Figure 2. MTAP-BRAF and MS4A6A-BRAF fusions are not suppressed by second generation RAF inhibitors but demonstrate sensitivity to LY3009120 and MEK inhibitors.
A. Western blot analysis (left) and soft agar colony counts (right) showing the effect of second generation RAFi, PLX8394, on NIH3T3 cells expressing MTAP-BRAF and MS4A6A-BRAF respectively. B. Western blot analysis (left) and soft agar colony counts (right) showing the effect of pan-RAF-dimer inhibitor, LY3009120, on NIH3T3 cells expressing MTAP-BRAF and MS4A6A-BRAF respectively. C. Co-immunoprecipitation (co-IP) assay assessing homo-dimerization of MTAP-BRAF as well as hetero-dimerization with wild-type BRAF and Trunc-MTAP in HEK293 cells under control, PLX8394, and LY3009120 treated conditions. D.Co-immunoprecipitation assay assessing homo-dimerization of MS4A6A-BRAF as well as hetero-dimerization with wild-type BRAF and Trunc-MS4A6A in HEK293 cells under control, PLX8394, and LY3009120 treated conditions.E. Competition co-IP assay assessing preferential interaction of Trunc. MTAP with MTAP-BRAF fusion versus homo-dimerization. Increasing doses of tetracycline (0, 0.1, 0.5, 1 ug/ml) used to regulate protein level of His-tagged Trunc-MTAP. F. Western blot analysis (left) and soft agar colony counts (right) showing the effect of MEK inhibitor, trametinib, on NIH3T3 cells expressing MTAP-BRAF and MS4A6A-BRAF respectively. Error bars represent SEM, n=3. No value on bar represents NS (non-significant), *p-value<0.05, **p-value< 0.01, ***p-value<0.001 compared with control conditions. ‘p-‘ and ‘t-‘represent phosphorylated and total versions of protein, respectively.