A Novel Drug Combination Suppresses KRAS-mutant Cancers

Oncogenic mutations in the GTPase protein RAS (NRAS, KRAS, and HRAS) occur in up to a third of all human cancers. Somatic mutations in KRAS, the principal isoform of RAS, comprise 86% of all RAS mutations, with a particularly high mutation frequency in pancreatic, colorectal, and lung cancers. Cancers harboring KRAS mutation seem to be extremely malignant and dangerous. However, no effective targeted therapies exist for mutant KRAS cancers yet, due to the intricate network of KRAS signaling cascades or disappointing efficacy in clinical testing. Recently, synthetic lethal screening did open up a new avenue to treat oncogene-driven cancers, especially in KRAS-mutation cancers. Furthermore, the benefits of single-agent therapy are limited and drug resistance may emerge after a period of administration. Thus, we developed a synthetic lethal drug screening plus combinatorial strategy using a panel of clinical agents in isogenic KRAS-mutant and wild-type cells to identify novel clinical drug pairs to treat KRAS-driven cancers. Such an approach could also provide immediately usable translational therapeutic strategies.In this study, we evaluated a panel of agents that directly or indirectly targeted KRAS effector pathways in a combinatorial screening, including inhibitors targeting signaling upstream of KRAS (receptor tyrosine kinases, RTKs), signalings downstream of KRAS (RAF/MAPK and PI3K/AKT/mTOR signaling pathway) and synthetic lethal genes of KRAS oncogene. We performed the primary screening in isogenic cell lines to evaluate the selective efficacy of single drugs, and the concentrations required to inhibit 50% of cell growth (IC50) were calculated. Then, we generated a mixture of two drugs at their equipotent ratio (at the ratio of their IC50’s) and serial dilutions of the mixture (1:1,1:2,1:4,1:8) to test drug combinations. The Combination Index (CI) was calculated using CalcuSyn software and CI<1 indicates synergism. The screening results showed that 71% of the combinations contained drugs whose targets were synthetic lethal genes of oncogenic KRAS in the synergy hits. Of all these drug pairs, the greatest synergistic inhibitory effects of the synthetic lethal genes was observed in the dual inhibition of PLK1 and ROCK, using BI-2536 and Fasudil (CI=0.33).To verify the synergistic effect of this novel drug combination, we evaluated its efficacy in a panel of cell lines from lung, colon and pancreatic cancers in which the KRAS mutation is the predominant oncogenic alteration. We found BI-2536/Fasudil exhibited a strong ability to induce apoptosis and cell cycle arrest in KRAS-mutant cancer cell lines at low doses (115 of the effective doses), while sparing the KRAS wild-type cells. To identify the potential mechanisms that underlie the enhanced activity of combined inhibition of PLK1 and ROCK, we conducted a microarray analysis to examine the gene expression profiles after BI-2536/Fasudil treatment. Microarray profiling revealed that the activated p53 signaling pathway was account for the synergistic efficacy of BI-2536/Fasudil. We then applied western blot and Q-PCR to further evaluated the mechanism in vitro. The results showed that this combinatory inhibition had little impact on p53, but significantly increased the protein level and the activity of cyclin-dependent kinase inhibitor p21WAF1/CIP1 in KRAS-mutant cancer cells. Overexpression of p21, either by cDNA transfection or drugs, selectively impaired the growth of KRAS-mutant cells, suggesting a novel synthetic lethal interaction between KRAS and CDKN1A (encodes p21), and a novel therapeutic target for cancers with KRAS mutations.Based on the broad efficacy of BI-2536/Fasudil against KRAS-mutant cells in vitro, we assessed the therapeutic efficacy of this combination in a series of KRAS-mutant lung cancer models in vivo by using a human cancer cell xenograft mouse model, an orthotopic tumor modeland a patient-derived tumor explant (PDTX) mouse model carrying G12D KRAS mutation. After a 4-6-weeks treatments, we found marked tumor growth inhibitions (P<0.0001-0.05) in the combination group compared to the vehicle control or single agent alone. To further evaluate the mechanism of this combination in vivo, PDTX tumor samples were collected for quantitative PCR and immunoblotting analyses. Consistent with the results in tumor cells in vitro, the combination of BI-2536 and Fasudil led to significant increases in the mRNA and protein expression of p21 in PDTX tumors. These findings indicated that this unique drug pair, combined inhibition of PLK1 and Rho signaling, exhibited genotype-selective efficacy and induced mitotic stress by elevating p21 expression, further suggesting a potential avenue for therapeutic treatment of KRAS-mutant cancers.In summary, the present study suggests a novel drug combination that disrupts KRAS synthetic lethal dependency, which could be therapeutically exploited to inhibit KRAS-driven cancers.