metabolic rewiring by oncogenic braf v600e links ketogenesis pathway to braf-mek1 signaling

Article

Metabolic Rewiring by Onc
ogenic BRAF V600E LinksKetogenesis Pathway to BRAF-MEK1 Signaling
Graphical Abstract

Highlights

d HMGCL is a synthetic lethal partner of BRAF V600E

d BRAF V600E upregulates HMGCL in human cancers

d HMGCL product acetoacetate selectively promotes BRAF

V600E-MEK1 binding

d Active BRAF upregulates HMGCL via Oct-1

Kang et al., 2015, Molecular Cell 59, 1–14August 6, 2015 ª2015 Elsevier Inc.http://dx.doi.org/10.1016/j.molcel.2015.05.037

Authors

Hee-Bum Kang, Jun Fan,

Ruiting Lin, ..., Chuan He, Sumin Kang,

Jing Chen

[email protected] (S.K.),[email protected] (J.C.)

In Brief

Many cancers share common metabolic

alterations, yet how such alterations

contribute to tumor development remains

unclear. Kang et al. demonstrate a

‘‘synthetic lethal’’ interaction between

oncogenic BRAF V600E and a ketogenic

enzyme 3-hydroxy-3-methylglutaryl-CoA

lyase (HMGCL) that promotes BRAF

V600E-dependent tumor development.

mailto:[email protected]


http://dx.doi.org/10.1016/j.molcel.2015.05.037

Please cite this article in press as: Kang et al., Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF-MEK1 Signaling,Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.05.037

Molecular Cell

Article

Metabolic Rewiring by Oncogenic BRAF V600ELinks Ketogenesis Pathway to BRAF-MEK1 SignalingHee-Bum Kang,1,14 Jun Fan,1,14 Ruiting Lin,1,14 Shannon Elf,1 Quanjiang Ji,2 Liang Zhao,1 Lingtao Jin,1 Jae Ho Seo,1

Changliang Shan,1 Jack L. Arbiser,3,4 Cynthia Cohen,5 Daniel Brat,5 Henry M. Miziorko,6 Eunhee Kim,7

Omar Abdel-Wahab,7 Taha Merghoub,7 Stefan Frohling,8 Claudia Scholl,9 Pablo Tamayo,10 David A. Barbie,10 Lu Zhou,11

Brian P. Pollack,3,4 Kevin Fisher,5 Ragini R. Kudchadkar,1 David H. Lawson,1 Gabriel Sica,1 Michael Rossi,1 Sagar Lonial,1

Hanna J. Khoury,1 Fadlo R. Khuri,1 Benjamin H. Lee,12 Titus J. Boggon,13 Chuan He,2 Sumin Kang,1,* and Jing Chen1,*1Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory, Emory University School of Medicine, Atlanta, GA

30322, USA2Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA3Department of Dermatology, Emory University, Atlanta, GA 30322, USA4Atlanta Veterans Administration Medical Center, Atlanta, GA 30322, USA5Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA6Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City, Kansas City, MO 64110, USA7Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA8Department of Translational Oncology, National Center for Tumor Diseases (NCT) Heidelberg and German Cancer Research Center (DKFZ),

Section for Personalized Oncology, Heidelberg University Hospital, German Cancer Consortium (DKTK), 69121 Heidelberg, Germany9Department of Translational Oncology, National Center for Tumor Diseases (NCT) Heidelberg, German Cancer Research Center (DKFZ),

69121 Heidelberg, Germany10Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA11School of Pharmacy, Fudan University, Shanghai 201203, China12Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA13Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA14Co-first author

*Correspondence: [email protected] (S.K.), [email protected] (J.C.)http://dx.doi.org/10.1016/j.molcel.2015.05.037

SUMMARY

Many human cancers share similar metabolic alter-ations, including the Warburg effect. However, it re-mains unclear whether oncogene-specific metabolicalterations are required for tumor development. Herewe demonstrate a ‘‘synthetic lethal’’ interactionbetween oncogenic BRAF V600E and a ketogenicenzyme 3-hydroxy-3-methylglutaryl-CoA lyase(HMGCL). HMGCL expression is upregulated inBRAF V600E-expressing human primary melanomaand hairy cell leukemia cells. Suppression of HMGCLspecifically attenuates proliferation and tumorgrowth potential of human melanoma cells express-ing BRAF V600E. Mechanistically, active BRAF upre-gulates HMGCL through an octamer transcriptionfactor Oct-1, leading to increased intracellular levelsof HMGCL product, acetoacetate, which selectivelyenhances binding of BRAF V600E but not BRAFwild-type to MEK1 in V600E-positive cancer cells topromote activation of MEK-ERK signaling. Thesefindings reveal a mutation-specific mechanism bywhich oncogenic BRAF V600E ‘‘rewires’’ metabolicand cell signaling networks and signals through theOct-1-HMGCL-acetoacetate axis to selectively pro-mote BRAF V600E-dependent tumor development.

INTRODUCTION

The importance of metabolic alterations in cancer has been

increasingly recognized over the past decade. Identification of

metabolic vulnerability of human cancers has informed develop-

ment of therapeutic strategies to treat cancer. However,

although increasing evidence emerges and suggests that

different human cancers may share common metabolic proper-

ties such as the Warburg effect, it remains unclear whether

distinct oncogenic backgrounds in different cancer types require

different metabolic properties for tumor development.

