LETTER dot:10.1038/nature09454 Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma Hoa Nguyen'Kam V.Zhang,Dean R.Artis nger,Fei Su Brian Higins Raman lyer,Kurt D'Andre B-RAF is the most frequently mutated protein kina e in humar related dimer (Fis 1).As pre iously described for the related RAF 实网 ano in that med a wides sis salt- Glu600 and Ly 07g trials usinga rystalline formulation of PLX4032(ref.5).In tra dos her exp not show tumour er drug expos harma okinetics in activity. d测gaiodbadtngdogpoachhec daily oral dos d approach allow DfB-RAF B-RAFCYS0OE) F(V6 aring colore De B-RA 200M as scen at 20 mg kg on ation ficr ma of ally affected were loun d th d beagle dogs were osed for 28 days with in easing dose d for the reatedB-RAFnb duration,26 weeks atsand 1 dogs The rat exposuresx tive site 名0 .37232.USA The University of lexas M.D ork New York 10065 USA tPr MA 02114.USA 596 I NATURE I VOL 467 1 30 SEPTEM millan Publishers Limited.All rights reserved LETTER doi:10.1038/nature09454 Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma Gideon Bollag1 , Peter Hirth1 , James Tsai1 , Jiazhong Zhang1 , Prabha N. Ibrahim1 , Hanna Cho1 , Wayne Spevak1 , Chao Zhang1 , Ying Zhang1 , Gaston Habets1 , Elizabeth A. Burton1 , Bernice Wong1 , Garson Tsang1 , Brian L. West1 , Ben Powell1 , Rafe Shellooe1 , Adhirai Marimuthu1 , Hoa Nguyen1 , Kam Y. J. Zhang1 , Dean R. Artis1 , Joseph Schlessinger2 , Fei Su3 , Brian Higgins3 , Raman Iyer3 , Kurt D’Andrea4 , Astrid Koehler3 , Michael Stumm3 , Paul S. Lin1 , Richard J. Lee3 , Joseph Grippo3 , Igor Puzanov5 , Kevin B. Kim6 , Antoni Ribas7 , Grant A. McArthur8 , Jeffrey A. Sosman5 , Paul B. Chapman9 , Keith T. Flaherty4 {, Xiaowei Xu4 , Katherine L. Nathanson4 & Keith Nolop1 B-RAF is the most frequently mutated protein kinase in human cancers1 . The finding that oncogenic mutations in BRAF are com￾mon in melanoma2 , followed by the demonstration that these tumours are dependent on the RAF/MEK/ERK pathway3 , offered hope that inhibition of B-RAF kinase activity could benefit mela￾noma 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 xeno￾grafts4 . Toxicology studies confirmed a wide safety margin consist￾ent 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 sub￾stantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumour regressions. At higher drug exposures afforded by a new amorphous drug formulation4,5, 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 mela￾noma patients treated at an oral dose of 960 mg twice daily5 . These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity. PLX4032 belongs to a family of mutant B-RAF kinase inhibitors discovered using a scaffold-based drug design approach6 . The crystal￾lography-guided approach allowed optimization of a compound with modest preference for the mutated form of B-RAF (B-RAF(V600E)) in comparison to wild-type B-RAF. Supplementary Table 1 summarizes the differential ability for PLX4032 to inhibit the activity of over 200 kinases. PLX4032 displays similar potency for B-RAF(V600E) (31 nM) and c-RAF-1 (48 nM) and selectivity against many other kinases, including wild-type B-RAF (100 nM). Whereas the vast majority of kinases are minimally affected, several were found that were also inhib￾ited at ,100 nM concentrations in biochemical assays; to date, inhibi￾tion of these non-RAF kinases such as ACK1 (also known as TNK2), KHS1 (also known as MAP4K5) and SRMS has not been tested in cellular assays. As previously demonstrated for the related B-RAF inhib￾itor PLX4720 (ref. 6), the biochemical selectivity of PLX4032 translates to cellular selectivity: potent inhibition of ERK phosphorylation and cell proliferation occurs exclusively in BRAF-mutant cell lines4 . PLX4032 was co-crystallized with a protein construct that contained the kinase domain of B-RAF(V600E). PLX4032 (Fig. 1a) binds in the active site of one of the