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RAF inhibitors that evade paradoxical MAPK pathway activation


Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling1. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma2,3,4,5. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers6,7,8,9. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation10,11,12. Here we have identified next-generation RAF inhibitors (dubbed ‘paradox breakers’) that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.

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Figure 1: Paradox breakers dissociate MAPK pathway inhibition from activation.
Figure 2: Molecular mechanisms of paradox breakers.
Figure 3: The structural determinant of paradox breakers can be transferred to another chemical series to drastically alter its biological profile.

Accession codes

Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4XV1 (BRAFV600E-PLX7904), 4XV9 (BRAFWT-PLX5568), 4XV3 (BRAFV600E-PLX7922) and 4XV2 (BRAFV600E-dabrafenib). Microarray data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE71109.


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X-ray diffraction data were collected at beamline ALS 8.3.1 at the Advanced Light Source (Lawrence Berkeley National Laboratory) and the Stanford Synchrotron Radiation Lightsource (a directorate of the SLAC National Accelerator Laboratory).

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Authors and Affiliations



C.Z., P.H., and G.B. designed the study and analysed data; W.S., J.Z., J.L., H. Cho, G.W., S.S., P.W., and M.N. designed and synthesized compounds, C.Z., Y.Z., T.E., and P.N.I. contributed to compound design, and G.T. and D.F. crystallized and collected data; Y.Z. and W.W. processed and refined X-ray data; E.A.B., Y.M., G.H., and B.M. performed assays; A.M., E.L., L.S., G.V., H.R., and P.N.I. performed absorption, distribution, metabolism, and excretion (ADME) and toxicity assays and formulation; H.N. conducted cloning and microarray experiments; R.S., H. Carias, and B.P. purified proteins; G.H., J.T., and B.W. assisted in pharmacology study design; J.W. managed compound inventory and platting; P.S.L., K.N., and P.N.I. were involved in overall study design; C.Z. and G.B. wrote the paper with input from the other authors.

Corresponding authors

Correspondence to Chao Zhang or Gideon Bollag.

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Competing interests

All authors are employees of Plexxikon Inc.

Extended data figures and tables

Extended Data Figure 1 Differential effects of PLX7904 and vemurafenib on MAPK signalling.

PLX7904 (black) and vemurafenib (red) show similar potency to block pERK signalling in human BRAFV600E melanoma cell COLO829 (a); but in RAS activated human melanoma cell line IPC-298 (NRASQ61L) (b) and human colorectal carcinoma cell line HCT116 (KRASG13D) (c), vemurafenib paradoxically activates MAPK signalling while PLX7904 causes negligible pERK increase. d, Expanded view of the pERK curves showing that PLX7904 inhibits pERK at high concentrations in three RAS mutant cell lines, with apparent IC50 (IC50app) values in the 100 μM range. Therefore, paradox breakers are not expected to affect the MAPK pathway in normal tissues (either paradoxical activation or inhibition) at therapeutic concentrations. The pERK curves were generated using an AlphaScreen assay. Mean ± s.d., n = 5 independent experiments.

Extended Data Figure 2 Gene expression analysis of B9 cells in response to either vemurafenib or PLX7904 treatment (both at 1 μM concentration).

a, b, Hierarchical clustering of the 236 Affymetrix mouse gene probes (see Supplementary Table 2 for a complete list) that were upregulated (a) or downregulated (b) by vemurafenib (233 probes) or PLX7904 (4 probes). The single overlap, Cyp1b1, and four representative MAPK pathway-responsive genes as well as three genes that encode EGFR ligands are marked. Two independent experiments are shown. MAPK pathway response genes Spry2, Fos, and Egr1 were upregulated by vemurafenib. The corresponding human genes are known to be suppressed by vemurafenib in BRAFV600E mutant human melanoma36. Opposing changes in expression were also observed with the Id2 gene. c, Changes in the messenger RNA levels of four EGFR ligands (amphiregulin, HB-EGF, TGF-α, and epiregulin) along with EGFR itself in B9 cells treated with vemurafenib or PLX7904. All four EGFR ligands abundantly expressed in B9 cells were induced by vemurafenib, but the expression of EGFR and other ERBB family members remained unchanged.

Extended Data Figure 3 EGFR ligands may mediate vemurafenib-induced cuSCC.

