Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A) Schematic of KRAS isogenic cell lines generation. KRAS mutations were introduced into the parental cell lines via r-AAV-mediated homologous recombination. A general structure of the targeting construct is represented. The resulting mutant KRAS allele is expressed from its endogenous promoter. The Neo cassette is removed from the genome of the targeted cells by Cre recombinase-mediated excision. AAV, adeno-associated virus; ITR, inverted terminal repeat; Neo, geneticin-resistance gene; P, SV40 promoter; triangles, loxP sites (Figure adapted from ). (B) RAS activation status of LIM1215 KRAS isogenic cell lines. Western blot showing active RAS (RAF1 GTP-bound) levels for LIM1215 KRAS isogenic cell lines. The RAF1 RAS binding domain (RBD) was used to precipitate GTP-RAS. The RAS activation status was tested for each clone with mutated KRAS. Precipitated RAS-GTP was detected by western blot using anti-RAS antibody. As a positive control, HeLa cells (RAS wild-type) were stimulated with epidermal growth factor (EGF) to activate the RAS pathway. HeLa and MCF7 cells (unstimulated) were used as negative controls. Total lysates were also immunoblotted with anti-β-Actin antibody as loading control. (C) and (D) KRAS dependence in the LIM1215 KRAS isogenic cell line models, obtained from the HT siRNA screen described in . Bar graph of KRAS siRNA Z-score values across the LIM1215 KRAS WT and mutant isogenic cell lines, C and D respectively. KRAS dependence was greater in the cell lines carrying KRAS mutations than in WT cells. Error bars represent SEM from three independent experiments. (E) Western blot of KRAS in SW48 cells expressing KRAS -specific siRNAs. Multiple KRAS siRNA oligos and a pool efficiently suppressed KRAS expression showing that the siRNAs were on-target.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Homologous Recombination, Construct, Mutagenesis, Virus, Activation Assay, Western Blot, Binding Assay, Positive Control, Control, Expressing

Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A) GTP-RAS assay showing the RAS activation status of SW48 KRAS isogenic cell lines. (B) KRAS dependence in the SW48 KRAS isogenic cell lines (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test for comparison between each KRAS mutant and the WT cell lines). (C) CDK1-specific siRNAs suppress CDK1 expression. Cell viability after CDK1 depletion in SW48 isogenic KRAS cell lines (ns, not statistically significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test for comparison between each KRAS mutant and the WT cell lines). Error bars represent SEM from three independent experiments. (D) Western blot of CDK1 in SW48 parental cells expressing CDK1-specific siRNAs.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Activation Assay, Comparison, Mutagenesis, Expressing, Western Blot

Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A) Exposure of SW48 isogenic cell lines to RO-3306 in a fifteen-day colony formation assay. (B) Exposure of SW48 isogenic cell lines to inhibitor AZD5438, in a fifteen-day colony formation assay. (C) Drug-dose response curves of CRC cells after AZD5438 exposure in a fifteen-day colony formation assay. ****P<0.0001, Two-way ANOVA. Error bars represent SEM of three technical replicates. All the experiments were performed two independent times with three technical replicates.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Colony Assay

Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A—C). CDK1 phosphorylation levels in KRAS mutant and WT cells as shown by Western blot analysis of total cell protein lysates from SW48 KRAS isogenic (A), non-isogenic pancreatic tumour cell lines (B) and non-isogenic colorectal cell lines (C). Western blots were probed for CDK1 (pThr161 CDK1 and total CDK1). β-actin detection was used as a loading control. (D and E) Bar graphs illustrating the percentage of cells in G1, S and G2/M cell cycle phases in SW48 KRAS WT or p.G12V mutant cell lines after AZD5438 exposure. SW48 KRAS WT (D) and p.G12V (E) were exposed to 0.3 μM AZD5438 or DMSO for 16, 24 and 48 hours after which cell cycle profiles were assessed by propidium iodide (PI) staining and flow cytometry. The KRAS p.G12V mutant cells showed a decrease in S and G2-fractions after exposure to AZD5438 when compared to control (DMSO) treated cells and to KRAS WT cells (AZD5438 and DMSO). (F—H) DNA synthesis in SW48 KRAS WT and p.G12V cell lines after AZD5438 exposure. (F) and (G) 5-ethynyl-2'-deoxyuridine (EDU)/ PI FACS plots in SW48 KRAS WT (F) and p.G12V mutant cells exposed to AZD5438 0.3 μM and 0.75 μM, or DMSO for 24 and 48 hours. After AZD5438 exposure, EDU/PI profiles were assessed by flow cytometry. EDU stained cells are represented in blue. (H) Bar graph illustrating the percentage of cells stained with EDU over time for both SW48 KRAS WT and p.G12V mutant cells. (I) Western blot illustrating the phosphorylation of Retinoblastoma protein (pRb) in SW48 KRAS WT and p.G12V mutant cell lines after AZD5438 exposure. Cells were exposed to AZD5438 for two hours after which total cell lysates were generated and western blotted as shown. Detection of β-Actin was used as a loading control. The levels of Rb phosphorylation on Ser807/811 were decreased in the KRAS p.G12V cells when compared to the WT cells, after AZD5438 2 hours exposure. (J) Western blot illustrating PARP1 cleavage in SW48 KRAS WT and p.G12V mutant cells after 72h of AZD5438 exposure. Cells were exposed to AZD5438 for two hours after which total cell lysates were generated and western blotted as shown. Exposure to camptothecin was used as a positive control.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Control, Staining, Flow Cytometry, DNA Synthesis, Generated, Positive Control