protomers in the non-crystallographic-symmetry related dimer (Fig. 1). As previously described for the related RAF inhibitor PLX4720 (PDB ID: 3C4C)6 , the PLX4032-bound protomer adopts the DFG-in conformation to enable the formation of a unique hydrogen bond between the backbone NH of Asp 594 and the sulfo￾namide nitrogen of PLX4032 (Fig. 1b). In addition, PLX4032-binding causes an outward shift in the regulatory aC helix, which may explain why the effect of PLX4720 and PLX4032 on RAF dimerization is in stark contrast to other RAF inhibitors such as AZD-628 and GDC- 0879 (Fig. 1c)7 . The apo-protomer displays the DFG-in conformation with the activation loop locked away from the ATP-binding site by a salt-bridge between Glu 600 and Lys 507 (Fig. 1d). In BRAF(V600E)-mutant xenograft studies, PLX4032 demonstrated dose-dependent inhibition of tumour growth, with higher exposures resulting in tumour regression (Fig. 2a and ref. 4). Efficacy could be demonstrated in cell lines and xenografts bearing either homozygous or heterozygous BRAF mutations. By contrast, no effect was observed on melanoma xenograft growth if both BRAF alleles were wild-type4,6. Due to their consistent pharmacokinetics in rodents, PLX4032 and PLX4720 were prioritized over a panel of related compounds that all had similar activities in vitro and in vivo. For further drug develop￾ment, PLX4032 was chosen (over PLX4720) because its pharmacoki￾netic properties scaled more favourably in beagle dogs and cynomolgus monkeys. In order to estimate PLX4032 exposures (as defined by AUC0–24, the area under the plasma concentration time curve over the dosing period of 24 h) that correlated with tumour response, conventionally formu￾lated daily oral doses of PLX4032 were administered in the BRAF(V600E)-bearing colorectal cancer COLO205 xenograft model. In this model, tumour growth inhibition was modest at 6 mg kg21 (AUC0–24 , 50 mM h), tumour stabilization was seen at 20 mg kg21 once daily (QD) (AUC0–24 , 200 mM h), and significant tumour regressions were observed at 20 mg kg21 twice daily (BID) (AUC0–24 , 300 mM h). BRAF(V600E)-bearing melanoma xenograft models, including NCI￾LOX and COLO829 are also sensitive to PLX4032 (ref. 4). Rats and beagle dogs were dosed for 28 days with increasing doses up to 1,000 mg kg21 day21 , and no toxicity was detected at any dose level. Likewise, no adverse effects were detected in a standard battery of safety pharmacology studies. Subsequent toxicology studies of longer duration, 26 weeks in rats and 13 weeks in dogs, further confirmed the tolerability of the compound. This safety profile was achieved in spite of very high compound exposures, reaching 2,600 mM h in rats and 820 mM h in dogs. The rat exposures exceeded those that were effective in patients (see below). Importantly, no histological changes were observed in the skin in any animal at any dose or duration of treatment, contrasting to results observed with other RAF inhibitors7 . 1 Plexxikon Inc., 91 Bolivar Drive, Berkeley, California 94710, USA. 2 Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA. 3 Roche Pharmaceuticals, 340 Kingsland Street, Nutley, New Jersey 07110, USA. 4 Departments of Medicine and Pathology and Laboratory Medicine, Abramson Cancer Center, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. 5 Vanderbilt University, 2220 Pierce Avenue, 777 PRB, Nashville, Tennessee 37232, USA. 6 The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 7 University of California, Los Angeles, 100 UCLA Medical Plaza, Los Angeles, California 90095, USA. 8 Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne 3002, Australia. 9 Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA. {Present address: Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA. 596 | NATURE | VOL 467 | 30 SEPTEMBER 2010 ©2010 Macmillan Publishers Limited. All rights reserved