Article Figures & Data
Figures
- Figure 1.
A whole-genome CRISPR screen implicates receptor tyrosine kinase and mTOR signaling networks as modulators of response to PI3K inhibition. A, Proliferation of 14 human pancreatic cell lines (all KRAS-mutant except BxPC3) and one breast cancer cell line (T47D KRAS wild-type) was measured after incubation with increasing concentrations of BYL719 for 72 hours. Proliferation was quantified using CellTiter-Blue and compound GI50 values were calculated using GraphPad Prism. Mean cell proliferation, relative to DMSO control, is plotted ± SE (n = 3). B, Phosphorylation of AKT and PRAS40 was assessed by Western blotting after incubation with increasing concentrations of BYL719 for 2 hours, in the cell lines MIAPACA2, PANC1, and T47D (n = 2). C, Overview of genome-wide CRISPR screening method. Cas9-expressing MIAPACA2 cells were infected with the Avana4 pooled CRISPR library and split into three arms. Cells were passaged for eight population doublings in the presence of DMSO, BYL719, or GDC0941. The abundance of each sgRNA was assessed by NGS, under each condition. D, Following successful transduction of MIAPACA2_Cas9 cells with the Avana4 sgRNA library, cells were cultured in the presence of DMSO, 10 μmol/L BYL719, or 1 μmol/L GDC0941 for at least eight population doublings. Cells were counted every 3–5 days and population doublings are the mean of four replicates ± SE. E, The average log-fold change (LFC) in sgRNA abundance was determined for BYL719-treated samples versus the DMSO controls. STARS analysis was used to rank genes that when targeted by CRISPR, enhanced the antiproliferative activity of BYL719. F, STARS analysis was used as in E to rank genes that when targeted by CRISPR, suppressed the antiproliferative activity of BYL719, and conferred resistance to the drug. G, Venn diagram analysis of hit genes to identify shared enhancers/suppressors of BYL719 and GDC0941 activity. H, GSEA highlights key pathways that modulate sensitivity to PI3K inhibition. Significantly enriched pathways from the Reactome and KEGG databases were nominated from a list of the hit genes (FDR < 0.3) that were enriched or depleted in the genome-wide CRISPR screen with either BYL719 or GDC0941.
- Figure 2.
The ERBB family can sustain pancreatic cancer cell proliferation and mediate resistance to PI3K inhibition associated with S6 phosphorylation. A, A secondary screen of top-ranking hits from the primary screen was conducted in MIAPACA2, PANC1, PATU8902, and PANC03.27 cells treated with BYL719 or GDC0941. For each cell line, STARS analysis was used to rank genes that enhanced or suppressed the antiproliferative activity of BYL719 or GDC0941. Genes were then ranked according to the mean STARS score across all four cell lines. Genes with a STARS score of 4 or more in three or more cell lines are highlighted in red. Gray boxes indicate the gene was not ranked by STARS. B, PANC03.27 and MIAPACA2 cells expressing Cas9 were transduced with lentiviral vectors encoding sgRNAs targeting GFP, UBE2H, or MEMO1 to generate cell populations with reduced expression of either UBE2H or MEMO1. Cells were lysed 7 days after transfection and cell lysates were analyzed by Western blotting (n = 3). C, Cells as in B were treated with increasing concentrations of BYL719 or GDC0941. After 4 days of drug treatment, cell proliferation was quantified using CellTiter-Blue. Mean cell proliferation, relative to the DMSO-treated control, is plotted ± SE (n = 3). D, PATU8988S cells were cultured in the presence of BSA, EGF, HRG, or IGF1 (100 ng/mL) with 10% FBS and treated with a range of concentrations of BYL719. Cell proliferation was quantified after 72 hours using CellTiter-Blue. The mean GI50 is plotted ± SE (n = 6) and statistical significance compared with the BSA control was determined by one-way ANOVA. E, PATU8988S cells were cultured in the presence of BSA, EGF, HRG, or IGF1 (100 ng/mL) with 10% FBS and treated with DMSO (0.1%) or BYL719 (2 μmol/L) for 72 hours. Cell lysates were analyzed by Western blotting (n = 3).
- Figure 3.
