Extended Data Fig. 5: CDK2 blockade enhances the effects of CDK4/6 inhibition in mouse KP and human lung adenocarcinoma cell lines.
From: RB constrains lineage fidelity and multiple stages of tumour progression and metastasis
a, BrdU/7AAD double labelling of KP clones expressing GFP (n = 1 biological replicate) or Cdk2 (n = 2 biological replicates) targeting sgRNAs with 0, 0.1 or 1.0 μM palbociclib. Percentage of cells in G1, S or G2/M phase are shown (2 technical replicates for each sample). Data are mean ± s.d. where appropriate. b, Top, cell cycle analysis (BrdU/7AAD double labelling) of KP clones expressing GFP (n = 1 clone per cell line) or Cdk2 (n = 2 clones per cell line) targeting sgRNAs. Bottom, the percentage of cells in each stage of the cell cycle is shown. c, d, Cell proliferation assay performed in duplicate showing number of cells 3 days after initial plating treated with 0, 0.1 or 1.0 μM palbociclib for either KP54 (c) or KP62 (d) cells. Two independent Cdk2 knockout clones and control GFP cells are shown. Data are means. e, f, Cell proliferation assay performed in triplicate showing number of cells 24, 48 and 72 h after initial plating for KP54 (e) or KP62 (f) cells. Three independent Cdk2 KO clones and one control is shown. Data are mean ± s.d. Significance was determined by ANOVA with Dunnett’s multiple comparisons test (n = 3 for all). e, Treatment for 24 h with GFP shRNA compared to CDK2-2 (P = 0.0013), CDK2-3 (P = 0.0029) or CDK2-8 (P = 0.0302) shRNA. Treatment for 48 h with GFP shRNA compared to CDK2-2 (P < 0.0001), CDK2-3 (P < 0.0001) or CDK2-8 (P = 0.0052) shRNA. Treatment for 72 h with GFP shRNA compared to CDK2-2 (P < 0.0001), CDK2-3 (P < 0.0001) or CDK2-8 (P < 0.0001) shRNA. f, Treatment for 48 h with GFP shRNA compared to CDK2-2 shRNA (P = 0.0415). Treatment for 72 h with GFP shRNA compared to CDK2-2 (P < 0.0001) or CDK2-5 (P < 0.0001) shRNA. g, Proliferation of KP (left) and KP;RbTR/TR (right) cells in quadruplicate, 72 h after addition of roscovitine (red outlines) and/or palbociclib (increasing grey tones) at the indicated concentrations. Cell numbers normalized to the average of the vehicle (DMSO) controls (white). Data are mean ± s.d. Significance was determined by ANOVA with Dunnett’s multiple comparisons test (n = 4 for all). KP control compared to 6 μM roscovitine (P < 0.0001) or 1 μM Palbociclib (P = 0.0016). KP 6 μM roscovitine compared to 6 μM roscovitine and 0.1 μM palbociclib (P < 0.0001) or 6 μM roscovitine and 1 μM palbociclib (P < 0.0001). KP;RbTR/TR control compared to 6 μM roscovitine (P = 0.0098). KP;RbTR/TR 6 μM roscovitine compared to 6 μM roscovitine and 0.1 μM palbociclib (P = 0.9811) or 6 μM roscovitine and 1 μM palbociclib (P = 0.0160). h, Proliferation of KP cells in quadruplicate, stably transduced with a tet-regulated dominant negative Cdk2 allele (CDK2DN) 72 h after addition doxycyclin (red) and/or palbociclib (increasing grey tones). Cell numbers normalized to the average of the vehicle (DMSO) controls (white). Data are mean ± s.d. Significance was determined by ANOVA with Dunnett’s multiple comparisons test (n = 4 for all). Control compared to 1 μM doxycycline to induce CDK2DN (P < 0.0001) or 1 μM palbociclib (P = 0.0194). 1 μM doxycycline compared to 1 μM doxycycline and 0.1 μM palbociclib (P = 0.4184) or 1 μM doxycycline and 1 μM palbociclib (P = 0.0020). i, Analysis of KP clones targeted with GFP-targeting or Cdk2-targeting sgRNAs. Western blot for Rb pathway component expression: RB, p-RB S807/S811, CDK2, CDK4, CDK6 and p-RB 780. Actin was used as loading control. j, Effects of CDK4/6 inhibition and CDK2 knockout on human lung adenocarcinoma cell lines. Data mined from the Sanger Center’s COSMIC database21 showing the relative sensitivity (IC50) of independent human lung adenocarcinoma cells lines to palbociclib. k, Western blot showing CDK2 loss following CRISPR-mediated knockout in indicated human lung adenocarcinoma cell lines. HSP90 was used as loading control. l, Representative images of clonogenic survival analysis of human lung adenocarcinoma cell lines performed in triplicate, treated every 3 days for 1.5 weeks with 0, 0.1 or 1.0 μM palbociclib. Cell lines were targeted with either an inert sgRNA or one targeting CDK2. m, Quantification of culture area covered by cells in l. Dark grey bars indicate inert sgRNA and red bars indicate CDK2 sgRNA. Data are mean ± s.d. Significance was determined by ANOVA with Sidak’s multiple comparisons test (n = 3 for all). A549 with inert sgRNA compared to CDK2 sgRNA in combination with 0 μM palbociclib (P > 0.9999), 0.1 μM palbociclib (P = 1.0 × 10−6) or 1 μM palbociclib (P = 0.0023). H1993 with inert sgRNA compared to CDK2 sgRNA in combination with 0 μM palbociclib (P = 2.7 × 10−7), 0.1 μM palbociclib (P = 0.0331) or 1 μM palbociclib (P = 0.6557). EKVX with inert sgRNA compared to CDK2 sgRNA in combination with 0 μM palbociclib (P = 0.5412), 0.1 μM palbociclib (P = 0.0003) or 1 μM palbociclib (P = 0.5929).
