly and modifications of the immunoglobulin gene products. CtIP is an essential gene implicated in end resection and DNA repair. Here, we show that CtIP is essential for early B cell development but dispensable in naive B cells. CtIP loss is well tolerated in G1-arrested B cells and during V(D)J recombination, but in proliferating B cells, CtIP loss leads to a progressive cell death characterized by ATM hyperactivation, G2/M arrest, genomic instability, and 53BP1 nuclear body formation, indicating that the essential role of CtIP during proliferation underscores its stage-specific requirement in B cells. B cell proliferation requires phosphorylation of CtIP at T847 presumably by CDK, but not its interaction with CtBP or Rb or its nuclease activity. CtIP phosphorylation by ATM/ATR at T859 (T855 in mice) promotes end resection in G1-arrested cells but is dispensable for B cell development and class switch recombination, suggesting distinct roles for T859 and T847 phosphorylation in B cell development.

scholarly journals B Lymphocytes Differentially Use the Rel and Nuclear Factor κB1 (NF-κB1) Transcription Factors to Regulate Cell Cycle Progression and Apoptosis in Quiescent and Mitogen-activated Cells

1998 ◽  
Vol 187 (5) ◽  
pp. 663-674 ◽  
Author(s):  
Raelene J. Grumont ◽  
Ian J. Rourke ◽  
Lorraine A. O'Reilly ◽  
Andreas Strasser ◽  
Kensuke Miyake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Cycle Progression ◽  
Nuclear Factor ◽  
Cycle Progression ◽  
Regulate Cell Cycle Progression

Rel and nuclear factor (NF)-κB1, two members of the Rel/NF-κB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-κB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1−/− mice, the level of apoptosis in cultures of quiescent nfkb1−/−, but not c-rel−/−, B cells is higher. The failure of c-rel−/− or nfkb1−/− B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel−/− and nfkb1−/− B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-κB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-κB–dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel−/− B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-κB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-κB factors to control cell cycle progression and prevent apoptosis.

scholarly journals The mTOR-Bach2 Cascade Controls Cell Cycle and Class Switch Recombination during B Cell Differentiation

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Toru Tamahara ◽  
Kyoko Ochiai ◽  
Akihiko Muto ◽  
Yukinari Kato ◽  
Nicolas Sax ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Class Switch Recombination ◽  
Cyclin D3 ◽  
Immunoglobulin Gene ◽  
B Cell Differentiation ◽  
Class Switch

ABSTRACT The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, including antibody class switching and regulatory T cell development in activated B and T cells, respectively. However, little is known about the molecular mechanisms of Bach2 regulation in response to signaling of cytokines and antigen. We show here that mammalian target of rapamycin (mTOR) controls Bach2 along B cell differentiation with two distinct mechanisms in pre-B cells. First, mTOR complex 1 (mTORC1) inhibited accumulation of Bach2 protein in nuclei and reduced its stability. Second, mTOR complex 2 (mTORC2) inhibited FoxO1 to reduce Bach2 mRNA expression. Using expression profiling and chromatin immunoprecipitation assay, the Ccnd3 gene, encoding cyclin D3, was identified as a new direct target of Bach2. A proper cell cycle was lost at pre-B and mature B cell stages in Bach2-deficient mice. Furthermore, AZD8055, an mTOR inhibitor, increased class switch recombination in wild-type mature B cells but not in Bach2-deficient cells. These results suggest that the mTOR-Bach2 cascade regulates proper cell cycle arrest in B cells as well as immunoglobulin gene rearrangement.

scholarly journals Increased Frequency of Pre–Pro B Cells in the Bone Marrow of New Zealand Black (NZB) Mice: Implications for aDevelopmental Block in B Cell Differentiation

2002 ◽  
Vol 9 (1) ◽  
pp. 35-45 ◽  
Author(s):  
Zhe-Xiong Lian ◽  
Hiroto Kita ◽  
Tomoyuki Okada ◽  
Tom Hsu ◽  
Leonard D. Shultz ◽  
...  
Keyword(s):  
New Zealand ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Differentiation ◽  
Differentiation Markers ◽  
Cell Stage

