CDK4/6 inhibition overcomes 5-FU resistance by suppressing TYMS-driven R-loop-associated DNA damage in colorectal cancer

Background: Resistance to 5-fluorouracil (5-FU) remains a significant obstacle in colorectal cancer (CRC) therapy. As an antimetabolite, 5-FU induces cytotoxicity by inhibiting thymidylate synthase (TYMS), thereby impairing DNA synthesis and repair. However, CRC cells frequently adapt through TYMS upregulation, thereby strengthening their DNA repair capacity and reducing the efficacy of drugs. R-loops, three-stranded nucleic acid structures formed by hybridization of RNA to its DNA template, are key contributors to replication stress and DNA damage. Since TYMS regulates nucleotide synthesis, it may influence R-loop-associated DNA damage. Cyclin-dependent kinases 4 and 6 (CDK4/6), which control G1-S cell cycle progression via E2F activation, also regulate TYMS transcription. Therefore, we hypothesized that CDK4/6 inhibition may downregulate TYMS and enhance R-loop-related DNA damage, restoring 5-FU sensitivity. Methods: We established 5-FU-resistant CRC cell lines (HCT116-5FUR and SW480-5FUR). Cell viability was assessed using the MTT assay, and protein and mRNA levels were quantified by Western blot and RT-PCR, respectively. Flow cytometry was used for cell cycle profiling. DNA damage was assessed by gammaH2AX immunofluorescence, and R-loop formation was detected via S9.6 and anti-nucleolin immunostaining. A luciferase reporter assay was used to monitor the efficiency of double-strand break (DSB) repair. Synergistic effects of 5-FU and the CDK4/6 inhibitor abemaciclib were also evaluated. Results: TYMS expression was markedly upregulated in HCT116 5FUR (p<0.001) and SW480 5FUR (p<0.01) compared to parental lines. CDK4 expression was upregulated in HCT116 5FUR and SW480 5FUR (p<0.01) compared to parental lines. CDK6 expression was also upregulated in HCT116 5FUR and SW480 5FUR (p<0.01) compared to their parental cell lines. Abemaciclib downregulated TYMS expression in HCT116 5 FUR (p = 0.011) and SW480 5FUR (p = 0.0002) compared to untreated 5-FU-resistant CRC cell lines. TYMS downregulation promoted R-loop-associated DNA damage, indicating a mechanistic link between nucleotide metabolism and genomic instability. Combination treatment with 5-FU and abemaciclib synergistically reduced the viability of resistant cells by inducing G1 arrest and enhancing DNA damage. Abemaciclib alone induced R-loop formation and DNA damage signaling, consistent with TYMS suppression and the amplification of replication stress. Conclusions: TYMS modulates R-loop-associated DNA damage, contributing to 5-FU resistance in CRC. CDK4/6 inhibition restores chemosensitivity by repressing TYMS and enhancing R-loop-driven genomic stress. Combining CDK4/6 inhibitors with 5-FU represents a promising therapeutic strategy to overcome drug resistance in colorectal cancer.