Abstract
The RAF family kinases function in the RAS–ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.
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Data availability
Coordinates and structure factors (6UUO) are available at the Protein Data Bank, www.rcsb.org. The kinase inhibitor competition data used for the protein kinase-biased analysis were also submitted as an incomplete submission to the MassIVE respository (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) and assigned the accession number MSV000085271. The dataset is available at ftp://massive.ucsd.edu/MSV000085271/. Data for the proteome-wide protein level analysis have been deposited as an incomplete submission to the MassIVE repository (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) and assigned the accession number MSV000084551. The dataset is available at ftp://massive.ucsd.edu/MSV000084551/.. The RNA-seq data reported in this paper are available at the Gene Expression Omnibus database with accession number GSE148500. All data supporting the findings are available in the paper and Supplementary Information files. Source data are provided with this paper.
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Acknowledgements
We thank G. Seabrook for technical assistance and access to the NMR Core Facility of Princess Margaret Cancer Center. Research was supported by grants from the Canadian Cancer Society (CCSRI-Impact grant no. 704116 to F.S. and grant no. 706165 to M. Therrien), the Canadian Institutes of Health Research (grant no. FDN 143277 to F.S.; grant no. FDN 388023 to M. Therrien; grant no. FRN 414829 to P. Maisonneuve; grant no. FDN 144301 to A.-C.G.; grant no. FDN 143343 to D.D.), the Ontario Research Fund (grant no. RE08-065 to F.S., D.D., J.W., A.-C.G.) and the Terry Fox Research Institute to F.S., J.W. and A.-C.G. M. Therrien, A.-C.G., D.D. and F.S. are recipients of Tier1 Canada Research Chairs. S.D. is a recipient of the Banting Postdoctoral Fellowship. Proteomics and RNA-seq work were performed at the Network Biology Collaborative Centre at the Lunenfeld-Tanenbaum Research Institute, a facility supported by Canada Foundation for Innovation funding, by the Ontarian Government and by Genome Canada and Ontario Genomics (grant no. OGI-139). Diffraction work conducted at the Northeastern Collaborative Access Team beamlines was funded by the National Institute of General Medical Sciences from the National Institutes of Health (grant no. P41 GM103403) and by an NIH-ORIP HEI grant (grant no. S10 RR029205).
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F.S., P. Maisonneuve, G. Posternak and X.T. conceived the idea and directed the project. G. Posternak, X.T., P. Maisonneuve, T.J., H.L., S.O., T.G.R., A.A., M.P., G. Poda, J.K., K.C., R.A.B., M. Taipale, D.U., J.W., A.-C.G., D.D., R.A.-A., M. Therrien and F.S. designed the experiments and interpreted results. G. Posternak, X.T., P. Maisonneuve, T.J., H.L., S.D., S.O., T.G.R., L.C., K.C., Z.Y., A.A., M.P., G. Poda, P. Mader, D.F.C., C.W., S.M., J.K., B.L. and I.K. performed experiments. F.S., P. Maisonneuve, G. Posternak, H.L. and M. Therrien wrote the manuscript with input from all other authors.
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F.S. and D.D. are founders and consultants for Repare Therapeutics. Host institutions LTRI and OICR have filed a patent relating to the PROTAC molecules presented in this study and their uses (pending USPA No. 62/812,567; applicants are G. Poda, G. Posternak and F.S.). No competing interests were disclosed by the other authors.
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Extended data
Extended Data Fig. 1 Structure of the BRAF kinase domain bound to P4B.
a, Stereo-view of an unbiased |Fo-Fc| electron density map contoured at 2.5σ colored green with P4B and the BRAF kinase domain colored orange and blue, respectively. b, Stereo-view of the superimposition of P4B (orange) bound to BRAF (dark blue) and BI 882370 (light green) bound to BRAF (light blue) (PDB of BRAF:BI 882370 structure is 5CSX). c, d, Comparison of the binding mode of P4B (c) and BI 882370 (d) to the BRAF kinase domain. P4B, BI 882370, glucose and the BRAF contacting residues are shown as sticks. e, f, Flat schematic representation of the binding mode of P4B (e) and BI 882370 (f) to the BRAF kinase domain. Blue eyelashes represent hydrophobic interactions and yellow dashed lines represent hydrogen bonds.
