Distinct Roles of Apobec3a and Apobec3b in Apobec3-Driven Mutagenesis in Urothelial Carcinoma

Approximately 70% of single nucleotide variant mutations seen in bladder cancer can be attributed to APOBEC3 enzymes, which catalyze the conversion of cytosine to uracil in both single-stranded DNA and RNA substrates. The APOBEC3 mutational signatures are primarily driven by the APOBEC3A (A3A) and APOBEC3B (A3B) family members. To investigate APOBEC3’s role in bladder cancer, we generated a novel, cre-inducible, pten and p53 knockout, to overexpress mouse Apobec3(mA3) in Upk3a+ (UPPA), compared to a model that does not overexpress mA3(UPP). Apobec3 expression was shown to accelerate bladder tumor formation, indicated by a shorter median time of tumor latency in the UPPA model compared to the UPP model. While both tumor models had presence of squamous differentiation, the extensiveness was more profound in the UPPA tumors and had an enrichment of squamous gene expression. Furthermore, DNA damage induced by mA3 is sufficient to drive squamous differentiation via signaling through IL-1a resulting in AP-1 transcription factor activation. Analysis of human bladder cancer samples revealed APOBEC3A to be the only APOBEC3 family member significantly associated with squamous gene expression. The use of immunologic agents to treat bladder cancer has been beneficial in both muscle-invasive and non-muscle invasive cases. While some patients with advanced or metastatic bladder cancer have experienced durable responses to immune checkpoint inhibitors (ICI), most patients do not respond to this therapeutic option. When examining RNA expression of A3A and A3B in human bladder tumors, A3A was significantly correlated with features of an inflamed tumor microenvironment, while A3B was significantly correlated with features of genomic instability. These distinct correlations suggest isoform-specific functions for the APOBEC3 enzymes. To further interrogate these findings in-vivo, we generated mouse BBN963 tumors to express A3A or A3B. The efficacy of anti-PD-1 therapy in this model was attenuated in BBN963 tumors expressing A3A. In contrast, A3B expression sensitized BBN963 tumors to anti-PD-1 therapy. These findings may help explain the differential responsiveness to ICI therapy in bladder cancer, given the widespread activity of APOBEC3 mutagenesis.