ELISA assays demonstrate increased levels of amphiregulin (a) and TGF-α (b) proteins in the supernatants and HB-EGF (c) in the cell lysates of B9 cells after vemurafenib treatment for 48 h. PLX7904 does not induce the expression of EGFR ligands. Like vemurafenib, exogenous amphiregulin (d), TGF-α (e), and HB-EGF (f) promote the anchorage-independent growth of B9 cells. B9 cells grown in soft agar were treated with EGFR ligands at the indicated concentrations for 3 weeks. Error bars, s.d. (ac), s.e.m. (df); n = 5 (ac) and 6 (df) independent experiments.

Extended Data Figure 4 Effect of BRAF inhibitors on EGFR signalling.

a, EGFR signalling measured by levels of phosphorylated EGFR and AKT after a brief (10 min) exposure of serum-starved B9 cells to supernatant collected from B9 cells treated with vemurafenib for the indicated time. b, Pre-treatment with EGFR inhibitor erlotinib (ERL) inhibited EGFR signalling induced by supernatants from vemurafenib (VEM)-treated B9 cells. Serum-starved B9 cells were pre-treated with 3 μM erlotinib before starting a 10 min exposure to the supernatants. Supernatants were collected from B9 cells treated with vemurafenib or PLX7904 for 3 days. c, Erlotinib inhibits the soft agar colony forming capacity of vemurafenib in B9 cells. Panels a and b are representative of results from three independent experiments. Error bars in c, s.e.m.; n = 6 independent experiments. Full scans of western blot data are presented in Supplementary Figure 1.

Extended Data Figure 5 Comparison of inhibitor-bound BRAF structures.

a, Perfect alignment between vemurafenib and PLX7904-bound BRAF structures (backbone root mean squared deviation 0.22 Å). b, An overlay of the structures of BRAF bound to four inhibitors: sorafenib, PLX5568, vemurafenib, and PLX7904 (colour schedule same as c). c, Outward movement of αC helix in response to different inhibitors. From sorafenib to PLX5568 to vemurafenib, the degree of outward shift correlates with increasing ERK pathway inhibition index (Table 1). d, Close-up view showing the Leu505 side-chain position in the four structures. PLX7904 pushes the tip of Leu505 side-chain away by 1 Å from its position in the vemurafenib-bound structure.

Extended Data Figure 6 The regulatory spine (R-spine) in BRAF.

a, b, R-spine refers to four conserved hydrophobic residues that form a column in the active state of a kinase, and the distortion or disassembly of the spine marks the transition to an inactive state22. The term was introduced using PKA as the template, and the four residues that compose the R-spine included Leu95, Leu106, Tyr164, and Phe185 (PKA numbering). The corresponding residues in BRAF are Leu505, Phe516, His574, and Phe595 (rendered here in spheres). Tyr164 of PKA, which is a histidine (for example, His574 in BRAF) in most other kinases, forms hydrogen bonds with the backbone of the DFG motif and packs against the side chain of DFG Phe185 (the corresponding residue in BRAF is Phe595). In the BRAF structure, Leu567 from αE helix also makes direct hydrophobic contacts with Phe595, an interaction that is conserved across the kinome. Leu567, Phe595, Leu505, along with another hydrophobic residue Ile527 that packs against Leu505, form a column (dubbed here as R-spine′) with an axis tilted 45° from that of the R-spine. Analyses of published kinase structures show that all four R-spine′ residues could be involved in kinase inhibitor binding whereas the two outer residues of R-spine rarely make direct contacts with inhibitors. Therefore, R-spine′ is more relevant for studying inhibitor-induced conformational change in kinases.

Extended Data Figure 7 Vemurafenib-resistant cells remain relatively sensitive to paradox breakers.

a, pMEK and b, growth IC50 curves (mean ± s.d., n = 5 experiments) for vemurafenib and PLX7904 in the SKMEL-239 parental cell line and a representative vemurafenib-resistant clone (C3) that expresses a spliced variant of BRAFV600E promoting dimerization.

Extended Data Table 1 Data collection and refinement statistics

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Supplementary Information

This file contains Supplementary Tables 1-3, Supplementary References and a Supplementary Figure showing uncropped scans. (PDF 1016 kb)

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Zhang, C., Spevak, W., Zhang, Y. et al. RAF inhibitors that evade paradoxical MAPK pathway activation. Nature 526, 583–586 (2015).

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