Abstract A10: MLL2 interactions in follicular and diffuse large B-cell lymphoma
Authors: Huff Ryan D. , Mendez-Lago Maria , Morin Ryan D. , Scott David W. , Connors Joseph M. , Gascoyne Randy D. , Marra Marco M. 
Published: August 2014

Introduction: Mixed-lineage leukemia 2 (MLL2) encodes a histone-lysine N-methyltransferase that tri-methylates histone H3 lysine 4 (H3K4), a marker of epigenetic transcriptional activation. Having identified MLL2 as a frequent target of somatic mutation in non-Hodgkin lymphomas (NHL), our objective is to characterize the gene targets and genetic interactions of MLL2.

Method: A total of 127 NHLs, including diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) transcriptomes, 12 genomes, and 2 exomes were sequenced to uncover recurrent mutations involved in NHL pathogenesis. ChIP-Seq and RNA-Seq will be utilized to examine gene expression and gene promoter H3K4 tri-methylation profiles of MLL2 wild type and knock out cell lines. The correlation of these data sets will identify MLL2 target genes. A Synthetic Lethal (SL) genetic interaction is characterized by the death of a cell following the disruption of any two genes, but not each gene alone. SL MLL2 partners will be identified by transducing MLL2 expressing cells and their knock out equivalents with a pooled genome-wide lentiviral shRNA library and comparing the resulting differences in shRNA sequence diversity post-transduction, propagation, and selection.

Results: Our genome wide study of NHL identified MLL2 as a target of somatic mutation in 89% of FL and 32% of DLBCL patient samples and 59% of DLBCL cell lines. Furthermore, 91% of all mutations discovered were predicted to result in a loss of function (LOF) phenotype. The biallelic status of 8 NHL cases harbouring two independent mutations in MLL2 was also assessed, and the MLL2 mutations were found to be in trans in all 8 cases. Currently, zinc finger nuclease technology has been utilized to create knock outs of MLL2 in the human embryonic kidney cell line HEK293, and NHL cell lines WSU-DLCL2 and RAMOS in preparation for MLL2 target gene and SL experiments.

Conclusions: MLL2 is a highly recurrent target of somatic mutation in NHL, with a strong selection for deleterious mutations such as nonsense, frame-shift insertions/ deletions and splice-site mutations. The high frequency of mutations predicted to result in LOF suggests inactivating MLL2 mutations are driver mutations in NHL.

Impact: Identification of MLL2 gene targets will expand our understanding of MLL2 biology through identification of gene networks under the control of MLL2 and could reveal how MLL2 is involved in malignant progression of NHL. Furthermore, the SL screen could reveal MLL2 synthetic lethal targets with potential to be exploited as candidate therapeutic targets in MLL2 mutated NHLs.

Citation Format: Ryan D. Huff, Maria Mendez-Lago, Ryan D. Morin, David W. Scott, Joseph M. Connors, Randy D. Gascoyne, Marco M. Marra. MLL2 interactions in follicular and diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A10.