Extended Data Fig. 2 Functional analysis of P4B in cells and in vitro.
a, P4B dose response analysis in A375 cells. Cells were treated for 24 h with P4B, PEG4-BI, or pomalidomide at the indicated concentrations. p27 accumulation is reflective of G1 cell cycle arrest upon BRAF degradation by P4B treatment. b, Immunoblot analysis of BRAF, CRAF, ARAF, KSR1, SRMS, pMEK and MEK levels in A375 cells after 24 h treatment cells with P4B, P4BME and pomalidomide at the indicated concentrations. c, Immunoblot analysis of BRAF, CRAF, pMEK and MEK levels in SKMEL-28 cells after 24 h treatment with P4B, P4BME and Pmd at the indicated concentrations. Tubulin served as a loading control. d, Time course analysis. A375 cells were treated with 100 nM P4B for the indicated time. e, Anti-BRAF immunoblot of in vitro ubiquitinatated full-length BRAF(V600E). Ubiquitination reactions were carried out + /- the indicated components. The white signal at the bottom of blot corresponds to saturation of BRAF signal. Data shown are representative of minimally two independent experiments.
Extended Data Fig. 3 Survey of PROTAC potency in cell lines with different RAS-ERK pathway lesions.
a–j, Dose-dependent inhibition of proliferation (left panel) and phospho-ERK (right panel) following P4B, P4BME and BI 882370* treatment in COLO-205 (a), RKO (b), HT-29 (c), MeWo (d), NCI-H508 (e), NCI-H1755 (f), HCT116 (g), SK-MEL-2 (h), MDA-MB-231 (i), and NCI-H2087 (j) cell lines. Data shown are representative of a minimum of three independent experiments performed in technical duplicates.
Extended Data Fig. 4 Immunoblot survey of PROTAC potency in cell lines with different RAS-ERK pathway lesions.
a-c, Immunoblot analysis of COLO-205 (a), HT-29 (b), and MeWo (c), cell lines after 24 h treatment at the indicated compound concentrations. (d), Washout analysis of A375 cells treated with P4B or non-PROTAC controls. Cells were treated with DMSO (0.1%), P4B (100 nM), P4BME (500 nM), or BI 882370* (500 nM) for 22 h followed by washing in medium to remove compounds. Note that a lower concentration of P4B was employed to enable similar starting points of pathway inhibition for comparison. Cells were incubated in fresh medium for the indicated time periods prior to harvesting and immunoblot analysis. Data shown in d are representative of minimally two independent experiments. e, Comparative immunoblot analysis of P4B, P4BME and BI 882370* function in HCT116 cells after 24 h treatment at the indicated compound concentrations. Data shown are representative of minimally two independent experiments.
Extended Data Fig. 5 Identification of BRAF-driven tumor cell lines sensitive to P4B.
a–c, Dose-dependent analysis of P4B and P4BME treatment on BRAF, MEK, ERK, pMEK and pERK levels in WM266–4 (a), NCI-H1666 (b) and A375-VR (c) cell lines. Data shown are representative of minimally three independent experiments performed in technical duplicates. Quantification of BRAF levels shown below blots.
Extended Data Fig. 6 Influence of RAS expression on the sensitivity of BRAF to degradation by P4B.
a, Over-expression of individual RAS(G12V) mutant isoforms in A375 cells. Lysates of A375 cells transduced with the indicated lentivirus were analysed by immunoblot. b, Immunoblot analyses of A375 cells transduced with the indicated lentivirus after 24 h treatment with P4B at the indicated concentrations. c, The MEF Hras-/-, Nras-/-, Kraslox/lox cell line (KrasWT-lox/lox) was treated with 1 µM 4-OHT for 18 days to knock out the KRAS isoform (RASless). Immunoblot was performed on whole cell extracts using the indicated antibodies. d, Immunoblot analyses of BRAF levels in KrasWT-lox/lox and RASless cells treated for 24 h with the indicated concentration of P4B. e, Dose-dependent inhibition of proliferation of KrasWT-lox/lox cells (left panel) and RASless cells (right panel) with increasing concentration of P4B and P4BME. f, Dose-dependent inhibition of proliferation (left panel) and phospho-ERK (right panel) following P4B and P4BME treatment of RASless cells stably expressing BRAF(V600E). g, Immunoblot analyses of BRAF(V600E) (anti-RM8) and total BRAF (WT + V600E) levels in RASless cells stably expressing BRAF(V600E) and treated for 24 h with the indicated concentration of P4B. Data shown in e and f are representative of minimally three independent experiments performed in technical duplicates.
