KMT2D acetylation by CREBBP reveals a cooperative functional interaction at enhancers in normal and malignant germinal center B cells.

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 Crebbp and Kmt2d 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 Crebbp/Kmt2d 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.

Super-enhancer hypermutation alters oncogene expression in B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

Biology of Germinal Center B Cells Relating to Lymphomagenesis.

The germinal center (GC) reaction is a key feature of adaptive humoral immunity. GCs represent the site where mature B cells refine their B-cell receptor (BCR) and are selected based on the newly acquired affinity for the antigen. In the GC, B cells undergo multiple cycles of proliferation, BCR remodeling by immunoglobulin somatic hypermutation (SHM), and affinity-based selection before emerging as effector memory B cells or antibody-secreting plasma cells. At least 2 histologically and functionally distinct compartments are identified in the GC: the dark zone (DZ) and the light zone (LZ). The proliferative burst and immunoglobulin remodeling by SHM occur prevalently in the DZ compartment. In the LZ, GC B cells undergo an affinity-based selection process that requires the interaction with the antigen and accessory cells. GC B cells are also targeted by class switch recombination, an additional mechanism of immunoglobulin remodeling that ensures the expression of diverse isotype classes. These processes are regulated by a complex network of transcription factors, epigenetic modifiers, and signaling pathways that act in concert with mechanisms of intra-GC B-cell trafficking. The same mechanisms underlying the unique ability of GC B cells to generate high affinity antibodies and ensure immunological memory are hijacked during lymphomagenesis and become powerful weapons for malignant transformation. This review will summarize the main processes and transcriptional networks that drive GC B-cell development and are relevant for human B-cell lymphomagenesis.

Genetic mechanisms of HLA-I loss and immune escape in diffuse large B cell lymphoma.

Fifty percent of diffuse large B cell lymphoma (DLBCL) cases lack cell-surface expression of the class I major histocompatibility complex (MHC-I), thus escaping recognition by cytotoxic T cells. Here we show that, across B cell lymphomas, loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. To identify the involved mechanisms, we performed whole exome and targeted HLA deep-sequencing in 74 DLBCL samples, and found somatic inactivation of B2M and the HLA-I loci in 80% (34 of 42) of MHC-INEG tumors. Furthermore, 70% (22 of 32) of MHC-IPOS DLBCLs harbored monoallelic HLA-I genetic alterations (MHC-IPOS/mono), indicating allele-specific inactivation. MHC-INEG and MHC-IPOS/mono cases harbored significantly higher mutational burden and inferred neoantigen load, suggesting potential coselection of HLA-I loss and sustained neoantigen production. Notably, the analysis of >500,000 individuals across different cancer types revealed common germline HLA-I homozygosity, preferentially in DLBCL. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multistep process of HLA-I loss in DLBCL development including both germline and somatic events, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

Tracking Immunoglobulin Repertoire and Transcriptomic Changes in Germinal Center B Cells by Single-Cell Analysis.

In response to T-cell-dependent antigens, mature B cells in the secondary lymphoid organs are stimulated to form germinal centers (GCs), which are histological structures deputed to antibody affinity maturation, a process associated with immunoglobulin gene editing by somatic hypermutation (SHM) and class switch recombination (CSR). GC B cells are heterogeneous and transition across multiple stages before being eliminated by apoptosis or committing to post-GC differentiation as memory B cells or plasma cells. In order to explore the dynamics of SHM and CSR during the GC reaction, we identified GC subpopulations by single-cell (sc) transcriptomics and analyzed the load of immunoglobulin variable (V) region mutations as well as the isotype class distribution in each subpopulation. The results showed that the large majority of GC B cells display a quantitatively similar mutational load in the V regions and analogous IGH isotype class distribution, except for the precursors of memory B cells (PreM) and plasma cells (PBL). PreM showed a bimodal pattern with about half of the cells displaying high V region germline identity and enrichment for unswitched IGH, while the rest of the cells carried a mutational load similar to the bulk of GC B cells and showed a switched isotype. PBL displayed a bias toward expression of IGHG and higher V region germline identity compared to the bulk of GC B cells. Genes implicated in SHM and CSR were significantly induced in specific GC subpopulations, consistent with the occurrence of SHM in dark zone cells and suggesting that CSR can occur within the GC.

Single-cell analysis of germinal-center B cells informs on lymphoma cell of origin and outcome.

