Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses KRAS-mutated non-small cell lung cancer

H23 cells progressed into S phase in the presence of CYH33. A549 and H23 cells were synchronized at G2/M phase by incubation with nocodazole (0.3 μM) for 24 h. Cells were further incubated in fresh medium in the absence or presence of CYH33 (1 μM) for indicated times and cell cycle distribution was analyzed with flow cytometry.

Combined treatment of CYH33 and PD0332991 is synergistic against KRAS-mutated NSCLC cells. KRAS-mutated NSCLC cells were treated with CYH33, PD0332991, or both CYH33 and PD0332991 for 72 h and cell proliferation was detected by SRB assay. (A) Inhibitory curves from three independent experiments are presented. (B) CI values at the GI₅₀ of seven NSCLC cancer cell lines were determined by the Chou-Talalay method. Gray bar: CI values indicating additive effect. R: CYH33-resistant cell lines; S: CYH33-sensitive cell lines. (C) GI₅₀ values for CYH33, PD0332991, or the combination of CYH33 and PD03329917 against KRAS-mutated NSCLC cells. Data are shown as mean ± SD.

Co-treatment with PI3K pathway inhibitor and CDK4/6 inhibitor synergistically suppress proliferation of Kras-mutated NSCLC cells. Combination Index (CI) values at GI₅₀ of Kras-mutated NSCLC cell lines were determined by Chou-Talalay method. Cell proliferation was detected by SRB assay after cells were treated with each single agent or indicated combination. (A) CI values of PI3Kα inhibitor CYH33 or BYL719 and CDK4/6 inhibitor PD0332991 or LEE011 in H1355 cells. CYH: CYH33, BYL: BYL719, PD: PD0332991, LEE: LEE011. Data shown are the mean + SD. (B) CI values of PD0332991 and PI3K pathway inhibitor AZD8055, BEZ235 or GDC0941 in Kras-mutated NSCLC A549, H460, H358 and H23 cells. Data shown are the mean.

Combined CYH33 and PD0332991 treatment induces significant G1 phase arrest in KRAS-mutated NSCLC cells. KRAS-mutated NSCLC cells were treated with vehicle, CYH33 (1 μM), PD0332991 (1 μM), or both CYH33 and PD0332991 for 72 h. (A) Cell cycle distribution was analyzed by flow cytometry. The cell population in G0/G1 phase is presented as the mean ± SD. Differences between the indicated groups were analyzed using a two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Cell lysates were subjected to Western blot with the indicated antibodies. Induction of apoptosis of A549 (C) and H23 (D) cells was determined by annexin-V-PI dual staining. Annexin-V positive cells were determined with flow cytometry. Data are shown as the mean ± SD from three independent experiments. (E) Lysates of A549 and H23 cells were subjected to Western blot with the indicated antibodies.

Co-treatment with CYH33 and PD0332991 failed to induce apoptosis in NSCLC cells. Kras-mutated NSCLC cells were treated with CYH33 (1 μM), PD0332991 (1 μM), or both for 72 h and apoptosis was determined by Annexin-V assay with flow cytometry. Data shown are mean + SD from three independent experiments.

