Simple SummaryDespite the clinical use of epidermal growth factor receptor (EGFR) inhibitors for non-small cell lung cancer (NSCLC) patients, this disease remains incurable due to the development of resistance mechanisms to treatment. We demonstrate that the inhibition of polo-like kinase 1 (PLK1), known as a master cell cycle regulator, decreases EGFR protein levels in NSCLC cell lines. Better inhibition of EGFR-mutant lung cancer cells was observed with the combination of EGFR and PLK1 inhibitors compared to EGFR inhibition alone. This might therefore be a more potent therapy option to improve the outcomes of patients with EGFR-mutated NSCLC.AbstractTyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) have significantly prolonged survival in EGFR-mutant non-small cell lung cancer patients. However, the development of resistance mechanisms prohibits the curative potential of EGFR TKIs. Combination therapies emerge as a valuable approach to preventing or delaying disease progression. Here, we investigated the combined inhibition of polo-like kinase 1 (PLK1) and EGFR in TKI-sensitive EGFR-mutant NSCLC cells. The pharmacological inhibition of PLK1 destabilized EGFR levels and sensitized NSCLC cells to Osimertinib through induction of apoptosis. In addition, we found that c-Cbl, a ubiquitin ligase of EGFR, is a direct phosphorylation target of PLK1 and PLK1 impacts the stability of c-Cbl in a kinase-dependent manner. In conclusion, we describe a novel interaction between mutant EGFR and PLK1 that may be exploited in the clinic. Co-targeting PLK1 and EGFR may improve and prolong the clinical response to EGFR TKI in patients with an EGFR-mutated NSCLC.