Deciphering the Network Architecture of APOBEC3-Driven Mutagenesis in HPV-Positive Head and Neck Cancers

1 Abstract APOBEC3A (A3A) and APOBEC3B (A3B) are cytosine deaminases that restrict viral infection and can also mutate the host genome. In human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC), expression of both enzymes is elevated, but bulk sequencing averages their effects across mixed cell populations. Here we profile single cells from HPV16-positive HNSCC tumors and matched normal tissue. We find that the APOBEC3 (A3) single base substitution mutational signature, SBS2, is enriched in cells expressing more A3A than A3B , whereas copy number alteration (CNA) burden is enriched in cells expressing more A3B than A3A . Tumor versus normal co-expression networks identify the A3 interactors RALY and HNRNPA2B1 as candidate A3 activators. We found that SBS2 and CNA mark the maintenance and productive stages of HPV16 lifecycle, and their ratio may offer a molecular estimate of tumor age. The neoantigens from immune-visible SBS2-HIGH and immune-evasive CNA-HIGH cells identify candidates for mRNA vaccines matched to a tumor’s viral state. 2 Significance A3A and A3B generate most SBS2 mutations in HPV-positive HNSCC, but their expression level alone only weakly predicts SBS2 burden. Single-cell profiling shows that the two enzymes instead track distinct stages of the HPV16 lifecycle, producing immunologically distinct tumor cell populations that yield stage-matched neoantigen vaccine candidates.