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Objective: Mcl-1 overexpression confers acquired resistance to Bcl-2 inhibitors in non-small cell lung cancer (NSCLC), but no direct Mcl-1 inhibitor is currently available for clinical use. Thus, novel therapeutic strategies are urgently needed to target Mcl-1 and sensitize the anti-NSCLC activity of Bcl-2 inhibitors.Methods: Cell proliferation was measured using sulforhodamine B and colony formation assays, and apoptosis was detected with Annexin V-FITC staining. Gene expression was manipulated using siRNAs and plasmids. Real-time PCR and West blot were used to measure mRNA and protein levels. Immunoprecipitation and immunofluorescence were used to analyze co-localization of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) and Mcl-1.Results: Suppression of DYRK1A resulted in reduced Mcl-1 expression in NSCLC cells, whereas overexpression of DYRK1A significantly increased Mcl-1 expression. Suppression of DYRK1A did not alter Mcl-1 mRNA levels, but did result in an accelerated degradation of Mcl-1 protein in NSCLC cells. Furthermore, DYRK1A mediated proteasome-dependent degradation of Mcl-1 in NSCLC cells, and DYRK1A co-localized with Mcl-1 in NSCLC cells and was co-expressed with Mcl-1 in tumor samples from lung cancer patients, suggesting that Mcl-1 may be a novel DYRK1A substrate. We showed that combined therapy with harmine and Bcl-2 antagonists significantly inhibited cell proliferation and induced apoptosis in NSCLC cell lines as well as primary NSCLC cells.Conclusions: Mcl-1 is a novel DYRK1A substrate, and the role of DYRK1A in promoting Mcl-1 stability makes it an attractive target for decreasing Bcl-2 inhibitor resistance.