Melanoma is one of the most common human cancers, which,

according to American Cancer Society, accounts for >76,600

cases in US in 2013 with �9,000 death each year. More than

50% of melanomas express BRAF V600E mutant, which repre-

sents a therapeutic target due to its pathogenic role. However,

despite the success of BRAF mutant and MEK inhibitors in clin-

ical trials for BRAF V600E-positive melanoma patients, clinical

resistance invariably develops (Bollag et al., 2012; Gibney

et al., 2013; Johnson and Sosman, 2013). Thus, identification

of alternative ‘‘targets’’ in BRAF V600E-positive melanomas

may inform effective long-term treatment strategies.

Herein we approached this question by identifying ‘‘metabolic

vulnerabilities’’ specifically required by oncogenic BRAF V600E

mutant, but not other oncogenes such as NRas Q61R/K in hu-

man melanomas. We found that HMG-CoA lyase (HMGCL), a

key enzyme in ketogenesis producing ketone bodies, was

Molecular Cell 59, 1–14, August 6, 2015 ª2015 Elsevier Inc. 1





selectively essential in melanoma cells expressing BRAF V600E,

but not in control cells containing NRas mutants or wild-type

(WT) BRAF and NRas. Ketogenesis mainly occurs in the mito-

chondria of liver cells, which normally produces ketone bodies

as a result of fatty acid breakdown to generate energy when

glucose levels in the blood are low (Balasse and Fery, 1989;

McPherson and McEneny, 2012). b-oxidation breaks down fatty

acids to form acetyl-CoA, which, under normal conditions, is

further oxidized in the TCA cycle. However, if TCA cycle activity

is low, or the acetyl-CoA generation rate of b-oxidation exceeds

the capacity of the TCA cycle, ketogenesis will be activated to

convert acetyl-CoA to ketone bodies via HMG-CoA. HMGCL

converts HMG-CoA to acetyl-coA and a ketone body, acetoace-

tate (AA), which can be further converted to two other ketone

bodies, including D-b-hydroxybutyrate (3-HB) and acetone. Ke-

tone bodies can be transported from liver to other tissues, where

AA and 3-HB but not acetone will be further oxidized via the TCA

cycle to produce acetyl-CoA for energy production. Organs

including heart and brain can use AA and 3-HB for energy. AA,

if not used for energy, will be decarboxylated to acetone, which

is removed as waste (Cotter et al., 2013; Morris, 2005). However,

although the ketogenic diet (high fat, adequate protein, and low

carbohydrate) has been evaluated for cancer prevention and

treatment purposes with the hope of attenuating tumor develop-

ment by limiting carbohydrate supply, it is unknown whether and

how ketogenesis and/or ketone bodies may contribute to cancer

metabolism and tumor growth.

Here we report that active BRAF upregulates HMGCL via an

octamer transcription factor Oct-1. Consistently, BRAF V600E

expression results in increased HMGCL gene expression in

cancer cells. HMGCL, however, selectively promotes BRAF

V600E-dependent phosphorylation and activation of MEK1 by

controlling intracellular levels of its product AA, which specif-

ically promotes BRAF V600E (but not BRAF WT) binding to

MEK1 and subsequent MEK1 phosphorylation in cancer cells.

RESULTS

HMGCL Is a ‘‘Synthetic Lethal’’ Partner of BRAF V600Ein Human Melanoma CellsTo identify ‘‘metabolic vulnerabilities’’ specific to oncogenic

BRAF V600E mutant, but not other oncogenes such as NRas

Q61R/K in human melanomas, we designed and constructed a

shRNA library that targets 1,361 out of 1,417 genes encoding

known metabolism-related enzymes and protein factors in

the human genome (search at http://www.phosphosite.org/

psrSearchAction.do by selecting ‘‘containing ‘metabolism’ ’’ in

‘‘Protein type’’ section), which are available in thewhole-genome

shRNA library that we purchased from OpenBioSystems. The li-

brary contains 6,872 lentiviral-based shRNA constructs, where

each gene is individually targeted by 1-5 different shRNA con-

structs that target different regions of the target gene (Figure 1A;

Table S1). We performed a systematic RNAi screen in two BRAF

V600D/E mutant expressing melanoma cells lines including

WM2664 (V600D) and A375 (V600E), as well as control BRAF

WT-expressing melanoma cells including PMWK, CHL-1,

HMCB (NRas Q61K) and SK-MEL-2 (NRas Q61R) (Figure 1B;

Table S2). We then used two RIGER methods (RIGER_SB

2 Molecular Cell 59, 1–14, August 6, 2015 ª2015 Elsevier Inc.

and RIGER_KS) and overlapped these against another

method, Gene Set Analysis R package, to analyze the normal-

ized B scores for each cell line (Barbie et al., 2009; Gould

et al., 2006; Malo et al., 2006; Sims et al., 2011). The top-ranked

100 genes identified by each method were overlapped, and 36

genes were enriched as top candidate synthetic lethal partners

of BRAF V600E (Figure 1B; Tables S3, S4, S5, and S6). In a sec-

ondary screen, we validated the 36 candidates (186 shRNAs)

using additional BRAF V600E-expressing SK-MEL-5 and

A2058melanoma cells, compared to control BRAFWT-express-

ing PMWK and HMCB cells (Figure 1B). Results were analyzed

by RIGER_SB, RIGER_KS, and Gene Set Analysis R package,

and eight genes were enriched using the top 15 genes identified

in the primary screen (Figure 1B; Table S7).

Among these candidates, we validated the two key ketogenic

enzymes, HMG-CoA lyase (HMGCL) (Figures 1C, S1A, and

S1B) and HMG-CoA synthase 1 (HMGCS1) (Figure S1C) as

synthetic lethal partners of BRAF V600E, using an alternative

cell-number-based cell proliferation rate assay. Suppression

of HMGCL and HMGCS1 resulted in more attenuated cell pro-

liferation rates in BRAF V600E-expressing melanoma cells

compared to cells expressing BRAF WT, suggesting selective

importance of ketogenesis in BRAF V600E induced melanoma

transformation.

