Fig 1.
Use of HTS to identify drugs that are synergistic with trametinib.
A, schematic of 2D HTS performed to identify drugs that are synergistic with trametinib. B, table of synergy score values for drugs combined with trametinib. A subjective cutoff value greater than 0.5 was considered to indicate synergy. The drugs tested (probes), their targets, and the synergy scores for the cell lines HCT116, SW480, and HCP-1 are shown. C, representative pairwise synergy plots for CRC cells. The left panels show the experimentally observed growth inhibitory/cytotoxic activity (observed response) of single agents and all pairwise combinations of concentrations of trametinib and dasatinib. Concentrations of trametinib (x-axis) and dasatinib (y-axis) are shown at log scale. The middle panels show the Bliss additivity surfaces subsequently calculated using the single-agent dose response. In both of these panels, blue denotes negative control-like activity, and yellow denotes strong activity. In the right panel, the difference between the experimental observed response and predicted Bliss surface is shown. Drug concentrations are shown at log scale. In this panel green denotes synergistic regions, and black denotes additive regions. D, results of validation studies of the combination of trametinib and dasatinib. Various CRC cell lines were grown in the presence of trametinib (x-axis) and dasatinib (y-axis) at extended concentration ranges. The top panels show the effects of various doses of single-agent and combination treatment on cell growth (observed response) with blue denoting negative control-like activity and yellow denotes strong activity. The bottom panels show the excess over Bliss as a measure of synergy at the same doses. The drug concentrations are shown at log scale. In the bottom panels green denotes synergistic regions, and black denotes additive regions.
Fig 2.
The combination of SRC and MEK inhibition is synergistic in multiple KRAS-mutated CRC cell lines.
A, the results of colony formation assays performed to demonstrate the synergy of trametinib and dasatinib in multiple KRAS-mutated CRC cell lines. Representative images of different cell lines are shown. The numbers in the boxes below the images indicate synergy (excess over Bliss). C, control; T, trametinib; D, dasatinib; T+D, trametinib and dasatinib. B, the results of MTT assays performed using different KRAS-mutated CRC cell lines to further validate the enhanced efficacy of combining trametinib with dasatinib as compared with either drug alone or DMSO (control). All cells were treated for 72 hours. The data were normalized to control (taken as 1.0) and relative cell growth were plotted. All data are presented as mean (± SD) values. P values shown are for combination treatments when compared with trametinib. All P values were generated using the student t-test. Note: the IC50 was determined for each drug for every cell line using MTT assays. Cells were treated with similar to or lower than the IC50 of each drug to demonstrate enhanced efficacy of the drug combinations.
Fig 3.
Combined targeting of SRC and MEK inhibits compensatory survival signaling pathways in multiple KRAS-mutated CRC cell lines treated with MEK inhibitors alone.
Western blots performed to detect changes in the levels of the signaling factors pSRC, pFAK, and pERK in multiple KRAS-mutated CRC cell lines are shown. Changes in phosphorylation levels were compared with their respective total levels. Vinculin was used as a loading control. C, control; T, trametinib; D, dasatinib; T+D, trametinib and dasatinib. The numbers below the blots denote the phospho-protein levels relative to total protein levels in each sample (Control cells standardized to 1).
Fig 4.
Combined SRC and MEK inhibition alters levels of multiple factors that regulate CRC cell proliferation and enhances apoptotic cell death in multiple KRAS-mutated CRC cell lines.
A, heat map of results of RPPA analyses done to detect factors that regulate CRC cell proliferation and apoptosis in different KRAS-mutated CRC cell lines following treatment with trametinib (T), dasatinib (D), or both (T+D) for 48 hours. C, control. B, Western blots performed to detect the apoptosis markers cleaved PARP, total PARP, cleaved caspase 3, and total caspase 3 in multiple KRAS-mutated CRC cell lines. Vinculin was used as a loading control. The numbers below the blots denote the cleaved protein levels relative to total protein levels in each sample (Control cells standardized to 1).
Fig 5.
Combination with dasatinib does not significantly enhance the efficacy of trametinib in vivo.
KRAS-mutated SW620 cells were grown subcutaneously in nude mice and subjected to treatment with vehicle (control), dasatinib (30 mg/kg), trametinib (0.3 mg/kg), or dasatinib and trametinib. KRAS-mutated CRC PDXs (C1138) were implanted subcutaneously in nude mice, which were given vehicle (control), dasatinib (10 mg/kg), trametinib (0.2 mg/kg), or dasatinib and trametinib. Tumor volumes were measured on the days shown on the x-axes (tumors/PDXs implanted on day 0). A and D, graphs of tumor growth. B and E, average weights of residual tumors plotted at the end of the experiments. C and F, images of tumors harvested at end of experiments. Note: no significant differences in either tumor volume or tumor weight between the combination therapy and trametinib groups were observed. G, Western blot performed using residual SW620 tumors at the end of in vivo experiments to determine the effects of trametinib and dasatinib on target inactivation. Vinculin was used as a loading control for multiple blots measuring different proteins. Only one representative vinculin blot is shown. C1-3, tumors harvested from 3 different control mice; T1-4, tumors harvested from 4 different trametinib treated mice; D1-4, tumors harvested from 4 different dasatinib treated mice; T+D 1–4, tumors harvested from 4 different trametinib and dasatinib treated mice. The numbers below the blots denote the phospho-protein levels relative to total protein levels in each sample (Set of control cells with highest phospho-protein standardized to 1).