BRAF Cooperates with PI3K Signaling, Independent of AKT, to Regulate

Published OnlineFirst January 14, 2014; DOI: 10.1158/1541-7786.MCR-13-0224-T

Molecular Cancer Signal Transduction Research

BRAFV600E Cooperates with PI3K Signaling, Independent of AKT, to Regulate Melanoma Cell Proliferation

Jillian M. Silva, Christina Bulman, and Martin McMahon

Abstract Mutationally activated BRAFV600E cooperates with PTEN silencing in the conversion of normal melanocytes to metastatic melanoma cells, but the mechanism underlying this cooperation is poorly understood. Here, the consequences of pharmacologic blockade of BRAFV600E or phosphoinositide 3-kinase (PI3K) signaling were explored using pathway-targeted inhibitors and a panel of human BRAF-mutated melanoma-derived cell lines. Blockade of BRAFV600E ! MEK1/2 ! ERK1/2 or class I PI3K inhibited melanoma proliferation, whereas inhibition of AKT had only modest effects, even in cells with mutated or ampliﬁed AKT. Although single-agent inhibition of either BRAFV600E or PI3K signaling elicited antiproliferative effects, combinatorial inhibition was more potent. Analysis of signaling downstream of BRAFV600E or PI3K revealed that these pathways cooperated to regulate protein synthesis through AKT-independent, mTOR complex 1 (mTORC1)-dependent effects on p70S6K, ribosomal protein S6, and 4E-BP1 phosphorylation. Moreover, inhibition of mTORC1/2 inhibited cell proliferation as profoundly as single-agent inhibition of either BRAFV600E or PI3K signaling. These data reveal a mechanism by which BRAFV600E and PI3K signaling cooperate to regulate melanoma proliferation through AKT- independent effects on protein translation. Furthermore, this study provides a potential foundation for pathway- targeted combination therapy designed to enhance the therapeutic beneﬁt to patients with melanoma that contain combined alterations in BRAF and PI3K signaling.

Implications: PI3K, but not AKT, represent potential targets for melanoma therapy. Mol Cancer Res; 12(3); 447–63. 2014 AACR.

Introduction cytes is frequently promoted by silencing of the tumor Melanoma is known for its rapidly increasing incidence, suppressor PTEN, a phosphatidylinositol-3,4,5-triphos- phate (PIP3) phosphatase that suppresses the production aggressive clinical behavior, and propensity for lethal metas- 0 – fi tasis (1). Mutational activation of BRAF, detected in 50% of PI3 -lipids in the cell (6 10). The suf ciency for these of patients, is one of the earliest and most common genetic alterations in melanomagenesis was demonstrated using alterations in melanomagenesis (2, 3). The importance of genetically engineered mouse (GEM) models of metastatic V600E melanoma built upon this same foundation (11–13). mutationally activated BRAF in melanoma mainte- ! ! ! nance was demonstrated by the clinical success of vemur- Recently, RAF MEK1/2 ERK1/2 and PI3K afenib and dabrafenib, ATP-competitive inhibitors of AKT signaling was demonstrated to cooperatively regulate V600E protein translation in carcinomas through inhibitory phos- BRAF activity (4, 5). – BRAF mutations are detected at high frequency in benign phorylation of 4E-BP1, a negative regulator of the eIF4E nevi, nonmalignant melanocytic lesions that display hall- mRNA complex and cap-dependent translation (14). In this study, using pharmacologic agents and a panel of melanoma marks of senescence, and rarely progress to melanoma (3). fi Malignant progression of BRAFV600E-expressing melano- cells, we con rm that phosphoinositide 3-kinase (PI3K) signaling is necessary to cooperate with BRAFV600E signaling in melanoma. However, inhibition of AKT had little or no Authors' Affiliation: Department of Cell and Molecular Pharmacology, antiproliferative effects on BRAF-mutated human melano- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California ma cell lines regardless of PTEN status. Similarly, the antiproliferative effects of pharmacologic blockade of AKT Note: Supplementary data for this article are available at Molecular Cancer BRAF Research Online (http://mcr.aacrjournals.org/). in -mutated melanoma cells expressing mutated or amplified AKT1 or AKT2, respectively, was not as profound Corresponding Author: Martin McMahon, Diller Cancer Research Build- ing, MC-0128, 1450 Third Street, Room HD-365, University of California, as PI3K inhibition. Although single-agent inhibition of V600E San Francisco, CA 94158. Phone: 415-502-5829; Fax: 415-502-3179; BRAF ! MEK1/2 ! ERK1/2 or PI3K often dis- E-mail: [email protected] played substantial antiproliferative activity, the effects of doi: 10.1158/1541-7786.MCR-13-0224-T combined inhibition of both BRAFV600E plus PI3K signal- 2014 American Association for Cancer Research. ing were significantly more potent. These data support the

