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.