Mutations Affecting RNA Binding Proteins Are a Novel Feature of Mantle Cell Lymphoma

Objectives Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. We sought to extend our understanding of the molecular etiology of this malignancy using an integrative genomic analysis of diagnostic biopsies. Methods We performed exome sequencing on 51 frozen MCL tumors and analyzed these alongside previously published exome cohorts. We sequenced tumour genomes and matched constitutional DNA from 34 frozen MCLs, along with matched constitutional DNA, to more broadly identify the pattern of non-coding mutations. Based on mutations identified in this discovery cohort, we re-sequenced 18 recurrently-mutated genes in 212 archival MCLs, each having clinical follow-up data. We also performed RNA-seq on 110 of these cases and analyzed these data for alternative splicing and differential expression, including the differential splicing of HNRNPH1 in the context of recurrent intronic mutations. We investigated the functional and phenotypic effect of mutations and deregulated HNRNPH1 protein through ectopic expression of full-length HNRNPH1 and a mini-gene containing the exons and introns affected by mutations. Using custom droplet digital PCR (ddPCR) assays, we validated alternative splicing patterns in HNRNPH1 itself and other targets identified through re-analysis of available CLIP-seq data. Results In addition to confirming the prognostic association of TP53 and NOTCH1 mutations in MCL, we identified two additional genes associated with outcome: EWSR1 with poor outcome (HR = 5.6) and MEF2B with good outcome (HR = 0.2). By comparing mutation patterns to diffuse large B-cell lymphoma (DLBCL), we identified an MCL-specific missense hot spot in MEF2B, non-specific truncating mutations in EWSR1, and truncating mutations affecting the DAZAP1 C-terminus in both MCL and DLBCL. The DAZAP1 mutations are predicted to alter protein sub-cellular localization and disrupt protein-protein interactions. We also identified the focal recurrence of non-coding mutations surrounding a single exon of the HNRNPH1 gene that were largely restricted to MCL. These mutations affected a region bound by HNRNPH1 protein and disrupted the preferred binding motif of this protein. Intronic mutations were significantly associated with alternative splicing of the HNRNPH1 mRNA and appear t