Date Published: February 16, 2016
Publisher: Public Library of Science
Author(s): Sara Costa-Cabral, Rachel Brough, Asha Konde, Marieke Aarts, James Campbell, Eliana Marinari, Jenna Riffell, Alberto Bardelli, Christopher Torrance, Christopher J. Lord, Alan Ashworth, Robert W Sobol.
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.
KRAS, also known as the Kirsten rat sarcoma viral oncogene homolog protein (V-Ki-ras2), is a member of the RAS superfamily [1, 2]. RAS proteins (HRAS, KRAS and NRAS) are small GTPases that cycle between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound conformations. RAS activity regulates a complex signalling network including the RAF-MEK-ERK cascade, the phosphatidylinositol 3-kinase (PI3K) pathway and the effector family of exchange factors for the RAL small GTPases [3–5]. Through the combined action of these signalling pathways, expression of activated mutant RAS is thought to promote several of the characteristics of malignant transformation. The KRAS oncogene is one of the most frequently mutated genes in human cancer , being altered in approximately 20% of all human tumours . Oncogenic forms of KRAS have profound effects on signalling, which can result in a hyper-proliferative and anti-apoptotic phenotype [3, 8–10]. In addition, KRAS mutations affecting amino acid position p.G12, cause resistance to EGFR targeted therapy in colorectal cancer (CRC) [11, 12].
Here, we describe a series of experiments aimed at identifying novel dependencies in KRAS mutant tumour cells. Using genetic screens in LIM1215 KRAS isogenic cell lines we identified novel KRAS synthetic lethal effects, including CDK1. One weakness of the HT screen might be the use of engineered models of KRAS mutation, rather than the use of models with naturally occurring KRAS mutations. It is possible that these engineered models do not fully replicate all of the KRAS synthetic lethal effects found in real human tumours. However, we do note that we also observed the KRAS selective effect of CDK1 inhibition in tumour cell lines with naturally occurring KRAS mutations (Fig 4, Fig 5 and Fig 7). CDK1 was validated using a different KRAS isogenic cell model (SW48) as well as a series of non-isogenic CRC and PDAC tumour cell models. This analysis suggested that the CDK1/KRAS synthetic lethal effect was not restricted to isogenic systems but also operated in a variety of different, genetically diverse, KRAS mutant tumour cells. One priority in identifying SL effects is to discriminate SLs that are easily abrogated by additional genetic and epigenetic alterations (soft SLs) from those that are somewhat more resistant to these changes (hard SLs) . The analysis in the non-isogenic tumour cell panel suggested that the CDK1/KRAS SL was a relatively hard synthetic lethal effect. CDK1 forms active complexes with A-, B-, E-, and D-type cyclins , as part of its critical role in cell cycle progression. Inhibition of CDK1 with AZD5438, led to a marked reduction in the proportion of cells in S and G2/M phases of the cell cycle in KRAS mutant cells. This was accompanied by an increased in the proportion of cells in G1. Moreover, western blotting revealed that AZD5438 decreased Rb phosphorylation levels in KRAS mutant cells compared to KRAS WT cells. As cells require hyperphosphorylation of Rb to pass the restriction point in G1 prior to entering S-phase, the reduction in Rb phosphorylation could provide an explanation as to the cell cycle arrest, which could be a possible cause for the reduced proportion of KRAS mutant cells in S-phase . We determined the mechanism of cell inhibition to be apoptosis, where western blotting showed increased PARP cleavage in the SW48 p.G12V cells. We also assessed the anti-tumour effect of AZD5438 on human tumour xenografts to access the KRAS selectivity of this compound in an in vivo setting. These experiments were performed in immunocompromised mice with SW620 cell together with SW48 KRAS isogenic p.G12V and WT cells xenografts, and have confirmed the KRAS selective inhibitory effect of AZD5438, supporting the in vitro results. Overall the results from this experiment suggested disease stabilisation after AZD5438 treatment, but in some cases complete tumour inhibition was observed. Already a number of other synthetic lethal interactions involving cyclin dependent kinases have been proposed. Perhaps the most notable synthetic lethal interaction involving CDK1 described to date is between CDK1 and the oncogenic transcription factor, MYC . In this particular case, MYC overexpressing tumour cells appear to be reliant upon the activity of the inhibitor of apoptosis protein (IAP), BIRC5 (survivin) a CDK1 target . This particular synthetic lethality can be elicited in triple negative breast tumour cells using the clinical CDK1,2,5,9 inhibitor dinaciclib . As well as providing a potential route to targeting MYC driven tumours, CDK1 inhibition has also been proposed as a route to causing chemosensitivity, enhancing the effects of PARP inhibitors  as well as PI3-kinase inhibitors . Here we show a KRAS/CDK1 synthetic lethality that can be elicited with small molecule CDK1 inhibitors might also be added to the list of potential utilities for small molecule CDK inhibitors in cancer.