33P Synergistic effects of alpelisib (PI3K inhibitor) and ribociclib (CDK 4/6 inhibitor) in preclinical colorectal cancer (CRC) models

Abstract

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. Ribociclib has 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. We explored the in vitro efficacy of drug combinations (Ribociclib and Alpelisib (R+A)) in four CRC cell lines with different mutational statuses; CACO-2 (PIK3CA/KRAS wildtype), LS-1034 (KRAS mutated), SNU-C4 (PIK3CA mutated), and DLD-1 (PIK3CA/KRAS mutated). The drug combination index (CI) was calculated by using Calcusyn Biosoft software. A western immunoblotting method was used for protein analysis. IC50s for R and A were calculated for all four cell lines. CACO2 and DLD-1 cells were resistant to R (IC50 >15μM). The cell lines had varying sensitivity to both drugs. Drug combination analysis showed that the combination of R+A has a synergistic anti-proliferative effect in all CRC cell lines tested. The combination of R+A is highly synergistic in LS1034 cells which harbor 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 and normalizing to β-actin. We demonstrated that 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. There was a marginal increment in the apoptotic marker (pBCL-2), possibly due to the shorter duration of treatment (24 hours).Table: 33PCell linesMolecular featuresRibociclib (R) IC50Alpelisib (A) IC50Drug combination doses R+ACombination Index CI ED50SNUC4PIK3CA E545G0.259μM0.4413μM2μM+400nM0.54LS1034KRAS A146T1.126μM0.3134μM2μM+400nM0.16DLD1KRAS G13D/PIK3CA E545K>15 μM1.307μM2μM +3μM0.78CACO2Wild type>15 μM1.547μM5μM+2μM0.28 Open table in a new tab A synergistic response to treatment with the combination of R+A is seen in all cell lines. We are currently investigating this combination in CRC animal models.