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Two thymidylate synthase inhibitory drugs induce markedly different genomic uracil patterns, mutagenic processes and cellular response in HCT116 colon cancer cells [re-analysis of GSE126822]

ABSTRACT: 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 in the genomic uracil profiles that associate with altered cytotoxicity and drug-induced mutagenesis. Both genome segmentation and U-score as a quantitative measure of gene-level uracil-content revealed biased uracilation upon the two drug-treatments. The corresponding genes are functionally related and characteristic for the applied drug, supporting the impact of genomic uracil patterns in the cellular responses. 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE285931 | GEO | 2026/01/31

REPOSITORIES: GEO

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Project description:HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used to treat B-cell-derived malignancies. Such malignancies regularly carry mutational signatures that conform to off-target induction of uracil by the AID/APOBEC family of cytidine deaminases, or downstream processing of uracil. . HDACi suppress thymidylate synthase increasing the cellular dUTP/dTTP ratio and leading to increased pressure on uracil repair machinery due to misincorporated uracil lesions. To investigate potential effect upon other enzymes involved in genomic uracil induction and processing, Jurkat (T-cell lymphoma) and SUDHL5 (B-cell lymphoma) cells were treated with pan-HDACi SAHA prior to SILAC based MS/MS investigation. HDACi treatment mediated significant differential expression of xx and xx proteins in Jurkat and SUDHL5, respectively, and had a substantial impact upon enzymes involved in in pyrimidine metabolism. Surprisingly, uracil N-glycosylase, UNG, was strongly downregulated by HDACi treatment. Further analysis in HEK and HeLa cells revealed that HDACis induce specific loss of the nuclear isoform UNG2 independent of transcription and cell-cycle alterations. More than 80% of UNG2 is degraded proteasomally after 24 hours treatment with SAHA, MS275, Valproate or Na-butyrate, indicating a universal ability of HDACis to mediate loss of UNG2. Targeted MS/MS analysis in HEK cells against a panel of proteins involved in DNA repair, translesion synthesis and nucleotide metabolism, revealed that UNG2 was the most pronounced differentially expressed among these after HDACi treatment. 48 hour treatment lead to a 30-40% increase in uracil lesions in the nuclear genome of HeLa and HEK cells and MS275 treatment in murine CH12F3 cell line mediated robust UNG2-loss accompanied by reduced class switch recombination. Furthermore, our analysis identified the PCNA-associated factor PAF15 among the downregulated proteins. PAF15 is overexpressed in many cancers and suppress TLS by inducing double monoubiquitinylation of PCNA and recruitment of the replicative polymerases. In summary, our findings demonstrate that HDAC inhibition affects the levels of proteins involved in DNA base excision repair, translesion synthesis and pyrimidine metabolism. These findings are important for a wide range of clinical applications of HDACi, such as in rheumatology, HIV-, and cancer treatment.

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Two thymidylate synthase inhibitory drugs induce markedly different genomic uracil patterns, mutagenic processes and cellular response in HCT116 colon cancer cells [re-analysis of GSE126822]

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