Synergistic Effects of Alpelisib (PI3K inhibitor) and Ribociclib (CDK4/6 inhibitor) Combination in Preclinical Colorectal Cancer Models

Bakcground: Multiple activating genetic mutations in the phosphatidylinositol-3 kinase (PI3K) and the mitogen activated protein kinase (MAPK) pathways have been implicated in the development of resistance to anti-cancer therapies.CDk4/6 inhibitors have limited activity as a single agent in CRC. However, combining Ribociclib with targeted therapies of the MAPK and PI3K pathways may be a promising treatment strategy in CRC.

Mehtods: We explored the in vitro efficacy of drug combinations (Ribociclib (CDK4/6 inhibitor and Alpelisib (PI3K inhibitor) in four CRC cell lines with different mutational status; CACO2 (PIK3CA/KRAS wild-type), LS1034 (KRAS mutated A146T), SNUC4 (PIK3CA mutated) and DLD1 (PIK3CA/KRAS mutated). Drug combination index (CI) was calculated using Calcusyn Biosoft software. A western immunoblotting method was used for protein analysis. The Chick Chorio-Allantoic Membrane (CAM) assay was used for in-vivo analysis of the effect of the drug combination.

Results: IC50s for Ribociclib and Alpelisib were calculated for all four cell lines. CACO2 and DLD1 cells were resistant to Ribociclib (IC50 >15µM).  The cell lines had varying sensitivity to both drugs. Drug combination analysis showed that the combination of Ribociclib and Alpelisib has a synergistic anti-proliferative effect in all CRC cell lines tested. The  combination of Ribociclib and Alpelisib is highly synergistic in LS1034 cells which harbour a KRAS mutation (CI=0.16). 

Relative expression of the proteins Cyclin D1, E2F-1, p-BCL-2, p-AKT, p-Rb and p-S6 was determined by measuring the density of each band from the western blot experiments and normalizing to β-actin. Combined inhibition of CDK4/6 and PI3Kα caused a simultaneous reduction of p-RB, p-AKT and p-S6 and a more complete inhibition of the PI3K/AKT/mTOR pathway in all four cell lines tested. There was also an increase in expression of  in the apoptotic marker pBCL2 in cells treated with the combination of Ribociclib and Alpelisib compared to vehicle control. Tumour viability was measured by presence or absence of tumour visually (macroscopically) before CAM extraction. A macroscopic reduction of visible tumour was observed in all cell lines treated with the Ribociclib and Alpelisib combination compared to control. Microscopic examination also showed less Ki67 staining and negative cytokeratin staining in the Ribociclib and Alpelisib combination treated groups. 

Conclusion: A synergistic response to treatment with the combination of Ribociclib and Alpelisib is seen in all cell lines and this combination may be a rational treatment strategy in colorectal cancer.