Expression of BRAF V600E Upregulates HMGCL in CellsKetogenesis mainly occurs in the mitochondria of liver cells,

which normally convert acetyl-CoA to ketone bodies via HMG-

CoA as a result of fatty acid breakdown to generate energy

when glucose levels in the blood are low. HMGCL converts

HMG-CoA to acetyl-coA and a ketone body, AA, which can be

further converted to two other ketone bodies, including 3-HB

and acetone (Balasse and Fery, 1989;McPherson andMcEneny,

2012). We found that HMGCL is upregulated in a group of BRAF

V600E-expressing humanmelanoma cell lines compared to cells

expressing BRAF WT (Figure 2A, left) and in the immortalized

melanocyte Mel-ST cells expressing BRAF V600E but not

BRAF WT (Figure 2A, right).

Consistent with these findings, HMGCL expression assessed

by immunohistochemistry (IHC) staining is significantly upregu-

lated in primary tumor samples of BRAF V600E-positive mela-

noma patients compared to control tumor tissue samples from

BRAF WT-expressing melanoma patients (Figure 2B). In addi-

tion, immunoblotting results confirmed upregulated HMGCL

protein andmRNA levels in primary human tumor tissue samples

from BRAF V600E-positive melanoma patients, which also

demonstrated enhanced phosphorylation and activation of

MEK1 and ERK, compared to primary melanoma sample from

one representative patient with BRAF WT (Figure 2C).

BRAF V600E mutation has been identified in other human ma-

lignancies such as colorectal cancer, multiple myeloma (Benl-

loch et al., 2006; Chapman et al., 2011), and hairy cell leukemia

(HCL). HCL is a chronic B cell lymphoproliferative disease, and

nearly 100% of classic HCL patients harbor somatic BRAF

V600E mutation (Arcaini et al., 2012; Golomb et al., 1982;

Schnittger et al., 2012; Tiacci et al., 2011). Consistently, protein

and mRNA levels of HMGCL and phosphorylation of MEK1 and

ERK were upregulated in primary human leukemia cells from

http://www.phosphosite.org/psrSearchAction.do


https://www.researchgate.net/publication/7864112_Cerebral_ketone_body_metabolism_J_Inherit_Metabol_Dis?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

https://www.researchgate.net/publication/235441882_Ketone_body_metabolism_and_cardiovascular_disease?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

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 onmelanoma 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.


HCL patients compared to control peripheral blood samples

from healthy donors (Figures 2D and 2E).

Furthermore, HMGCL protein levels were increased in tumor

and bone marrow samples of BRAF V600E conditional knockin

mousemodels ofmelanoma (Dankort et al., 2009) or HCL (Chung

et al., 2014) compared to control skin or bone marrow samples

from normal mice, respectively (Figure S2A). These results

together suggest that HMGCL is upregulated in BRAF V600E-

positive human malignancies.

HMGCL Is Required for BRAF V600E-InducedTransformationWenext examined whether HMGCL is required for BRAF V600E-

induced transformation.We found that stable knockdown (KD) of

HMGCL or BRAF resulted in attenuated colony formation poten-

tial of BRAF V600E-expressing A2058 and A375 cells but not

control PMWK cells (Figure 3A). Moreover, in a xenograft nude

mouse model, tumors derived from HMGCL KD A375 cells

demonstrated decreased growth rate and masses with

decreased cell proliferation rate assessed by reduced IHC stain-

ing of Ki-67, compared to those derived from control A375 cells

(Figure 3B). Similar results were obtained using A2058 cells,

whereas tumors derived from BRAF WT-expressing PMWK

and PMWK HMGCL KD cells, or HMCB and HMCB HMGCL

KD cells, in xenograft mice were indistinguishable (Figures

S2B–S2D).

We next generated ‘‘rescue’’ A375 cell lines with stable KD of

endogenous HMGCL and rescue expression of WT or an

enzyme-dead (ED) R41M mutant (Fu et al., 2010) of an shRNA-

resistant, FLAG-tagged human HMGCL form. Rescue expres-

sion of FLAG-HMGCL WT, but not R41M mutant, significantly

reversed the reduced cell proliferation upon HMGCL KD in

A375 cells but not control PMWK cells (Figures S3A and S3B).

Moreover, rescue A375 cells expressing HMGCL WT but not

R41M demonstrated restored potential of tumor formation and

growth (Figure 3C). These data together suggest that the syn-

thetic lethal interaction between BRAF V600E and HMGCL re-

quires HMGCL activity.

HMGCL Selectively Enhances Activation of MEK-ERKin BRAF V600E-Expressing CellsWe next sought to explore the underlying molecular mechanism.

Although HMGCL KD did not selectively confer any metabolic

alteration to BRAF V600E-expressing melanoma cells (Figures


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 3 percentage of strong staining + 2 3 percentage of

moderate staining + 13%of weak staining + 03%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 (203). 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 increasedMEK1 and ERK phosphorylation in human primary melanoma (C) and

HCL tissue samples ([D] and [E]). PB: peripheral blood; BM: bonemarrow. Data aremean ± SD; n = 3 each; p valueswere obtained by a two-tailed Student’s t test.

Also see Figure S2.



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


(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.

(legend continued on next page)




S3C–S3H), silencing HMGCL resulted in significantly decreased

cell proliferation of Mel-ST cells expressing BRAF V600E but not

BRAF WT (Figure 3D) with decreased phosphorylation of MEK1

and ERK1/2 but not AKT and AMPK (Figure 3E). These results

suggest not only that BRAF V600E conferred HMGCL reliance

to Mel-ST cells but also that HMGCL is specifically involved in

BRAF V600E-dependent activation of MEK-ERK signaling.

