Abstract 4345: A large-scale RNA-seq screen to identify regulators of alternative splicing in cancer
Authors: Lo April , McSharry Maria , Berger Alice 
Published: August 2019

RNA splicing is dysregulated in a widespread manner in cancers including lung adenocarcinoma. In some cases, splicing changes can be attributed to cis-acting splice site mutations or trans-acting mutations in splicing factors. However, in most cases, the underlying causes of splicing changes are unknown. We hypothesized that upstream signaling inputs to alternative splicing regulation can explain some of these unknowns. Specifically, we studied how oncogenic lung cancer signaling pathways (EGFR/Ras, KEAP1/NRF2, MYC, and others) regulate alternative splicing and the expression and activity of splicing factors. We focus on signaling pathways because they can be readily therapeutically modulated with small molecule inhibitors (e.g. tyrosine kinase inhibitors), offering opportunities for therapeutic suppression of downstream splicing effects.

To experimentally determine how signaling pathway perturbation affects alternative splicing, we perturbed A549 lung cancer cells with each of 82 alleles of 27 genes (n = 4 to 8 biological replicates per allele). Genes and variants were selected based on the occurrence of the variants in lung adenocarcinoma tumors. In total, 417 whole transcriptome profiles were generated using the Smart-Seq v4 method (Clontech) and Nextera XT library preparation (Illumina). We performed differential expression analysis using edgeR and differential splicing analysis using MISO. Using this approach, we identified a high-confidence set of 2430 alternative splicing events differentially spliced in the perturbed samples compared to controls. Of these alternative splicing events, 1219 are skipped exons, 469 are mutually exclusive exons, 235 are alternative 5’ splice sites, 255 are alternative 3’ splice sites, and 252 are retained introns.

Among the perturbations tested, overexpression of the RBM45 wild-type allele and the RBM45 D434Y variant allele resulted in the greatest number of alternatively spliced events, with 367 and 323 events respectively. A closer look at these events reveals that when the RBM45 wild-type allele is overexpressed, exon 3 of the cyclin gene CCNG1 is skipped. Interestingly, when RBM45 variant alleles (D434Y, M126I) are overexpressed instead of the wild-type, the exon is not skipped, suggesting loss or change of function. These results suggest that, among other roles, RBM45 may regulate cell cycle patterns by regulating alternative splicing.

In sum, our screen identifies splicing events which are regulated by oncogenic signaling pathways. With this information, it will be possible to propose therapeutic options that mitigate aberrant splicing driven by signaling pathway components. Such therapy may already exist in the form of drugs for other targeted uses but could be repurposed to address aberrant splicing. Importantly, therapy of this type has the potential to be more tolerable compared to treatments directly aimed at splicing factors and splice sites.

Citation Format: April Lo, Maria McSharry, Alice Berger. A large-scale RNA-seq screen to identify regulators of alternative splicing in cancer [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 4345.