Fig. 5: CDK4/6 inhibitors were carefully selected for LKB1 mutant lung cancer based on the mechanistic link between LKB1-RB-CDK4 interaction.

a Combined drug screening using drugs from both the Genomics of Drug Sensitivity in Cancer (GDSC) database based on sensitivity to LKB1 mutation and an FDA-approved immunology compound library. b Quantitative RT-qPCR of relative ICAM1 expression in A549 and H460 cells treated with 5 selected drugs (cyclophosphamide monohydrate; vinorelbine tartrate; vorinostat; rapamycin; palbociclib). n = 4 biologically independent samples examined over 1 independent experiment. ****p < 0.0001, ****p < 0.0001. A549 cells transfected with lentivirus expressing the indicated genes (LV-Ctrl, LV-LKB1-WT, LV-LKB1-Mut), treated ± 500 nM palbociclib were harvested for quantitative RT-qPCR (c) and flow cytometry analysis (d). n = 3 biologically independent samples examined over 1 independent experiment. ***p = 0.0002, ****p < 0.0001, ***p = 0.007 (c). n = 4 biologically independent samples examined over 1 independent experiment. *p = 0.023, *p = 0.0378 (d). e, f Impact of CDK4/6i on LKB1 mutant lung cell line profiles. e Expression of immune resistance program genes (columns) that were most differentially expressed in palbociclib-treated (green) versus control (pink) cell lines (rows). Expression is normalized in each cell line. f Immune resistance scores in cell lines (A549, H460, and H2030) treated with palbociclib (“Pal”) or with DMSO vehicle (“con”) (GSE110397). Middle line: median; box edges: 25th and 75th percentiles. n = 2 biologically independent samples. g A549 cells transfected with lentivirus expressing the indicated genes (LV-Ctrl, LV-LKB1-WT, LV-LKB1-Mut), treated ± 500 nM palbociclib, transfected ± siRB were harvested for immunoblot. n = 3 independent experiments. h Immunoprecipitation analysis of the interaction among LKB1, RB, and CDK4 performed in H1299 cells expressing intact LKB1. n = 3 independent experiments. i Immunoblot analysis of the indicated proteins performed in A549 or H460 cells expressing the indicated genes in plasmids. n = 3 independent experiments. j ChIP assay was performed with cell lysates from A549/vector, A549/LKB1-OE, A549/CDK4-OE and A549/vector-palbociclib cells. A pair of primers flanking the p65 binding site within the ICAM1 promoter were used in PCR. Real-time PCR was employed to the ChIP assay. n = 4 biologically independent samples examined over 1 independent experiment. **p = 0.0015, *p = 0.0249, ****p < 0.0001. (Results are presented as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparisons test or two-tailed Student’s t-test was used to analyze the data. *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a Source Data file.).