Furthermore, we found that HMGCL KD selectively attenuated

phosphorylation of MEK1 and ERK1/2 only in BRAF V600E-ex-

pressing melanoma cells (Figure 3F), while rescue expression

of HMGCL WT, but not R41M mutant, reversed the decreased

phosphorylation of MEK1 and ERK1/2 in A375 HMGCL KD cells

(Figure 3G). Consistently, expressing a constitutively active (CA)

S222D (Mansour et al., 1994) form of MEK1, but not a catalyti-

cally inactive dominant negative (DN) K97M mutant of MEK1

(Mansour et al., 1994), reversed the reduced cell proliferation in

A375 HMGCL KD cells but did not affect control A375 cells

(Figure 3H). Similar results were obtained using A2058 cells

(Figure S3I).

HMGCLSignals through Its Product AA toPromoteBRAFV600E Activated MEK-ERK Signaling CascadeHMGCL KD resulted in decreased intracellular concentration of

its product, AA, in melanoma cells (Figure 4A). Note that the

intracellular levels of AA are lower in melanoma cells expressing

BRAF WT compared to BRAF V600E-expressing cells (Fig-

ure 4A), which correlates with the differential expression levels

of HMGCL in these cells (Figure 2A). We next tested whether

AA mediates the synthetic lethal importance of HMGCL only in

BRAF V600E-expressing melanoma cells. Consistent with litera-

ture, both AA and its subsequent ketone body 3-HB can readily

enter cells (Patel et al., 1981) (Figures 4B and S4A), and we also

found that addition of AA up to 10 mM in the culture media even-

tually significantly reversed the decreased intracellular AA levels

in HMGCL KD cells (Figures 4C and S4B).

Interestingly, addition of AA but not 3-HB in the culture media

selectively promoted cell proliferation of BRAF V600E-express-

ing A375 and A2058 cells, but not control PMWK and HMCB

cells (Figures 4D, S4C, and S4D), and significantly reversed the

reduced cell proliferation of A375 and A2058 rescue cells with

HMGCL KD (Figure S4D) or cells expressing HMGCL ED mutant

R41M (Figures 4E and S4E). In addition, AA treatment only pro-

moted proliferation of Mel-ST cells expressing BRAF V600E

(Figure 4F).

AA treatment reversed the decreased phosphorylation of

MEK1 and ERK1/2 due to HMGCL KD only in BRAF V600E-ex-

pressing cells (Figure 4G), whereas 3-HB treatment had no effect

on MEK1 and ERK phosphorylation (Figure S5A). Moreover,

(D) Effect of HMGCL KD on cell proliferation rates of Mel-ST cells expressing BRA

shRNA-mediated KD. FLAG-BRAF WT and V600E expression in Mel-ST cells is s


(E) Immunoblotting of phosphorylation levels of MEK1, ERK1/2, AKT, and AMPK

(F) Effect of HMGCL KD on phosphorylation levels of MEK1 and ERK1/2 in mela

(G) Effect of rescue expression of HMGCL WT or R41M on phosphorylation leve

(H) Left: Immunoblotting of expression of GST-taggedMEK1 CA or DN forms, as w

Right: Effect of expressing MEK1 CA or DN forms on cell proliferation of parent


Also see Figures S2–S4.


despite the fact that AA levels were commonly decreased in tu-

mors derived from HMGCL KD melanoma cells (Figures 4H and

S5B, left panels), we found decreased phosphorylation levels of

MEK1 and ERK1/2 only in tumors derived from A2058 and A375

HMGCL KD cells and A375 rescue cells expressing R41M

mutant, but not in tumors derived from control PMWK and

HMCB HMGCL KD cells (Figures 4H and S5B, right panels).

To examine the clinical impact of our findings, we measured

the intracellular levels of ketone bodies in human primary tissues

from HCL patients. We observed increased intracellular levels of

ketone bodies including AA and 3-HB in primary human leukemia

cells from HCL patients compared to control peripheral blood

samples from a healthy donor (Figure S5C, left two panels),

which is consistent with the fact that HCL cells are BRAF

V600E positive with upregulated HMGCL. In addition, increased

AA levels were also detected in tumor and bonemarrow samples

from BRAF V600E conditional knockin mouse models of mela-

noma or HCL compared to control skin or bone marrow samples

from normal mice, respectively (Figure S5C, right two panels).

These findings are consistent with the results showing increased

HMGCL expression in primary leukemia cells from HCL patients

(Figures 2D and 2E) and tumors expressing BRAF V600E

(Figure S2A).

AA Selectively Enhances BRAF V600E-MEK1 BindingWe next tested the hypothesis that AA might affect BRAF V600E

kinase activity. We thus performed cell-free in vitro kinase as-

says using purified recombinant BRAF V600E or BRAF WT incu-

bated with purified myelin basic protein (MBP) as a non-specific

substrate or purified MEK1 in the presence of increasing

amounts of AA. We found that not BRAF WT but BRAF V600E-

dependent phosphorylation of MEK1 was increased in the pres-

ence of AA (Figure S5D). Although both BRAF V600E or BRAF

WT phosphorylates MBP, such phosphorylation was not

affected in the presence of increasing concentrations of AA or

3-HB (Figures 5A, S5E, and S5F), suggesting that AAmay specif-

ically affect BRAF V600E-dependent phosphorylation of MEK1.

