GC-derived Diffuse Large B Cell Lymphoma Research Articles

Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes.

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

Compromised counterselection by FAS creates an aggressive subtype of germinal center lymphoma.

Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B cell lymphoma (DLBCL). Although GC-derived DLBCL has better overall outcomes than other DLBCL types, some cases are refractory, and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes cell death in the light zone, likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. FAS alterations occurred most commonly in GC-derived DLBCL, were associated with inferior outcomes and an enrichment of Tfh cells, and co-occurred with deficiency in HVEM and PD-L1 that regulate the Tfh-B cell interaction. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in GC-derived lymphoma.

Abstract PO-22: Compromised counterselection by FAS creates a lethal subtype of germinal center lymphoma

Abstract Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B-cell lymphoma (DLBCL); however, the function of Fas in the GC remains controversial. Although GC-derived DLBCL has better overall outcomes to therapy than other DLBCL types, some cases are refractory and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes B-cell death in the light zone likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. We found that FAS alterations occurred most commonly in the GC-derived genetic subtype of DLBCL, EZB. FAS alterations in EZB were associated with inferior outcomes and an enrichment of Tfh cells. Alterations in FAS co-occurred with deficiency in HVEM and PD-1 ligands that regulate the Tfh-B cell interaction and were associated with increased diversity of B-cell receptor variable genes across samples. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in a distinct subtype of GC-derived lymphoma. Citation Format: Raud Razzaghi, Shreya Agarwal, Nikita Kotlov, Olga Plotnikova, Krystle Nomie, Da Wei Huang, George W. Wright, Moyi Li, Katsuyoshi Takata, Chen Yao, John J. O'Shea, James D. Phelan, Stefania Pittaluga, David W. Scott, Jagan R. Muppidi. Compromised counterselection by FAS creates a lethal subtype of germinal center lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-22.

Molecular Features of Germinal Cell Derived B-Cell Lymphomas Using miRNA Signatures

Background:B-cell development is a complex biological process that initiates in bone marrow and matures in lymph nodes. These hierarchical stages are tightly regulated by transcription factors and microRNAs (miRNAs). B-cells are prone to genetic alteration during these maturation stages due to DNA breaks required for B-cell receptor re-arrangement and additional changes that occur during the germinal center (GC)-reaction for class switch recombination and somatic hypermutation. The unwarranted genomic changes resulting from these processes can lead to B-cell malignancies with varied clinical and pathological characteristics. Of the lymphoma subtypes derived from mature B-cells, a subset of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large cell lymphoma (PMBL), follicular lymphoma (FL) and Burkitt lymphoma (BL) are putatively derived from GC B-cells, with varied morphological, pathological and clinical characteristics. These tumor are characterized by distinct genetic abnormalities,[ t(14;18) in GCB-DLCBL, and FL or t(8;14)] in BL, and mutational profile. We postulate miRNA expression signatures, may also explain the wide-ranging clinico-pathological characteristics of these GC B- cell derived tumors.Methods:We performed a meta-analysis of miRNA profiles of DLBCL, BL, PBML, FL and EBV+DLBCL performed by us or others. (Iqbal 2015; 2012; Leich 2011; Andrade 2014). We also included lymphomas derived from naïve B-cells (MCL, SLL) and post GC B-cells (activated B cells like ABC-DLBCL), EBV (+) DLBCL, normal B-cell subsets and cell lines of distinct B-cell lineages. The majority of the cases have been characterized by gene expression profiling (GEP) with corresponding pathological clinical characteristics in above studies. The data was obtained using Taqman® human microRNA array (Applied Biosystems, CA) containing 380 miRNAs and analyzed using BRB-array Tools.ResultsIn our initial miRNA expression profile of 249 lymphomas including GCB-DLBCL (n=34), ABC-DLBCL(n= 29), PMBL(n= 9), BL(n= 33), FL (n= 32), EBV+DLBCL (n=8); MCL(n=30) and SLL(n=12)showed distinct hierarchical clusters enriched with GEP defined subgroup, with few interspersed cases from other subgroups suggesting that miRNAs can complement and delineate GEP defined molecular subgroups, an observation consistent with earlier findings (Iqbal 2015).When we examined miRNAs, that are specifically associated with GC B- derived lymphomas, we observed a subset of miRNA including miR-146a, miR-142-3p, miR-17, miR19b, and miR106a were present at higher levels (top 5% in abundance by CT values) in GC B lymphomas. Of these, somatic mutations in miR142 have been recently reported in GC derived lymphomas (Alyssa, Leukemia 2016). These miRNAs also represented the most abundant miRNAs in centroblast, suggesting lineage specificity of miRNA signature in these lymphomas. However, miR222 highly expressed in centroblast was not expressed at higher levels in GCB-DLBCL and BL. A number of miRNAs specifically abundant in distinct lymphomas included (BL: miR-20a, miR-16), (GCB-DLBCL: miR-223, miR-24) and (FL: miR222). MiR-150 was highly expressed in NHLs putatively derived from naive B-cells like MCL or SLL, but absent from GC-derived NHLs. Consistent with this, miR-150 was present in naive B-cells, but not in centroblast, suggesting that miR-150 may have discrete function during early B-cell differentiation and late maturation stages.Within GC derived lymphoma, we examined miRNA profiles in clinically aggressive (BL); indolent lymphomas (FL) and intermediate aggressive (GCB-DLBCL) and their association with normal B-cell subsets, and indeed a number of miRNAs (ex. miR-150) were associated with indolent lymphomas, whereas a subset of miRNAs was present in aggressive lymphomas (ex. miR-16), suggesting these miRNAs may contribute the distinct clinical characteristics. Though we have identified unique miRNA profiles in these GC derived NHLs, the target genes of these miRNAs remains to be identified.Conclusion:We identified microRNAs that were differentially expressed between GC-derived DLBCL and other lymphomas, which may help explain distinguishing features of these lymphomas. DisclosuresNo relevant conflicts of interest to declare.

