17 research outputs found
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
Correction: Correction: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.
[This corrects the article DOI: 10.1371/journal.pone.0176578.]
Candidate drug therapies for molecularly defined subgroups of esophageal cancer identified from high-throughput drug screening.
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
Genome-wide Profiling of Genetic Synthetic Lethality Identifies CDK12 as a Novel Determinant of PARP1/2 Inhibitor Sensitivity
Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconi's anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest
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
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
Validation of the CDK1 hit from the LIM1215 siRNA screen in SW48 isogenic cell lines.
<p>(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 <i>KRAS</i> 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 <i>KRAS</i> 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.</p