Further mechanistic studies revealed that AA treatment re-

sulted in increased MEK1 binding to BRAF V600E as well as

increased phosphorylation of V600E-bound MEK1 in A2058,

A375, and WM2664 cells, but not to BRAF WT in control

PMWK and HMCB cells (Figures 5B and S6A) or CRAF in

A2058 cells (Figure S6B). In contrast, 3-HB treatment did not

affect BRAF-MEK1 binding or MEK1 phosphorylation (Fig-

ure S6C). Moreover, KD of HMGCL resulted in decreased

BRAF V600E/D-MEK1 binding and MEK1 phosphorylation only

in A2058, A375, andWM2664 (V600D) cells, whichwere reversed

by treatment with AA (Figure 5C) but not 3-HB (Figure S6D).

F WT or V600E by daily cell counting. Right: Immunoblotting of HMGCL upon

hown in Figure 2A on the right. Data are mean ± SD; n = 3 each; p values were

in Mel-ST cells expressing BRAF WT or V600E upon HMGCL KD.

noma cell lines.

ls of MEK1 and ERK1/2 in melanoma cell lines with HMGCL KD.

ell as phosphorylation levels of ERK1/2 in BRAF V600E-expressing A375 cells.

al or HMGCL KD A375 cells. Data are mean ± SD; n = 3 each; p values were

https://www.researchgate.net/publication/15146105_Mansour_SJ_et_al_Transformation_of_mammalian_cells_by_constitutively_active_MAP_kinase_kinase_Science_265_966-969?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

https://www.researchgate.net/publication/15146105_Mansour_SJ_et_al_Transformation_of_mammalian_cells_by_constitutively_active_MAP_kinase_kinase_Science_265_966-969?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

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 humanmelanoma cells cultured

in the presence of 14C-labeled AA or 3-HB for 12 hr.





To determine whether AA directly or indirectly affects the

BRAF V600E-MEK1 complex, we performed cell-free in vitro

binding and kinase assays using purified recombinant BRAF

V600E or BRAF WT incubated with recombinant purified MEK1

as substrate, in the presence of increasing amounts of AA. We

found that AA treatment promoted MEK1 binding to BRAF

V600E and phosphorylation of V600E-bound MEK1 in a dose-

dependent manner, whereas such phenotypes were absent in

MEK1 incubated with BRAF WT (Figure 5D). Notably, increasing

AA concentrations from 200 mM to 300 mM caused a significant

increase in BRAF V600E-bound and phosphorylated MEK1 (Fig-

ure 5D), which were physiologically consistent with the AA levels

determined in HMGCL KD A375 and A2058 cells compared to

control cells (Figure 4A).

Moreover, we found that purified BRAF V600E pre-treated

with increasing concentrations of AA (Figure 5E, left) but not

3-HB (Figure S6E, left) showed increased binding ability to

MEK1, whereas neither AA (Figure 5E, right) nor 3-HB (Fig-

ure S6E, right) pre-treated MEK1 demonstrated increased

binding ability to BRAF V600E. These results are consistent

with our observation that AA might directly bind to BRAF

V600E but not BRAF WT in a thermal melt shift assay using

purified BRAF WT or V600E incubated with increasing concen-

trations of AA (Figure 5F). Furthermore, we performed a

radiometric metabolite-protein interaction analysis using 14C-

labeled AA incubated with purified BRAF variants. Labeled

AA specifically binds to BRAF V600E and a V600E mutant of

an active, truncated C-terminal domain of BRAF (tBRAF,

416–766 aa) (Brummer et al., 2006), but not to control proteins

including BRAF WT, tBRAF WT, or a N-terminal domain of

BRAF (BRAF-N, 1–415 aa), which further suggest the direct as-

sociation between AA and V600E mutant of BRAF (Figure 5G).

Consistently, treatment with 300 or 400 mM of AA resulted in

increased Vmax and slightly decreased Km of BRAF V600E us-

ing MEK1 as a substrate, whereas treatment with lower con-

centrations (0, 100, and 200 mM) of AA did not affect Vmax

or Km of V600E (Figure 5H, left panels). In contrast, treatment

with AA did not affect Vmax or Km of BRAF V600E using MBP

as a substrate (Figure 5H, right panels). Together, these data

suggest an important role of AA in BRAF V600E-dependent

transformation.

Active BRAF Signals through Oct-1 to UpregulateHMGCL and Its Product AA, which, however, SelectivelyEnhances BRAF V600E-Dependent Activation of MEK1We found that stable KD of BRAF V600E but not BRAF WT in

melanoma cells resulted in decreased HMGCL protein (Fig-

ure 6A, upper) and mRNA (Figure S7A, upper) levels. Similar

results were obtained using treatment with BRAF mutant small

(C) Effect of adding increasing concentrations of AA on A375 (upper) or HMCB (low

(D–F) Effect of adding AA or 3-HB in culture media on cell proliferation rates of m

expressing BRAF WT or V600E (F). Cell proliferation rates were determined by d

(G) Effect of adding AA in culture media on phosphorylation levels of MEK1 and

(H) Intracellular AA levels (left panels) and immunoblotting results detecting MEK1

and ERK1/2 (right panels) using tumor lysates are shown. The tumorswere from xe

Figure 4 are represented as mean ± SD; n = 3 each; p values were obtained by a

Also see Figures S4 and S5.


molecule inhibitor PLX-4032 (Figures 6A and S7A, lower

panels), whereas treatment with MEK1 inhibitors U0126

(Figure 6B), selumatinib, and trametinib (Figure S7B) did not

affect HMGCL mRNA levels in PMWK, A375, and A2058 cells.

These results suggest that active BRAF V600E but not its

downstream MEK1-ERK signaling is involved in HMGCL

upregulation.

