ro-3306 and Colorectal-Neoplasms

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.

Topics: Animals; Antineoplastic Agents; CDC2 Protein Kinase; Cell Line, Tumor; Colorectal Neoplasms; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genes, Lethal; High-Throughput Screening Assays; Humans; Imidazoles; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Pyrimidines; Quinolines; RNA, Small Interfering; Signal Transduction; Survival Analysis; Thiazoles; Xenograft Model Antitumor Assays

Inhibition of type 1 IGF receptor (IGF-1R) sensitizes to DNA-damaging cancer treatments, and delays repair of DNA double strand breaks (DSBs) by non-homologous end-joining and homologous recombination (HR). In a recent screen for mediators of resistance to IGF-1R inhibitor AZ12253801, we identified RAD51, required for the strand invasion step of HR. These findings prompted us to test the hypothesis that IGF-1R-inhibited cells accumulate DSBs formed at endogenous DNA lesions, and depend on residual HR for their repair. Indeed, initial experiments showed time-dependent accumulation of γH2AX foci in IGF-1R -inhibited or -depleted prostate cancer cells. We then tested effects of suppressing HR, and found that RAD51 depletion enhanced AZ12253801 sensitivity in PTEN wild-type prostate cancer cells but not in cells lacking functional PTEN. Similar sensitization was induced in prostate cancer cells by depletion of BRCA2, required for RAD51 loading onto DNA, and in BRCA2(-/-) colorectal cancer cells, compared with isogenic BRCA2(+/-) cells. We also assessed chemical HR inhibitors, finding that RAD51 inhibitor BO2 blocked RAD51 focus formation and sensitized to AZ12253801. Finally, we tested CDK1 inhibitor RO-3306, which impairs HR by inhibiting CDK1-mediated BRCA1 phosphorylation. R0-3306 suppressed RAD51 focus formation consistent with HR attenuation, and sensitized prostate cancer cells to IGF-1R inhibition, with 2.4-fold reduction in AZ12253801 GI50 and 13-fold reduction in GI80. These data suggest that responses to IGF-1R inhibition are enhanced by genetic and chemical approaches to suppress HR, defining a population of cancers (PTEN wild-type, BRCA mutant) that may be intrinsically sensitive to IGF-1R inhibitory drugs.

Topics: Blotting, Western; Boron Compounds; BRCA1 Protein; BRCA2 Protein; CDC2 Protein Kinase; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; DNA Breaks, Double-Stranded; DNA Repair; Histones; Homologous Recombination; Humans; Isoxazoles; Male; Microscopy, Fluorescence; Phosphorylation; Prostatic Neoplasms; PTEN Phosphohydrolase; Pyrimidines; Quinolines; Rad51 Recombinase; Receptor, IGF Type 1; RNA Interference; Thiazoles