Marc A. Weniger

DNA methylome analysis in Burkitt and follicular lymphomas identifies differentially methylated regions linked to somatic mutation and transcriptional control

Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG-MYC translocation–positive Burkitt lymphoma, nine BCL2 translocation–positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas.

MINCR is a MYC-induced lncRNA able to modulate MYC’s transcriptional network in Burkitt lymphoma cells

Significance Gains of the MYC gene are the most common imbalances in cancer and are associated with poor prognosis, particularly in B-cell lymphoma. Recent advances in DNA sequencing have revealed the existence of thousands of long noncoding RNAs (lncRNAs) with unknown functional relevance. We have here identified a MYC-regulated lncRNA that we named MYC-induced long noncoding RNA (MINCR) that has a strong correlation with MYC expression in cancer. We show that MINCR is functional and controls cell cycle progression by influencing the expression of MYC-regulated cell cycle genes. MINCR is, therefore, a novel player in MYC’s transcriptional network, with the potential to open new therapeutic windows in the fight against malignant lymphoma and, possibly, all cancers that rely on MYC expression. Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

Highly recurrent mutations of SGK1 , DUSP2 and JUNB in nodular lymphocyte predominant Hodgkin lymphoma

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)—a subtype of Hodgkin lymphoma (HL)—is characterized by a low content of tumor cells, the lymphocyte predominant (LP) cells. Transformation into diffuse large B-cell lymphoma (DLBCL) occurs in about 10% of patients. We performed whole-genome mutation analysis of the DLBCL components from two composite lymphomas consisting of clonally related NLPHL and DLBCL as a means to identify candidate tumor suppressor genes and oncogenes in NLPHL. The analysis of LP cells for selected mutations of the DLBCL revealed that most mutations are also present in the LP cells, indicating a close relationship between the two components. The analysis of 62 selected genes in NLPHL by targeted ultra-deep sequencing revealed three novel highly recurrently mutated genes (each mutated in ~50% of cases), that is, DUSP2, SGK1 and JUNB. SGK1 was expressed in the LP cells of primary NLPHL cases and in the NLPHL cell line DEV. Administration of an SGK1 inhibitor induced apoptosis in the NLPHL cell line DEV and the DLBCL cell line Farage, suggesting a pathogenetic role of SGK1 in the LP and DLBCL cells. In summary, the present study identifies SGK1, DUSP2 and JUNB as novel key players in the pathogenesis of NLPHL.

NF-κB deregulation in Hodgkin lymphoma

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (HL) show constitutive activity of both the canonical and non-canonical NF-κB signaling pathways. The central pathogenetic role of this activity is indicated from studies with HL cell lines, which undergo apoptosis upon NF-κB inhibition. Multiple factors contribute to the strong NF-κB activity of HRS cells. This includes interaction with other cells in the lymphoma microenvironment through CD30, CD40, BCMA and other receptors, but also recurrent somatic genetic lesions in various factors of the NF-κB pathway, including destructive mutations in negative regulators of NF-κB signaling (e.g. TNFAIP3, NFKBIA), and copy number gains of genes encoding positive regulators (e.g. REL, MAP3K14). In Epstein-Barr virus-positive cases of classical HL, the virus-encoded latent membrane protein 1 causes NF-κB activation by mimicking an active CD40 receptor. NF-κB activity is also seen in the tumor cells of the rare nodular lymphocyte predominant form of HL, but the causes for this activity are largely unclear.

An oncogenic axis of STAT-mediated BATF3 upregulation causing MYC activity in classical Hodgkin lymphoma and anaplastic large cell lymphoma

Classical Hodgkin lymphoma (cHL) and anaplastic large cell lymphoma (ALCL) feature high expression of activator protein-1 (AP-1) transcription factors, which regulate various physiological processes but also promote lymphomagenesis. The AP-1 factor basic leucine zipper transcription factor, ATF-like 3 (BATF3), is highly transcribed in cHL and ALCL; however, its functional importance in lymphomagenesis is unknown. Here we show that proto-typical CD30+ lymphomas, namely cHL (21/30) and primary mediastinal B-cell lymphoma (8/9), but also CD30+ diffuse large B-cell lymphoma (15/20) frequently express BATF3 protein. Mass spectrometry and co-immunoprecipitation established interactions of BATF3 with JUN and JUNB in cHL and ALCL lines. BATF3 knockdown using short hairpin RNAs was toxic for cHL and ALCL lines, reducing their proliferation and survival. We identified MYC as a critical BATF3 target and confirmed binding of BATF3 to the MYC promoter. JAK/STAT signaling regulated BATF3 expression, as chemical JAK2 inhibition reduced and interleukin 13 stimulation induced BATF3 expression in cHL lines. Chromatin immunoprecipitation substantiated a direct regulation of BATF3 by STAT proteins in cHL and ALCL lines. In conclusion, we identified STAT-mediated BATF3 expression that is essential for lymphoma cell survival and promoted MYC activity in cHL and ALCL, hence we recognized a new oncogenic axis in these lymphomas.

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.