Reductions in populations of both Pre-B cell (Hardy fractions D) and Pro-B cells (Hardy fractions B–C) have been described in association with murine lupus. Recent studies of B cell populations, based on evaluation of B cell differentiation markers, now allow the enumeration and enrichment of other stage specific precursor cells. In this study we report detailed analysis of the ontogeny of B cell lineage subsets in New Zealand black (NZB) and control strains of mice. Our data suggest that B cell development in NZB mice is partially arrested at the fraction A Pre–Pro B cell stage. This arrest at the Pre-Pro B cell stage is secondary to prolonged lifespan and greater resistance to spontaneous apoptosis. In addition, expression of the gene encoding the critical B cell development transcription factor BSAP is reduced in the Pre–Pro B cell stage in NZB mice. This impairment may influence subsequent B cell development to later stages, and thereby accounts for the down-regulation of the B cell receptor componentIgα(mb-1). Furthermore, levels of expression of theRug2, λ5andIgβ(B29) genes are also reduced in Pre–Pro B cells of NZB mice. The decreased frequency of precursor B cells in the Pre–Pro B cell population occurs at the most primitive stage of B cell differentiation.

scholarly journals Complete Block of Early B Cell Differentiation in Mice Lacking the Endosomal Adaptor Protein p14

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1026-1026
Author(s):  
Marcin Lyszkiewicz ◽  
Daniel Kotlarz ◽  
Natalia Zietara ◽  
Gudrun Brandes ◽  
Jana Diestelhorst ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Differentiation ◽  
Knock Out ◽  
And Function

Abstract Human primary immunodeficiency caused by a point mutation in the 3' untranslated region of the endosomal adaptor protein p14 (also known as Lamtor2) resulted in severely impaired function of neutrophils, B cells, T cells and melanocytes. However, complexity of the phenotype and scarcity of human material preclude in-depth studies. Therefore, to gain insight into the role of p14 in B cell development and function, we generated loxP conditional knock-out mice. Using mb-1-Cre mice we demonstrated that loss of p14 at the preB1 stage lead to a complete block of B cell development, resulting in the absence of IgM-positive B cells. Further, to test the significance of p14 deficiency in peripheral organs, we took advantage of CD19-Cre mice, which have limited efficiency in deleting target genes in the bone marrow, but reach up to 95% efficiency in spleen. Thus, we could demonstrate that later in B cell development, p14 was essential for the generation and activation of mature B lymphocytes. While B1 cell development was maintained, splenic follicular B cells were massively reduced in the absence of p14. Furthermore, activation of B cell receptor (BCR) resulted in impaired intracellular signalling and proliferation of p14 deficient B cells. In particular, lack of p14 lead to delayed internalization of BCR and endosomal processing associated with impaired mobilization of Ca++ from intracellular stores as well as aberrant phosphorylation of BCR-associated kinases. In conclusion, our data revealed that p14 is a critical regulator of B cell development and function, which acts by modulating BCR signalling. Disclosures No relevant conflicts of interest to declare.

scholarly journals Chronic GvHD Is Characterized By Impaired B Cell Development in the Bone Marrow

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1883-1883
Author(s):  
Oleg Kolupaev ◽  
Michelle West ◽  
Bruce R. Blazar ◽  
Stephen Tilley ◽  
James Coghill ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
B Lymphopoiesis