A combinatorial drug screen identifies mTOR and ERBB family inhibitors as potent enhancers of PI3K inhibition. A, MIAPACA2 cells were treated with a library of 487 FDA-approved drugs and tool compounds at a concentration of 800 nmol/L in the presence or absence of 10 μmol/L BYL719 or 1 μmol/L GDC0941. After 96 hours, cell proliferation was quantified by CellTiter-Blue assay. Proliferation was normalized to a DMSO-treated control on each plate and the Bliss independence model was used to calculate synergy with BYL719 or GDC0941 for each of the compounds in triplicate. The mean Bliss score for each compound in combination with BYL719 was plotted against the mean Bliss score for each compound in combination with GDC0941. B, The mean Bliss score for each compound in combination with BYL719 and GDC0941 was calculated and used to rank compounds. The top 20 compounds are shown. C, Cells were incubated with a matrix of increasing concentrations of BYL719 and either pelitinib or AZD2014 for 144 hours. Cell viability was measured using CellTiter-Blue and normalized to DMSO-treated wells (a shift from blue to red indicates loss of proliferation). The Bliss independence model was used to calculate synergy where a shift from green to red indicates increasing synergy (n = 3). D, Cells were incubated with the indicated compounds or the combinations in triplicate for 14 days. Cells were fixed and stained with 0.5% crystal violet. E, Spheroids consisting of a coculture of MIAPACA2 cells and PSCs (1:1) were allowed to establish for 24 hours and were then treated with the indicated compounds or the combination for 9 days. Dual calcein AM (viable cells) and propidium iodide (nonviable cells) staining was performed. Images were obtained using the Celigo imaging cytometer. F, The diameter of the spheroid was measured every 3–4 days by quantification of images recorded on the Celigo. Colony diameter is plotted as the mean of at least three independent spheroids ± SE (n = 2). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. G, Propidium iodide staining of spheroids was performed after 4 days and fluorescence intensity of the spheroid was quantified on the Celigo (n = 6). Mean fluorescence intensity is represented by the horizontal bar. Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test.
- Figure 4.
Inhibition of S6 phosphorylation is associated with synergistic, antiproliferative effects of combined PI3K and mTOR or pan-ERBB family inhibition. A, MIAPACA2 and T47D cells were exposed to the indicated concentrations of BYL719 for 2 hours or MIAPACA2_Cas9 cell clones lacking expression of p110α (knocked out via CRISPR-Cas9) were analyzed by Western blotting for PI3K pathway activation using the indicated antibodies. NIC, no infection control. B, Percentage cell proliferation after incubation with 10 μmol/L BYL719 for 72 hours relative to DMSO (n = 3) is plotted against percentage S6 phosphorylation, relative to DMSO, quantified from Western blots after incubation with 10 μmol/L BYL719 for 2 hours (n = 2). Each point represents a different cell line from a panel of 12 PDAC cells. Linear regression analysis in GraphPad Prism was used to determine the correlation between the two variables. C, MIAPACA2 cells were incubated with DMSO (0.1%) BYL719 (10 μmol/L), AZD2014 (250 nmol/L), pelitinib (1 μmol/L), or the indicated combinations for 72 hours and cell lysates were analyzed by Western blotting (n = 2).
- Figure 5.
Alterations in genes encoding the ERBB family and selected downstream signaling mediators is associated with reduced pancreatic cancer patient survival. A, Genetic alteration of the ERBB–PI3K signaling axis was assessed using cBioPortal and the TCGA “provisional dataset” for 149 pancreatic cancer patients. Sixty of 149 patients (40%) displayed alterations in one or more of the indicated genes. B, Overall survival of patients with pancreatic cancer with or without genetic alterations of the genes shown in A. Significance was assessed by Log-rank Mantel–Cox test.
Additional Files
Supplementary Data
- Supplementary Data Figure S1-S8 - Figure S1. KRAS-dependency and effect of Cas9 expression on response to PI3K inhibition. Figure S2. Whole-genome CRISPR screen shows good replicate correlation and identifies enhancers and suppressors of GDC0941 antiproliferative activity. Figure S3. Expression of Cas9 does not alter response to BYL719 or GDC0941. Figure S4. Population doublings, replicate correlation and alternative analysis of the minipool screen. Figure S5. PI3K signalling is reduced following serum starvation. Figure S6. Combined inhibition of PI3K and mTOR results in potent antiproliferative activity in pancreatic cancer cell lines. Figure S7. Combined p110� and mTOR or ERBB-family inhibition leads to sustained suppression of S6 phosphorylation. Figure S8. CRISPR and drug combination screens implicate the ERBB-PI3K-MTOR signalling axis as a key vulnerability of pancreatic cancer cells.
- Supplementary Tables S1-S26 - Tables S1-S26. Includes details of antibodies, shRNAs and sgRNAs used in this study. CRISPR and drug screening data.
Volume 19, Issue 7