In response to T cell-dependent antigens, mature B cells are stimulated to form germinal centers (GCs), the sites of B cell affinity maturation and the cell of origin (COO) of most B cell lymphomas. To explore the dynamics of GC B cell development beyond the known dark zone and light zone compartments, we performed single-cell (sc) transcriptomic analysis on human GC B cells and identified multiple functionally linked subpopulations, including the distinct precursors of memory B cells and plasma cells. The gene expression signatures associated with these GC subpopulations were effective in providing a sc-COO for ∼80% of diffuse large B cell lymphomas (DLBCLs) and identified novel prognostic subgroups of DLBCL.

Unique and Shared Epigenetic Programs of the CREBBP and EP300 Acetyltransferases in Germinal Center B Cells Reveal Targetable Dependencies in Lymphoma.

Inactivating mutations of the CREBBP and EP300 acetyltransferases are among the most common genetic alterations in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). Here, we examined the relationship between these two enzymes in germinal center (GC) B cells, the normal counterpart of FL and DLBCL, and in lymphomagenesis by using conditional GC-directed deletion mouse models targeting Crebbp or Ep300. We found that CREBBP and EP300 modulate common as well as distinct transcriptional programs implicated in separate anatomic and functional GC compartments. Consistently, deletion of Ep300 but not Crebbp impaired the fitness of GC B cells in vivo. Combined loss of Crebbp and Ep300 completely abrogated GC formation, suggesting that these proteins partially compensate for each other through common transcriptional targets. This synthetic lethal interaction was retained in CREBBP-mutant DLBCL cells and could be pharmacologically targeted with selective small molecule inhibitors of CREBBP and EP300 function. These data provide proof-of-principle for the clinical development of EP300-specific inhibitors in FL and DLBCL.

Repurposing dasatinib for diffuse large B cell lymphoma.

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

MEF2B Instructs Germinal Center Development and Acts as an Oncogene in B Cell Lymphomagenesis.

The gene encoding the MEF2B transcription factor is mutated in germinal center (GC)-derived B cell lymphomas, but its role in GC development and lymphomagenesis is unknown. We demonstrate that Mef2b deletion reduces GC formation in mice and identify MEF2B transcriptional targets in GC, with roles in cell proliferation, apoptosis, GC confinement, and differentiation. The most common lymphoma-associated MEF2B mutant (MEF2BD83V) is hypomorphic, yet escapes binding and negative regulation by components of the HUCA complex and class IIa HDACs. Mef2bD83V expression in mice leads to GC enlargement and lymphoma development, a phenotype that becomes fully penetrant in combination with BCL2 de-regulation, an event associated with human MEF2B mutations. These results identify MEF2B as a critical GC regulator and a driver oncogene in lymphomagenesis.

The CREBBP Acetyltransferase Is a Haploinsufficient Tumor Suppressor in B-cell Lymphoma.

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, Crebbp-deficient B cells exhibit enhanced response to mitogenic stimuli and perturbed plasma cell differentiation. Although GC-specific loss of Crebbp was insufficient to initiate malignant transformation, compound Crebbp-haploinsufficient/BCL2-transgenic mice, mimicking the genetics of FL and DLBCL, develop clonal lymphomas recapitulating the features of the human diseases. These findings establish CREBBP as a haploinsufficient tumor-suppressor gene in GC B cells and provide insights into the mechanisms by which its loss contributes to lymphomagenesis.Significance: Loss-of-function mutations of CREBBP 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. Cancer Discov; 7(3); 322-37. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Clinical impact of miR-223 expression in pediatric T-Cell lymphoblastic lymphoma.

Although probability of event-free survival in pediatric lymphoblastic T-cell lymphoma (T-LBL) is about 75%, survival in relapsed patients is very poor, so the identification of new molecular markers is crucial for treatment optimization. Here, we demonstrated that the over-expression of miR-223 promotes tumor T-LBL cell growth, migration and invasion in vitro. We found out that SIK1, an anti-metastatic protein, is a direct target of miR-223 and consequently is significantly reduced in miR-223-overexpressing tumor cells. We measured miR-223 expression levels at diagnosis in tumor biopsies from 67 T-LBL pediatric patients for whom complete clinical and follow up data were available, and we found that high miR-223 expression (above the median value) is associated with worse prognosis (PFS 66% vs 94%, P=0.0036). In addition, the multivariate analysis, conducted taking into account miR-223 expression level and other molecular and clinical characteristics, showed that only high level of miR-223 is an independent factor for worse prognosis. MiR-223 represents a promising marker for treatment stratification in pediatric patients with T-LBL and we provide the first evidence of miR-223 potential role as oncomir by SIK1 repression.

