Figure 1.
BAY61-3606 affects viability in cells expressing mutant K-RAS or B-RAF through a MAPK-independent pathway.
(a) Cell viability quantified by Syto60 after 72 hours of AZ-628, CI-1040 or BAY61-3606 treatment in HCT-116 (K-RASG13D/+, red) or HKe-3 (K-RAS−/+, blue) cell lines. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. Cells expressing mutant K-RAS were relatively sensitive to AZ-628 and BAY61-3606, but not CI-1040. (b) Cell cycle profiles, as determined by propidium iodide staining, of colorectal cancer cells with mutant B-RAF (HT-29) treated with CI-1040 or mutant K-RAS (DLD-1) treated with BAY61-3606. While inhibition of MEK induces G1 arrest in HT-29 cells, as evidenced by the loss of the 4N peak, BAY61-3606 did not appear to alter the profile of DLD-1 cells. (c) Cell viability of colorectal cancer cells expressing mutant B-RAF (V600E) after 72 hours of treatment with BAY61-3606 and/or CI-1040. Cells expressing mutant B-RAF (HT-29 – solid outline and RKO – dotted outline) were sensitive to both BAY61-3606 and CI-1040 and these two inhibitors cooperated to produce an enhanced response. (d) Phospho-MEK1 (Ser217/Ser221) and phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) levels in K-RAS wild-type and mutant cells after 45 minute exposure to 1 µM AZ-628, 1 µM BAY61-3606, or vehicle control. Signals were measured using Bio-Plex assays. Relative signal was normalized to a master control lysate. AZ-628 treatment reduced the level of phospho-MEK and phospho-ERK, but BAY61-3606 did not.
Figure 2.
Inhibition of SYK is not responsible for the BAY61-3606 effect on cell viability in colorectal cancer cells.
(a) Knockdown of SYK protein in DLD-1 cells via shRNA, as shown by Western Blotting. The Ramos (R) cell line, a hematopoetic cell line with a high expression of SYK, was used as a positive control. (b) Cell viability of DLD-1 cells (red), and its K-RAS wild-type (DKs-8 – blue) and SYK knockdown (red shaded) derivatives after 72 hours of treatment with 1 μM of BAY61-3606 or R406, a distinct SYK inhibitor. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. DLD-1 cells and its K-RAS wild-type derivative did not exhibit sensitivity to R406, while knocking down SYK in DLD-1 cells only minimally affected its sensitivity to BAY61-3606.
Figure 3.
Identification and attempted validation of BAY61-3606 targets.
(a) TREEspot image representing the inhibitory activity of BAY61-3606. Kinases that are inhibited for ATP binding by BAY61-3606 are indicated by red dots on the phylogenetic tree of kinases. The size of the red dot corresponds to the amount of inhibition by 1 μM BAY61-3606. The identity of the individual kinases can be found in Table S1. (b) Cell viability quantified by Syto60 after shRNA-mediated knockdown of the potential BAY61-3606 targets in DLD-1 (red) or DKs-8 (blue) cell lines. Relative cell viability is normalized to the parental cell lines infected with vector only. Error bars represent SEM for 2 independent experiments.
Table 1.
Activity of BAY61-3606 against selected kinases.
Figure 4.
Biological activities of BAY61-3606 derivatives.
(a) GI50 values for BAY61-3606 derivatives performed in HCT-116 (red) or HKe-3 (blue) cells. Derivative 6 was chosen for further study for its increased potency and specificity for K-RAS mutant cells. (b) Cell viability quantified by Syto60 after 72 hours exposure to BAY derivative 6 in HCT-116 (red) or HKe-3 (blue) cells. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. HCT-116 cells were relatively sensitive to BAY derivative 6.
Figure 5.
MAP4K2 is a target for BAY61-3606 that modulates the response of wild-type cells to AZ-628.
(a) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with varying concentrations of BAY61-3606 and 1 µM AZ-628 in HCT-116 (red line) or HKe-3 (blue lines) cells. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. The two inhibitors cooperate in wild-type cells, but not in cells expressing mutant K-RAS. (b) Cell viability after 72 hours of combinatorial treatment with varying concentrations of BAY derivative 6 and 1 µM AZ-628 in HCT-116 and HKe-3 cells. BAY derivative 6 does not confer additional sensitivity to AZ-628 upon HKe-3 cells. (c) Cell viability quantified by Syto60 after 72 hours of AZ-628 treatment in HCT-116 or HKe-3 cell lines with MAP4K2 knockdown. Loss of MAP4K2 does not affect AZ-628 response in cells expressing mutant K-RAS, but enhances the effect of AZ-628 in cells expressing wild-type K-RAS. (d) Cell viability after 72 hours of combinatorial treatment with 1 µM BAY61-3606 and 1 µM AZ-628 (shaded) or 1 µM AZ-628 alone (clear) in HKe-3 cells with MAP4K2 knockdown. Relative cell viability was normalized to 1 µM AZ-628 treated samples. In parental HKe-3 cells, BAY61-3606 confers sensitivity to AZ-628. Upon loss of MAP4K2, BAY61-3606 no longer sensitizes.
Figure 6.
MAP4K2 modulates NFκβ signaling.
(a) Time course of phospho-Iκβα (Ser32/Ser36) activation after 1 µM AZ-628 treatment in HCT-116 (red lines) or HKe-3 (blue line) cells with MAP4K2 knock down, as measured via Bio-Plex. Relative signal was normalized to a master control lysate. Error bars represent SEM for 3 independent experiments. NFκβ signaling was enhanced in HKe-3 cells after exposure to AZ-628 and this was dependent upon MAP4K2. (b) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with IKK inhibitor VII and 1 µM AZ-628. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Like BAY61-3606, IKK inhibitor VII enhanced the effect of AZ-628 specifically in K-RAS wild-type cells. (c) Cell viability after 72 hours of combinatorial treatment of 1 µM IKK inhibitor VII and 1 µM AZ-628 (shaded) or 1 µM AZ-628 alone (clear) in HKe3 cells with MAP4K2 knockdown. Relative cell viability was normalized to 1 µM AZ-628 treated samples. Loss of MAP4K2 abrogated the ability of IKK inhibitor VII to sensitize HKe-3 cells to AZ-628.