Background: Combinative inhibitors with multiple actions to target downstream effectors are good strategy to reduce resistance occurrence. This is a preclinical study to evaluate efficacy of Palbociclib (P; CDK4/6 inhibitor) in combination with either Gedatolisib (G; P13K/mTOR dual inhibitors) or PD0325901 (PD; MEK 1/2 inhibitor) in CRC cell models. Methods: Five CRC cell lines with hot-spots mutations in PI3K and MAPK pathways genes are used. Cell growth assay is used to evaluate antiproliferative response to P, G and PD alone and in combination. RPPA is used to clarify the role actions of combinative drugs on resistance development and new druggable proteins. Paired t-test is used. Results: All cell lines except LS1034 (IC50=7.2µM) were sensitive to G. LS411N and SNUC4 were sensitive to P whereas Caco2, DLD1 and LS1034 were resistant (IC50>15µM). All cells lines were sensitive to PD. When tested in combination, P+G has synergistic response in all cells lines, except for LS411N (CI=0.9; p=0.02). P+G is highly synergistic in LS1034 with KRAS mutation (CI=0.1; p=0.11). P+G is also synergistic in DLD1 with co-occurring PIK3CA and KRAS mutations (CI=0.6; p=0.04). P+PD is synergistic in all cells lines except for LS411N. In SNUC4 with PIK3CA mutation, both drug combinations have equal synergism (CI=0.5; p<0.05). RPPA analysis is in progress and will be included in final abstract. Conclusions: This offers good rationale for further clinical development of P+G as beneficial therapy in treatment-resistant CRC patients. P+G offers better alternative to P+PD since MEK inhibitor resistance had been associated with tumours harbouring KRAS with(out) PIK3CA mutations. BRAFV600E and ERBB2 mutations are useful negative predictors in this cohort.