APOBEC3-mediated mutagenic processes in DNA-repair-deficient HCT116 cells selectively induced by high-dose 5FdUR treatment.

The presence of genomic uracil and its respective repair play key roles in colorectal, gastric, and other solid tumor therapies targeting thymidylate biosynthesis. Previously, we established that the treatment of HCT116 colon cancer cell lines with either raltitrexed (RTX) or 5-fluoro-2'-deoxyuridine (5FdUR), two potent inhibitors of thymidylate synthase, results in characteristic uracil patterns (PMID: 32956035). Here, we focus on drug-specific differences manifested in the drug-induced mutagenesis. Therefore, whole genome sequencing was performed in drug-treated (5FdUR or RTX) HCT116 cells that are deficient and stably express the UNG inhibitor protein, UGI. Mutational analysis revealed a significant increase in the frequency of C-to-T somatic transitions selectively upon high-dose 5FdUR treatment in DNA-repair deficient cells. The mutational spectra and the clustered nature of these transitions pointed to the action of DNA cytidine deaminases. Indeed, an induction of some APOBEC3 enzymes was experimentally confirmed upon the high-dose 5FdUR treatment, in parallel with decreased cytotoxicity, as compared to the low-dose, or any efficient doses of RTX. The observed drug-induced mutagenicity and decreased cytotoxicity might be relevant for personalized cancer therapy. Overall design: We performed whole genome sequencing experiments from UGI expressing HCT116 cells treated with either 0.1 or 20 microM of RTX or 5FdUR for 48 hours. Samples are as follows: 5FdUR_0.1_UGI_HCT116, RTX_0.1_UGI_HCT116, 5FdUR_20_UGI_HCT116, RTX_20_UGI_HCT116, and the non-treated control: NT_UGI_HCT116). Genomic DNA was isolated from each sample and fragmented to approximately 300 bp fragments by sonication. Sequencing libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, Ipswitch, MA, USA), and were sequenced by Illumina paired-end reads (PE)150 bp in a Novaseq 6000 platform by iBioScience (Pécs, Hungary). Reads were filtered and aligned to the reference human genome (GRCh38.d1.vd1, (https://gdc.cancer.gov/about-data/data-harmonization-and-generation/gdc-reference-files)). For details, see the Supplementary Method of the related publication. Genomic variants called using mutect2 (GATK4) from treated vs. non-treated input samples in several arrangements defined in the names of each vcf file. For details, see the Supplementary Method of the related publication.