Human CD30+ B cells represent a unique subset related to Hodgkin lymphoma cells

&NA; Very few B cells in germinal centers (GCs) and extrafollicular (EF) regions of lymph nodes express CD30. Their specific features and relationship to CD30‐expressing Hodgkin and Reed/Sternberg (HRS) cells of Hodgkin lymphoma are unclear but highly relevant, because numerous patients with lymphoma are currently treated with an anti‐CD30 immunotoxin. We performed a comprehensive analysis of human CD30+ B cells. Phenotypic and IgV gene analyses indicated that CD30+ GC B lymphocytes represent typical GC B cells, and that CD30+ EF B cells are mostly post‐GC B cells. The transcriptomes of CD30+ GC and EF B cells largely overlapped, sharing a strong MYC signature, but were strikingly different from conventional GC B cells and memory B and plasma cells, respectively. CD30+ GC B cells represent MYC+ centrocytes redifferentiating into centroblasts; CD30+ EF B cells represent active, proliferating memory B cells. HRS cells shared typical transcriptome patterns with CD30+ B cells, suggesting that they originate from these lymphocytes or acquire their characteristic features during lymphomagenesis. By comparing HRS to normal CD30+ B cells we redefined aberrant and disease‐specific features of HRS cells. A remarkable downregulation of genes regulating genomic stability and cytokinesis in HRS cells may explain their genomic instability and multinuclearity.

A large fraction of human tonsillar B cells expressing CD27 are germinal center B cells.

We read with great interest the recent article by Perez et al.1 reporting on a comparative analysis of human memory B cells derived from peripheral blood (PB) and tonsils. The authors analyzed the functional differences of memory B cells in vitro, stimulating either bulk or sorted memory B cells over several days. Although the PB memory B cells largely survived and proliferated when stimulated for up to 10 days, the cells sorted as tonsillar memory B cells failed to expand, showed massive cell death and a larger fraction of them differentiated into antibody-secreting cells. The experimental setup of the work was either based on the analysis of total B cells with subsequent staining for CD27 (Figure 1a of Perez et al.1) or based on the isolation of PB and tonsillar memory B cells by magnetic- or fluorescence-activated cell sorting as CD19+ and CD27+ cells for the further studies.

Complex immune evasion strategies in classical Hodgkin lymphoma

Classical Hodgkin lymphoma uses multiple strategies to evade the immune system. cHL cells promoted regulatory T-cell features in CD4+ T cells and invoked additional strategies, such as purinergic signaling, to suppress immune detection. The cellular microenvironment in classical Hodgkin lymphoma (cHL) is dominated by a mixed infiltrate of inflammatory cells with typically only about 1% Hodgkin and Reed/Sternberg (HRS) tumor cells. T cells are usually the largest population of cells in the cHL microenvironment, encompassing T helper (Th) cells, regulatory T cells (Tregs), and cytotoxic T cells. Th cells and Tregs presumably provide essential survival signals for HRS cells. Tregs are also involved in rescuing HRS cells from antitumor immune responses. An understanding of the immune evasion strategies of HRS cells is not only relevant for a characterization of the pathophysiology of cHL but is also clinically relevant, given the current treatment approaches targeting checkpoint inhibitors. Here, we characterized the cHL-specific CD4+ T-cell infiltrate regarding its role in immune evasion. Global gene expression analysis of CD4+ Th cells and Tregs isolated from cHL lymph nodes and reactive tonsils revealed that Treg signatures were enriched in CD4+ Th cells of cHL. Hence, HRS cells may induce Treg differentiation in Th cells, a conclusion supported by in vitro studies with Th cells and cHL cell lines. We also found evidence for immune-suppressive purinergic signaling and a role of the inhibitory receptor-ligand pairs B- and T-cell lymphocyte attenuator–herpesvirus entry mediator and CD200R–CD200 in promoting immune evasion. Taken together, this study highlights the relevance of Treg induction and reveals new immune checkpoint-driven immune evasion strategies in cHL. Cancer Immunol Res; 5(12); 1122–32. ©2017 AACR.

Composite Lymphomas and the Relationship of Hodgkin Lymphoma to Non-Hodgkin Lymphomas

Composite lymphomas are rare combinations of two distinct types of lymphomas occurring simultaneously in one patient, often a Hodgkin lymphoma (HL) and a non-Hodgkin lymphoma (NHL). In other instances, a HL and a NHL may occur subsequently in a patient. Some composite lymphomas likely represent chance occurrences of two independent tumors. However, in a large fraction of combined HL and NHL, the two lymphomas are clonally related and hence have a common origin. Detailed analysis of the rearranged immunoglobulin V region genes of such related lymphomas provided strong evidence that in many cases, the two lymphomas in composite as well as consecutive HL and B-cell NHL developed from distinct daughter cells of a mutating germinal center (GC) B-cell clone. Clonally related composite lymphomas carry both shared early genetic lesions as well as later separate transforming events, supporting a multi-step transformation process in such cases. The pattern of clonally related immunoglobulin V region genes and shared genetic lesions points to a close relationship of HL to B-cell NHL.