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: Molecular features of the GSC lines suggest associations with sensitivity and resistance(A) Immunoblot analysis of CDK4, CDK6, Rb1, and E2F1 in the GSC lines. a-Tubulin was used as a loading control. (B) qPCR analysis of p15, p16, p18, and p19 expression levels in the GSC lines and NHA.

Fig 4: Restoration of GIST oncogenic signalling pathways and proliferation after TKI withdrawal. a Immunoblotting evaluations show reactivation of KIT and AKT, as assessed by phospho-KIT and phospho-AKT, and show reactivation of the cell cycle, as assessed by phospho-RB1 and Cyclin A. b Ki-67 staining shows recovery of proliferation after drug withdrawal. c, Mitotic counts (per 5 mm2) show recovery of proliferation after drug withdrawal

Fig 5: ZBTB46 overexpression results in a broad suppression of multiple cell cycle genes and correspondingly a reduction in phosphorylated retinoblastoma (pRb) protein content in HAECs. a Relative mRNA expression of various genes involved in cell cycle regulation in HAECs overexpressing ZBTB46 compared to controls by qPCR, grouped by their predominant known roles in various phases of the cell cycle. b Representative Western blots showing reduced total Rb and pRb protein content in ZBTB46 transducing HAECs compared to control cells. c Quantification of Rb and pRb protein content based on Western blots. d Calculated ratio of pRb to total Rb showed decreased Rb protein phosphorylation. *P < 0.05, ***P < 0.001 compared to GFP-AV, one-way ANOVA, n = 4–6/group