Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma

Abstract

B-RAF is the most frequently mutated protein kinase in human cancers. The finding that oncogenic mutations in BRAF are common in melanoma, followed by the demonstration that these tumours are dependent on the RAF/MEK/ERK pathway, offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients. Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity. Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts. Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032 (ref. 5). In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment. This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumour regressions. At higher drug exposures afforded by a new amorphous drug formulation, greater than 80% inhibition of ERK phosphorylation in the tumours of patients correlated with clinical response. Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily. These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.

Fig 1. Three-dimensional structure of PLX4032 binding to B-RAF^V600E

A: The chemical structure of PLX4032. B: Structure highlights the interactions of azaindole with the kinase hinge and the sulfonamide with the DFG loop, with F595 rendered in balls and other key protein residues shown as sticks. C: The structure of the asymmetric dimer of B-RAF^V600E is shown with the PLX4032-protomer bound to PLX4032 colored yellow (consistent with panel B). The surface outline of the other protomer (blue) is shown lightly shaded. Highlighted residues are R509 to reflect its role in anchoring the dimer and F595 to show that both protomers are in the DFG-in state. The α_c-helix shown in magenta is overlaid on the PLX4032-bound protomer to show its typical configuration in an unoccupied protomer; the binding of PLX4032 causes a shift of the α_c-helix as noted by the arrow. D. Magnified view of the salt bridge between Lys-507 and Glu-600 that helps prevent compound binding to the apo protomer.

Fig 2. Effect of PLX4032 on COLO205 xenograft tumor growth

Tumor volume measurements of mice treated by oral gavage with the indicated doses of PLX4032 or vehicle (n=10 for all groups, error bars indicate standard error) are shown. A. Administration in conventional formulation occurred daily. Exposures measured on day 7 are shown. At the 6, 20 and 20 BID doses, 1/10, 1/10, and 8/10 animals achieved CR, respectively. B. Administration in the MBP formulation occurred twice daily. At the 25 mg/kg BID dose (blue), 7/10 animals achieved CR and 3/10 animals achieved PR; at the 75 mg/kg BID dose (red), all animals achieved CR.

Fig 3. Semi-quantitative immunohistochemistry in paired tumor biopsies

Matched baseline and day 15 tumor samples are at the same magnification; the measurement bar is 70 μm. A. Representative IHC for Ki67 and pERK staining is shown for patient 12. B. Representative IHC for Ki67, pERK and H&E staining is shown for patient 42. The arrow indicates tumor breakdown with macrophages engulfing the released melanin in the day 15 sample. C. Summary graph showing correlation of reduction in cytoplasmic pERK with tumor responses (data from Supplementary Table 2). D. Summary graph indicating weak correlation of reduction in nuclear pERK with tumor responses.

Fig 4. Representative PET scans for patients taken pre-dose and following 2 weeks of dosing with PLX4032

Each of these image pairs demonstrates significant reduction in FDG uptake following PLX4032 treatment. Note that tumor regressions were later documented for each of these patients: best responses were 70% for patient 45, 70% for patient 59, 68% for patient 61 and 37% for patient 69.