Cancer research has traditionally focused on protein-coding genes, but approximately 98% of the human genome is non-coding, comprising gene regulatory elements such as enhancers, silencers, and promoters. Mutations within these regions can foster tumorigenesis by modulating the expression of oncogenes. Recent whole-genome sequencing studies have revealed several recurrently mutated non-coding genomic regions, including in bladder cancer, but their role in tumorigenesis remains unknown. In this study, we delved into whole-genome sequencing data from the bladder cancer TCGA cohort and an independent set of bladder tumors, uncovering hundreds of recurrent mutations. Mutational signature analysis identified over 65% of these mutations as APOBEC3-induced. We substantiated the involvement of APOBEC3 enzymes by using conventional deamination assays and novel approach dubbed as APOBEC3-mediated mutation capture assay. Predominantly, these mutations occurred in the hairpin loop, a favored motif for APOBEC3 enzymes. To systematically assess which of the mutations are functional and prioritize them for biological characterization, we developed Massively Parallel Reporter Assays (MPRA). Two types of MPRA libraries were constructed, one for evaluating promoter activity and the other for assessing enhancer activity of the mutations. Each library construction entailed 36,000 oligonucleotides, comprehensively representing all mutation hotspots and their corresponding wild-type alleles. The application of MPRA resulted in the identification of a substantial number of functionally relevant mutations. Zooming in on the top 20 genomic regions, we employed CRISPR-based approaches to pinpoint target genes and elucidate their functional consequences. Most of these regions co-localized with enhancers, as determined by analysis of ATAC-seq, ChIP-seq, and Hi-C data from bladder cancer cell lines. Perturbation of hotspots regions resulted in dysregulation of multiple genes. The co-regulation patterns of genes were further supported by strong correlation of their mRNA expression in RNA-seq datasets (TCGA, SRA, etc.). Notable examples included twin hotspot mutations upstream of the LEPROTL1 gene, elevating the expression of LEPROTL1, DCTN6 and SARAF, resulting in aggressive tumor growth in in vitro functional experiments. These three genes emerged as novel oncogenes in cancer. Another functional hotspot influenced the expression of two known long non-coding RNAs, NEAT1 and MALAT1, previously characterized as cancer genes. Additionally, some recurrently mutated regions or their target genes were associated with worse overall survival in the TCGA cohort. In conclusion, our findings propose that non-coding mutations likely drive tumorigenesis, contributing to tumor heterogeneity. Employing a combination of functional, multi-omics, and in vivo approaches, we aim to comprehensively characterize the role of hotspot non-coding mutations in bladder cancer and assess their clinical significance.

Citation Format: Rouf Banday, Bilal Lone, Kelly Butler, Dhanusha Yesudhas, Alexandra Dobbins, Arup Chakraborty. Exploring the functional consequences of APOBEC3-induced non-coding hotspot mutations in bladder cancer using massively parallel reporter assays and CRISPR-mediated base editing [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR009.