Down-regulation of cyclin D1 sensitizes A549 and H23 cells to CYH33. A549 and H23 cells transfected with siRNAs targeting cyclin D1 or a negative control (NC) siRNA were treated with serially diluted CYH33 (A, B) or CYH33 at 1 μM for 72 h (C, F). (A, B) The effect of CYH33 on cell proliferation was determined by SRB assay. GI₅₀ values obtained from A549 (A) and H23 (B) cells are presented. (C, D) The cell cycle distribution of A549 (C) and H23 (D) cells was analyzed by flow cytometry. The cell population in G0/G1 phase is presented. (E, F) Lysates of A549 (E) and H23 (F) cells were subjected to Western blot with the indicated antibodies. Data are shown as the mean + SD or representatives from at least three independent experiments. Differences between cells transfected with an NC siRNA and siRNAs targeting cyclin D1 were analyzed using a two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Down-regulation of cyclin D1 had no effect on CYH33-induced apoptosis or the anti-proliferative activity of PD0332991. A549 and H23 cells transfected with siRNAs targeting cyclin D1 or negative control were treated with CYH33 (1 μM) for 72 h (A–D). Induction of apoptosis in A549 (A) and H23 (B) cells was determined by Aneexin-V-PI dual staining. Annexin-V positive cells were measured with flow cytometry. Data shown are mean + SD from three independent experiments. (C–D) Cell lysates of A549 (C) and H23 (D) cells were subjected to Western blot with indicated antibodies. A549 (E) and H23 (F) cells transfected with SiRNA targeting cyclin D1 or negative control were treated with PD0332991 (1 μM) for 72 h, and inhibition of cell proliferation by PD0332991 was calculated. Data shown are mean + SD. Differences between cells transfected with siRNAs targeting cyclin D1 and negative control were analyzed using two-tailed Student’s t test. *: P < 0.05.

CYH33 abrogates PD0332991-induced AKT phosphorylation and cyclin D1 expression. (A, B) A549 cells were treated with vehicle or PD0332991 (1 μM) for 72 h and total RNA were extracted. Genome-wide gene expression profiling was performed by microarray analysis. (A) GSEA enrichment plot of differentially expressed genes in the gene set of Hallmark of E2F targets (HALLMARK_E2F_TARGETS) after treatment with PD0332991. (B) Heatmap of the mRNA levels of genes involved in the PI3K-AKT signaling pathway. The fold change (log 2) in the expression of the listed genes after treatment with 1 μM PD0332991 is shown. (C) A549 cells were treated with 1 μM PD0332991 for the indicated times and cell lysates were subjected to Western blot with the indicated antibodies. (D) A549 cells were treated with PD0332991 (0, 1 or 5 μM) alone or in combination with CYH33 (1 μM) for 72 h and cell lysates were subjected to Western blot with the indicated antibodies. Data are shown as representatives from at least three independent experiments.

Genome-wide gene expression profiling in A549 cells after PD0332991 treatment. A549 cells were treated with vehicle or PD0332991 (1 μM) for 72 h and total RNA was extracted. Genome-wide gene expression profiling was performed with microarray. (A) The genome-wide gene expression profile of A549 cells treated with vehicle control or PD0332991. Three biological replicates were employed in each group. (B) GSEA enrichment plot of differentially expressed genes in the gene set of hallmark of G2/M checkpoint (HALLMARK_G2/M_Checkpoints) and hallmark of mitotic spindle (HALLMARK_MITOTIC_SPINDLE) upon PD0332991 treatment.

Combined inhibition of PI3Kα and CDK4/6 displays synergistic activity against A549 and H23 xenografts. Randomly grouped nude mice bearing A549 (A) or H23 (B) xenografts were administrated orally with a vehicle control, CYH33 (5 mg/kg), PD0332991 (50 mg/kg for mice bearing A549 xenografts, 100 mg/kg for mice bearing H23 xenografts), or a combination of CYH33 and PD0332991, once a day for the indicated times. Tumor volume and body weight were measured twice a week. Data are depicted as the mean + SD (n = 6). Differences between the indicated groups were analyzed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) The combination ratios of CYH33 and PD0332991 against A549 and H23 xenografts were calculated using the fractional product method. FTV (fractional tumor volume) = mean final tumor volume of treated group/mean final tumor volume of control group. Expected FTV = (FTV of PD0332991) * (FTV of CYH33). Observed FTV = mean final tumor volume of combinatorial treatment/mean final tumor volume of control group. Combination ratio = expected FTV/observed FTV. (D) Tumor sections from A549 xenografts after treatment for 21 d were stained with Ki-67 (tawny) or phosphorylated Rb (Ser780, tawny). Cell nuclei were stained with hematoxylin (calamine blue, 40 ×). Representative images are shown for each group.