The Mutagenic Effects of APOBEC3A and APOBEC3B in Urothelial Carcinoma

PROJECT SUMMARY & ABSTRACT Abstract As the most common malignancy of the urinary tract among men and women, bladder cancer is estimated to surpass 80,000 new cases and 17,000 deaths is 2023. Accounting for most bladder cancers cases, urothelial carcinoma has a dismal survival rate of approximately 5% in the metastatic setting. APOBEC3A (A3A) and

APOBEC3B (A3B) are members of a family of cytidine deaminase enzymes that catalyze the removal of an amino group from cytosine nucleotides generating a uracil in its place that serve as a source of mutations. A3A and A3B enzymes are commonly overexpressed in numerous cancer types, including urothelial carcinoma,

with the APOBEC mutation signature seen in most cases of this cancer type. Due to their mutagenic activity, A3A and A3B have been implicated in altering the genomic landscape of urothelial carcinoma tumors over time. These alterations have the potential to augment tumor cell clonality that can lead to an enrichment of

cells that are refractory to treatment and drive disease progression. Currently, it is unclear if both A3A and A3B drive APOBEC-induced mutagenesis, or if one enzyme plays a larger mutagenic role than the other. This proposal seeks the address the hypothesis that A3A and A3B enzymes differ in mutagenic activity leading to

differences in promotion of intratumoral heterogeneity and response to anti-PD-1 immune checkpoint blockade inhibition. To address this hypothesis, two mouse bladder cancer cell lines entitled BBN963 and BBN976, that are representative of human muscle invasive bladder cancer, will be developed to express either HA-epitope

tagged A3A or A3B, in a doxycycline inducible manner. Utilization of this model will allow for a direct comparison of A3A and A3B induced mutagenic activity, tumor cell clonality diversification, and modulation of response to treatment. Experiments detailed in Aim 1 will elucidate the driver of the APOBEC mutational

signatures in urothelial carcinoma using in-vitro assays to analyze the predicted target sequences of APOBEC3 enzymes. Subsequently, Aim 2 will use DNA barcoding technology to compare the promotion of intratumoral heterogeneity by A3A and A3B. Lastly, Aim 3 will determine the influence of A3A and A3B on

treatment response to anti-PD-1 therapy and the role these enzymes play in altering the tumor-immune microenvironment utilizing an in-vivo approach. Completion of the work outlined in these aims will result in the identification of the driver of APOBEC3 mutagenesis in urothelial carcinoma and highlight a potential target for

inhibition for combination therapy with immune checkpoint blockade in patients with elevated APOBEC3 mutagenic activity.