Background <p>CDK4 alterations are common in lung adenocarcinoma, but recent clinical trials only demonstrated modest therapeutic responses to CDK4/6 inhibitors. The mechanism of CDK4/6 inhibitor resistance has not been fully characterized.</p> Methods <p>Patient-derived organoids and cell lines were tested for their sensitivity to CDK4/6 inhibitors. The drug resistance mechanism relating to stemness pathway was tested by in-vitro and in-vivo assays. Screening of small molecule library was performed to explore potential therapeutic agents that could potentiate the efficacy of CDK4/6 inhibitors.</p> Results <p>CDK4/6 inhibitors could inhibit the growth of lung adenocarcinoma patient-derived organoids and cell lines, and its drug resistance was associated with HES1 overexpression. We identified the vital role of HES1 in promoting cancer cell stemness through SOX9 upregulation via the phosphorylation of transcription factor STAT3. Inhibition of HES1 impaired lung cancer spheroid formation, reduced stemness marker expression, and enhanced the sensitivity of the spheroids towards the CDK4/6 inhibitor palbociclib. Knockdown of SOX9 recapitulated the functional effect of HES1 inhibition, confirming its role as a downstream effector mediating the stemness properties of lung cancer cells. Screening of small molecules revealed VR23 as a potent HES1 inhibitor, and it could suppress lung cancer growth and patient-derived organoids in a synergistic manner with palbociclib in-vitro and in-vivo. Moreover, the pSTAT3 inhibitor napabucasin could also afford synergy with palbociclib as well, further confirming the therapeutic vulnerability conferred by the HES1-pSTAT3-SOX9 pathway in potentiating CDK4/6 inhibitors.</p> Conclusions <p>Our findings revealed a signalling pathway in which lung adenocarcinoma regulates stemness and tumourigenesis through HES1, and the targeting of this pathway by VR23 or napabucasin supports further preclinical development for CDK4/6 inhibitor combination therapy.</p>