Immunosuppression allows for proliferation of alternative splicing events in a murine model of tobacco-associated oral cavity squamous cell carcinoma

Abstract Background: Non-canonical alternative splicing events have been demonstrated to occur in head and neck squamous cell carcinoma, and result in functionally active aberrant isoforms that contribute to carcinogenesis. Our preliminary data also demonstrates that high splicing burden is associated with higher recurrence rates in oral cancer as well as relative immunosuppression. These splicing events are hypothesized to generate immunogenic neoantigens given non-self sequences that are generated from these novel isoforms. However, the role of the immune system in regulating the proliferation of aberrant splicing is not well understood. Therefore, we evaluated splicing alteration in the setting of immunosuppression in a murine model of oral cancer. Study Design: An established murine cell line with genetic signature resembling human tobacco-associated oral cavity SCC (4MOSC1) was injected into the oral tongue of wild type (C57/Blk6) and immunocompromised (SCID) mice. At takedown, specimens were collected for bulk RNA sequencing (RNASeq). Fastq files were aligned to mm39 using STAR v.2.7.1a. Splicing analysis was performed using OutSplice. Somatic variants in tumor samples were determined from RNASeq data using the GATK v4.4.0 tool Mutect2. Mutational burden was estimated using the number of single nucleotide variants (SNVs) called in each sample (CountVariants, GATK). Trinity v2.12.0 and NetMHCPan v4.1 were used to define PHBR scores to predict MHC binding affinity of neopeptides. Results: Seven of ten mice had measurable tumor by day 5, for an engraftment rate of 70% (3/5 C57/Blk6; 4/5 SCID). At day 15, tumors in wild type mice had a mean volume of 14.7mm3 compared to 28.9mm3 in SCID mice (one-sided p=0.132). Sequencing analysis demonstrated that splicing burden was higher in tumors developed in SCID mice compared to Blk6 mice (mean 475.25 vs 381.67, p=0.022). The mean number of SNVs were 144.2 mt/mb in SCID mice compared to 81.2 mt/mb in Blk6 mice (p=0.044). Amongst predicted splice derived neopeptides, those in SCID tumors had lower PHBR scores versus wild type tumors (median: 3.13 vs 4.23, p<0.001), correlating with higher predicted MHC binding affinity. However, amongst very strong binders (PHBR score ≤0.5), there was no difference in PHBR score between groups (0.30 vs 0.22, p=0.97). Conclusion: As expected, immunosuppression allowed for increased growth of tumors in the murine oral cancer model. We show that immunosuppression allows for the proliferation of both alternative splicing events and mutations. These preliminary data demonstrate that the immune system may play a role in regulating aberrant splicing events in oral cancer. Aberrant splice derived peptides which stimulate the immune system may be more likely to develop in an immunosuppressed setting. Citation Format: Mitchell Victor, Sophie Jang, Joseph Bendik, Silvio Gutkind, Theresa Guo. Immunosuppression allows for proliferation of alternative splicing events in a murine model of tobacco-associated oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-075.