To decipher how BRAF V600E upregulates HMGCL in cells in-

dependent of MEK1, using a series of luciferase reporter con-

structs, we identified �929 to �665 as the functional region of

HMGCLpromoter (Figure 6C). Sequence analysis by TFSEARCH

(https://archive.is/ANbf4) revealed seven potential transcription

factors that may bind directly to �929 to �665 and modulate

HMGCL promoter activity. Among these factors, ChIP assay

suggested that both Oct-1 and IKAROS could bind to HMGCL

promoter (Figure 6D). Further experiments revealed that KD of

BRAF V600E in A2058 cells resulted in decreased binding of

Oct-1 but not IKAROS to HMGCL �929 to �665 promoter re-

gion, whereas KD of BRAF WT in PMWK cells did not affect

either Oct-1 or IKAROS binding to this region (Figure 6E). Similar

results were obtained using A375 and HMCB cells with BRAF KD

(Figure S7C), or those cells treated with PLX-4032 (Figures 6F

and S7D).

Consistent with these findings, we found that Oct-1 KD re-

sulted in decreased HMGCL expression and intracellular AA

levels in BRAF V600E-expressing A2058 and A375 cells but

not in control PMWK and HMCB cells expressing BRAFWT (Fig-

ures 6G and S7E). Moreover, Oct-1 KD resulted in decreased

phosphorylation of MEK1 and ERK1/2 in A2058 and A375 cells

but not in control PMWK and HMCB cells (Figures 6H and

S7F), which was reversed by adding AA in culture media (Figures

6I and S7G). In addition, treatment with AA also reversed

reduced protein amount and phosphorylation of MEK1 bound

to BRAF V600E in A2058 and A375 cells with Oct-1 KD, but

not in control PMWK and HMCB cells (Figures 6J and S7H).

Consistently, AA treatment also partially reversed decreased

cell proliferation in A2058 and A375 cells with Oct-1 KD, but

not in control PMWK and HMCB cells (Figures 6K and S7I).

This suggests that Oct-1 plays an essential role in BRAF

V600E-dependent transformation, and HMGCL is one of the

important downstream effectors of Oct-1.

In order to determine whether active BRAF activates the Oct-

1-HMGCL-AA axis in cells, we included an active, N-terminally

truncated BRAF (tBRAF, 416–766 aa). We found that both

BRAF V600E and tBRAF demonstrated comparably increased

kinase activity in an in vitro kinase assay compared to BRAF

WT (Figure 7A). In addition, stable expression of BRAF V600E

and tBRAF in Mel-ST cells resulted in comparably enhanced

cell proliferation (Figure 7B), increased HMGCL expression

er) cells in culturemedia on reduced intracellular levels of AA upon HMGCLKD.

elanoma cell lines (D), HMGCL R41M rescue A375 cells (E), and Mel-ST cells

aily cell counting.

ERK1/2 in melanoma cell lines and cells with HMGCL KD.

, ERK1/2, and HMGCL protein levels as well as phosphorylation levels of MEK1

nograft nudemice presented in Figures 3B, 3C, and S2D. Datawith error bars in

two-tailed Student’s t test.

https://archive.is/ANbf4

https://www.researchgate.net/publication/7078589_Functional_analysis_of_the_regulatory_requirements_of_B-Raf_and_the_B-RafV600E_oncoprotein?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

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 andC) Effect of adding cell-permeable AA in culturemedia on BRAF-MEK1 binding andMEK1 phosphorylation inmelanoma cells (B) and cells with HMGCLKD

(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 andMEK1 phosphorylation in cell-

free in vitro assays.

(F) Thermal melt shift assay was performed to examine the protein (BRAFWT or V600E) and ligand (AA) interaction. Change of melting temperature (Tm) in a dose-

dependentmanner at concentrations from 0 mM to 400 mMdemonstrates that AAmay directly bind to BRAF V600E but not BRAFWTprotein. Arrows in each panel

indicate melting temperatures at 0 mM (left) and 300 mM (right), since 300 mM 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.


(Figure 7C) along with increased Oct-1 binding to HMGCL pro-

moter region (Figure 7D), and elevated intracellular AA levels

(Figure 7E) compared to cells expressing control BRAF WT or

an ED (K482M) (Sievert et al., 2013) form of tBRAF.

However, only expression of BRAF V600E and tBRAF but not

the EDmutant of tBRAF resulted in increased phosphorylation of

MEK1 and ERK1/2 (Figure 7C). Moreover, treatment with AA

selectively promotes BRAF V600E but not tBRAF to bind and


https://www.researchgate.net/publication/236084807_Paradoxical_activation_and_RAF_inhibitor_resistance_of_BRAF_protein_kinase_fusions_characterizing_pediatric_astrocytomas?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

Figure 6. BRAF V600E Upregulates HMGCL Expression through an Octamer Transcription Factor Oct-1

(A) Western blot to detect HMGCL protein levels in humanmelanoma cells with stable KD of BRAFWT 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).





phosphorylate MEK1 (Figure 7F), leading to increased cell prolif-

eration in Mel-ST cells (Figure 7G). These results together sug-

gest that active BRAF commonly activates Oct-1, leading to

upregulated HMGCL and consequently intracellular AA levels,

whereas AA selectively promotes BRAF V600E-dependent acti-

vation of MEK-ERK signaling cascade.

