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Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype worldwide, accounting for 40% of all non-Hodgkin lymphomas. DLBCL presents as an aggressive disease requiring immediate treatment. Although significant improvement in outcome has been achieved, ~40% of patients still experience treatment failure. Here, we characterized the recurrent genetic alterations and transcriptomic signatures in diagnostic biopsies from a population registry-based cohort of 347 patients with de novo DLBCL uniformly treated with R-CHOP. This analysis revealed bi-allelic loss of function mutations of TMEM30A that were associated with favorable treatment outcome. TMEM30A is a chaperone protein, involved in maintaining the asymmetric distribution of phosphatidylethanolamine and phosphatidylserine, an integral component of the plasma membrane and “eat-me” signal recognized by macrophages. Using TMEM30A knockout systems by CRISPR genome editing techniques, we have functionally characterized this loss-of-function mutation in representative human and mouse DLBCL cell line models. We have discovered that TMEM30A loss is associated with increased B-cell signaling following antigen stimulation, including a two-fold increase in the diffusion rate of B-cell receptor (BCR) clustering, using high resolution Single Particle Tracking (SPT) technology. In addition, we have measured three-fold increase in chemotherapeutic drug accumulation in both knockout cell lines and randomly selected patient biopsies with TMEM30A biallelic loss. This observation was validated in a xenograft mouse model, which presented improved survival and limited tumor growth following vincristine treatment in mice injected with TMEM30A null DLBCL cell lines compared with native cell lines. This phenotype explains the improved prognosis observed in DLBCL patients following R-CHOP treatment. Furthermore, we have observed over two fold higher numbers of tumor-associated macrophages in B-cell lymphoma syngeneic mouse models with Tmem30a loss-of-function, prior to any form of treatment, suggesting the existence of “hot” and primed tumors. Our data highlight a multi-faceted role for TMEM30A and plasma membrane physiology in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Characterization of these mechanisms will address a missing link in the cancer field as related insights in lymphoma will outline therapeutic approaches that can be extended to cancer therapy in general. Citation Format: Shannon Healy, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Christoffer Hother, Anja Mottok, Fong Chun Chan, Lauren Chong, Robert Kridel, Merrill Boyle, Barbara Meissner, Tomohiro Aoki, Katsuyoshi Takata, Bruce W. Woolcock, Elena Vigano, Libin Abraham, Michael Gold, Adele Telenius, Pedro Farinha, Graham Slack, Susana Ben-Neriah, Daniel Lai, Allen W. Zhang, Sohrab Salehi, Hennady P. Shulha, Derek S. Chiu, Sara Mostafavi, Alina S. Gerrie, Diego Villa, Laurie H. Sehn, Kerry J. J. Savage, Andrew J. J. Mungall, Andrew P. Weng, Marcel Bally, Ryan D. Morin, Gabriela V. Cohen Freue, Joseph M. Connors, Marco A. Marra, Sohrab P. Shah, Randy D. Gascoyne1, David W. Scott, Christian Steidl, Ulrich Steidl. TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3480.

Genomic studies have revealed surprising new insights into the genetics of human B-cell lymphomas. Of particular note the lysine-specific methyl transferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and in diffuse large B-cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations in lymphoma are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and promotes lymphoma development in vivo. Integrative analyses of KMT2D function indicate specific and focal effects on H3K4 methylation and gene expression. These include B-cell growth and differentiation signals such as the CD40, JAK/STAT, and B-cell receptor pathways. Moreover, KMT2D targets include frequently mutated tumor suppressor genes such as TNFAIP3/A20, SOCS3, and TNFRSF14/HVEM. In this manner, KMT2D deficiency disrupts the physiological process of B-cell differentiation, facilitates malignant outgrowth, and can impede therapies acting on the CD40 and B-cell receptor pathways. Citation Format: Ana Ortega-Molina, Isaac W. Boss, Andres Canela, Heng Pan, Yanwen Jiang, Chunying Zhao, Man Jiang, Deqing Hu, Ji-Eun Lee, Hua-Tang Chen, Daisuke Ennishi, David W. Scott, Christoffer Hother, Shichong Liu, Xing-Jun Cao, Wayne Tam, Rita Shaknovich, Benjamin A. Garcia, Randy D. Gascoyne, Kai Ge, Ali Shilatifard, Olivier Elemento, Andre Nussenzweig, Ari M. Melnick, Hans-Guido Wendel. Functional characterization of the tumor suppressor lysine-specific methyltransferase KMT2D in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA18.

