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ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma

Abstract

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1,2,3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

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Fig. 1: Belvarafenib is a potent RAF dimer inhibitor.
Fig. 2: Generation of belvarafenib-resistant cell lines results in ARAF mutations.
Fig. 3: ARAF mutations confer belvarafenib resistance in a kinase- and dimer-dependent manner.
Fig. 4: Clinical data confirm belvarafenib activity in BRAFV600E- and NRAS-mutant melanoma and the mechanism of resistance to monotherapy.

Data availability

The coordinates of the BRAFKD–belvarafenib complex have been deposited in the Protein Data Bank (PDB) with accession code 6XFP. Raw data from exome sequencing were deposited in the European Genome-phenome Archive (EGA) hosted by EBI, with accession number EGAS00001005086. An overview of the clinical protocol (also found at https://clinicaltrials.gov/) and details of patient demographics, subject information and dose information are included in the Supplementary Information. Information on the full kinase selectivity data, cell line profiling IC50 values, raw gels for all western blot figures, and all mouse experiments have been provided. Source data are provided with this paper.

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Acknowledgements

We thank patients, their families, and investigators for participation in this trial. We also thank members of the Hanmi and Genentech clinical study teams. We are grateful to S. A. Foster and S. Gendreau for discussions and critical reading of the manuscript, and B. N. Park at NALA CnT and J. S. Lim at the Clinical Research Center of Asan Medical Center for medical writing assistance. We thank the BioMolecular Resources (BMR) group at Genentech for construct generation. We thank the Next Generation Sequencing (NGS) group at Genentech for support with exome sequencing. We thank the gCell and Genentech Cell Screening Initiative (gCSI) groups at Genentech for cell line validation and screening.

Author information

Affiliations

Authors

Contributions

S.M. conceived the project, and T.W.K. coordinated the clinical trial. I.Y. and F.S. led the project, designed experiments, and interpreted the results. J.L., Y.S.H., S.J.S. and T.W.K. were the lead clinical investigators on the study, collected plasma samples from patients for ctDNA analysis, and reviewed and interpreted the clinical computerized axial tomography scans. S.M. and I.Y. wrote the manuscript. A.R.M., F.S., J.L. and T.W.K. provided revisions of the manuscript. F.S., I.Y., A.R.M., A.V., N.P.D.L., Y.S.N., Y.-H.H., I.B., E.L., J.Y., X.Y., E.S., D.D.C., T.H., S.E.M. and J.S. established the experimental systems, performed laboratory experiments, and analysed the results. J.Y. and J.S. conducted the structural studies, and J.S. conducted the structural analysis. Z.M., C.K., M.T.C. and R.P. acquired and analysed the in vitro sequencing data. C.K., M.T.C. and R.P. conducted the bioinformatics analyses. J.-S.K., K.-P.K., Y.J.K., H.S.H., S.J.L., S.T.K. and M.J. were clinical investigators on the study. Y.-H.H., Y.S.N., M.C. and O.H. established the trial design, analysed the clinical data, and interpreted the results. H.-S.L., M.N. and S.S. analysed the clinical pharmacokinetic data and interpreted the results. L.Z. and Y.Y. analysed the ctDNA data.

Corresponding authors

Correspondence to Tae Won Kim or Shiva Malek.

Ethics declarations

Competing interests

Hanmi Pharmaceutical Co., Ltd. funded the clinical study and assisted in preparation of the manuscript. S.M., I.Y. and Y.Y. are employees and stockholders of Genentech/Roche, and inventors of the patent application on belvarafenib. F.S., A.R.M., A.V., C.K., J.Y., N.P.D.L., E.L., L.Z., X.Y., E.S., D.D.C., T.H., Z.M., S.E.M., J.S., M.T.C., R.P., M.N. and S.S. are employees and stockholders of Genentech/Roche. T.W.K. is an inventor of the patent application on belvarafenib and received research funding from Sanofi-Aventis. J.L. is a consultant for Oncologie and Seattle Genetics and received research funding from AstraZeneca, Merck Sharp & Dohme, and Lilly. J.-S.K. is a stockholder of Dae Hwa Pharmaceutical and a consultant for Lilly and CJ Healthcare, provided expert testimony for CJ Healthcare, received honoraria from Merck, CJ Healthcare, Lilly, Boehringer Ingelheim, AstraZeneca, and Dae Hwa Pharmaceutical, and received research funding from AstraZeneca, Boehringer Ingelheim, Sanofi, Lilly, CJ Healthcare, Hanmi Pharmaceutical, Chong Kun Dang Pharmaceutical, Ono Pharmaceutical, Pfizer, Novotech, Astellas Pharma, Merck, Aslan Pharmaceuticals, Alphabiopharma, Yuhan, MSD, and Il-Yang Pharmiceutical. Y.-H.H. and Y.S.N. are employees of Hanmi Pharmaceutical and inventors of the patent application on belvarafenib. M.C. and O.H. are employees of Hanmi Pharmaceutical. The remaining authors have no conflicts of interest to disclose.

