| Reference | [1]. Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):3041-6. doi: 10.1073/pnas.0711741105. Epub 2008 Feb 19.<br />
Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.<br />
Tsai J(1), Lee JT, Wang W, Zhang J, Cho H, Mamo S, Bremer R, Gillette S, Kong J, Haass NK, Sproesser K, Li L, Smalley KS, Fong D, Zhu YL, Marimuthu A, Nguyen H, Lam B, Liu J, Cheung I, Rice J, Suzuki Y, Luu C, Settachatgul C, Shellooe R, Cantwell J, Kim SH, Schlessinger J, Zhang KY, West BL, Powell B, Habets G, Zhang C, Ibrahim PN, Hirth P, Artis DR, Herlyn M, Bollag G.<br />
Author information: (1)Plexxikon, Inc., 91 Bolivar Drive, Berkeley, CA 94710, USA.<br />
BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.<br />
DOI: 10.1073/pnas.0711741105 PMCID: PMC2268581 PMID: 18287029 [Indexed for MEDLINE]<br />
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[2]. Cancer Cell. 2016 Sep 12;30(3):485-498. doi: 10.1016/j.ccell.2016.06.024. Epub 2016 Aug 11.<br />
An Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling.<br />
Karoulia Z(1), Wu Y(2), Ahmed TA(1), Xin Q(2), Bollard J(3), Krepler C(4), Wu X(1), Zhang C(5), Bollag G(5), Herlyn M(4), Fagin JA(6), Lujambio A(3), Gavathiotis E(7), Poulikakos PI(8).<br />
Author information: (1)Department of Oncological Sciences, Department of Dermatology, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. (2)Department of Biochemistry, Department of Medicine, Albert Einstein Cancer Center, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (3)Department of Oncological Sciences, Liver Cancer program, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. (4)Molecular and Cellular Oncogenesis Program, Tumor Microenvironment and Metastasis Program, and Melanoma Research Center, The Wistar Institute, Philadelphia, PA 19014, USA. (5)Plexxikon Inc., Berkeley, CA 94710, USA. (6)Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. (7)Department of Biochemistry, Department of Medicine, Albert Einstein Cancer Center, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address: [email protected]. (8)Department of Oncological Sciences, Department of Dermatology, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address: [email protected].<br />
Erratum in Cancer Cell. 2016 Sep 12;30(3):501-503.<br />
Comment in Pigment Cell Melanoma Res. 2017 Mar;30(2):177-178.<br />
The complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms of resistance to these drugs, but a unified mechanistic model has been lacking. Here we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC helix stabilized by inhibitor, whereas inhibitor-induced RAF priming and dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect of RAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAF(V600E) cancers, in which first-generation RAF inhibitors have been ineffective.<br />
DOI: 10.1016/j.ccell.2016.06.024 PMCID: PMC5021590 PMID: 27523909 [Indexed for MEDLINE]<br />
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[3]. Nature. 2010 Dec 16;468(7326):973-7. doi: 10.1038/nature09626. Epub 2010 Nov 24.<br />
Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.<br />
Nazarian R(1), Shi H, Wang Q, Kong X, Koya RC, Lee H, Chen Z, Lee MK, Attar N, Sazegar H, Chodon T, Nelson SF, McArthur G, Sosman JA, Ribas A, Lo RS.<br />
Author information: (1)Division of Dermatology/Department of Medicine, UCLA's Jonsson Comprehensive Cancer Center, 52-121 CHS, Los Angeles, California 90095-1750, USA.<br />
Comment in Nature. 2010 Dec 16;468(7326):902-3. Nat Rev Cancer. 2011 Jan;11(1):2.<br />
Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.<br />
DOI: 10.1038/nature09626 PMCID: PMC3143360 PMID: 21107323 [Indexed for MEDLINE]<br />
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[4]. Nature. 2010 Sep 30;467(7315):596-9. doi: 10.1038/nature09454.<br />
Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma.<br />
Bollag G(1), Hirth P, Tsai J, Zhang J, Ibrahim PN, Cho H, Spevak W, Zhang C, Zhang Y, Habets G, Burton EA, Wong B, Tsang G, West BL, Powell B, Shellooe R, Marimuthu A, Nguyen H, Zhang KY, Artis DR, Schlessinger J, Su F, Higgins B, Iyer R, D'Andrea K, Koehler A, Stumm M, Lin PS, Lee RJ, Grippo J, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, Chapman PB, Flaherty KT, Xu X, Nathanson KL, Nolop K.<br />
Author information: (1)Plexxikon Inc., 91 Bolivar Drive, Berkeley, California 94710, USA. [email protected]<br />
Comment in Cancer Cell. 2010 Oct 19;18(4):301-2. Nat Rev Drug Discov. 2010 Nov;9(11):841.<br />
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.<br />
DOI: 10.1038/nature09454 PMCID: PMC2948082 PMID: 20823850 [Indexed for MEDLINE]<br />
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[5]. Curr Opin Investig Drugs. 2010 Jun;11(6):699-706.<br />
PLX-4032, a small-molecule B-Raf inhibitor for the potential treatment of malignant melanoma.<br />
Smalley KS(1).<br />
Author information: (1)The Moffitt Cancer Center, Programs of Molecular Oncology and Cutaneous Oncology, 12902 Magnolia Drive, Tampa, FL, 33612, USA. [email protected]<br />
PLX-4032 is a small-molecule, orally available B-Raf kinase inhibitor being developed by Plexxikon Inc and Hoffman-La Roche Ltd for the treatment of cancers harboring activating BRAF mutations. The primary focus of development is in melanoma (> 50% harbor activating BRAF mutations) with other solid tumors, such as colorectal carcinoma (> 10% harbor BRAF mutations), also under investigation. Purified kinase assays have demonstrated that PLX-4032 and its related analogs are highly potent inhibitors of B-Raf activity, with 3-fold selectivity for the V600E mutation over the wild-type kinase. In preclinical models, PLX-4032 and its analogs inhibited the growth of BRAFV600E-positive melanoma cell lines both in vitro and in vivo. In phase I clinical trials, PLX-4032 was well tolerated and objective responses were observed in several patients with BRAFV600E-positive tumors. Responses correlated well with inhibition of intratumoral phospho-ERK and cell proliferation, and reductions in fluorodeoxyglucose uptake on PET scanning. A preliminary analysis of this phase I data suggested that progression-free survival was approximately 7 months, and phase II and III clinical trials are now underway. These studies provide the proof-of-concept for B-Raf as a therapeutic target in melanoma.<br />
PMID: 20496265 [Indexed for MEDLINE]
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