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Fig. 5 | Molecular Cancer

Fig. 5

From: Loss of FBXW7-mediated degradation of BRAF elicits resistance to BET inhibitors in adult T cell leukemia cells

Fig. 5

FBXW7 wild type and mutants selectively target BRAF for proteasome degradation. a 293 T cells were co-transfected with FBXW7 and increasing amounts of BRAF for 48 h. FBXW7-mediated degradation of BRAF was then determined by Western blot. 293 T cells were co-transfected with FBXW7 and BRAF or BRAF V600E mutant for 48 h. FBXW7-mediated degradation of BRAF or BRAF V600E was determined by Western blot. b FBXW7-mediated ubiquitination of BRAF. 293 T cells were co-transfected with FBXW7, K48-Ub and BRAF. Cells were treated with MG132 for 6 h before harvest. Immunoprecipitated BRAF and Western blot Ub showed the ubiquitination level of BRAF. c Comparison of FBXW7 wild type and mutants W406R, W425R and S462P in their ability to target BRAF for degradation following transient transfection in 293 T cells. The interaction between FBXW7 WT/mutants and BRAF was analyzed by co-immunoprecipitation. 293 T cells co-transfected with wild-type or mutant FBXW7 and BRAF were lysed and immunoprecipitated with BRAF. Western blot was used to analyze the interaction between FBXW7 WT/mutants and BRAF. d ATL cells MT1 stably transfected with a TET-inducible FBXW7 wild type or W425R were induced with doxycycline and degradation (WT) or lack thereof (W425R) of endogenous BRAF was confirmed by Western blot. Endogenous degradation of BRAF by wild type FBXW7 was confirmed by immunohistochemistry following induction of FBXW7 with doxycycline. e Graphical representation showing the correlation between FBXW7 gene alternation in T-ALL cell lines and JQ1 IC 50. f Data from RPPA was used to quantify BRAF and c-MYC protein expression in MT1 cells expressing FBXW7 WT, R465C, R505C and R479Q. g FBXW7-mediated degradation of BRAF by wild type FBXW7 and R505C but not R465C was confirmed in transient transfection assays and Western blot analyses. h FBXW7 R505C retains wild type ability to add ubiquitin to BRAF. 293 T cells were co-transfected with FBXW7, K48-Ub and BRAF. Cells were treated with MG132 for 6 h before harvest. Immunoprecipitated BRAF and Western blot Ub showed the ubiquitination level of BRAF. i Combination therapy with Ulixertinib or PLX8394 prevents tumor cell resistance to BET inhibitors. MT1 cells carrying an inducible wild type FBXW7 or S462P mutant were cultured in presence of doxycycline and in the presence of JQ1 or JQ1 in combination with BRAF inhibitor PLX8394 or ERK inhibitor Ulixertinib. After 48 h hours cells were collected, and cellular proliferation measured by XTT assays. FBXW7 WT cells treated with JQ1 alone was set as 100% reference. j Following the same settings as in (i), cellular proliferation was measured by XTT assays every 2 days for 8 days and Doxycycline was added to the media every 2 days

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