Additional information

Peer review information Nature thanks Rene Bernards, Helen Rizos and Frank Sicheri for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Belvarafenib effectively inhibits NRAS-mutant melanoma cells.

a, Cell lysate treated with 10 μM belvarafenib or DMSO for 1 h before performing thermal-shift CETSA assay. b, Inhibition of pMEK by belvarafenib after treatment for 24 h in A549 cells engineered to express a single RAF isoform. Ratio of phosphorylated and total MEK plotted after treatment. Data are mean ± s.e.m., n = 2 replicates. c, Representation of RAF inhibitor binding for vemurafenib (left) or belvarafenib (right). d, e, Inhibition of pMEK by belvarafenib or vemurafenib after 24 h in Hec-1-A BRAFnull cells transiently transfected with BRAF(V600E) or BRAF(V600E/E568K). Ratio of phosphorylated and total MEK plotted. Data are mean ± s.e.m., n = 2 replicates. fh, MAPK signalling in SK-MEL-28 (f), SK-MEL-2 (g), or human melanocytes, HEMn-LP (h) after treatment with serial titration of vemurafenib or belvarafenib for 24 h. i, Sensitivity of melanoma cell lines to vemurafenib or belvarafenib. In vitro IC50 screening data for a panel of 25 melanoma cell lines. j, Cell viability of a panel of melanoma cell lines treated with belvarafenib (left) or cobimetinib (right) for 3 days. Data are mean ± s.e.m., n = 2 replicates. k, Clonogenic assay of panel of BRAFV600E, NRAS, and BRAF non-canonical mutant melanoma cells treated with vemurafenib or belvarafenib. Cells were cultured for 8 days then stained with crystal violet. l, Cell viability of Ba/F3 cells expressing RAS-mutations treated with belvarafenib (left) or vemurafenib (right) for 3 days. Data are mean ± s.e.m., n = 6 replicates. m, Confirmation of RAS overexpression in Ba/F3 cells by western blot. Tubulin was stained on a separate gel as a sample processing control. n, Mice with established A431 tumours treated with vemurafenib, dabrafenib or belvarafenib. Data are mean ± s.e.m., n = 6 mice per group. *P = 0.0374 (Belva 30 versus vehicle), **P = 0.0003 (Belva versus vem), one-way ANOVA, followed by Dunnett’s multiple-comparisons test.

Source data

Extended Data Fig. 2 ARAF p.G387 mutations confer mutational specific belvarafenib resistance.

a, Cellular viability of IPC-298 belvarafenib-resistant clones (round 2) treated with belvarafenib for 3 days. Data are mean ± s.e.m., n = 3 replicates. b, Clonogenic assay of IPC-298 parental, BRC9 and BRC9-WO cells treated with increasing concentrations of belvarafenib. Cells were cultured for 8 days then stained with crystal violet. c, Cell growth in IPC-298 parental or BRC9 cells treated with DMSO or 10 μM belvarafenib. d, MAPK signalling in parental, BRC9 or BRC9-WO cells after 24 h treatment with 10 μM belvarafenib. e, Relative ARAF expression in BRC cells compared to parental IPC-298 cells. Data are mean ± s.e.m. (ΔΔCt), n = 4 replicates. ***P < 0.0001, one-way ANOVA, followed by Dunnett’s multiple-comparisons test. f, Cellular viability of BRC or parental cells treated with vemurafenib for 3 days, n = 5 BRCs. Data are mean ± s.e.m., n = 3 replicates. g, IGV view of exome sequencing reads from BRCs around ARAF p.Gly387 (c.1168G>C or c.1169G>A). Wild-type G in orange, mutant C allele in blue, mutant A allele in green. h, Allelic frequency of ARAF p.G387 in a panel of BRCs and BRC9-WO. i, IGV view of exome sequencing reads from IPC-298 parental, BRC9 and BRC9-WO cells around ARAF p.Gly387 (c.1169G>A). j, Clonality of belvarafenib-resistant cells assessed by high-complexity genomic barcoding using Cellecta CloneTracker 50M library. k, Sanger sequencing reads of ARAF p.Gly387 of BRC1-1 and BRC6-3. l, Cellular viability of IPC-298 belvarafenib-resistant clones (Cellecta CloneTracker) treated with belvarafenib for 3 days. Data are mean ± s.e.m., n = 3 replicates. m, n, Deep sequencing nucleotide reads of ARAF p.Gly387 in BRC9, IPC-298 parental, and MelJuso. o, Cellular growth of IPC-298 or BRC9 doxycycline-inducible shRNA knockdown cells against ARAF, BRAF or CRAF for 128 h in the presence of doxycycline treated with DMSO. p, MAPK signalling and RAF protein levels after shRNA knockdown in the presence of doxycycline for 24 h (pre-seeding levels for o, q). q, Cellular growth of doxycycline-inducible IPC-298 or BRC9 cells after shRNA knockdown of ARAF, BRAF or CRAF for 128 h in the presence of doxycycline treated 10 μM belvarafenib. r, MAPK signalling and RAF protein levels in doxycycline-inducible IPC-298 or BRC9 cells after shRNA knockdown of ARAF and treatment with serial titrations of belvarafenib for 24 h in the presence of doxycycline. s, ARAF protein levels after shRNA knockdown, as shown in Fig. 2e.

