Fig 2: B-Raf signaling, which is activated by MDM2, can promote glioma xenograft tumor progression. (A) Representative images of tumors of NOD/SCID mice bearing U251 ×enografts stably expressing empty vector, MDM2, MDM2 together with 14-3-3 or MDM2 together with an ERK inhibitor. Tumors isolated on day 28 after subcutaneous injection. Ctrl referred to tumors, which were injected with the stable overexpression of plenti-CMV-puro empty vector U251 cell line. Data are from one representative animal of 5–6 animals from each group. (B) Tumor weight from experiment in (A) upon autopsy at day 28 post injection. Ctrl referred to tumors, which were injected with the stable overexpression of plenti-CMV-puro empty vector U251 cell line. Results are presented as the mean weight (g) ± SD for 5–6 mice per group. (C) Tumor volume of xenografts formed after subcutaneous injection of NOD/SCID mice with U251 cells stably expressing indicated plasmids. Ctrl referred to tumors, which were injected with the stable overexpression of plenti-CMV-puro empty vector U251 cell line. Results are presented as the mean volume ± SD for 5–6 mice per group per time point. (D) Immunohistochemical staining of E-cadherin and N-cadherin in indicated U251 ×enograft tumor genotypes (magnification, ×400). Data are from 1 animal that was representative of the 5–6 animals from each group (n=5-6 mice per group). Ctrl referred to tumors, which were injected with the stable overexpression of plenti-CMV-puro empty vector U251 cell line. For all quantifications, data are presented as the mean ± SD derived from three independent experiments. Comparisons were made using Student's t-test. ***P<0.001; n.s., not significant. 14-3-3, tyrosine 3-monooxygenase activation protein ε; Ctrl, control; GFP, green fluorescent protein; MDM2, MDM2 proto-oncogene, E3 ubiquitin protein ligase.

Fig 3: Significant association between RIT2 and PD, and the effect of dabrafenib on RIT2-knockout (KO) cells(A) RT-PCR data for the RIT2, BRAF and TH expression in the mouse substantia nigra. (B) Regional association plot of the RIT2 locus. (C) Immunoblot of phosphorylated ERK and Rit2. Phospho-ERK was reduced in RIT2-KO (clone 42) compared to control cells (clone 7), and overexpression of RIT2 in RIT2-KO cells rescued the phosphorylation of ERK. (Upper) representative immunoblots and (bottom) summarized data of phospho-ERK/total ERK levels in each cell line. (D) Dabrafenib upregulated the expression of phospho-ERK in RIT2-KO cells (clone 42). (Left) representative immunoblots and (right) summarized data of phospho-ERK/total ERK levels in each cell line. Data in (C) and (D) represent the mean ± s.e.m. (*, P-value < 0.05 and **, P-value < 0.01; one-way ANOVA with the Tukey post-hoc test; n = 3 per group).

Fig 4: Chemical structures of BRAF inhibitors and HDAC inhibitors and the pharmacophore hybrid strategy for BRAF/HDAC inhibitors.

Fig 5: Cells expressing BRAF WT are more sensitive to nutrient availability than cells expressing BRAF V600E. (A) Schematics of Seahorse metabolic flux analysis for both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The poisons used (10 µM each) are oligomycin (oligo), (4-(trifluoromethoxy) phenyl) carbonohydrazonoyl dicyanide (FCCP), antimycin (ant), and rotenone (rot). In OCR (left), red represents non-mitochondrial respiration, teal represents basal respiration, purple represents ATP-linked production, yellow represents proton leak, green represents maximal respiratory capacity, and the reserve respiratory capacity is calculated from [maximal respiratory capacity] − [basal respiration]. In ECAR (right), red represents non-glycolytic acidification, teal represents glycolysis, green represents glycolytic capacity, and the reserve glycolytic capacity is calculated from [glycolytic capacity] − [glycolysis]. OCR and ECAR were measured one hour after BRAF WT (blue lines), BRAF V600E (teal lines), control (green lines), and parental (red lines) cells were cultured in media supplemented with either (B,F) 25 mM glucose, (C,G) 25 µM palmitic acid, (D,H) 25 µM oleic acid, or (E,I) 25 µM α-linolenic acid. The ANOVA statistical test with post-hoc analysis was performed where * p < 0.05, ** p < 0.005, **** p < 0.00005 indicates the level of statistical significance.