Abstract 2173: Targeting CDK1 and MEK/ERK overcome mutant BRAF-mediated apoptosis resistance in human colorectal cancer cells

Abstract

Abstract BRAFV600E mutation occurs in ~10% of human colorectal cancers (CRC) where it is associated with treatment resistance and poor prognosis. Data from TCGA and a clinical trial cohort identified a distinct subset of BRAF mutant colon cancers with deregulation of the cell cycle and overexpression of CDK1 (Barras D, et al, Clin Cancer Res. 2016). We tested the hypothesis that CDK1 inhibition can enhance apoptosis in BRAFV600E CRC cells. Since BRAF mutant cells show p-ERK activation, combined inhibition of CDK1 and MEK/ERK was evaluated. Using isogenic colon cancer cells, BRAF mutant alleles were shown to confer resistance to the CDK1 inhibitor, R0-3306, in a gene dose-dependent manner that was associated with reduced cleavage of caspase-3 and downstream PARP, and decreased pH2AX (DNA fragmentation marker). Ectopic BRAFV600E or constitutively active MEK mutant also conferred resistance to R0-3306. CDK1 siRNA was shown to increase cobimetinib-induced apoptosis, including annexin V labeling, and increased pH2AX. Furthermore, ERK siRNA or cobimetinib treatment attenuated CDK1 protein expression and increased R0-3306-induced pH2Ax. Moreover, treatment with R0-3306 plus cobimetinib significantly enhanced a caspase-dependent apoptosis and markedly reduced colony formation vs either drug alone in two CRC cell lines. Caspase-3 cleavage by R0-3306 + cobimetinib was dependent upon caspase-8. Mechanistically, CDK1 inhibition by R0-3306 suppressed the phosphorylation of procaspase-8 at Ser-387 shown by R0-3306 withdrawal which restored p-C8-S387 coincident with expression of the mitotic marker, pH3S10. In conclusion, inhibition of CDK1 can significantly augment apoptosis induction by MEK/ERK inhibition in BRAFV600E CRC cells, suggesting a novel therapeutic strategy to overcome mutant BRAF-mediated resistance. Apoptosis induction by CDK1 inhibitor (R0-3306) ± MEK/ERK inhibitor cobimetinibTreatmentTreatmentTreatementTreatmentCRC cell linesDMSOR0-3306 (5 µM)cobimetinib (5 µM)combinationAnnexin V+ (%)RKO13.43 ± 1.4132.55 ± 2.9315.04 ± 0.2652.72 ± 3.16Annexin V+ (%)HT-2911.22 ± 1.8442.44 ± 2.7025.29 ± 0.1665.62 ± 4.11 Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. Targeting CDK1 and MEK/ERK overcome mutant BRAF-mediated apoptosis resistance in human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2173. doi:10.1158/1538-7445.AM2017-2173