Figure 4.
Type II RAFi and MEKi coordinate RAF/MEK stabilization, sequestering MEK from ERK.
A, Proximity ligation assays (PLAs) detecting CRAF/MEK and MEK/ERK proximity complexes in NRASMUT melanoma SDR sub-lines withdrawn from trametinib for 12 days or treated for 12 days with trametinib (0.1 μM), SCH-772984 (0.1 μM), or BVD-523 (1.0 μM), ± BGB-283 co-treatment (1 μM). Last fresh dose of inhibitor(s), 12 hours before analysis. DAPI, nuclear stain. Ruler, 20 μm.
B, Quantifications of PLA signals in (a) expressed as the fold change (FC) of PLA dots (per nucleus) of BGB-283 co-treatment over DMSO or single inhibitor treatment. N=5 fields; mean ± SDs; all comparisons with respect to FC (BGB-283+DMSO/DMSO); *p<0.1, **p<0.05 and ***p <0.01 based on t-test.
C, Western blots (Wbs) of lysates from culture conditions in A using indicated antibodies.
D, PLA detecting CRAF/CRAF, BRAF/BRAF, BRAF/MEK and MEK1/MEK1 proximity complexes in two NRASMUT melanoma SDR sub-lines treated with vehicle or trametinib (0.1 μM) for 48 hours, ± BGB-283 (1 μM, last 2 hours). DAPI, nuclear stain. Ruler, 20 μm.
E, Quantification of PLA data in c. N=5 fields; mean ± SDs. FCs: black, BGB-283+DMSO/DMSO; grey, BGB-283+tram/tram. Latter FC is compared to the former FC; **p<0.05 and ***p <0.01 based on t-test.
F, Tumor volumes of NRAS_PDX1 R2 (with acquired trametinib resistance) in mice that were (starting on day 78, indicated by an arrow) maintained on trametinib (5 mg/kg/day PO), switched to BGB-283 (20 mg/kg/day PO), or treated with BGB-283 (20 mg/kg/day PO) on top of trametinib (5 mg/kg/day PO). N=5 tumors per group; means ± SEMs.
G, Immunofluorescence (IF) of p-ERK levels (top row) and PLA of indicated protein/protein proximity complexes (bottom three rows) for three groups of PDX tumors in e. Tumors were collected on days 3 or 5 (from day 78). Images representative of five tumors per group.
H, Quantifications of PLA signals of CRAF/BRAF, CRAF/MEK and MEK/ERK complexes expressed as FC of PLA dots per nucleus of each indicated treatment condition vs. continuous trametinib monotherapy. Mean ± SDs; **p<0.05 and ***p <0.01 based on pairwise t-test.
I, IP-WBs or direct WBs of WCLs after treatment of indicated trametinib-resistant sub-lines with vehicle (DMSO) or indicated inhibitor(s) as in a. IgG, isotype control for IP.
J, Predicted conformational rearrangements of MEK1 and BRAF in complex upon binding to trametinib and BGB-283, respectively. Light brown, BRAF in its MEK1-bound tetrameric conformation; dark brown, BRAF in its BGB-283-bound conformation (BRAF P-loop in orange). Dark blue, MEK1 in BRAF-bound tetrameric conformation; light blue, MEK1 in its Tak-733-bound conformation (MEK1 activation loop in green). Red arrows, predicted and actual rearrangements of MEK1 and BRAF upon trametinib and BGB-283 binding, respectively.