Extended Data Fig. 7 Analysis of PROTAC induced ternary complexes between the isolated protein kinase domain of BRAF and CRBN/DDB1 in vitro.
a, Binding of CRBN/DDB1 to a fluorescein-labelled pomalidomide probe assessed by fluorescence polarization. b-h, Competitive displacement of fluorescein-labelled pomalidomide probe from CRBN-DDB1 (at 100 nM and 400 nM, respectively, corresponding to 80% saturated binding) by pomalidomide (Pmd) (b), P4B (c), 2 (d), 4 (e), 5 (f), 6 (g), 7 (h) assessed in the absence or in the presence of BRAF16mut(WT) kinase domain, or BRAF16mut(V600E) kinase domain. IC50 values were obtained by fitting FP signal using a variable slope - four parameter equation in GraphPad Prism. Data shown are representative of two independent experiments each performed in triplicate. Data represent mean values ± s.d.
Extended Data Fig. 8 P4B induced ternary complexes in cells.
a, A375 cells ectopically expressing flag-tagged CRBN/DDB1 were treated with P4B or BI 882370* at the indicated experimental conditions. Flag-tagged CRBN was immunoprecipitated using anti-Flag magnetic beads. Lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. * indicates nonspecific signal. Data are representative of two independent experiments. b. GFP-fluorescence images of HEK293T cells expressing CRBN-EGFP-HO-Tag6 and BRAF-EGFP-HO-Tag3 (WT or V600E) at the indicated time point after treatment with 0.5 µM of P4B, BI 882370* and P4BME or equivalent volume of DMSO. Data are representative of two independent experiments. Scale bars represent 10 µm.
Extended Data Fig. 9 Kinase specificity profiles of P4B and BI 882370*.
a, Summary table of the competition assay. A375 cell lysate was preincubated with DMSO (control), CTx-0294885, P4B or BI 882370* prior to CTx-0294885-based affinity purification and mass spectrometric analysis; only proteins (and kinases) passing the 1% FDR threshold set in MaxQuant are reported (as number of unique genes). The threshold for reporting kinase inhibitor efficiency was a minimal reduction to 60% of its initial recovery value at the highest dose (30 µM). b–d, Individual curves for those kinases that meet the 60% threshold of displacement with both P4B and BI 882370* (b), only with P4B (c) or only with BI 882370* (d). Both Swiss-Prot protein names and NCBI gene names are listed.
Extended Data Fig. 10 Proteome wide protein level analysis.
a, Immunoblot analysis of A375 whole cell lysates used in proteome wide analyses in b and c. Data are representative of two independent duplicates. Changes in protein levels between P4B and P4BME treatments (b) and between P4B and DMSO treatments (c). A375 cells were incubated for 24 h with 200 µM of P4B, P4BME or DMSO only, prior to lysis, TMT labeling, and mass spectrometric analysis. Blue and red vertical lines are representative of log2 fold change cutoffs of - and + 1.5 and the dotted horizontal line is representative of a p-value cutoff of 0.01. Coloured points represent proteins that are considered significantly decreased (blue) and increased (red), respectively. Only proteins passing these thresholds are listed.
Supplementary information
Supplementary Information
Supplementary Figs. 1–8, Tables 1–4 and Note, and Source Data Supplementary Figs. 1–3, 5, 7 and 8.
Supplementary Table 5
Lists of genes identified in the transcriptome-wide analyses
Source data
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Posternak, G., Tang, X., Maisonneuve, P. et al. Functional characterization of a PROTAC directed against BRAF mutant V600E. Nat Chem Biol 16, 1170–1178 (2020). https://doi.org/10.1038/s41589-020-0609-7
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DOI: https://doi.org/10.1038/s41589-020-0609-7
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