Abstract Background. Chronic-graft-versus-host disease (cGvHD) continues to be a major complication following allogeneic hematopoietic stem cell transplantation (HSCT). Despite significant progress, mechanisms underlying development of the pathology are yet to be fully understood. Recent studies utilizing mouse models and patient samples have demonstrated a critical role for B cells in GvHD pathogenesis. Bone marrow (BM)-derived B cells can produce auto-reactive antibodies causing tissue fibrosis and multiorgan cGvHD. Impaired B cell homeostasis in the periphery, activation due to abnormally high levels of B cell-activating factor (BAFF), increased survival of auto-reactive B cells and aberrant BCR signaling are shown to be important for disease progression in cGvHD patients. Murine models also highlighted the critical role of germinal center reactions, particularly interactions between T follicular helper (Tfh) cells and B cells for generation of auto-antibodies which are responsible for triggering immune responses and cell-mediated toxicity. A growing body of evidence has emerged highlighting the fact that BM itself is a target organ during acute GvHD (aGvHD) with recent work suggesting a role for donor CD4+ T cells in BM specific aGvHD. Our group has shown that patients with higher numbers of BM B cell precursors were less likely to develop cGvHD after allogeneic HSCT (Fedoriw et al., 2012). These observations indicate clinical relevance of impaired BM B lymphopoiesis for cGvHD development. Methods. In order to investigate the effect of cGvHD on BM B cell development, we used the well-characterized major mismatch B6 into B10.BR model of systemic cGvHD. Recipient mice were treated with cyclophosphamide on day -3 and -2, irradiated with 700 cGy on day -1, and injected with 107 T cell depleted (TCD) BM with or without total splenic T cells (0.5-1x105). Mice were monitored for 30 days, and BM and spleen was harvested and analyzed using flow cytometry. Results. Consistent with patient data, we observed a decrease in the frequency and number of donor-derived uncommitted common lymphoid progenitors (CLP) and B cell progenitors in the BM+ allogeneic T cells group (CLP: 0.17±0.03% vs. 0.06±0.01%, p <0.01; pro B: 2.2 ± 0.5% vs. 0.7 ± 0.3%, p<0.05; pre B: 15.3±1.8% vs. 6.3±2.4%, p<0.05; immature B cells: 5.7±0.7% vs. 2.1±0.7%, p<0.01) (Fig.1). As previously reported for this model, we also found a decrease in the frequency of follicular (FO) B cells (Flynn et al., 2014). We hypothesized that during cGvHD the B cell progenitor BM niche is affected by donor CD4+ T cells leading to impaired B lymphopoiesis. Bone marrow from BM+T cell animals had a significantly higher frequency of CD4+ cells compared to the control group (0.45±0.06% vs. 0.2±0.02%). Depletion of CD4+ T cells using anti-CD4 antibody during the first two weeks after transplant improved pathology scores and prevented weight loss in BM+T cells mice. We also observedpartial recovery of B cell progenitors and Lin-CD45-CD31-CD51+ osteoblasts (OB) in animals treated with anti-CD4 antibodies (pre B 3.5±1.1% vs. 20.4±4.5%, p<0.05; immature B: 1.9±0.9% vs. 3.5±0.3%; OB: 0.8±0.1% vs.1.2±0.2%). A recent study showed that activation and proliferation of conventional T cells in aGvHD model can be prevented by in vivo expansion of regulatory T cells (Tregs) using αDR3 antibody (4C12). We adopted this approach to determine whether Tregs can suppress the cytotoxic effect of donor CD4+ T cells in BM in cGvHD model. Animals that received T cells from 4C12-treated donors had an increase in survival and lower cGvHD pathology scores. These mice also had higher frequency of pro B, pre B, and immature B cells compared to the mice infused with T cells from isotype-treated donors. Conclusions. These studies demonstrate that BM development of B lymphocytes is impaired in a mouse model of systemic cGvHD. Our data suggests that donor-derived CD4+ T cells are involved in the destruction of hematopoietic niches in BM, particularly OB, which support B lymphopoiesis. Moreover, depletion of CD4+ T cells and infusion with in vivo expanded Tregs reduced the severity of cGvHD. Thus, Treg therapy in patients with cGvHD may be important for BM B cell development, and improvement of clinical outcomes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions

2020 ◽  
Author(s):  
Silke E. Lindner ◽  
Colt A. Egelston ◽  
Stephanie M. Huard ◽  
Peter P. Lee ◽  
Leo D. Wang
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Peripheral Blood ◽  
Cell Development ◽  
B Cell Development ◽  
Dependent Manner ◽  
B Cell Differentiation ◽  
Nucleotide Exchange

ABSTRACTRho family GTPases are critical for normal B cell development and function and their activity is regulated by a large and complex network of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, the role of GAPs in B cell development is poorly understood. Here we show that the novel Rac-GAP ARHGAP25 is important for B cell development in mice in a CXCR4-dependent manner. We show that Arhgap25 deficiency leads to a significant decrease in peripheral blood B cell numbers, as well as defects in mature B cell differentiation. Arhgap25-/- B cells respond to antigen stimulation in vitro and in vivo but have impaired germinal center formation and decreased IgG1 class switching. Additionally, Arhgap25-/- B cells exhibit increased chemotaxis to CXCL12. Taken together, these studies demonstrate an important role for Arhgap25 in peripheral B cell development and antigen response.

scholarly journals EZH2 Enables the Proliferation of Germinal Center B Cells and DLBCL through a Rb-E2F1 Positive Feedback Loop Involving Repression of CDKN1A

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 734-734
Author(s):  
Wendy Béguelin ◽  
Martin A Rivas ◽  
María Teresa Calvo Fernández ◽  
Ari Melnick
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Transcriptional Repression ◽  
Feedback Loop ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Conditional Deletion ◽  
Direct Target