Extended Data Fig. 3 Corresponding glycine substitutions in BRAF and CRAF confer belvarafenib resistance.

a, Homology model of the ARAF kinase domain (KD) bound to belvarafenib based on the BRAFKD–belvarafenib co-crystal structure. Highlighted residues are mutated in ARAF in belvarafenib-resistant cells. b, Confirmation of BRAF- and CRAF-mutant overexpression in doxycycline-inducible IPC-298 cell lines treated with doxycycline, assayed by Flag–BRAF or Flag–CRAF western blot. c, Cellular viability of doxycycline-inducible IPC-298 cells expressing wild-type BRAF, BRAF(G534D), wild-type CRAF, or CRAF(G426D) and treated with belvarafenib for 3 days ± doxycycline. Data are mean ± s.e.m., n = 3 replicates. d, MAPK signalling in doxycycline-inducible IPC-298 cells expressing wild-type BRAF, BRAF(G534D), wild-type CRAF, or CRAF(G426D) and treated with 0.1, 1 or 10 μM belvarafenib for 24 h. e, Dimer interface of BRAF (PDB code 4MNF). E586 from each BRAF monomer (yellow and cyan) forms hydrogen bonds across the dimer interface with E586 and T589 from the interacting protomer in trans. E586 and T589 are shown as stick models, G534 is shown as spheres. f, MAPK signalling in IPC-298 cells transiently transfected with ARAF(G387D), ARAF(G387D/R362H), ARAF(G387D/E439K), ARAF(G387D/K336M), wild-type ARAF or empty vector (EV). After 24 h, cells were treated with 0.1, 1 or 10 μM belvarafenib for 24 h. g, MAPK signalling in IPC-298 and BRC9 cells after 24-h treatment with 1 μM belvarafenib.

Extended Data Fig. 4 Participant selection, baseline characteristics, and safety for belvarafenib phase I study.

a, b, Flow diagram indicating selection of study participants for dose-escalation (a) and dose-expansion (b) phases. In the dose-escalation phase (a), the full analysis set (FAS) included 67 out of 72 patients. Five patients without any post-dose tumour response assessments due to withdrawal of consent (n = 2), adverse event (n = 2), or progression of disease or lack of treatment effect (n = 1) were excluded from the FAS. In the dose-expansion phase (b), FAS included 59 out of 63 patients. Four patients without any post-dose tumour response assessments due to violation of inclusion/exclusion criteria (n = 1) or confirmed progressive disease or lack of efficacy in the judgement of the investigator (n = 3) were excluded from the FAS. QD, once daily; DLT, dose-limiting toxicity. c, Patient demographics and baseline characteristics. ECOG, Eastern Cooperative Oncology Group; GIST, gastrointestinal stromal tumour. d, Overall safety summary and treatment-emergent adverse events occurring in >10% of patients. TEAE, treatment-emergent adverse event. Result analysed with pooled data from dose-escalation and dose-expansion phases.

Extended Data Fig. 5 Pharmacokinetic properties of belvarafenib.

a, b, Pharmacokinetic assessment of belvarafenib after first administration (day 1) (a) or multiple administration (day 22*) (b). Area under the curve (AUC) was calculated based on the concentrations measured from 0 (pre-dose) to 48 h in cohort 1, and from 0 to 168 h in other cohorts. Mean and coefficient of variation are presented except where indicated (*day 17 for cohort 1). c, Plasma AUC of belvarafenib on multiple doses by cohort. *N = 83, dose-escalation and dose-expansion phases; **plasma AUC of all 450 mg BID patients from dose-escalation and dose-expansion phases. Yellow box indicates target exposure of belvarafenib, 50,000–100,000 μg h l−1 of AUC0~24.