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hypothesis that PI3K signaling cooperates with BRAFV600E nylidene difluoride (PVDF) membrane using an iBlot transfer for melanoma maintenance through the regulation of pro- apparatus (Invitrogen). Membranes were blocked in Odyssey tein translation and provides a biochemical basis for the blocking buffer (LI-COR Biosciences) and probed with the simultaneous targeting of these two pathways in patients primary antibodies as described in Supplementary Tables S3 with BRAF-mutated melanoma (15–17). and S4. Antigen–antibody complexes were detected using fluorescent goat anti-rabbit IRDye 800 or goat anti-mouse Materials and Methods IRDye 680 secondary antibodies (LI-COR Biosciences) and visualized with a LI-COR infrared imaging system (Odyssey Cell culture and drug treatments Classic or Fc). Immunoblot data were analyzed using either Human melanoma cell lines, WM793, WM9, and A375, the Odyssey application software v3.0.30 or Image Studio were kindly provided from the well-curated cell line repos- v2.0 software (LI-COR Biosciences; ref. 22). itories established by Dr. Meenhard Herlyn (Wistar Insti- tute, Philadelphia, PA), and genomic sequencing of these cells was performed in the laboratory of Dr. Katherine Protein synthesis assays 5 Nathanson (University of Pennsylvania, Philadelphia, PA; Melanoma cells, plated at approximately 6 10 cells in Supplementary Table S1; refs. 18–20). The cell lines were 6-well plates, were treated with the indicated agents for 18 cultured in DME-H16 media containing 3 mg/mL glucose, hours and then deprived of methionine for 1 hour before m 35 0.584 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, culturing in DME-H16 media containing 20 Ci of [ S]- and 3.7 mg/mL NaHCO supplemented with 10% FBS, 5 methionine for 1 hour. Cells were prepared as previously 3 m mg/mL of insulin, L-glutamine, penicillin/streptomycin, described and 30 g of cell extracts were electrophoresed and and fungizone. M249 and M262 melanoma cells were then transferred to PVDF membrane. Radiolabeled proteins fi fi kindly provided by Dr. Antoni Ribas (University of Cali- were visualized by exposure to X-ray lm and quanti ed by fornia, Los Angeles) and authenticated by genomic densitometric analysis using ImageJ 1.45 software (National sequencing as previously described (Supplementary Table Institute of Health, Bethesda, MD). S1; ref. 21). These cells were maintained in RPMI-1640 7 supplemented with 10% FBS, L-glutamine, penicillin/ m GTP-sepharose cap-binding assays streptomycin, and fungizone. Pathway-targeted pharmaco- Following the indicated drug treatments for 24 hours, logic agents were obtained from various colleagues in the melanoma cells were lysed in buffer A (10 mmol/L Tris, 140 private sector or commercial sources and drug concentra- mmol/L KCl, 4 mmol/L MgCl2, 1 mmol/L EDTA, 1% NP- tions used for each treatment are listed in Supplementary 40 with protease and phosphatase inhibitors). Four hundred Table S2. micrograms of protein were incubated with 40 mLofm7 GTP-sepharose beads (GE Healthcare) in buffer B (10 Proliferation and growth assays mmol/L Tris, 140 mmol/L KCl, 4 mmol/L MgCl2,1 Melanoma cell proliferation was assessed by seeding 105 mmol/L EDTA with protease and phosphatase inhibitors) cells in 12-well plates. Cells were treated with the various overnight at 4C. eIF4E-associated complexes were washed pharmacologic agents as described in Supplementary Table twice with buffer C (10 mmol/L Tris, 140 mmol/L KCl, 4 S2 for 24, 48, and 72 hours. Viable cells were enumerated mmol/L MgCl2, 1 mmol/L EDTA, 0.5% NP-40 containing using a Countess automated cell counter (Invitrogen). Data protease and phosphatase inhibitors) and then twice with presented are representative of three independent experi- cold PBS containing 5 mmol/L EDTA. m7GTP-sepharose ments. To complement short-term proliferation assays, affinity precipitated proteins were boiled in 4 LDS Sample replicate cultures of melanoma cells were plated in 6-well Buffer (Invitrogen) and analyzed by immunoblotting with plates and cultured in the absence or presence of drug for 4 to the indicated antibodies. 11 days with viable cells fixed and stained with crystal violet. Cell proliferation was quantified by solubilizing the crystal Statistical analysis violet-stained cells in 33% acetic acid and measuring the All quantitative data are represented as mean SEM. absorbance at 562 nm using a plate reader. GraphPad Prism 6 statistical software was used to determine P values by performing either a two-way ANOVA Immunoblot analysis analysis with Bonferroni multiple comparisons test for the proliferation graphs or paired, two-tailed t tests for the Cells were lysed using radioimmunoprecipitation assay fi buffer (50 mmol/L Tris, 150 mmol/L NaCl, 0.5 mmol/L quanti cation of cell proliferation by crystal violet-stained EDTA, 10 mmol/L NaF, 0.1% SDS, 0.5% sodium deox- cells. ycholate, 1% NP-40) containing protease and phosphatase inhibitors (Pierce/Thermo Scientific) and then centrifuged at Results 14,000 rpm for 5 minutes at 4oC to generate postnuclear Pharmacologic blockade of either BRAFV600E or PI3K lysates with protein concentrations measured using the signaling inhibits proliferation and signaling of human bicinchoninic acid assay (Pierce/Thermo Scientific; ref. 22). BRAF-mutated melanoma cells Thirty micrograms of protein were separated using NuPAGE To determine the signal pathway dependencies of Novex Bis-Tris gels (Invitorgen) and transferred to polyvi- BRAF-mutated melanoma cells, we examined the effects

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BRAFV600E Cooperates with PI3K in Melanomagenesis Independent of AKT

of pharmacologic blockade of BRAFV600E ! MEK1/2 ! inhibitory effects of the other pathway, at least over a 24- ERK1/2 or PI3K ! AKT signaling using human mela- hour exposure period. noma-derived cell lines that were selected solely on the MEK1/2 or ERK1/2 inhibition resulted in decreased P- basis of their BRAF mutation status, some of which are p70S6K and P-rpS6 in all three cell lines at 24 hours, proficient or deficient for PTEN expression (Supplemen- whereas P-4E-BP1 was only significantly reduced in tary Table S1; refs. 18–20). Agents used to inhibit MEK1/2-inhibited WM793 and A375 cells (Fig. 1D and BRAFV600E ! MEK1/2 ! ERK1/2 or PI3K ! AKT SupplementaryFig.S2).AlthoughMEK1/2orERK1/2 signaling were chosen because of their reported specificity inhibition for 4 hours potently suppressed P-p70S6K,there and selectivity against the relevant targets (Supplementary was no effect on P-rpS6 in any of the cell lines at this time Fig. S1 and Supplementary Table S2; refs. 23–35). point (Fig. 1C). Interestingly, inhibition of PI3K for Where possible, results were verified by targeting multiple either 4 or 24 hours significantlyreducedP-rpS6inall components of the same pathway using structurally cell lines, which occurred in the absence of changes in P- unrelated, mechanistically dissimilar inhibitors of the p70S6K (Fig. 1C and D and Supplementary Fig. S2). same target. Inhibition of PI3K also led to reduced P-4E-BP1 at both Treatment of WM793, WM9, and A375 cells with either 4 and 24 hours with the most profound effect at the later a MAP–ERK kinase (MEK1/2; 1 mmol/L PD0325901/ time point (Fig. 1C and D and Supplementary Fig. S2). MEKi1) or an extracellular signal-regulated kinase (ERK1/ Strikingly, AKT inhibition with either of two inhibitors 2; 1 mmol/L SCH772984/ERKi) inhibitor for 24, 48, and had no discernible effect on either P-p70S6K or P-4E-BP1 72 hours led to robust inhibition of cell proliferation (Fig. and only reduced P-rpS6 by approximately 50% in WM9 1A; 72 hours; P < 0.001); however, we did not observe a cells at both time points (24 hours: P < 0.05; Fig. 1C and significant decrease in cell number, which is consistent with D and Supplementary Fig. S2). Taken together, these data the negligible proapoptotic effects of MEK inhibition on suggestthatBRAFV600E ! MEK1/2 ! ERK1/2 and cultured melanoma cells (36). Similarly, inhibition of class I PI3K signaling regulates mTOR complex 1 (mTORC1)- PI3K (5 mmol/L GDC-0941/PI3Ki1) inhibited melanoma dependent phosphorylation of rpS6 and 4E-BP1 signaling cell proliferation without a measureable decrease in cell events through distinct mechanisms that are largely inde- number at 72 hours (WM793: P < 0.05; WM9 and pendent of AKT activity. A375: P < 0.0001). Surprisingly, inhibition of AKT with either of two structurally unrelated and mechanistically Temporal regulation of rpS6 and 4E-BP1 dissimilar inhibitors (MK-2206/AKTi1 or GSK690693/ phosphorylation by BRAFV600E ! MEK1/2 ! ERK1/2 AKTi2) was largely without effect in WM793 and A375 or PI3K cells. In contrast, the proliferation of WM9 cells at 72 hours To further assess differences in the regulation of rpS6 was inhibited by approximately 50% by both AKT inhibi- and 4E-BP1 phosphorylation downstream of BRAFV600E tors (AKTi1: P < 0.01; Fig. 1A). In addition to short-term and PI3K signaling, WM793 or A375 cells were treated growth assays, the effect of these agents on longer term cell with inhibitors of either MEK1/2 (PD0325901/MEKi1) proliferation (4–6 days) was examined (Fig. 1B and Sup- or PI3K (GDC-0941/PI3Ki1)for1to10hours(Fig.2A). plementary Fig. S4A). These data largely confirmed the Both of these agents exerted robust and selective inhib- short-term assays, in which single-agent inhibition of itory effects against P-ERK1/2 or P-AKT after one hour of BRAFV600E, MEK1/2, ERK1/2, or PI3K had potent anti- treatment. Although MEK1/2 inhibition led to a rapid proliferative activity compared with modest effects of AKT decrease of P-p70S6K, this did not result in an immediate inhibition. decrease in rpS6 phosphorylation as we noted a 4- to 8- To examine the consequences of pathway-targeted inhi- hour delay between effects of MEK1/2 inhibition on P- bition on signaling downstream of BRAFV600E or PI3K, p70S6K and dephosphorylation of rpS6. As before, extracts were prepared from cells treated with the various MEK1/2 inhibition had negligible effects on P-4E-BP1 agents for 4 or 24 hours and analyzed by immunoblotting at all time points evaluated. Although PI3K inhibition (Fig. 1C and D). Information about the significance of each had no effect on p70S6K phosphorylation at any time phosphorylation site is included in Supplementary Table S4. point, there was a notable decrease in P-rpS6 in both cell Control (CTL) WM793, WM9, and A375 cells displayed lines within 4 hours, which was most striking in WM793 readily detectable phosphorylation of ERK1/2 (P-T202/ cellsafter10hours(Fig.2A).Inaddition,PI3Kinhibition Y204), AKT (P-S473), p70S6K (P-T389), ribosomal protein also led to decreased P-4E-BP1 that was detected within 1 S6 (rpS6; P-S235/236 and P-S240/244), and 4E-BP1 hour in both cell lines (Fig. 2A). Thus, inhibition of either (P-T37/46 and P-S65). Inhibition of MEK1/2 (1 mmol/L MEK1/2 or PI3K resulted in decreased phosphorylation PD0325901/MEKi1) or ERK1/2 (1 mmol/L SCH772984/ of rpS6, but elicited their effects with different kinetics ERKi) led to decreased P-ERK1/2 with little or no effect on and contrasting effects on the activation status (P-T389) P-AKT. Similarly, inhibition of class 1 PI3K (5 mmol/L of p70S6K. GDC-0941/PI3Ki1) or AKT (5 mmol/L MK-2206/AKTi1) Evidence suggests that phosphorylation of rpS6 and 4E- potently suppressed P-AKT, but had no effect on P-ERK. BP1 is downstream of mTORC1 (37). However, Thus, BRAFV600E ! MEK1/2 ! ERK1/2 and PI3K ! BRAFV600E ! MEK1/2 ! ERK1/2 signaling can also AKT signaling seems to be largely insulated from the contribute to rpS6 phosphorylation through p90RSK (38).

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WM793 A 1 1 (BRAFV600E , PTENNull) B Control BRAFi MEKi

WM793 (BRAFV600E, PTENNull)

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A375 Control BRAFi1 MEKi1 (BRAFV600E , PTENWT)

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Figure 1. Sensitivity of human melanoma-derived cell lines to pharmacologic inhibition of BRAFV600E or PI30K signaling. A, WM793 (BRAFV600E, PTENNull), WM9 (BRAFV600E,PTENNull), and A375 (BRAFV600E, PTENWT) cells were treated with DMSO (CTL) or inhibitors of MEK1/2 (1 mmol/L PD0325901/MEKi1), ERK1/2 (1 mmol/L SCH772984/ERKi), PI3K (5 mmol/L GDC-0941/PI3Ki1), or AKT (5 mmol/L MK-2206/AKTi1 or 5 mmol/L GSK690693/AKTi2) with cell proliferation assessed for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as mean SEM (n ¼ 3). B, cells were plated at a 30% conﬂuency and cultured in the continuous presence of inhibitors of BRAFV600E (1 mmol/L GSK2118436/BRAFi1), MEK1/2 (MEKi1), ERK1/2 (ERKi), PI3K (PI3Ki1), or AKT (AKTi1 or AKTi2) for 4 (A375) or 6 (WM793 and WM9) days with viable cells ﬁxed and stained with crystal violet. (Continued on the following page.)

To determine the role of mTORC1 signaling and the S6 mTORC1 inhibitor, rapamycin, also inhibited P-rpS6, kinase, p70S6K or p90RSK, responsible for sustaining rpS6 but had no effect on 4E-BP1 phosphorylation (Supple- phosphorylation, WM793 and A375 cells were treated mentary Fig. S3A). Although inhibition of p90RSK had no with either PP242 (1 or 5 mmol/L mTORi), a potent effect on either P-rpS6 or P-4E-BP1, inhibition of p70S6K ATP-competitive inhibitor of mTORC1/2 signaling; selectively reduced P-rpS6. These data are consistent with FMK (1 mmol/L RSKi), a covalent inhibitor of p90RSK; a model in which phosphorylation of both rpS6 and 4E- or DG2 (10 mmol/L S6Ki), a p70S6K inhibitor (Fig. 2B BP1 is mTORC1 dependent and the phosphorylation and Supplementary Table S2; refs. 23, 24, 30). As of rpS6 is p70S6K dependent but p90RSK independent expected, PP242-mediated inhibition of mTORC1/2 (Fig. 2B). resulted in decreased phosphorylation of P-rpS6 and P- In parallel, we assessed the effects of mTORC1/2 inhi- 4E-BP1 (Fig. 2B). In addition, a mechanistically distinct bition (PP242/mTORi) on melanoma cell proliferation

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BRAFV600E Cooperates with PI3K in Melanomagenesis Independent of AKT

C WM793 WM9 A375 (BRAFV600E, PTENNull) (BRAFV600E, PTENNull) (BRAFV600E, PTENWT)

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CTL MEKi1 ERKi PI3Ki1 AKTi1 CTL MEKi1 ERKi PI3Ki1 AKTi1 CTL MEKi1 ERKi PI3Ki1 AKTi1

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P-p70S6K T389

P-rpS6 S235/236

P-rpS6 S240/244

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P-4E-BP1 S65

T-rpS6

Figure 1. (Continued.) C and D, WM793, WM9, and A375 cells were treated with DMSO (CTL) or inhibitors of MEK1/2 (MEKi1), ERK1/2 (ERKi), PI3K (PI3Ki1), or AKT (AKTi1 or AKTi2) for 4 (C) or 24 (D) hours and cell lysates were immunoblotted with the indicated antibodies.

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A WM793 A375 (BRAFV600E, PTENNull) (BRAFV600E, PTENWT)

1 h 4 h 8 h 10 h 1 h 4 h 8 h 10 h

CTL MEKi1 PI3Ki1 CTL MEKi1 PI3Ki1 CTL MEKi1 PI3Ki1 CTL MEKi1 PI3Ki1 CTL MEKi1 PI3Ki1 CTL MEKi1 PI3Ki1 CTL MEKi1 PI3Ki1 CTL MEKi1 PI3Ki1

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WM793 A375 WM793 A375 (BRAFV600E, PTENNull) (BRAFV600E, PTENWT) (BRAFV600E, PTENNull) (BRAFV600E, PTENWT)

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Figure 2. Kinetics of rpS6 and 4E-BP1 dephosphorylation following inhibition of BRAFV600E or PI3K signaling. A, WM793 and A375 melanoma cells were treated with MEK1/2 (1 mmol/L PD0325901/MEKi1)orPI3K(5mmol/L GDC-0941/PI3Ki1) inhibitors for 1, 4, 8, and 10 hours and cell lysates were analyzed by immunoblotting with the indicated antibodies. B, cells were treated with inhibitors of mTORC1/2 (1 or 5 mmol/L PP242/mTORi), p90RSK (1 mmol/L FMK/RSKi), or p70S6K (10 mmol/L DG2/S6Ki) for 24 hours and cell lysates were immunoblotted with the indicated antibodies. (Continued on the following page.)

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C WM793 D (BRAFV600E, PTENNull) 6

Control 4 1 µmol/L mTORi 5 µmol/L mTORi Control 1 µmol/L mTORi 5 µmol/L mTORi 2 Relative cell number WM793 0 (BRAFV600E, PTENNull) 02448 72 Hours WM9 (BRAFV600E, PTENNull) 10 Control 1 µmol/L mTORi 5 µmol/L mTORi

8 Control 1 µmol/L mTORi 6 WM9 5 µmol/L mTORi (BRAFV600E, PTENNull) 4

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Figure 2. (Continued.) C, melanoma proliferation was assessed in WM793, WM9, and A375 cells treated with DMSO (CTL) or the mTORC1/2 inhibitor (mTORi) for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as mean SEM (n ¼ 3). D, WM793, WM9, and A375 cells were also plated at a 30% confluency and cultured in the continuous presence of the mTORC1/2 inhibitor (mTORi) for 11 days with viable cells fixed and stained with crystal violet. using both short-term (1–3 days) and long-term (11 days) cell lines (Supplementary Fig. S3B; ref. 11). Thus, dual proliferation assays (Fig. 2C and D and Supplementary control of mTORC1 signaling by cooperative BRAFV600E Fig. S4B). Proliferation of WM793, WM9, and A375 and PI3K signaling appears important for melanoma cell cells was significantly inhibited by either 1 or 5 mmol/L proliferation. PP242 at all time points analyzed (24 hours: WM793 P < 0.05, WM9 and A375 P < 0.01; 48–72 hours: P < BRAFV600E and PI3K signaling cooperate to regulate 0.0001); however, the antiproliferative effect of 1 rpS6 and 4E-BP1 phosphorylation and melanoma cell mmol/L PP242 was less robust against WM793 and proliferation A375 cells (Fig. 2C). These results are consistent with Given that BRAFV600E and PI3K signaling cooperate to a longer term proliferation assay in which 5 mmol/L regulate mTORC1 signaling output, we tested whether PP242 elicited more potent antiproliferative effects against combined inhibition of these pathways might elicit more all three cell lines than 1 mmol/L PP242 (Fig. 2D and striking effects on intracellular signaling events leading to Supplementary Fig. S4B). Similartopreviousanalysesof more profound effects on melanoma cell proliferation. BRAFV600E,PTENNull melanoma cells derived from a Extracts of WM793, WM9, or A375 cells were treated with GEM model, rapamycin was less potent in inhibiting the inhibitors of BRAFV600E, MEK1/2, or PI3K, either alone proliferation of all three human BRAF-mutated melanoma or in combination, and analyzed by immunoblotting

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A B WM793 (BRAFV600E, PTENNull) 5 WM793 WM9 A375 (BRAFV600E, PTENNull) (BRAFV600E, PTENNull) (BRAFV600E, PTENWT) 4 CTL MEKi1 MEKi1+ MEKi1+ MEKi1+ Control MEKi1 PI3Ki1 Control MEKi1 PI3Ki1 Control MEKi1 PI3Ki1 3 PI3Ki1 PI3Ki1 PI3Ki1 PI3Ki1 MEKi1+PI3Ki1 2 P-ERK1/2 T202/Y204 1 Relative cell number 0 T-ERK1/2 02448 72 Hours P-AKT WM9 S473 (BRAFV600E, PTENNull) 8

P-p70S6K T389 6 CTL MEKi1 PI3Ki1 P-rpS6 4 MEKi1+PI3Ki1 S235/236 2 P-rpS6

S240/244 Relative cell number 0 0244872 P-4E-BP1 Hours T37/46 A375 (BRAFV600E, PTENWT) P-4E-BP1 10 S65 8 CTL MEKi1 T-rpS6 6 PI3Ki1 MEKi1+PI3Ki1 4 β-Actin 2

Relative cell number 0 0244872 Hours

Figure 3. Effects of single-agent or combined inhibition of MEK1/2 or PI3K on melanoma cell signaling and proliferation. A, WM793, WM9, and A375 cells were treated with inhibitors of MEK1/2 (1 mmol/L PD0325901/MEKi1) or PI3K (5 mmol/L GDC-0941/PI3Ki1) either alone or in combination for 18 hours with cell lysates analyzed by immunoblotting with the indicated antibodies. B, single-agent or combined inhibition of MEK1/2 (MEKi1) or PI3K (PI3Ki1) on WM793, WM9, and A375 cell proliferation for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as mean SEM (n ¼ 3). (Continued on the following page.)

(Figs. 3A and 4A). As previously demonstrated, MEK1/2 this was not superior to single-agent inhibition at any time (PD0325901/MEKi1) or BRAFV600E (PLX-4032/BRAFi2) point (Fig. 3B). In the longer-term assays (9 days), single- inhibition led to decreased P-ERK, P-p70S6K, and P-rpS6 agent MEK1/2 inhibition had more potent antiprolifera- with only modest inhibition of P-4E-BP1. PI3K inhibition tive activity compared with single-agent PI3K inhibition (GDC-0941/PI3Ki1) robustly suppressed P-AKT and P- (Fig.3CandD).However,inallthreecelllines,combined rpS6, had no effect on P-p70S6K,andledtoamodest inhibition of both MEK1/2 and PI3K significantly inhib- decrease of P-4E-BP1 in WM793 cells with a more ited melanoma proliferation compared with the corre- profound inhibition in WM9 and A375 cells. Although sponding single agents with the magnitude of the observed PI3K inhibition reproducibly inhibited P-4E-BP1, we effects being greatest in WM9 and A375 cells (Fig. 3C and noted some experiment-to-experiment variation in the D). These results were confirmed using a short-term magnitude of this effect. Most importantly, combined proliferation assay (4 days), in which combined inhibition inhibition of either BRAFV600E or MEK1/2 plus PI3K of BRAFV600E (BRAF2)plusPI3K(PI3K1)morepotently routinely resulted in a more potent suppression of P-rpS6 suppressed melanoma cell growth compared with single- and P-4E-BP1 compared with the corresponding single agent inhibition alone (Fig. 4B and Supplementary Fig. agents (Figs. 3A and 4A). S4C). These data suggest that, at least in vitro,BRAFV600E In parallel, the effects of combinedMEK1/2plusPI3K and PI3K signaling cooperate to promote key signaling inhibition on cell proliferation were assessed in short- and events downstream of mTORC1, which in turn promote long-term assays (Fig. 3B–D). In the short term, single- melanoma cell proliferation. agent inhibition of MEK1/2 (MEKi1)orPI3K(PI3Ki1) significantly inhibited cell growth at 48 (P < 0.01) and 72 Coordinate control of melanoma protein synthesis by (P < 0.01) hours. As predicted by the single-agent effects, BRAFV600E and PI3K signaling combined inhibition of MEK1/2 plus PI3K also led to a In conjunction with previous experiments, we assessed the significant decrease in melanoma cell proliferation, but effects of combined inhibition of MEK1/2 (PD0325901/

454 Mol Cancer Res; 12(3) March 2014 Molecular Cancer Research

BRAFV600E Cooperates with PI3K in Melanomagenesis Independent of AKT

WM793 WM9 A375 (BRAFV600E, PTENNull) (BRAFV600E, PTENNull) (BRAFV600E, PTENWT)

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Figure 3. (Continued.) C, triplicate cultures of WM793, WM9, or A375 cell lines were treated with inhibitors of MEK1/2 (MEKi1) or PI3K (PI3Ki1) as single agents or in combination for 9 days with viable cells ﬁxed and stained with crystal violet. DMSO-treated controls were split twice to prevent overgrowth and therefore staining of these wells is an underestimate of actual cell number (12-fold WM793, 22-fold WM9, 60-fold A375). D, quantiﬁcation of the crystal violet staining of control versus drug treated WM793, WM9, and A375 cells. Results are expressed as a fold-change of the DMSO-treated control and presented as mean SEM (n ¼ 3). Paired, two-tailed t tests were used to determine P values (, P < 0.05; , P < 0.01).

MEKi1) plus PI3K (GDC-0941/PI3Ki1) on protein syn- Hypophosphorylated 4E-BP1 binds to the eIF4E ini- thesis in WM793, WM9, and A375 cells (Fig. 5A and B). tiation complex at the 50 cap of mRNAs to inhibit cap- Single-agent inhibition of MEK1/2 or PI3K alone led to a dependent translation by preventing eIF4G from binding 40% to 80% inhibition of [35S]-methionine incorporation to this complex (39). Phosphorylation of 4E-BP1 by (Fig. 5B). The effect of MEK1/2 inhibition on protein mTORC1 releases it from eIF4E allowing eIF4G to bind synthesis was most striking in WM793 cells, whereas PI3K to the eIF4E–mRNA complex and initiate cap-dependent inhibition had the equivalent effects on protein synthesis in translation (40). Thus, hypophosphorylated 4E-BP1 and all three cell lines. Importantly, combined inhibition of both eIF4G directly compete for the same binding site on MEK1/2 plus PI3K resulted in the most profound inhibitory eIF4E, whereas hyperphosphorylated 4E-BP1 is not a effects on protein translation in all three cell lines. Consistent strong competitor for this site (40). Because activation of with previous observations, combined inhibition of MEK1/ BRAFV600E and PI3K signaling promotes hyperphosphor- 2 and PI3K also had the most striking effects on P-rpS6 and ylation of 4E-BP1, we examined the effects of MEK1/2 or P-4E-BP1 compared with the corresponding single agents PI3K inhibition on cap-dependent protein translation, (Fig. 5A). speciﬁcally the association between 4E-BP1 and the eIF4E

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A WM793 WM9 A375 (BRAFV600E, PTENNull) (BRAFV600E, PTENNull) (BRAFV600E, PTENWT)

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Figure 4. Effects of single-agent or combined inhibition of BRAFV600E or PI3K on melanoma cell growth and signaling. A, WM793, WM9, and A375 cells were treated with inhibitors of BRAFV600E (5 mmol/L PLX-4032/BRAFi2) or PI3K (5 mmol/L GDC-0941/PI3Ki1) as single agents or in combination for 24 hours with cell lysates analyzed by immunoblotting with the indicated antibodies. B, melanoma cell proliferation was assessed by treating all three cell lines with inhibitors of BRAFV600E (BRAFi2) or PI3K (PI3Ki1) alone or in combination for 4 days with viable cells ﬁxed and stained with crystal violet.

complex. Extracts of melanoma cells treated with inhibi- this assay, eIF4E present in melanoma lysates binds to tors of MEK1/2 (MEKi1)orPI3K(PI3Ki1), either alone sepharose beads coupled to the cap analog 7-methyl GTP or in combination, were used in a cap pull-down assay. In (m7GTP) to determine whether there is an increase or

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A B

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Figure 5. Cooperation of BRAFV600E and PI3K signaling on the regulation of protein translation. A, protein synthesis was measured by [35S]-methionine incorporation of newly synthesized proteins in WM793, WM9, and A375 cells treated with inhibitors of MEK1/2 (1 mmol/L PD0325901/MEKi1) or PI3K (5 mmol/L GDC-0941/PI3Ki1) as single agents or in combination for 18 hours (n ¼ 2). These same cell extracts were also analyzed for rpS6 and 4E-BP1 phosphorylation as indicated. B, densitometric quantiﬁcation of [35S]-methionine incorporation of newly synthesized proteins in all three cell lines. Results are represented as a fold-change of the DMSO-treated control. C, WM793, WM9, and A375 cells were treated with inhibitors of MEK1/2 (MEKi1)or PI3K (PI3Ki1) alone or in combination for 24 hours with cell lysates precipitated with m7GTP sepharose beads followed by immunoblotting for 4E-BP1, eIF4E, or with the indicated antibodies. www.aacrjournals.org Mol Cancer Res; 12(3) March 2014 457

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Figure 6. Sensitivity of melanoma cell proliferation and signaling to either single-agent or combined inhibition of MEK1/2 or AKT. A, proliferation of melanoma cells treated with MEK1/2 (1 mmol/L GSK1120212/MEKi2) or AKT (5 mmol/L GSK690693/AKTi2) inhibitors either as single agents or in combination was assessed for 5 (WM9 and A375) or 6 (WM793) days with viable cells ﬁxed and stained with crystal violet. B, WM793, WM9, and A375 cells were treated with inhibitors of MEK1/2 (MEKi2) or AKT (AKTi2) either alone or in combination for 24 hours and cell lysates were analyzed by immunoblotting with the indicated antibodies.

decrease in 4E-BP1 bound to the eIF4E–m7GTP cap than the corresponding single-agent treatments in the crude complex. cell extracts used for the cap pull-down assays (Fig. 5C). As observed previously, combined inhibition of MEK1/2 However, despite the effects of MEK1/2 inhibition on P-4E- plus PI3K more potently suppressed P-rpS6 and P-4E-BP1 BP1, we only observed increased association of 4E-BP1 with

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the eIF4E–m7GTP cap complex in WM793 cells. In all may modestly contribute to rpS6 phosphorylation, its three cell lines, PI3K inhibition led to increased association contribution is substantially less than what is observed of 4E-BP1 with the eIF4E–m7GTP cap complex. Interest- following inhibition of BRAFV600E ! MEK1/2 ! ingly, and contrary to expectation, combined inhibition of ERK1/2 or PI3K signaling. Taken together, these data MEK1/2 plus PI3K did not lead to a further increase in 4E- emphasize the importance of BRAFV600E and PI3K sig- BP1 association with eIF4E–m7GTP cap complexes. These naling in the regulation of protein translation leading to data suggest that the regulation of melanoma protein syn- melanoma cell proliferation in a substantially AKT-inde- thesis downstream of BRAFV600E and PI3K activation is pendent manner. more complex than the simple regulation of the stoichiom- etry of 4E-BP1 phosphorylation detected in cell lysates and Effects of AKT inhibition on melanoma cells with its capacity for interaction with eIF4E. mutated or amplified AKT1/2 A small percentage of human melanoma cells are reported Inhibitors of MEK1/2 and AKT display little or no to express either amplified or mutated AKT in addition to cooperative effects against melanoma proliferation or mutated BRAF (21). In addition, AKT1 mutation is asso- signaling ciated with BRAFV600E inhibitor resistance in melanoma The ineffectiveness of the AKT inhibitors in suppressing (42, 43). Consequently, we tested the effects of AKT melanoma cell proliferation was surprising given the prom- inhibition on the proliferation of melanoma cells with inence ascribed to AKT as an effector of PI3K signaling in alterations in AKT1 or 2 (Supplementary Table S1; ref. 21). cancer (14, 31, 41). Because AKT inhibition had modest M249 (BRAFV600E, PTENNull, AKT2Amp) or M262 effects on melanoma cells, we assessed whether combined (BRAFV600E, PTENWT, AKT1E17K/Amp) cells were treated inhibition of MEK1/2 plus AKT might provide a more with inhibitors of BRAFV600E (GSK2118436/BRAFi1), robust inhibition of melanoma cell proliferation and signal- MEK1/2 (PD0325901/MEKi1), PI3K (GDC-0941/ ing. To test the effects of single-agent versus combined PI3Ki1), or AKT (MK-2206/AKTi1 or GSK690693/ MEK1/2 or AKT inhibition, melanoma cells were treated AKTi2) with proliferation measured at 24, 48, or 72 hours with MEK1/2 (1 mmol/L GSK1120212/MEKi2) or AKT (5 (Fig. 7A). In short-term cell proliferation assays, neither of mmol/L GSK690693/AKTi2) inhibitors, either alone or in the AKT inhibitors was as effective as single-agent inhibition combination, for 5 to 6 days with viable cells stained with of MEK1/2 or PI3K at inhibiting cell proliferation (Fig. 7A). crystal violet (Fig. 6A and Supplementary Fig. S4D). Similar In the longer-term assays, the allosteric AKT inhibitor MK- to our previous results, single-agent MEK1/2 inhibition 2206 inhibited the growth of M249 cells (AKT2Amp)as profoundly inhibited the proliferation of all three melanoma effectively as single-agent inhibition of BRAFV600E, MEK1/ cell lines; however, the antiproliferative effects of AKT 2, or PI3K (Fig. 7B and Supplementary Fig. S4E). In inhibition were modest (35%–70% inhibition) and sub- contrast, the ATP-competitive AKT inhibitor, GSK690693, stantially less than that observed in response to single-agent was substantially less effective in these same cells. Both AKT PI3K inhibition (Fig. 1A and B). Moreover, inhibition of inhibitors inhibited cell growth by 60% to 65% in M262 AKT did not substantially enhance the antiproliferative cells (AKT1E17K/Amp), but were not as effective as single- effect of MEK1/2 inhibition in these cells (Fig. 6A and agent blockade of BRAFV600E, MEK1/2, or PI3K (Fig. 7B Supplementary Fig. S4D). and Supplementary Fig. S4E). Although these data suggest To assess the effect of combined inhibition of MEK1/2 that AKT inhibition in melanoma cells with mutated or and AKT on melanoma signaling, melanoma cells were amplified AKT1/2 may predict for more robust antiproli- treated with MEK1/2 (GSK1120212/MEKi2)orAKT ferative effects on cell growth, these effects generally fall short (GSK690693/AKTi2) inhibitors, either alone or in com- of what is observed with inhibitors of BRAFV600E ! MEK1/ bination, for 24 hours with extracts analyzed by immu- 2 ! ERK1/2, or PI3K signaling. noblotting (Fig. 6B). As expected, MEK1/2 inhibition led We next assessed the effects of the various agents on to decreased P-ERK, P-rpS6, and P-4E-BP1 with no effect signaling downstream of BRAFV600E ! MEK1/2 ! on P-AKT. AKT inhibition greatly enhanced AKT phos- ERK1/2 or PI3K ! AKT (Fig. 7C). As previously observed phorylation, as predicted by this agent's mechanism of with other melanoma cells, inhibition of BRAFV600E ! action, and reduced P-PRAS40 (T246), a direct down- MEK1/2 ! ERK1/2 signaling in either M249 or M262 cells stream substrate of AKT. Consistent with previous data, led to substantially decreased P-ERK, P-p70S6K, and P-rpS6 AKT inhibition had no effect on P-4E-BP1 in any of the with only modest effects on P-4E-BP1. Blockade of PI3K cell lines, but moderately decreased P-rpS6 in WM9 cells with two different inhibitors (GDC-0941/PI3Ki1 or BKM- with little or no discernible inhibitory effect in WM793 or 120/PI3Ki2) suppressed P-AKT and P-rpS6, modestly A375 cells. Combined MEK1/2 plus AKT inhibition had reduced P-4E-BP1, but did not alter P-p70S6K. Although only a modest effect on P-rpS6 in WM9 and A375 cells, both AKT inhibitors had their predicted effects on P-AKT whereas P-rpS6 in WM793 cells was not further decreased (decreased with MK-2206/AKTi1, increased with compared with single-agent inhibition with MEK1/2. GSK690693/AKTi2), these agents had only modest effects Likewise, the combination of MEK1/2 and AKT inhibi- on P-rpS6 and negligible effects on either P-p70S6K or P-4E- tion was also ineffective in further reducing P-4E-BP1 BP1 compared with single-agent blockade of BRAFV600E, compared with MEK1/2 inhibition alone. Although, AKT MEK1/2, ERK1/2, or PI3K.

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A M249 M262 (BRAFV600E, PTENNull, AKT2Amp) (BRAFV600E, PTENWT, AKT1E17K/Amp)

B M249 M262 (BRAFV600E, PTENNull, AKT2Amp) (BRAFV600E, PTENWT, AKT1E17K/Amp)

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Discussion signaling through ERK1/2 regulation of the TSC1/2 com- The conversion of melanocytes to metastatic melanoma plex as previously proposed (55). In contrast, PI3K blockade cells is accompanied by alterations in key signaling pathways also led to diminished rpS6 phosphorylation, but this that cooperate in melanomagenesis (Supplementary Fig. S1; occurred in a largely AKT-independent manner and without refs. 1, 7, 11, 44–46). The most common alterations in the any change in the key activating phosphorylation of p70S6K 0 PI3K ! AKT pathway are silencing of the PTEN, INPP4B, (P-T389). One possibility is that PI3 -lipid signaling or PIB5PA lipid phosphatases and, less commonly, point may regulate rpS6 phosphorylation through effects on PP1, mutation/amplification of PIK3CA or AKT1-3 (21, 47–51). the protein phosphatase that dephosphorylates rpS6 (56). Thus, cooperating alterations in PI3K signaling seem to be Although we propose that PI3K cooperates with BRAFV600E essential for progression of BRAFV600E-initiated melanoma. to regulate melanoma protein synthesis through cooperative Here, we show that melanoma cells, regardless of their effects on translation initiation, there are likely additional PTEN or AKT mutational status, were sensitive to single- regulatory events in protein synthesis under the control of agent inhibition of BRAFV600E or PI3K signaling and even these pathways as suggested by the results of the m7GTP cap- more susceptible to combined pathway inhibition. This binding experiments (57). suggests that melanoma cells, even those lacking genetically Despite effective suppression of AKT phosphorylation defined alterations in PI30-lipid signaling (such as A375 by either of the two structurally unrelated and mechanis- cells), have a requirement for PI3K signaling for their tically dissimilar agents, we observed only modest effects maintenance (52, 53). on melanoma cell proliferation and failed to detect the Regulation of protein translation is a process critically expected effects on downstream signaling events. The required for the aberrant behavior of cancer cells (54). effects of these agents on cell proliferation or signaling Considerable attention has focused on the mechanisms by in cells expressing either amplified or mutated AKT1/2 which PI3K signaling regulates mTORC1 activity to initiate were less robust than expected on the basis of current cap-dependent protein translation. Here, we demonstrate literature (21). Moreover, thecombinedblockadeof that, in BRAF-mutated melanoma cells, these signaling MEK1/2 and AKT had little or no effect on the inhibition events are under the coordinate control of both on rpS6 and 4E-BP1 phosphorylation, which stood in BRAFV600E and PI3K signaling in a manner that appears contrast with the substantive cooperative effects of PI3K largely AKT independent. Others have previously reported and BRAFV600E or MEK1/2 inhibition. Consequently, dual control of these signaling events in tumor cells with the lack of potent single-agent activity and evidence of coincident KRAS and PIK3CA mutations; however, in that strong cooperation with the BRAFV600E pathway inhibi- case AKT was reported to be a key effector of PI30-lipid tors, suggest that AKT is not an attractive target for signaling (14). Moreover, in these cells, single-agent block- melanoma therapy. This is consistent with the inability ade of MEK1/2 or AKT had little effect on proliferation, of the allosteric AKT inhibitor (MK-2206) to prevent the rpS6 or 4E-BP1 phosphorylation, or cap-dependent trans- growth of BRAFV600E,PTENNull melanomas in a GEM lation, and combined inhibition of both pathways was model (58). However, because evidence suggests that required to suppress these processes (14). In contrast, we AKT1 mutation can promote resistance of BRAF-mutated show that single-agent inhibition of either BRAFV600E or melanomas to vemurafenib, AKT inhibitors might fore- PI3K signaling was sufficient to inhibit both protein syn- stall the onset of drug resistance in patients (42, 43). 0 thesis and melanoma cell proliferation, but that combined Although alternative effectors of PI3 -lipid signaling in pathway blockade led to more robust suppression of prolif- melanomagenesis remain to be elucidated, there are 0 eration. Interestingly, single-agent inhibition of mTORC1/ numerous proteins with PH, PX, or FYVE PI3 -lipid– 2 with PP242 inhibited cell proliferation as potently as the binding domains that are potential candidates (59). blockade of either BRAFV600E or PI3K signaling, indicating Indeed, parsing out which of these effectors is important the potential importance of mTORC1/2 signaling as an for the cooperation of oncogenic BRAFV600E and PI3K effector of cooperating BRAFV600E and PI3K activity for signaling will help to decipher the underlying biochemical melanoma cell proliferation. and signaling mechanisms that exist in the melanoma cell Mechanistically, it remains unclear how BRAFV600E or and could lead to the identification of new potential PI3K influences the phosphorylation of rpS6 and 4E-BP1. It biomarkers and molecular targets for the treatment of is possible that BRAFV600E signaling influences mTORC1 patients with melanoma.

Figure 7. Pharmacologic inhibition of BRAFV600E or PI3K signaling in melanoma cells with mutated or amplified AKT. A, M249 (BRAFV600E, PTENNull, AKT2Amp) and M262 (BRAFV600E, PTENWT, AKT1E17K/Amp) melanoma cells were treated with DMSO (CTL) or inhibitors of BRAFV600E (1 mmol/L GSK2118436/BRAFi1), MEK1/2 (1 mmol/L PD0325901/MEKi1), PI3K (5 mmol/L GDC-0941/PI3Ki1), or AKT (5 mmol/L MK-2206/AKTi1 or 5 mmol/L GSK690693/AKTi2) with cell proliferation assessed for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as mean SEM (n ¼ 3). B, in parallel, these cells were plated at a 30% confluency and cultured in the continuous presence of DMSO or inhibitors of BRAFV600E (BRAFi1), MEK1/2 (MEKi1), PI3K (PI3Ki1) or AKT (AKTi1 or AKTi2) for 4 (M249) or 6 (M262) days with viable cells fixed and stained with crystal violet. C, M249 and M262 cells were treated with DMSO (CTL) or inhibitors of BRAFV600E (BRAFi1), MEK1/2 (MEKi1), ERK1/2 (ERKi), PI3K (PI3Ki1 or 5 mmol/L BKM-120/PI3Ki2) or AKT (AKTi1 or AKTi2) for 24 hours and cell lysates were analyzed by immunoblotting with the indicated antibodies.

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Disclosure of Potential Conﬂicts of Interest protein synthesis and m7GTP cap-binding assays and advice on the regulation of M. McMahon has commercial research grant from Novartis and Plexxicon and is a protein translation, and Kevan Shokat (UCSF) and Jack Taunton (UCSF) for DG2, consultant/advisory board member of AbbVie. No potential conﬂicts of interest were PP242, and FMK as well as Drs. Meenhard Herlyn (Wistar Institute) and Antoni Ribas disclosed. (UCLA) for providing melanoma cell lines. The authors also thank their many colleagues in the private sector for providing compounds and information on their use: Leisa Johnson and Lori Friedman (Genentech) for GDC-0941; Tona Gilmer and Authors' Contributions Kirin Patel (Glaxo-Smith-Kline) for GSK2118436 and GSK1120212; Steven Town- Conception and design: J.M. Silva, M. McMahon son, Heike Keilhack, and Ahmed Samatar (Merck & Co.) for MK-2206 and Development of methodology: J.M. Silva, M. McMahon SCH772984; Janet Lyle, Darrin Stuart, and Emmanuelle di Tomaso (Novartis) for Acquisition of data (provided animals, acquired and managed patients, provided BKM-120; and Fei Su (formerly of Roche) for PLX-4032. facilities, etc.): J.M. Silva, C. Bulman Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- tational analysis): J.M. Silva, M. McMahon Grant Support Writing, review, and/or revision of the manuscript: J.M. Silva, M. McMahon This work was supported by grants from the Melanoma Research Alliance, an NIH/ Administrative, technical, or material support (i.e., reporting or organizing data, NCI R01 CA176839 (to M. McMahon), and an Institutional Research and Academic constructing databases): J.M. Silva Career Development Award (IRACDA; to J.M. Silva). Study supervision: M. McMahon The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Acknowledgments 18 U.S.C. Section 1734 solely to indicate this fact. The authors thank all the members of the McMahon laboratory for advice and guidance on this project with special thanks to Victoria Marsh Durban and Marian Received May 8, 2013; revised December 11, 2013; accepted December 27, 2013; Deuker, their colleagues Craig Stumpf and Davide Ruggero (UCSF) for assistance with published OnlineFirst January 14, 2014.

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www.aacrjournals.org Mol Cancer Res; 12(3) March 2014 463

BRAFV600E Cooperates with PI3K Signaling, Independent of AKT, to Regulate Melanoma Cell Proliferation

Jillian M. Silva, Christina Bulman and Martin McMahon

Mol Cancer Res 2014;12:447-463. Published OnlineFirst January 14, 2014.

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