Abstract Many B cell lymphomas arise from germinal center (GC) B cells of the humoral immune system, which are unique in their ability to replicate at an accelerated rate, which requires attenuation of replication checkpoints. Upon activation, GC B cells upregulate EZH2, a Polycomb protein that mediates transcriptional repression by trimethylating histone 3 lysine 27 (H3K27me3). Conditional deletion of EZH2 results in failure to form GCs. EZH2 is often highly expressed or affected by somatic gain of function mutations in GC B cell-derived diffuse large B cell lymphoma (DLBCL) and is required to maintain lymphoma cell proliferation and survival. Our previous research identified CDKN1A (p21 Cip1) as a direct target of EZH2 in GC B cells and DLBCLs. EZH2 causes promoter H3K27 trimethylation and transcriptional repression of CDKN1A in GC B cells and DLBCL cells. Treatment of DLBCLs with a specific EZH2 inhibitor (GSK343) or EZH2 shRNA caused CDKN1A H3K27me3 demethylation and derepression. Based on these considerations we hypothesized that silencing of CDKN1Athrough H3K27me3 might explain the proliferative GC and DLBCL phenotype. To test this notion, we crossed GC-specific conditional Cg1Cre;Ezh2fl/fl mice with Cdkn1a-/- mice. We assessed GC formation after T cell-dependent immunization in double vs. single Cdkn1a or Ezh2 KO mice. Cdkn1a-/- mice manifested perfectly normal GC formation, whereas there was complete absence of GCs in Cg1Cre-Ezh2fl/fl mice. In contrast, Cg1Cre;Ezh2fl/fl;Cdkn1a-/- double KO mice exhibited normal GC formation as measured by immunohistochemistry and flow cytometry. While conditional deletion of Ezh2 in GCs abrogates immunoglobulin affinity maturation, the double KO mice manifested normal development of high affinity antibodies after specific antigen exposure (NP-KLH). Cell cycle analysis of double KO mice showed a similar proportion of GC B cells in S phase as WT or Cdkn1a-/- controls, as measured by BrdU incorporation, indicating that loss of p21 allows progression of cell cycle. These effects were linked to the methyltransferase function of EZH2 since Cdkn1a-/- also rescued the loss of GCs driven by administration of EZH2 inhibitor observed in WT mice. We observed a similar phenomenon in DLBCL cells since shRNA-mediated depletion of CDKN1A rescued the growth suppressive effect of EZH2 shRNA or specific EZH2 inhibitors. Therefore H3K27me3 and repression of CDKN1Aexplains to a large extent how EZH2 enables GC formation and maintains growth of DLBCL cells. To further understand the role of EZH2 as a driver of the cell cycle we explored its relation to the G1/2 checkpoint regulated by p21Cip1. We found that GC B cells from Cg1Cre;Ezh2fl/fl;Cdkn1a-/- double KO mice exhibited high levels of phospho Rb by IHC, similar to the levels found in WT or Cdkn1a-/- control mice. Hyperphosphorylation of Rb induces its inactivation, allowing the release of E2F transcription factors and cell cycle progression. EZH2 was previously shown to be a direct target of E2F1, E2F2 and, to a lesser extent E2F3. Among these we found that E2F1 mRNA and protein expression are especially highly expressed and upregulated in GC B cells vs. naïve B cells. By qChIP we show that E2F1 is bound to the EZH2 promoter in GC-derived DLBCL cell lines. Moreover, E2F1 gene expression is positively correlated with EZH2 (R=0.35, p<0.0001) and moderately inversely correlates with CDKN1A (R=-0.22, p<0.0001) in a cohort of 757 DLBCL patient samples. Therefore, we explored the function of E2F1 in GC formation. We found that E2f1-/- mice developed reduced number and size of GCs as compared to control mice (E2f1-/- vs. WT, p<0.01). To determine if this phenotype was due to a lack of induction of EZH2 by E2F1, we transduced bone marrow of E2f1-/- or WT donor mice with retrovirus encoding EZH2-GFP or GFP alone, transplanted them into lethally irradiated recipients and assessed the GC reaction after immunization. Notably, EZH2 expression successfully rescued E2f1-/- phenotype (E2f1-/-+GFP vs.E2f1-/-+EZH2, p<0.001), indicating that the pRb-E2F1 pathway drives the GC reaction by inducing EZH2. In summary we identified a positive feedback loop required for GC formation and DLBCL whereby EZH2 controls GC B cell proliferation by suppressing the critical cell cycle checkpoint gene CDKN1A, allowing cell cycle progression with a concomitant phosphorylation of Rb. This causes the release of E2F1, which positively regulates the expression of EZH2. Disclosures Melnick: Janssen: Research Funding.

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