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30 reviewsPolo-like kinase 1 (PLK1) signaling drives tumor malignancy and chemotherapy resistance, which is an unmet clinical need.Recruiting PLK1 to the central spindle during anaphase is necessary for its function in promoting cancer cell proliferation, whichis achieved by binding to microtubule-associated protein regulating of cytokinesis (PRC1) located in the spindle. However, therole of PLK1/PRC1 signaling in chemotherapy resistance is unknown. In this study, we identified a small molecule B4 whichinhibited PLK1/PRC1 signaling through disrupting the formation of PLK1/PRC1 protein complexes. In the presence of blockingPLK1/PRC1 signaling, enhanced sensitivity of drug-resistant tumors to traditional chemotherapy was found. Suppression of PLK1activity by B4 inhibited disease progression in allograft models, and combination with cisplatin elicited dramatic regression of1234567890();,:drug-resistant tumors. Our findings provide a promising strategy to target the PLK1 signalingpotential modality to enhance sensitivity to chemotherapy in non-small cell lung cancer (NSCLCell Death and Disease (2025) 16:374 ;