CDK1 is a synthetic lethal target for KRAS mutant tumours.

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation

Small molecules as modulators of mitotic arrest and senescence in cancer

Manipulation of the cell cycle is an extensively used and promising strategy for cancer therapy. To identify novel cell cycle modulators, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic arrest and senescence. 8-azaguanine, IC 261, erysolin and SKF 96365 were identified as chemicals that stimulate senescence, a state of prolonged growth arrest, in a p53-mutated growth arrest-deficient cell line. Microtubule-targeting cancer therapies such as paclitaxel block cell cycle progression at mitosis by prolonged activation of the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time, or undergo mitotic slippage through degradation of cyclin B1 in the presence of an active mitotic checkpoint and enter interphase without having separated their chromosomes. Regulation of extended mitotic arrest and mitotic slippage and their contribution to subsequent cell death or survival is incompletely understood. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic exit through mitotic slippage. Using these drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. SU6656 and geraldol were identified as chemicals that induce mitotic slippage. Cells arrested at mitosis with paclitaxel or vinblastine and induced by these compounds to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death. Mitotic slippage induced by SU6656 or geraldol involved proteasome-dependent degradation of cyclin B1, but also required proteasome- and caspase-3-dependent inactivation of the mitotic checkpoint through degradation of the mitotic checkpoint protein BubR1. Caspase-3 and p53, both apoptotic effectors, did not affect cell death after exposure to paclitaxel, with or without mitotic slippage induction. The requirement for caspase-3 for chemically induced mitotic slippage reveals a new mechanism for mitotic exit and a link between mitosis and apoptosis that has implications for the outcome of cancer chemotherapy.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Correction: Correction: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

[This corrects the article DOI: 10.1371/journal.pone.0176578.]

Characterization of LIM1215 isogenic cell lines.

<p>(A) Schematic of KRAS isogenic cell lines generation. <i>KRAS</i> 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 <i>KRAS</i> allele is expressed from its endogenous promoter. The <i>Neo</i> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149099#pone.0149099.ref027" target="_blank">27</a>]). (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149099#pone.0149099.g002" target="_blank">Fig 2</a>. Bar graph of <i>KRAS</i> siRNA Z-score values across the LIM1215 <i>KRAS</i> 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 <i>KRAS</i>-specific siRNAs. Multiple <i>KRAS</i> siRNA oligos and a pool efficiently suppressed KRAS expression showing that the siRNAs were on-target.</p

Candidate <i>KRAS</i> synthetic lethal genes identified in LIM1215 KRAS isogenic cell lines siRNA screens.

<p>The table shows the median Z scores for the KRAS WT and mutant cells and the respective Delta median Z scores.</p

Response to three second-generation CDK inhibitors in CRC cell lines.

<p>(A) AT7519, (B) dinaciclib and (C) PD023309 median of drug-dose response curves of <i>KRAS</i> WT and mutant cells from a five-day cell viability assay to assess the KRAS selectivity of the CDK inhibitors in ten colorectal cell lines, four KRAS WT (black) and six mutant (pink) cell lines. (ns not-significant, Two-way ANOVA) Experimental conditions were repeated in triplicate. Error bars represent SEM. Only AT7519 and dinaciclib showed KRAS selectivity in the CRC cell lines.</p

<i>In vivo</i> efficacy of AZD5438 in SW620 cell xenografts.

<p>KRAS mutant xenografts were treated with vehicle (DMSO in black) or AZD5438 (20mg/kg/day (in pink)). (A) Mean of increase in tumour volume relative to initial tumour volume. A one-way ANOVA was performed to compare both arms and the difference in the SW620 xenografts was statistically significant (p = 0.017). (B) Survival curves showing a statistically significant difference between the treated and vehicle arms, where the mice in the drug arm had an increase in overall survival using a Log-rank Mantel-Cox test (p = 0.0018 in SW620 xenografts). (C) Average final tumour weight. There is a significant difference between the vehicle and treatment arms (**p < 0.01, t-test). Error bars represent SEM. (D) Photograph of a mouse treated with AZD5438, where the tumour disappeared completely, after 37 days of treatment.</p

CDK inhibitors sensitivity profile in SW48 KRAS isogenic cell lines.

<p>(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.</p

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours - Fig 6

<p>(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.</p

Identification of CDK1 as a KRAS synthetic lethality using functional genomic screens in the LIM1215 KRAS isogenic models.

<p>(A) Schematic describing the screen format. LIM1215 cells plated in 384 well plates were transfected with siRNA. Each transfection plate contained 300 experimental siRNAs supplemented with wells of non-targeting siCONTROL (siCON) and siRNA targeting PLK1 (positive control). Transfected cells were divided into three replica plates. Cell viability was assessed after six days using CellTiter-Glo Luminescent Cell Viability Assay (Promega). (B) Heatmap of siRNA z-scores for 15 screens used in the analysis. The rows of the heatmap correspond to siRNAs and are ordered by the difference in the KRAS mutant and non-mutant group median z-scores. The heatmap inset shows the six siRNAs selected for andditional analyses. These siRNAs were selected because they caused reduced viability in the KRAS mutant isogenic models (median z ≤ -2) but not in the WT and neo cell lines (median z ≥ -1).</p