DISCUSSION

Thecurrent understandingofmetabolic alterations in humancan-

cers is befuddling. We propose to distinguish metabolic ‘‘rewir-

ing’’ and ‘‘reprogramming’’ in cancer cells, wherein metabolic re-

programming describes ‘‘software’’ changes induced by growth

factors in normal proliferative cells that are ‘‘hijacked’’ by onco-

genic signals in cancer cells, while metabolic rewiring represents

‘‘hardware’’ changes that are ‘‘forged’’ due to ‘‘neo-function’’ of

oncogenic mutants, but are not found in normal cells. Our find-

ings suggest a ‘‘mutation-specific’’ function in which BRAF

V600E upregulates HMGCL, leading to increased intracellular

levels of AA that specifically promote BRAF V600E binding to

MEK1and subsequentMEK1phosphorylation (Figure 7H). These

results also support the emerging ‘‘metabolic rewiring’’ concept

describing metabolic alterations in cancer cells that are required

for a group of oncogenes but are not found in normal cells. In our

case, we demonstrated that oncogenic BRAF V600E links keto-

genesis to BRAF-MEK-ERK signaling cascade, representing a

‘‘wiring’’ between metabolic and cell signaling pathways. This

in part shares conceptual similarity with the mutations in isoci-

trate dehydrogenase (IDH) 1 and 2 identified in glioma and acute

myeloid leukemia, which enable the enzymes to produce onco-

metabolite 2-hydroxyglutamate (Dang et al., 2009; Mardis et al.,

2009; Parsons et al., 2008; Yan et al., 2009).

In addition, our findings provide evidence that supports a

concept suggesting that a group of oncogenes may require

different metabolic alterations for tumor growth. We found that

active BRAF commonly activates Oct-1 to promote HMGCL

gene expression, which in turn leads to increased intracellular

levels of AA in cells. However, it appears that only BRAF proteins

with substitution of V600 or mutations within the V600 flanking

region may benefit from the elevated levels of AA, which selec-

tively promotes MEK1 binding. Thus, although AA levels are

commonly elevated upon activation of BRAF, only cancer cells

expressing BRAF V600E/D mutant can benefit from increased

intracellular AA, where increased AA specifically promotes

V600E-MEK1 signaling, providing an ‘‘evolutionary advantage’’

that may explain why V600E is the predominant mutation of

BRAF identified in human malignancies. Further studies are war-

ranted to determine whether other cancer-associated active

BRAF mutants may similarly upregulate HMGCL expression

(D) ChIP results detecting binding ability of a group of transcription factors to the f

indicated with ‘‘+’’ in red color.

(E and F) ChIP results detecting binding ability of Oct-1 or IKAROS to HMGCL prom

inhibitor PLX-4032 (F).

(G and H) Effect of Oct-1 KD on HMGCL expression and intracellular AA levels (

(I–K) Effect of adding AA in culture on phosphorylation levels of MEK1 and ER

proliferation (K) inmelanoma cells with Oct-1 KD. Data with error bars in Figure 6 ar

Student’s t test.

Also see Figure S7.

and AA levels but cannot respond to AA in terms of MEK1 bind-

ing and phosphorylation.

In addition, future structural studies are also warranted to

explore the molecular basis underlying how AA specifically pro-

motes BRAF V600E-MEK1 binding and elucidate the molecular

mechanism by which active BRAF mutants, which, however,

may not be necessarily limited to BRAF V600E, upregulate

HMGCL gene expression in cancer cells. It will also be inter-

esting to explore the molecular mechanism by which active

BRAF activates Oct-1 in a MEK1-independent manner, which

may involve direct or indirect phosphorylation of Oct-1, since

BRAF kinase activity is important to promote Oct-1 binding to

HMGCL promoter. In addition, there may be additional Oct-1

transcription targets besides HMGCL that are important to

mediate BRAF V600E-dependent transformation in cancer cells,

which may explain why AA treatment only partially reversed the

decreased cell proliferation in BRAF V600E-positive melanoma

cells with Oct-1 KD (Figures 6K and S7I).

Our findings also link the ketogenic pathway to oncogenic

BRAF V600E-MEK-ERK signaling cascade, suggesting a

signaling function of AA that is independent of its role in cell meta-

bolism. These findings add to emerging evidence that supports a

concept suggesting that metabolites could function as signaling

molecules to allow crosstalk between metabolic pathways and

cell signaling networks. For example, we previously reported

that glycolytic intermediate 3-phosphoglycerate is a competitive

inhibitor of 6-phosphoglyconate dehydrogenase (6PGD) in the

oxidative pentose phosphate pathway (Hitosugi et al., 2012),

while others have found that AMP is an allosteric activator for

AMPK.On theother hand, there has beenaccumulating evidence

showing that post-translational modifications including tyrosine

phosphorylation (Fan et al., 2011; Hitosugi et al., 2011; Hitosugi

et al., 2009; Hitosugi et al., 2013) and lysine acetylation (Choudh-

aryet al., 2009; Fanet al., 2014;Kimet al., 2006;Wanget al., 2010;

Zhao et al., 2010) of metabolic enzymes are common and impor-

tant to link cell signaling pathways to metabolic pathways in can-

cer cells. These findings together represent a realm of crosstalk

with ‘‘back and forth’’ signal flows between metabolic and cell

signaling networks that ‘‘acutely’’ regulate cell metabolism and

proliferation,which, unfortunately, are ‘‘hijacked’’ bycancer cells.

Lastly, our results suggest that HMGCL inhibitor or non-

metabolizable AA derivatives that can compete with AA for

BRAF V600E binding may represent alternative therapies to treat

oncogenic BRAF V600E driven cancers. However, organs

including heart and brain can use ketone bodies including AA

and 3-HB for energy (Cotter et al., 2013; Morris, 2005). This war-

rants further detailed toxicity and pharmacokinetics studies to

evaluate the proposed anti-HMGCL or anti-AA therapies in can-

cer treatment.

unctional region of HMGCL promoter. Positive binding of Oct-1 and IKAROS is

oter region inmelanoma cells with BRAF KD (E) or treatment with BRAF V600E

G) and phosphorylation levels of MEK1 and ERK1/2 (H).

