Subtype-Specific and Co-Occurring Genetic Alterations in B-cell Non-Hodgkin Lymphoma

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis

Glucose Limitation Alters Glutamine Metabolism in MUC1-Overexpressing Pancreatic Cancer Cells

Pancreatic cancer cells overexpressing Mucin 1 (MUC1) rely on aerobic glycolysis and, correspondingly, are dependent on glucose for survival. Our NMR metabolomics comparative analysis of control (S2–013.Neo) and MUC1-overexpressing (S2–013.MUC1) cells demonstrates that MUC1 reprograms glutamine metabolism upon glucose limitation. The observed alteration in glutamine metabolism under glucose limitation was accompanied by a relative decrease in the proliferation of MUC1-overexpressing cells compared with steady-state conditions. Moreover, glucose limitation induces G1 phase arrest where S2–013.MUC1 cells fail to enter S phase and synthesize DNA because of a significant disruption in pyrimidine nucleotide biosynthesis. Our metabolomics analysis indicates that glutamine is the major source of oxaloacetate in S2–013.Neo and S2–013.MUC1 cells, where oxaloacetate is converted to aspartate, an important metabolite for pyrimidine nucleotide biosynthesis. However, glucose limitation impedes the flow of glutamine carbons into the pyrimidine nucleotide rings and instead leads to a significant accumulation of glutamine-derived aspartate in S2–013.MUC1 cells

Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice

CREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, andshowedMycbinding. Through the analysis ofCREBBPmutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/ frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.Research in the M.R.G. group is supported by grants from the Nebraska Department of Health and Human Services (LB506 2016-16) and the National Institutes of Health, National Cancer Institute (1R01CA201380). Research in the I.S.-G. group is partially supported by Fondo Europeo de Desarrollo Regional (FEDER) and by the Ministry of Economy and Competitiveness (SAF2012-32810, SAF2015-64420-R, and Red de Excelencia Consolider OncoBIO SAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), Instituto de Salud Carlos III Plan de Ayudas Institute of Biomedical Research of Salamanca 2015 Proyectos Integrados (IBY15/00003), by Junta de Castilla y Leon (BIO/SA51/15, CSI001U14, UIC-017, andCSI001U16), Fundacion Inocente Inocente, by the German Carreras Foundation (Organisation der Deutsche Jose Carreras Leukamie-Stiftung eV R13/26), and by the Advanced Research on Interaction Mechanisms of electro Magnetic Exposures with Organisms for Risk Assessment project (European Union’s Seventh Framework Programme [FP7/2007-2013] under grant agreement no. 282891). The Nebraska Lymphoma Study Group tissue bank is supported by the Fred & Pamela Buffett Cancer Center’s National Cancer Institute Cancer Center Support Grant (P30CA036727). The I.S.-G. laboratory is a member of the EuroSyStem and the Developing Evidence to Inform Decisions about Effectiveness Network funded by the European Union under the FP7 program. Research in the C.V.-D. group is partially supported by a “Miguel Servet” Grant (CP14/00082, AES 2013-2016, FEDER) from the Instituto de Salud Carlos III (Ministerio de Economia y Competitividad). I.G.-R. was supported by BES-Ministerio de Economia y Competitividad (BES-2013-063789). G.R.-H. and L.R.-R. were supported by Fondo Social Europeo-Consejeria de Educacion de la Junta de Castilla y Leon. R.D. was supported by a fellowship from Region Ile de France (Appel hors DIM 2013). Research in the F.R.-L. group was supported by grants from Universite Paris Diderot, CNRS, Institut National du Cancer (2012-1-PL-BIO), and the Plan Cancer Environnement 2013. Ultrafast liquid chromatography analyses were done on the platform “Bioprofiler” (Unite de Biologie Fonctionnelle et Adaptative).Peer Reviewe

Targetable genetic alterations of TCF4 (E2-2) drive immunoglobulin expression in diffuse large B cell lymphoma

The activated B cell (ABC-like) subtype of diffuse large B cell lymphoma (DLBCL) is characterized by chronic activation of signaling initiated by immunoglobulin m (IgM). By analyzing the DNA copy number profiles of 1000 DLBCL tumors, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data, we found that the TCF4 (E2-2) transcription factor gene was the target of these alterations. Overexpression of TCF4 in ABC-like DLBCL cell lines led to its occupancy on immunoglobulin (IGHM) and MYC gene enhancers and increased expression of these genes at the transcript and protein levels. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting these gains as an attractive potential therapeutic target. Furthermore, the TCF4 gene was one of the top BRD4-regulated genes in DLBCL cell lines. BET proteolysistargeting chimera (PROTAC) ARV771 extinguished TCF4, MYC, and IgM expression and killed ABC-like DLBCL cells in vitro. In DLBCL xenograft models, ARV771 treatment reduced tumor growth and prolonged survival. This work highlights a genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease

TET2 Deficiency Causes Germinal Center Hyperplasia, Impairs Plasma Cell Differentiation, and Promotes B-cell Lymphomagenesis.

TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. SIGNIFICANCE: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors