1. Volume 30, Issue 3, Pages 485-498 (September 2016)
An Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling 
Zoi Karoulia, Yang Wu, Tamer A. Ahmed, Qisheng Xin, Julien Bollard, Clemens Krepler, Xuewei Wu, Chao Zhang, Gideon Bollag, Meenhard Herlyn, James A. Fagin, Amaia Lujambio, Evripidis Gavathiotis, Poulikos I. Poulikakos 
Cancer Cell 
Volume 30, Issue 3, Pages (September 2016)
DOI: /j.ccell
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2. Figure 1
The OUT Position of the αC Helix Stabilized by Inhibitor Is the Structural Basis of Resistance of Dimeric RAF to RAF Inhibitors
(A) Parental SKMEL239 cells (PAR) and the clone C3 were treated with increasing concentrations of the indicated RAF inhibitors, and cell lysates were immunoblotted for pMEK and pERK.
(B) Bar graph showing the fold differences of IC50s (ERK signaling) between PAR and C3 for each RAF inhibitor, derived from (A).
(C) Bar graph showing growth proliferation difference in GI50 between PAR and C3 for the indicated RAF inhibitors.
(D) Parental (P) or VEM-resistant (R) M249 cells were treated with increasing concentrations of the indicated RAF inhibitors, and cell lysates were immunoblotted for pMEK and pERK.
(E) Bar graph derived from growth proliferation assays after treatment of M249 (P) and M249 (R) cells with the indicated RAF inhibitors.
(F) Ribbon representations of BRAF structures bound to TAK (PDB: KSP4, left) and VEM (PDB: 3OG7, right). The blue oval highlights the predicted steric hindrance for binding of a hypothetical VEM molecule in protomer II (insert).
(G) Overlay of the two protomers for each BRAF structure bound to inhibitor showing the difference in the position of the αC helix (red and yellow for each protomer) stabilized by each inhibitor.
See also Figure S1; Tables S1 and S2.
Cancer Cell  , DOI: ( /j.ccell )
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3. Figure 2
A Chimeric Compound Derived from the αC-IN Inhibitor AZ that Stabilizes the αC Helix in the OUT Position Is an Inefficient Inhibitor of Dimeric RAF
(A) Chemical structures of AZ and AZ-VEM. The headgroup (in red) of AZ-VEM structure contains the propane-1-sulfonamide of VEM instead of the cyanopropan-2-yl benzamide in AZ.
(B) Overlay of the first BRAF protomer from BRAF structures bound to AZ-VEM, AZ, and VEM highlighting the position of αC helix.
(C) Superimposition of the two protomers for the BRAF structure bound to AZ-VEM indicates different position of the αC helix toward the IN or the OUT position.
(D) SKMEL239 cells were treated with the indicated concentrations of AZ, AZ-VEM, or VEM for 1 hr. Cell lysates were immunoblotted with the indicated antibodies.
(E) Bar graph showing growth of SKMEL239, M249, and M397 parental and resistant cells after treatment with the indicated RAF inhibitors.
See also Figure S2.
Cancer Cell  , DOI: ( /j.ccell )
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4. Figure 3
αC-IN RAF Inhibitors Induce the Activated State of RAF by Promoting the Formation of the RAF/RAS-GTP Complex
(A) Lysates from SKMEL2 cells treated with the indicated concentrations of RAF inhibitors were either immunoblotted with the indicated antibodies or subjected to immunoprecipitation with a CRAF antibody followed by immunoblotting for BRAF.
(B) HCT116 (KRASG13D) cells engineered using CRISPR/Cas9 technology to express V5-tagged CRAF from the endogenous promoter were treated with the indicated inhibitors. Lysates were either immunoblotted with the indicated antibodies or subjected to immunoprecipitation with a V5 antibody, and either subjected to kinase assay with recombinant catalytically inactive MEK as substrate or immunoblotted for BRAF to detect CRAF/BRAF interaction.
(C) Lysates from 293H cells ectopically expressing either FLAG-tagged CRAF-Dim or FLAG-tagged CRAFWT were treated with increasing concentrations of AP and immunoblotted with the indicated antibodies.
(D) PC9 cells were treated with the indicated RAF inhibitors for 1 hr and lysates were subjected to either immunoblotting with the indicated antibodies or RAS pulldown assay.
(E) Lysates from SKMEL2 cells treated with either AZ or PB were subjected to immunoprecipitation with a BRAF antibody followed by immunoblotting for RAS and CRAF.
See also Figure S3.
Cancer Cell  , DOI: ( /j.ccell )
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5. Figure 4
The Biochemical Effect of RAF Inhibitor Is the Combined Outcome of Distinct Allosteric Mechanisms
(A) Lysates from PC9 cells treated or not with GEF (100 nM for 1 hr) followed by the indicated RAF inhibitors for 1 hr were immunoblotted with the indicated antibodies.
(B) Lysates from SKMEL2 cells treated with the indicated concentrations of RAF inhibitors were either immunoblotted with the indicated antibodies or subjected to immunoprecipitation with a CRAF antibody followed by immunoblotting for BRAF.
(C) Lysates from SKMEL2 cells treated with AZ, AZ-VEM, or VEM (1 μM for 1 hr) were either immunoblotted with the indicated antibodies or subjected to immunoprecipitation with a BRAF antibody followed by immunoblotting for CRAF or MEK.
(D) Overlay of the BRAF structures bound to RAF inhibitors showing the degree of the αC-helix movement toward OUT or IN position and the position of the side chain of residue R506 stabilized by each inhibitor. PB stabilizes the R506 side chain in an outward position, VEM in a slightly more IN position, followed by DAB and AZ-VEM in a midway position. AZ, TAK, LY, GDC, and SB stabilize R506 in a fully IN position.
(E) SKMEL2 cells were treated with the indicated RAF inhibitors, and lysates were immunoblotted for pMEK and pERK.
(F) SKMEL2 cells were treated with the indicated RAF inhibitors, and the extent of formation of the RAF/MEK complex was determined by co-immunoprecipitation.
(G) Summary of the biochemical effects of RAF inhibitors based on their structural properties. VEM is included in the R506-IN inhibitors because it stabilizes R506 in a slightly more IN position, compared with the fully OUT position stabilized by PB.
See also Figure S4.
Cancer Cell  , DOI: ( /j.ccell )
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6. Figure 5
αC-IN RAF Inhibitors Are Effective Inhibitors of ERK Signaling in Cells Expressing Mutant BRAF Other than V600
(A) PC9 cells ectopically expressing the indicated V5-tagged BRAF mutants were treated with GEF (100 nM for 1 hr) followed by either VEM or TAK for 1 hr. Whole-cell lysates were immunoblotted for pMEK and pERK.
(B) PC9 cells ectopically expressing the indicated V5-tagged BRAF mutants were treated with GEF (100 nM for 1 hr) followed by either VEM or TAK for 1 hr. Whole-cell lysates were immunoblotted for pMEK and pERK.
(C) WM266 (BRAFV600D) cells were treated with increasing concentrations of the indicated RAF inhibitors and cell lysates were immunoblotted for pMEK and pERK.
(D) Cell growth curves of WM266 cells treated with the indicated RAF inhibitors.
(E) SKMEL208 (BRAFG466E) cells were treated with increasing concentrations of the indicated RAF inhibitors. Lysates were immunoblotted for pMEK and pERK.
(F) Cell growth curves of SKMEL208 cells treated with the indicated RAF inhibitors.
(G) NCI-H2087 (BRAFL597VNRASQ61K) were treated with increasing concentrations of the indicated RAF inhibitors. Lysates were immunoblotted for pMEK and pERK.
(H) Cell growth curves of NCI-H2087 cells treated with the indicated RAF inhibitors.
In (D), (F), and (H), data represent mean ± SEM. See also Figure S5.
Cancer Cell  , DOI: ( /j.ccell )
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7. Figure 6
αC-IN RAF Inhibitors Overcome Feedback-Induced Reactivation of BRAF/ERK Signaling and Suppress Growth of Colorectal and Thyroid BRAFV600E Tumor Cells
(A) Lysates from the indicated melanoma (A375, SKMEL239) or colorectal (RKO, HT-29) cell lines were subjected to immunoprecipitation with a CRAF antibody and immunoblotted for BRAF.
(B) The indicated BRAFV600E melanoma (A375, SKMEL28), thyroid (SW176, Hth104, 8505C), or colorectal (HT-29, RKO, SW1417) tumor cells were treated with 2 μM VEM for 24 hr. Cell lysates were subjected to immunoprecipitation with a CRAF antibody and immunoblotted for either BRAF or BRAFV600E.
(C) A375 cells were treated with either VEM or TAK, and reactivation of ERK signaling was monitored for 48 hr by immunoblotting with the indicated antibodies.
(D) RKO cells were treated with either VEM or TAK, and reactivation of ERK signaling was monitored for 48 hr by immunoblotting with the indicated antibodies.
(E) Cell proliferation assays comparing the growth inhibitory effect of VEM or TAK in the indicated melanoma and colorectal cancer cell lines.
(F) 8505C cells were treated with either VEM or TAK, and reactivation of ERK signaling was monitored for 48 hr by immunoblotting with the indicated antibodies.
(G) Cell growth assay comparing the growth inhibitory effect of VEM and TAK in 8505C cells.
In (E) and (G), data represent mean ± SEM. See also Figure S6.
Cancer Cell  , DOI: ( /j.ccell )
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8. Figure 7
Therapeutic Strategies Including αC-IN Inhibitors Are Effective in BRAFV600E Models
(A) The indicated BRAFWTRASWT cells were treated with RAF inhibitors, and inhibition of ERK signaling was determined by immunoblotting.
(B) Crystal violet assay after treatment of human keratinocytes with the indicated RAF inhibitors for 7 days.
(C) Cell growth assays comparing 72-hr treatments of WM1382 cells with the indicated RAF inhibitors.
(D) Colony formation of the indicated BRAFV600E melanoma cells after treatment for 4 weeks with VEM, TAK, or the combination of TAK and VEM. Colonies were stained with crystal violet.
(E) Crystal violet staining of the indicated thyroid or colorectal BRAFV600E-expressing tumor cells after treatment for 7 days with the indicated RAF inhibitors.
(F) Mice bearing the RKO xenografts were treated with PLX4720 (50 mg/kg, once daily), TAK-632 (20 mg/kg, twice daily), or the combination for 24 hr, and tumors were collected after an additional 4 or 24 hr. ERK activity was determined by immunoblotting.
(G) RKO cells were injected subcutaneously into the flanks of nude mice (10 million cells per injection). When tumors reached 100–120 mm3 in size, indicated treatments started. The graph shows the size of each tumor compared with control after 21 days of treatment (p values calculated using unpaired t test).
In (C) and (G), data represent mean ± SEM. See also Figure S7.
Cancer Cell  , DOI: ( /j.ccell )
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9. Figure 8
Model of the Mechanism of Action of RAF Inhibitors in Different Cellular Contexts
(A) Growth factor-induced RAF activation. Under conditions of low RAS-GTP, RAF is cytosolic and inactive (1). Growth factor stimulation upregulates the levels of RAS-GTP, promoting the formation of the RAF/RAS-GTP complex in the membrane, followed by activating CRAFS338 phosphorylation (2) and dimerization for full RAF activation (3).
(B) RAF inhibitor-induced RAF activation and allosteric hindrance. RAF inhibitors promote the activated state of RAF by binding to inactive RAF (1) and promoting its interaction with RAS-GTP (2). CRAF bound to RAS-GTP is phosphorylated at S338 and dimerized (3). Inhibitors that bind to one protomer and stabilize the αC helix closer to the OUT position (shown here) are sterically prevented from binding the second monomer (negative allostery). RAF inhibitors that stabilize the αC helix closer to the IN position will induce potent RAF priming and RAF dimerization, and will show minimal negative allosteric to inhibit the dimer. Conversely, αC-OUT RAF inhibitors will promote less RAF priming and RAF dimerization, but will also be less potent inhibitors of dimeric RAF. Thus, the biochemical effect of a RAF inhibitor on ERK signaling will be the combined outcome of the two mechanisms.
(C) In the context of elevated RAF dimerization and low RAS-GTP, such as in cells expressing splicing variants of BRAFV600E (shown here) or non-V600 mutant forms of BRAF that form dimers, the effect of RAF inhibitors is determined only by negative allostery. (1) Monomeric BRAFV600E is inhibited by RAF inhibitor. (2) Inhibition of the second protomer of dimeric BRAFV600E is allosterically hindered when the first protomer is occupied by inhibitor. INH indicates inhibitor.
Cancer Cell  , DOI: ( /j.ccell )
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