Exploring Splicing Factor Aberrations As a Therapeutic Target in Mature B-Cell Malignancies

Objectives Cancers originating from mature B cells at various stages of terminal differentiation represent a diverse set of cancers. There are numerous pathological subtypes, each comprising genetically and clinically heterogeneous malignancies with distinct biology and clinical outcomes. We have previously identified the recurrence of mutations in splicing factor HNRNPH1 in mantle cell lymphoma (MCL), which were associated with inferior outcomes. Based on growing evidence for the role of splicing factors in mature B cell neoplasms, we sought to extend our characterization of splicing factor aberrations across additional B cell malignancies. We hypothesize that regulation of alternative splicing is essential for B cell differentiation and maturation, and aberrations of splicing factors in B cell malignancies are related to these normal patterns of alternative splicing. Ultimately, we are pursuing alternative splicing as a potential novel therapeutic vulnerability common among mature B cell neoplasms.

Methods We leveraged whole genome, exome, and RNA sequencing data from 2999 tumors assembled from controlled-access repositories and ongoing projects at BC Cancer as part of our Genomic Analysis of Mature B-cell Lymphomas (GAMBL) project. RNA-seq data was aligned with STAR and splicing events were quantified with RMATS. Novel splicing events were quantified following de novo transcriptome assembly with StringTie. Simple somatic mutations were detected using a workflow comprised of four algorithms to identify high-confidence variants. We used additional RNA-seq data from distinct B-cell maturation states (naïve, centroblast, centrocyte, memory, bone marrow plasma, and tonsillar plasma cells) to identify annotated and novel splicing events which are differentially regulated throughout B cell maturation. Annotated and novel events were subsequently used to query alternative splicing in a subset of tumor samples using a combination of Illumina-based and Oxford Nanopore sequencing.

Results More than 30% of patient samples harboured somatic mutations in at least one of 67 queried splicing factors. HNRNPU, PCBP1, SF3B1, DAZAP1, and HNRNPH1 were most commonly mutated, 13% of cases (Figure 1A). Although only reported in MCL thus far, we noted an abundance of HNRNPH1 mutations in DLBCL showing the same pattern of non-coding mutations, most commonly in the activated B cell subgroup. In non-malignant samples, we identified 129 known and 267 novel splicing events which display differential regulation during B cell maturation and warrant further investigation within subtypes of B cell malignancies (Figure 1B). Based on this observation, we hypothesize that the RNA isoform repertoire may vary across B cell differentiation states and its perturbation may contribute to oncogenesis in a context-dependent manner. Furthermore, the importance of alternative splicing during B cell maturation implies that perturbation of splicing in B cell malignancies could play a role in pathogenesis.

We previously demonstrated that non-coding mutations in HNRNPH1 contribute to overexpression of the hnRNP H protein and are associated with poor patient outcomes in MCL. Based on the predominance of these mutations in DLBCL, we sought to evaluate hnRNP H overexpression as a potential therapeutic target. We identified a putative exonic splicing enhancer (ESE) with minimal secondary structure in the essential exon of HNRNPH1 using ESEfinder and designed locked nucleic acid antisense oligos (ASOs) complementary to the ESE. Using ddPCR, we showed that the ESE ASO can effectively decrease the proportion of the productive HNRNPH1 splice variant at nanomolar concentrations in HEK cells; however, a splice site ASO does not display the same efficacy.

Conclusions We have identified a growing number of splicing factors in mature B cell malignancies as recurrently mutated, indicating that deregulation of the RNA isoform repertoire in B cells can contribute to oncogenesis. Characterizing the landscape of alternative splicing in normal and malignant B cells will allow us to identify opportunities to exploit this reliance in the development of novel therapeutic strategies. Our data highlight antisense oligonucleotides as a promising therapeutic avenue for targeting splicing aberrations caused by common mutations in B cell malignancies.

Coyle:Dren Bio, Inc.: Consultancy; Allakos, Inc.: Consultancy. Rushton:SAGA Diagnostics: Consultancy.