PMC

Dasatinib enhances the cytotoxicity of PLX4032 in cancer cells resistant to dasatinib. Dasatinib-resistant NSCLC cells were incubated with single agent dasatinib (50–600 nM) or single agent PLX5032 (1000- 12000 nM) or in combination of both (dasatinib:PLX4032 = 1:20) for 72 h, and their viability was estimated using an MTT assay.

Expression of kinase inactive and active BRAF leads to sensitivity and resistance to dasatinib, respectively, in NSCLC cells. (A, B, D) Cell viability was assayed following dasatinib treatment in NSCLC cells transfected with BRAF constructs. Following transfection with the noted BRAF constructs (A) H661, (B) H226, and (D) H1666 cells were and incubated with dasatinib at increasing doses for 72 h and cell viability was estimated using the MTT assay. The half-maximal inhibitory concentration (IC₅₀) of dasatinib was calculated for H661. C) Apoptosis in H661 cells expressing BRAF with the noted mutations as measured using the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling assay.

Dasatinib induces RAF dimerization in NSCLC cells which is necessary for sensitivity in cells expressing kinase impaired BRAF. (A) Dasatinib induced RAF dimerization. NSCLC cells were incubated with 150 nM dasatinib for 72h then CRAF was immunoprecipitated. The resulting lysate was split with part used for blotting with antibodies to BRAF or CRAF (to measure dimerization) and part used to measure CRAF kinase activity using MEK as a substrate. (B–C) Introduction of a mutation that interferes with RAF dimerization abrogates the effects of kinase deficient BRAF on dasatinib sensitivity. H661 cells transfected with the BRAF constructs as indicated were subjected to Western blotting (B) and an MTT assay (C).

^Y472CBRAF is biochemically similar to ^G466VBRAF but different from ^V600EBRAF. A) Kinase activity in Flag-tagged BRAF proteins with the noted mutations. After immunoprecipitation (IP) from COS7 cells and incubation with kinase-dead MEK (substrate) and ATP, MEK phosphorylation was measured using Western blotting and quantitated with ImageJ. (B–D) The effect of the expression of mutated BRAF proteins on MEK and ERK activity in intact cells. Western blotting was performed in (B) COS7, (C) H661, and (D) H226 cells transfected with BRAF constructs. E) The activity of CRAF in H661 cells transfected with mutated BRAF, as measured by incubating immunoprecipitated CRAF with kinase-dead MEK (substrate) and ATP. F) CRAF kinase activity and BRAF-CRAF binding, as measured in COS7 cells transfected with FLAG-tagged BRAF with the indicated mutations by kinase activity (IVKA) or IP of CRAF followed by blotting with anti-Flag and anti-BRAF antibodies (binding). WT, wild-type.

NSCLC cells with an inactivating BRAF mutation undergo senescence when exposed to dasatinib. A–D) Biological effects of dasatinib in NSCLC cells with BRAF mutations. NSCLC cells were incubated with 150 nM dasatinib or a control vehicle for 72 h followed by β-galactosidase (A) staining and (B) quantification, (C) cell cycle analysis using propidium iodide staining and FACS analysis, or (D) BrdU incorporation. E) The effect of dasatinib on downstream signaling as measured by Western blotting of NSCLC cells incubated with 150nM dasatinib for 24 h. (F) The reversibility of dasatinib-induced senescence. Cal12T cells were incubated with 150nM dasatinib or fresh medium (vehicle, but no drug) for the indicated times (left panel) and senescence was measured with β-galactosidase staining using light microscopy. *P <0.05. **P <0.001.

Dasatinib indirectly inhibits CRAF activity in cells with an inactivating BRAF mutation but not in cells with ^wtBRAF or ^V600EBRAF. A) Kinase activity of isolated BRAF and CRAF incubated with dasatinib. Purified recombinant BRAF and CRAF were incubated with 100 nM dasatinib in the presence of inactive MEK-1 and ATP. Kinase activity was measured by phosphorylation of MEK-1. B) The effect of dasatinib on the kinase activity of CRAF in intact cells expressing various BRAF constructs. COS-7 cells were transfected with Flag-tagged CRAF along with HA-tagged BRAF constructs with the noted mutations 24 h prior to dasatinib treatment. Transfected cells were incubated with 150 nM dasatinib or vehicle for 24 h followed by immunoprecitation with an anti-Flag antibody. The resulting lysate was subjected to a Western blot with anti-Flag and anti-HA antibodies (lower two rows) and CRAF kinase activity was measured by incubating Flag-immunoprecipitated CRAF with kinase-dead MEK-1 (substrate) and ATP (IVKA, top row). MEK activation was quantitated and normalized with total FLAG. (C) The effect of CRAF knock down (KD) on NSCLC cell number in cells with endogenous BRAF mutations. Untransfected Cat12T, H1666, H322, and H661 cells were incubated with CRAF siRNA, and their viability was estimated using an MTT assay. In all cases, Western blotting was used to confirm CRAF knockdown. At 120h Cal12T cells lost dasatinib sensitivity and CRAF expression recovered.