Introduction: Primary mediastinal large B cell lymphoma (PMBCL) is a distinct subtype of aggressive B cell lymphoma that arises from thymic medullary B cells and characteristically presents as a mass in the anterior mediastinum. A proportion of PMBCL patients suffer from refractory or relapsing disease, and subsequent salvage therapies are frequently ineffective. Development of targeted therapies is impeded by the lack of knowledge about the mutational landscape in the lymphoma genomes and mutation-associated phenotypes. We recently reported somatic mutations in the transcriptome of 7 PMBCL patients and 3 cell lines by next-generation sequencing and found a hotspot mutation in exon 8 of IL4R (Gunawardana et al, Nat Genet, 2014). Interleukin (IL)-4 is a type II cytokine that regulates immune responses through binding to a receptor complex consisting of the IL4 receptor alpha (IL4Rα) chain and IL13Rα or the common gamma chain (γc). Activation of IL4R by IL-4 and/or IL-13 initiates intracellular signal transduction mediated by the phosphorylation of JAnus Kinase-Signal Transducer and Activation of Transcription (JAK-STAT) pathway. We hypothesize that constitutively active JAK-STAT observed in PMBCL is in part due to gain-of-function IL4R mutations signaling through this oncogenic pathway. Methods and samples: A sequencing cohort consisting of fresh-frozen pretreatment lymph node biopsies from 62 PMBCL cases were selected from the BCCA tissue archive. DNA from PMBCL tumors and 3 PMBCL-derived cell lines were extracted for IL4R exonic PCR amplification and Sanger sequencing or high-throughput sequencing on an Illumina MiSeq instrument. WT and mutant (I242N) IL4R cDNA were cloned into mammalian expression vectors and expressed in the lymphoma cell line DEV and in engineered HEK293 cells expressing STAT6. Supernatant from cultured cells were used to measure the activity of STAT6-dependent expression of secreted embryonic alkaline phosphatase (SEAP). Extracted RNA and protein from transfected cells were used for qRT-PCR and Western blotting, respectively. Results: Somatic IL4R mutations were found in 18 of 65 (28%) cases confirming a hotspot mutation (I242N) in exon 8 in 11 of 18 (61%) mutated cases. Ectopic expression of the mutant in HEK293 cells showed increased SEAP levels compared to WT (534% vs. 100%) indicating STAT6 activation independent of cytokine stimulation. Introduction of the mutant into DEV cells showed IL4 independent hyperphosphorylation of JAK2, STAT5 and STAT6 proteins and upregulation of the B cell activation marker CD23 (963% vs. 100% in WT). Conclusions: IL4R is frequently mutated in PMBCL with a recurrent hotspot affecting the transmembrane domain of the protein. Functional analyses characterize these mutations as gain-of-function leading to constitutive activation of the JAK-STAT pathway and downstream target genes. These data suggest IL4R mutations as novel driver alterations in PMBCL and may provide a rational therapeutic target. Citation Format: Jay Gunawardana, Tessa Van Tol, Katina Mak, David Twa, Elizabeth Chavez, Bruce Woolcock, Robert Kridel, Anja Mottok, Shannon Healy, Adele Telenius, Merrill Boyle, Susana Ben-Neriah, Stacy Hung, Christoffer Hother, Randy Gascoyne, Christian Steidl. Recurrent IL4R mutations in primary mediastinal large B cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3941. doi:10.1158/1538-7445.AM2015-3941