Volume 59, Issue 3, Pages 345-358 (August 2015) Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF- MEK1 Signaling  Hee-Bum Kang, Jun Fan, Ruiting Lin, Shannon Elf, Quanjiang Ji, Liang Zhao, Lingtao Jin, Jae Ho Seo, Changliang Shan, Jack L. Arbiser, Cynthia Cohen, Daniel Brat, Henry M. Miziorko, Eunhee Kim, Omar Abdel-Wahab, Taha Merghoub, Stefan Fröhling, Claudia Scholl, Pablo Tamayo, David A. Barbie, Lu Zhou, Brian P. Pollack, Kevin Fisher, Ragini R. Kudchadkar, David H. Lawson, Gabriel Sica, Michael Rossi, Sagar Lonial, Hanna J. Khoury, Fadlo R. Khuri, Benjamin H. Lee, Titus J. Boggon, Chuan He, Sumin Kang, Jing Chen  Molecular Cell  Volume 59, Issue 3, Pages 345-358 (August 2015) DOI: 10.1016/j.molcel.2015.05.037 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 “Metabolism-Targeted” RNAi Screens Identify HMGCL as a Synthetic Lethal Partner of BRAF V600E (A) Construction of a shRNA library systematically targeting human genes related to metabolism. (B) Primary and secondary screening strategy. Supervised analysis of viability data (B score) identified candidate genes that, when knocked down by shRNAs, distinguish BRAF V600E human melanoma cells (BRAFM) from mutant NRas cells (NRasM) and cells expressing WT BRAF and NRas (WT). Overlapped results of indicated statistical methods identified top eight candidate genes. (C) Effect of BRAF or HMGCL KD on melanoma cell proliferation rates assessed by daily cell counting. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. Also see Figure S1 and Tables S1, S2, S3, S4, S5, S6, and S7. Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Expression of BRAF V600E Upregulates HMGCL in Cells (A) Left: RT-PCR and western blot results show increased HMGCL expression in human melanoma cells expressing BRAF V600E/D compared to cell expressing BRAF WT. Right: Western blot results of HMGCL expression in Mel-ST cells with FLAG-BRAF WT or V600E. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. (B) HMGCL IHC. Left: Positive staining of HMGCL was determined by histochemical score (H score = 3 × percentage of strong staining + 2 × percentage of moderate staining + 1 × % of weak staining + 0 × % of no staining; score range 0–300). Representative IHC staining images for 0 (WT; no staining), 1+ (WT; weak staining), 2+ (V600E; moderate staining), and 3+ (V600E; strong staining) scores of human melanoma tissue samples are shown (20×). Right: H scores are presented by box-and-whisker plots. Medians, interquartile, maximum, and minimum are shown. (C–E) Western blot and RT-PCR results show increased HMGCL expression with increased MEK1 and ERK phosphorylation in human primary melanoma (C) and HCL tissue samples ([D] and [E]). PB: peripheral blood; BM: bone marrow. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. Also see Figure S2. Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 HMGCL Promotes MEK-ERK Activation and Is Specifically Required for Cell Proliferation and Tumor Growth Potential of BRAF V600E-Expressing Melanoma Cells (A) Anchorage-independent growth of melanoma cells with or without stable KD of BRAF or HMGCL. Duplicate experiment; data are mean ± SEM; p values were obtained by a two-tailed Student’s t test. (B and C) Left two panels: Tumor growth and size of xenograft mice injected with parental or HMGCL KD BRAF V600E-positive A375 cells (B) or HMGCL KD cells with rescue expression of FLAG-HMGCL WT or enzyme deficient R41M mutant (C). Middle panels show the dissected tumors in representative mice. Right panels show representative images of IHC staining of Ki-67 of tumors (brown color). Data are mean ± SEM; p values were obtained by a paired two-tailed Student’s t test. (D) Effect of HMGCL KD on cell proliferation rates of Mel-ST cells expressing BRAF WT or V600E by daily cell counting. Right: Immunoblotting of HMGCL upon shRNA-mediated KD. FLAG-BRAF WT and V600E expression in Mel-ST cells is shown in Figure 2A on the right. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. (E) Immunoblotting of phosphorylation levels of MEK1, ERK1/2, AKT, and AMPK in Mel-ST cells expressing BRAF WT or V600E upon HMGCL KD. (F) Effect of HMGCL KD on phosphorylation levels of MEK1 and ERK1/2 in melanoma cell lines. (G) Effect of rescue expression of HMGCL WT or R41M on phosphorylation levels of MEK1 and ERK1/2 in melanoma cell lines with HMGCL KD. (H) Left: Immunoblotting of expression of GST-tagged MEK1 CA or DN forms, as well as phosphorylation levels of ERK1/2 in BRAF V600E-expressing A375 cells. Right: Effect of expressing MEK1 CA or DN forms on cell proliferation of parental or HMGCL KD A375 cells. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. Also see Figures S2–S4. Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 HMGCL’s Product AA Specifically Promotes MEK-ERK Activation in BRAF V600E-Expressing Cells (A) Intracellular concentration of AA in melanoma cells with HMGCL KD. (B) Cell permeability of AA (upper) or 3-HB (lower) was examined by increased scintillation counting of 14C in the cell lysates using human melanoma cells cultured in the presence of 14C-labeled AA or 3-HB for 12 hr. (C) Effect of adding increasing concentrations of AA on A375 (upper) or HMCB (lower) cells in culture media on reduced intracellular levels of AA upon HMGCL KD. (D–F) Effect of adding AA or 3-HB in culture media on cell proliferation rates of melanoma cell lines (D), HMGCL R41M rescue A375 cells (E), and Mel-ST cells expressing BRAF WT or V600E (F). Cell proliferation rates were determined by daily cell counting. (G) Effect of adding AA in culture media on phosphorylation levels of MEK1 and ERK1/2 in melanoma cell lines and cells with HMGCL KD. (H) Intracellular AA levels (left panels) and immunoblotting results detecting MEK1, ERK1/2, and HMGCL protein levels as well as phosphorylation levels of MEK1 and ERK1/2 (right panels) using tumor lysates are shown. The tumors were from xenograft nude mice presented in Figures 3B, 3C, and S2D. Data with error bars in Figure 4 are represented as mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. Also see Figures S4 and S5. Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 AA Selectively Enhances BRAF V600E-MEK1 Binding (A) Effect of AA on phosphorylation of MBP in an in vitro kinase assay using purified recombinant BRAF V600E or BRAF WT incubated with purified MBP as a substrate. (B and C) Effect of adding cell-permeable AA in culture media on BRAF-MEK1 binding and MEK1 phosphorylation in melanoma cells (B) and cells with HMGCL KD (C). IP: immunoprecipitates. (D) Effect of AA on BRAF-MEK1 binding and MEK1 phosphorylation in cell-free in vitro assays using purified recombinant BRAF (rBRAF) and MEK1 (rMEK1). (E) Effect of pre-treatment of rBRAF V600E (left) or rMEK1 (right) with increasing concentrations of AA on BRAF-MEK1 binding and MEK1 phosphorylation in cell-free in vitro assays. (F) Thermal melt shift assay was performed to examine the protein (BRAF WT or V600E) and ligand (AA) interaction. Change of melting temperature (Tm) in a dose-dependent manner at concentrations from 0 μM to 400 μM demonstrates that AA may directly bind to BRAF V600E but not BRAF WT protein. Arrows in each panel indicate melting temperatures at 0 μM (left) and 300 μM (right), since 300 μM represents the physiological AA level in BRAF V600E-expressing human melanoma cells. (G) Radiometric metabolite-protein interaction analysis using 14C-labeled AA incubated with purified BRAF variants. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. (H) Vmax and Km of BRAF V600E were measured using purified BRAF V600E protein (100 ng) incubated with increasing concentrations of ATP in the presence and absence of increasing concentration of AA, using excessive amount of purified MEK1 (left) or MBP (right) as substrates. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. Also see Figures S5 and S6. Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 BRAF V600E Upregulates HMGCL Expression through an Octamer Transcription Factor Oct-1 (A) Western blot to detect HMGCL protein levels in human melanoma cells with stable KD of BRAF WT or V600E/D mutant (upper) or treatment with BRAF V600E inhibitor PLX-4032 (lower). (B) RT-PCR to examine the effect of treatment with MEK1 inhibitor U0126 on HMGCL mRNA levels in PMWK (BRAF WT) and A2058 (V600E) melanoma cells. (C) Luciferase reporter assay revealed a functional HMGCL promoter region (−929 to −665). (D) ChIP results detecting binding ability of a group of transcription factors to the functional region of HMGCL promoter. Positive binding of Oct-1 and IKAROS is indicated with “+” in red color. (E and F) ChIP results detecting binding ability of Oct-1 or IKAROS to HMGCL promoter region in melanoma cells with BRAF KD (E) or treatment with BRAF V600E inhibitor PLX-4032 (F). (G and H) Effect of Oct-1 KD on HMGCL expression and intracellular AA levels (G) and phosphorylation levels of MEK1 and ERK1/2 (H). (I–K) Effect of adding AA in culture on phosphorylation levels of MEK1 and ERK1/2 (I), phosphorylation of MEK1 and BRAF-MEK1 association (J), and cell proliferation (K) in melanoma cells with Oct-1 KD. Data with error bars in Figure 6 are represented as mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. Also see Figure S7. Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Active BRAF Signals through Oct-1 to Upregulate HMGCL and Its Product AA, which, however, Selectively Enhances BRAF V600E-Dependent Activation of MEK1 (A) Results of an in vitro kinase assay using purified recombinant BRAF (rBRAF) WT, V600E, or a truncated form of BRAF (tBRAF) that is constitutively active (CA) incubated with recombinant MEK1 (rMEK1) as exogenous substrate. (B–E) Effects of stable expression of BRAF variants in Mel-ST cells on cell proliferation (B), HMGCL expression and phosphorylation of MEK1 and ERK1/2 (C), Oct-1 binding ability of promoter region of HMGCL (D), and intracellular AA levels (E). ED: enzyme dead. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. (F and G) Effects of adding AA in culture media on protein amount and phosphorylation of MEK1 bound to BRAF (F) and cell proliferation (G) in Mel-ST cells expressing BRAF WT, or CA V600E or tBRAF. Data are mean ± SD; n = 3 each; p values were obtained by a two-tailed Student’s t test. (H) Proposed working model: CA, oncogenic BRAF V600E activates Oct-1 to upregulate HMGCL (reprogramming), leading to increased levels of AA that specifically binds to BRAF V600E and promotes BRAF V600E-MEK1 binding (rewiring). Molecular Cell 2015 59, 345-358DOI: (10.1016/j.molcel.2015.05.037) Copyright © 2015 Elsevier Inc. Terms and Conditions