K1/2 (I), phosphorylation of MEK1 and BRAF-MEK1 association (J), and cell

e represented asmean ± SD; n = 3 each; p valueswere obtained by a two-tailed


https://www.researchgate.net/publication/7864112_Cerebral_ketone_body_metabolism_J_Inherit_Metabol_Dis?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

https://www.researchgate.net/publication/235441882_Ketone_body_metabolism_and_cardiovascular_disease?el=1_x_8&enrichId=rgreq-13ef005b-cc47-4b59-b62e-0977dda603c2&enrichSource=Y292ZXJQYWdlOzI3NzY2NjcwNTtBUzoyNTExNjU5MzM4MzAxNDVAMTQzNjg5NDAzMzYyMg==

Figure 7. Active BRAF Signals throughOct-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).


EXPERIMENTAL PROCEDURES

RNAi Screens and Data Analysis

The identities of known metabolism-related enzymes and protein factors in

the human genome were provided by the Phosphosite Plus website of Cell

Signaling Technology (http://www.phosphosite.org/psrSearchAction.do).

We constructed a ‘‘metabolism-targeted’’ shRNA library that targets

1,361 out of 1,417 genes encoding known enzymes and protein factors


in the human genome, which are available in the whole-genome shRNA li-

brary that we purchased from OpenBioSystems. The library contains 6,872

lentiviral-based shRNA constructs, where each gene is individually targeted

by one to five different shRNA constructs that target different regions of

the target gene (Table S1). In brief, RNAi screens were performed in 96-

well format such that each well contained a single shRNA species, and

each transcript was covered, on average, by one to five different shRNAs.

Assay conditions (cell number per well, viral dose, and puromycin



concentration) were optimized for each cell line prior to screening. Cells

were seeded, incubated for 24 hr, infected with lentivirus, and incubated

for 5 days. All lentiviral infections were performed with two replicates

selected with puromycin during the final 5 days of incubation and the other

two replicates left untreated. Cell viability and proliferation were measured

5 days after lentiviral infection using a CyQUANT Direct Cell Proliferation

Assay kit (Invitrogen). For both primary and secondary screens, we used

two RIGER methods (RIGER_KS and RIGER_SB) and another method,

Gene Set Analysis R package (Barbie et al., 2009; Gould et al., 2006;

Malo et al., 2006; Sims et al., 2011), to analyze the normalized B scores

for each cell line.

Xenograft Studies and Primary Tissue Samples from Patients with

Melanoma or HCL and Healthy Donors

Approval of use of mice and designed experiments was given by the Insti-

tutional Animal Care and Use Committee of Emory University. Approval of

use of human specimens was given by the Institutional Review Board of

Emory University School of Medicine. All clinical samples were obtained

with informed consent with approval by the Emory University Institutional

Review Board. Clinical information for the patients was obtained from the

pathologic files at Emory University Hospital under the guidelines and

with approval from the Institutional Review Board of Emory University

School of Medicine and according to the Health Insurance Portability and

Accountability Act. Detailed experimental procedures using mice and hu-

man primary tissue samples are described in the Supplemental Experi-

mental Procedures.

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures, seven tables, and Supple-

mental Experimental Procedures and can be found with this article online at

http://dx.doi.org/10.1016/j.molcel.2015.05.037.

AUTHOR CONTRIBUTIONS

H.-B.K., J.F., and R.L. contributed equally to this work. J.L.A., C.C., D.B.,

H.M.M., E.K., O.A.-W., T.M., B.P.P., K.F., R.R.K., D.H.L., G.S., M.R., S.L.,

H.J.K., and F.R.K. provided critical reagents. Q.J. and C.H. performed

biochemical analysis of purified proteins incubated with AA and analyzed

the data. T.J.B. performed structural analysis. B.H.L. performed the histopath-

ological analyses. S.F., C. Scholl, P.T., and D.A.B. contributed to experimental

design of screens and statistical analyses. H.-B.K., J.F., R.L., S.E., L.Z., L.J., C.

Shan, and J.H.S. performed all other experiments. H.-B.K., J.F., R.L., S.K., and

J.C. designed the study. S.K. and J.C. are senior authors and jointly managed

the project and wrote the paper.

ACKNOWLEDGMENTS

We thank Cancer Tissue and Pathology Core of Emory University for providing

primary cancer patient tissue samples. This work was supported in part by NIH

grants CA140515, CA183594, CA174786 (J.C.), CA175316 (S.K.), GM071440

(C.H.), and AR47901 (J.L.A.); the Pharmacological Sciences Training Grant

T32 GM008602 (S.E.); DoD grant W81XWH-12-1-0217 (J.C.); National Natural

Science Funds of China No.20902013 (L.Z.); the Jamie Rabinowitch Davis

Foundation (J.L.A.); the Charles Harris Run For Leukemia, Inc. (H.J.K.); and

the Hematology Tissue Bank of the Emory University School of Medicine

and the Georgia Cancer Coalition (H.J.K.). H.J.K., F.R.K., S.K., and J.C. are

Georgia Cancer Coalition Distinguished Cancer Scholars. S.K. and J.C. are

American Cancer Society Basic Research Scholars. J.C. is a Scholar of the

Leukemia and Lymphoma Society.

Received: February 13, 2015

Revised: April 17, 2015

Accepted: May 28, 2015

Published July 2, 2015

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metabolic rewiring by oncogenic braf v600e links ketogenesis pathway to braf-mek1 signaling

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