Fig 2: Physical and functional interaction of tumour suppressor STK11 with CDK4.(a) The OncoPPi network links STK11 to CDK4 and palbociclib. (b) Mutual exclusivity map for the STK11 and CDK4 pathways in LUAD patient samples. Missense and truncation mutations are shown in green and black, respectively. DNA amplification is shown in magenta, deletions are shown in blue. Alterations of the PRKAA1 gene coding alpha 1 catalytic subunit of AMPK are shown. (c) Heatmap with expression signatures for FUSION reporter genes with individual knockdowns of STK11, AMPK, mTOR, CDK4, CDKN2A and CDKN2B based on Pearson correlation values from highest (blue) to lowest (magenta). (d) Interaction of STK11 with CDK4 and CCND2 using Renilla luciferase-PCA. Representative results of three independent experiments are shown. The error bars show the mean±s.d. of three replicates. (e,f) Endogenous STK11 was co-immunoprecipitated with CDK4 in lung cancer (e) H1299 and (f) H1792 cells. (g) Effect of STK11 status on CDK4 and STK11 activity as shown by pRB and pAMPK levels in isogenic H1299 lung cancer cells (STK11 wild-type and knockdown). (h) Effect of CDK4 inhibition by palbociclib on pAMPK status in isogenic H1792 cells with wild-type (STK11-WT) and STK11 knockdown (STK11-KD). (i) CDK4 inhibition disrupts the interaction of CDK4 with STK11. HEK293T cells transfected with indicated plasmids were treated with palbociclib at the indicated concentrations. GST-CDK4 pull-down assay was carried out and protein expression was examined by western blot with the indicated antibodies. (j) Silencing of STK11 (H1792-408 cells) enhanced lung cancer cell response to phenformin and to (k) palbociclib in a cell viability assay. (l) Overexpression of STK11 in STK11 null cells (H157) reduced sensitivity of H157 cells to palbociclib. Western blots are representative of three independent experiments. Cell viability is expressed as % of control (mean±s.d.).

Fig 3: Palbociclib specifically inhibited nasopharyngeal carcinoma (NPC) cell growth in vitro by inhibiting cell cycle progression. a. Western blot analysis of cell cycle-related proteins in two- and three-dimensional cultures of NPC cells (C666–1, NPC43, and C17) and two-dimensional culture of nasophayngeal epithelial cells (NP69). The levels of markers predictive of palbociclib sensitivity were examined. b. Western blot analysis of total RB, phosphorylated (p) RB (Ser780), and cyclin A in NPC and NPE cells after treatment with 0.2 µM palbociclib for 24 h. c. Cell cycle distribution analysis of NPC and NPE cells after treatment with 0.2 µM palbociclib for 24 h. A prominent accumulation of cells in the G1 phase was observed in the NPC but not the NPE cell cultures after palbociclib treatment. d. IC50 values of palbociclib in NPC cell lines were determined at Days 1, 3 and 5 using a resazurin assay (e). Western blot analysis of cell cycle-related proteins [total RB, pRB (Ser780), cyclin A, cyclin D1, CDK4] and an apoptosis marker (cleaved PARP) in NPC cell lines after treatment with palbociclib at different doses for 24 h. Quantitative protein expression data are presented in Supplementary Figure S2. f. Cell cycle distribution analysis of NPC43 cells after treatment with different doses of palbociclib for 1, 3 and 5 days. g. RNA transcriptome analysis of the three NPC cell lines after treatment with palbociclib for 24 h. Differentially expressed genes (DEG) were subjected to a KEGG pathway analysis. The top 20 KEGG pathways with the most significant gene ratios are plotted in the order of gene ratio. The sizes of the dots are scaled according to the numbers of significant genes revealed by the DEG analysis. The colors of the dots represent the p-adjusted significance values. Genes involved in cell cycle regulation were significantly enriched in all three cell lines after treatment with palbociclib

Fig 4: PI3K inhibition sensitizes non-basal-like BC PDX to CDK4/6i.A Waterfall plot showing the growth of n = 23 PDX treated with ribociclib 75 mg/kg plus alpelisib 35 mg/kg (bars and black dots) and vehicle (white circles). The percentage change from the initial volume is shown at day 35 of treatment. Dashed lines indicate the range of PD (>20%), SD (20% to -30%) and PR/CR (<-30%). The number of tumors treated per model is indicated in the brackets (n). Hashtags (#) indicate models harboring mutations in PIK3CA. Data represent mean values and error bars ± SEM. Boxes underneath show the molecular and intrinsic tumor subtype as well as their responses to the indicated treatments. The preclinical benefit to each drug is indicated as percentage in brackets. n.d, not determined. B IHC analysis of KI67 (left graph; p-value < 0.001) and phospho-pRb S807/811 (right graph; p-value < 0.01) in vehicle and alpelisib plus ribociclib-treated PDXs according to the PDX response to alpelisib plus ribociclib. Data are presented as mean values of each PDX. Two-tailed p-values are based on Mann-Whitney U test for all biological replicates. Different colors represent the tumor’s intrinsic subtype. R, resistant; S, sensitive; Alp, alpelisib; Rib, ribociclib. C Relative tumor growth of PDX039 and PDX191 after treatment with the indicated drugs and time. Dashed lines indicate the range of PD (>1.2), SD (1.2 to -0.7) and PR/CR (<-0.7). Two-tailed p-values are based on the two-way ANOVA test with Bonferroni’s method correction. V, vehicle; R, ribociclib; A, alpelisib. D, E Relative tumor growth of the ribociclib-resistant PDX244LR#18R and PDX450 after treatment with the indicated drug(s) for the indicated period of time, respectively. Dashed lines indicate the range of PD (>1.2), SD (1.2 to -0.7) and PR/CR (<-0.7). F Immunoblot of the indicated proteins in PDC476.2 treated with vehicle or 500 nM palbociclib as single-agent or combined with 100 nM fulvestrant and/or 2.5 µM alpelisib in ex vivo cultures for 48 h. At least two independent experiments were conducted. Source data are provided as a Source Data file.

Fig 5: Effect of orientin on cell cycle regulatory proteins. HT29 cells were treated with orientin (6.25 and 12.5 µM) and irinotecan for 24 h. The immunoblot analysis of cyclins, CDKs and their inhibitors involved in G0/G1 phase were carried out. Orientin reduced the expression of cyclins, pRb and CDKs, in contrast, increased the level of p21WAF1/CIP1. ß-actin was used as an internal control. Quantitative expression of proteins shown after normalization to ß-actin. The data presented are the mean ± SD of results from three independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control).