FBXO11 inactivation leads to abnormal germinal-center formation and lymphoproliferative disease.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for the germinal center (GC) reaction and is implicated in lymphomagenesis. BCL6 protein stability is regulated by F-box protein 11 (FBXO11)-mediated ubiquitination and degradation, which is impaired in ∼6% of diffuse large B-cell lymphomas that carry inactivating genetic alterations targeting the FBXO11 gene. In order to investigate the role of FBXO11 in vivo, we analyzed GC-specific FBXO11 knockout mice. FBXO11 reduction or loss led to an increased number of GC B cells, to an altered ratio of GC dark zone to light zone cells, and to higher levels of BCL6 protein in GC B cells. B-cell receptor-mediated degradation of BCL6 was reduced in the absence of FBXO11, suggesting that FBXO11 contributes to the physiologic downregulation of BCL6 at the end of the GC reaction. Finally, FBXO11 inactivation was associated with the development of lymphoproliferative disorders in mice.

The FOXO1 Transcription Factor Instructs the Germinal Center Dark Zone Program.

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.

Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis.

Mutations in the gene encoding the KMT2D (or MLL2) methyltransferase are highly recurrent and occur early during tumorigenesis in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the functional consequences of these mutations and their role in lymphomagenesis are unknown. Here we show that FL- and DLBCL-associated KMT2D mutations impair KMT2D enzymatic activity, leading to diminished global H3K4 methylation in germinal-center (GC) B cells and DLBCL cells. Conditional deletion of Kmt2d early during B cell development, but not after initiation of the GC reaction, results in an increase in GC B cells and enhances B cell proliferation in mice. Moreover, genetic ablation of Kmt2d in mice overexpressing Bcl2 increases the incidence of GC-derived lymphomas resembling human tumors. These findings suggest that KMT2D acts as a tumor suppressor gene whose early loss facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of KMT2D-deficient cells may thus represent a rational therapeutic approach for targeting early tumorigenic events.

Germinal centres and B cell lymphomagenesis.

Germinal centres (GCs) are involved in the selection of B cells secreting high-affinity antibodies and are also the origin of most human B cell lymphomas. Recent progress has been made in identifying the functionally relevant stages of the GC and the complex trafficking mechanisms of B cells within the GC. These studies have identified transcription factors and signalling pathways that regulate distinct phases of GC development. Notably, these factors and pathways are hijacked during tumorigenesis, as revealed by analyses of the genetic lesions associated with various types of B cell lymphomas. This Review focuses on recent insights into the mechanisms that regulate GC development and that are relevant for human B cell lymphomagenesis.

MicroRNA 28 controls cell proliferation and is down-regulated in B-cell lymphomas.

Burkitt lymphoma (BL) is a highly aggressive B-cell non-Hodgkin lymphoma (B-NHL), which originates from germinal center (GC) B cells and harbors translocations deregulating v-myc avian myelocytomatosis viral oncogene homolog (MYC). A comparative analysis of microRNAs expressed in normal and malignant GC B cells identified microRNA 28 (miR-28) as significantly down-regulated in BL, as well as in other GC-derived B-NHL. We show that reexpression of miR-28 impairs cell proliferation and clonogenic properties of BL cells by modulating several targets including MAD2 mitotic arrest deficient-like 1, MAD2L1, a component of the spindle checkpoint whose down-regulation is essential in mediating miR-28-induced proliferation arrest, and BCL2-associated athanogene, BAG1, an activator of the ERK pathway. We identify the oncogene MYC as a negative regulator of miR-28 expression, suggesting that its deregulation by chromosomal translocation in BL leads to miR-28 suppression. In addition, we show that miR-28 can inhibit MYC-induced transformation by directly targeting genes up-regulated by MYC. Overall, our data suggest that miR-28 acts as a tumor suppressor in BL and that its repression by MYC contributes to B-cell lymphomagenesis.

RNAs with multiple personalities.

In the last decade, advances in sequencing technology and a renewed focus on the regulatory potential of RNA molecules have combined to stimulate an enormous expansion in the catalog of known eukaryotic RNAs. Beyond the sheer numerical diversity of RNA species, recent studies have begun to uncover hints of even greater functional complexity. An increasing number of RNA molecules, including those from classic, well-studied classes, have been found to act in previously unanticipated regulatory roles, or as substrate for the biogenesis of functionally distinct RNA molecules, or both. Thus, these molecules can fulfill multiple, parallel functions, compounding the already rich landscape of RNA biology, and potentially connecting disparate biological regulatory networks in unexpected ways. In this article, we review recently discovered instances of RNA multifunctionality, with a particular focus on regulatory small RNAs.