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

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 (p21Cip1) 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. DisclosuresMelnick:Janssen: Research Funding.

GNA13 loss in germinal center B cells leads to impaired apoptosis and promotes lymphoma in vivo

AbstractGNA13 is the most frequently mutated gene in germinal center (GC)-derived B-cell lymphomas, including nearly a quarter of Burkitt lymphoma and GC-derived diffuse large B-cell lymphoma. These mutations occur in a pattern consistent with loss of function. We have modeled the GNA13-deficient state exclusively in GC B cells by crossing the Gna13 conditional knockout mouse strain with the GC-specific AID-Cre transgenic strain. AID-Cre+ GNA13-deficient mice demonstrate disordered GC architecture and dark zone/light zone distribution in vivo, and demonstrate altered migration behavior, decreased levels of filamentous actin, and attenuated RhoA activity in vitro. We also found that GNA13-deficient mice have increased numbers of GC B cells that display impaired caspase-mediated cell death and increased frequency of somatic hypermutation in the immunoglobulin VH locus. Lastly, GNA13 deficiency, combined with conditional MYC transgene expression in mouse GC B cells, promotes lymphomagenesis. Thus, GNA13 loss is associated with GC B-cell persistence, in which impaired apoptosis and ongoing somatic hypermutation may lead to an increased risk of lymphoma development.

Lectin-like transcript 1 is a marker of germinal center-derived B-cell non-Hodgkin's lymphomas dampening natural killer cell functions

Non-Hodgkin's lymphomas (NHLs) are malignant neoplasms which are clinically and biologically diverse. Their incidence is constantly increasing and despite treatment advances, there is a need for novel targeted therapies. Here, we identified Lectin-like transcript 1 (LLT1) as a biomarker of germinal center (GC)-derived B-cell NHLs. LLT1 identifies GC B cells in reactive tonsils and lymph nodes and its expression is maintained in B-cell NHLs which derive from GC, including Burkitt lymphoma (BL), follicular lymphoma (FL), and GC-derived diffuse large B-cell lymphoma (DLBCL). We further show that LLT1 expression by tumors dampens natural killer (NK) cell functions following interaction with its receptor CD161, uncovering a potential immune escape mechanism. Our results pinpoint LLT1 as a novel biomarker of GC-derived B-cell NHLs and as a candidate target for innovative immunotherapies.