Figure 4. Allosteric MEK inhibitors induce RAF-MEK complex formation in KRAS mutant tumors.
(A) HEK293 cells were transfected with FLAG-MEK1, followed by treatment with PD0325901 for 3 hrs. MEK1 was immunoprecipitated using an anti-FLAG antibody followed by immunoblotting with the indicated antibodies to determine RAF-MEK interactions. A representative example of at least two independent experiments with each drug is shown.
(B) Whole cell lysates from A375 cells were subjected to immunoprecipitation with IgG or a MEK1 antibody and then immunoblotted for the indicated proteins. Two MEK specific IP replicates are shown.
(C) Cells were treated with PD0325901 for 3 hrs and endogenous MEK1 was immunoprecipitated to determine its interaction with RAF kinases. A representative example of three independent experiments is shown.
(D) HEK293 cells were transfected with WT MEK1 or a MEK1 mutant with impaired RAF interaction (IRI, M308A/I310A). RAF-MEK1 complexes were determined as in C.
(E) A375 cells were transfected with the indicated constructs and then treated with PD0325901 (50 nM), selumetinib (500 nM) or RDEA119 (100 nM) for 1 hr to determine the effect on ERK phosphorylation. A representative example of at least two independent experiments for PD0325901 and selumetinib are shown.
(F) A schematic diagram modeling the role of CRAF in the differential adaptation of KRAS and BRAF mutant tumors to MEK inhibitor treatment.
See also Figure S4