Harringtonine (HT) targets PARP-1 to inhibit TS mRNA m6A modification, blocks STAT3-induced miR-4521/TS axis and attenuates 5-fluorouracil (5-FU) resistance in NSCLC.

Background: Harringtonine (HT), derived from the genus Cephalotaxus, has demonstrated anti-proliferative effects against non-small cell lung cancer (NSCLC). 5-Fluorouracil (5-FU), a cost-effective chemotherapy, faces resistance issues in NSCLC. Thus, exploring HT's mechanisms in inhibiting NSCLC proliferation and overcoming 5-FU resistance is clinically important.

Purpose: This study aims to investigate for the first time the mechanism by which HT, a novel poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor, attenuates 5-FU resistance in NSCLC.

Methods: Enzyme activity assay and surface plasmon resonance (SPR) were used to verify the HT's effects on PARP-1 activity. CCK-8, colony formation, drug affinity responsive target stability (DARTS), and cellular thermal shift assay (CETSA) were used to explore HT's ability to target PARP-1. Molecular docking, molecular dynamics, and one-point mutation assays explored the mode of action of HT with PARP-1. The anti-resistance effect of HT was assessed in vivo using regular and 5-FU-resistant NSCLC xenograft mice. The PARP-1-promoted 5-FU resistance mechanism in NSCLC was further explored by RNA immunoprecipitation (RIP), RNA pull-down, and co-immunoprecipitation (Co-IP) assays. Additional mechanisms of 5-FU resistance were explored through dual-luciferase assays and microRNA sequencing.

Results: HT effectively inhibited PARP-1 activity, downregulating thymidylate synthase (TS) in both regular and 5-FU-resistant xenograft models, thereby reducing NSCLC proliferation and resistance to 5-FU. Mechanistically, PARP-1 promoted the m6A modification of TS mRNA by binding to methyltransferase-like 3 (METTL3), thus increasing the stability of TS mRNA. Moreover, PARP-1 could inhibit the Activator of Transcription 3 (STAT3)-mediated expression of miR-4521 and promote the high expression of TS mRNA. This dual action mediated by PARP-1 ultimately promotes the resistance of NSCLC to 5-FU.

Conclusion: These results demonstrated for the first time that HT was a novel PARP-1 inhibitor that inhibits 5-FU resistance in NSCLC, providing mechanistic clues and rational dosing recommendations for clinical treatment.