Differential expression of splice variants between responders and non-responders to immunotherapy in merkel cell carcinoma

Summary: Post-transcriptional splicing of mRNA leads to a variety of protein isoforms of a gene, each with distinct functions. Although alternative splicing events have been studied in many cancers and associated with outcomes, the role of alternative splicing in Merkel cell carcinoma (MCC) is not well understood, especially in the context of resistance to immune checkpoint inhibition (ICI). We evaluated bulk RNA-seq data from 9 primary MCC tumors from patients treated with neoadjuvant nivolumab (4 tumors that completely resolved complete response and 5 tumors that persisted at 4 weeks after ICI start) for differential expression of alternative splicing events. We used the well-established bioinformatic tool called Replicate Multivariate Analysis of Transcript Splicing (rMATS)-turbo bioinformatic tool. Our analysis focused on skipped exon events, because these splice variants can be detected with highest confidence. 215 splice variants were significantly more highly expressed in responders to ICI, while 172 splice variants were more highly expressed in non-responders. To further eliminate false positive, we filtered by difference in expression of ≥25% with FDR ≤ 0.01 and focused on genes specifically expressed in tumor cells in single cell RNA-Seq data from primary MCC tumors. This filtering yielded 10 gene variants. Non-responders showed higher expression of a 130 bp exon in PLEKHA5, a phosphorylation target of MET receptor tyrosine kinase involved in metastasis of various cancers, including melanoma. MET antagonism is a treatment for melanoma though not explored in MCC. Samples with complete response, on the other hand, showed higher expression of a 50 bp exon in ITGB3BP, which is upregulated by TGF-beta pathway and associated with poor prognosis in several cancers. Our results suggest alternative splicing plays a role in MCC ICI resistance and there may be existing therapeutic strategies that could overcome the identified resistance mechanisms. Joshua D. Bloomstein¹, Tomas Bencomo^{2, 3}, Paul Nghiem^{2, 3} 1. Internal Medicine, University of Washington, Seattle, WA, United States. 2. Dermatology, University of Washington, Seattle, WA, United States. 3. Fred Hutchinson Cancer Center, Seattle, WA, United States. Translational Studies: Cell and Molecular Biology