Extended Data Fig. 6 Clinical activity of belvarafenib.

a, Tumour responses in the dose-escalation phase. Best percentage changes in size of target lesions from baseline and specific genetic mutations in each evaluable patient are shown. Others include: NSCLC, bladder, GIST, and sarcoma. Two patients with only non-target lesions at baseline were excluded. b, Tumour response in efficacy-evaluable patients from the dose-escalation and dose-expansion phases. DCR, disease control rate; PFS, progression-free survival; DOR, duration of response; NE, not estimable. Note that BORR (%) = (number of subjects with best overall response as complete or partial response/total number of subjects) × 100. ORR (%) = (number of subjects with confirmed best overall response as complete or partial response/total number of subjects) × 100. DCR (%) = (number of subjects with best overall response as complete or partial response or stable disease/total number of subjects) × 100. cf, Progression-free survival plot of all patients (c) and patients with NRAS-mutant melanoma (d) in dose-escalation phase, and all patients (e) and patients with NRAS-mutant melanoma (f) in dose-expansion phase. g, Patients with NRAS-mutant melanoma with previous immunotherapy treatments. BOR, best overall response; CPI, check-point inhibitor; PD, progression of disease; PR, partial response; SD, stable disease; uPR, unconfirmed partial response. Confirmed partial response is claimed only if patient achieved partial or complete responses at a subsequent time point as specified in the protocol. h, Patients with BRAFV600E-mutant melanoma and CRC with previous BRAFV600E inhibitor treatments. i, j, Responses and treatment durations of patients in dose-escalation (i) and dose-expansion (j) phases. In the dose-escalation and dose-expansion phases, one patient with both BRAF and NRAS mutations enrolled in each phase as indicated in the swimmer plot.

Extended Data Fig. 7 Patient ARAF mutations confirm resistance in BRAF- and NRAS-mutant cell lines.

a, Confirmation of ARAF-mutant overexpression in doxycycline-inducible cell lines (IPC-298, A375, WM-266-4, MelJuso) treated with doxycycline, assayed by Flag–ARAF western blot. be, Cellular viability of IPC-298 (b), A375 (c), WM-266-4 (d), or MelJuso (e) doxycycline-inducible expression of patient derived ARAF mutations (G387N, P462L, G377R) or wild-type treated with belvarafenib for 3 days ± doxycycline. Data are mean ± s.e.m., n = 3 replicates. IC50 values are indicated. f, Confirmation of ARAF double-mutation overexpression in doxycycline-inducible IPC-298 cells treated with doxycycline, assayed by Flag–ARAF western blot. g, Cellular viability of IPC-298 doxycycline-inducible expression of ARAF double mutations treated with belvarafenib for 3 days ± doxycycline. Data are mean ± s.e.m., n = 3 replicates. IC50 values are indicated. h, MAPK signalling in IPC-298 cells transiently transfected with ARAF constructs, treated with serial titration of belvarafenib for 24 h. i, Cellular viability of IPC-298 doxycycline-inducible cells expressing ARAF patient-derived mutations or wild-type ARAF, treated with AZ-628, LXH-254, cobimetinib or GDC-0994 for 3-days ± doxycycline. Data are mean ± s.e.m., n = 3 replicates.

Extended Data Fig. 8 Belvarafenib and cobimetinib combination in NRAS- and BRAF-mutant models delays resistance.

ac, Clonogenic assay of IPC-298 (a), MelJuso (b) or WM-266-4 (c) cells treated with 100 nM belvarafenib, 50 nM cobimetinib, 100 nM belvarafenib + 50 nM cobimetinib, 100 nM vemurafenib, or 100 nM vemurafenib + 50 nM cobimetinib. Cells were cultured for 7, 14 or 21 days then stained with crystal violet. d, Body weight change of mice xenografted with IPC-298 tumours treated with belvarafenib, cobimetinib, or a combination of both. n = 10 mice per group; data are mean ± s.e.m. e, Percentage change in mRNA of DUSP6 and SPRY4 of IPC-298 tumours treated with belvarafenib, cobimetinib, or their combination. n = 5 mice per group; data are mean ± s.e.m.

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Extended Data Table 1 Single point screening data of 187 kinases with 1 μM belvarafenib (percentage inhibition of in vitro kinase panel)
Extended Data Table 2 Data collection and refinement statistics for the BRAFKD–belvarafenib complex

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Supplementary Information

This file contains: Supplementary methods; Clinical study information; Supplementary Figure 1 (Uncropped and unprocessed western blots); and Preliminary full wwPDB X-ray structure validation report.

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Yen, I., Shanahan, F., Lee, J. et al. ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma. Nature 594, 418–423 (2021). https://doi.org/10.1038/s41586-021-03515-1

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