Dabrafenib; preclinical characterization, increased efficacy when combined with trametinib, while BRAF/MEK tool combination reduced skin lesions.

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAF(V600E) kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAF(V600E)-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.

Discovery of Dabrafenib: A Selective Inhibitor of Raf Kinases with Antitumor Activity against B-Raf-Driven Tumors.

Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf(V600E) mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf(V600E) human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma.

GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition.

PURPOSE: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life. EXPERIMENTAL DESIGN: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models. RESULTS: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27(Kip1/CDKN1B), and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. CONCLUSIONS: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors.

Discovery of a Highly Potent and Selective MEK Inhibitor: GSK1120212 (JTP-74057 DMSO Solvate).

Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) represents a promising strategy for the discovery of a new generation of anticancer chemotherapeutics. Our synthetic efforts, beginning from the lead compound 2, were directed at improving antiproliferative activity against cancer cells as well as various drug properties. These efforts led to the discovery of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethylsulfoxide solvate (GSK1120212, JTP-74057 DMSO solvate; 1), a selective and highly potent MEK inhibitor with improved drug properties. We further confirmed that the antiproliferative activity correlates with cellular MEK inhibition and observed significant antitumor activity with daily oral dosing of 1 in a tumor xenograft model. These qualities led to the selection of 1 for clinical development.

Evaluation of antitumor properties of novel saframycin analogs in vitro and in vivo.

Novel analogs of (-)-saframycin A are described. The analogs are shown to be potent inhibitors of the in vitro growth of several tumor cells in a broad panel and promising as leads for further optimization. The first in vivo studies in a solid tumor model (HCT-116) reveal potent antitumor activity with associated toxicity of daily administration.

Contribution of individual targets to the antitumor efficacy of the multitargeted receptor tyrosine kinase inhibitor SU11248.

Recent achievements in the development of multitargeted molecular inhibitors necessitate a better understanding of the contribution of activity against individual targets to their efficacy. SU11248, a small-molecule inhibitor targeting class III/V receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors, KIT and FLT3, exhibits direct effects on cancer cells as well as antiangiogenic activity. Here, we investigated the contributions of inhibiting individual SU11248 target receptors to its overall antitumor efficacy in tumor models representing diverse signaling paradigms. Consistent with previous results, SU11248 was highly efficacious (frequently cytoreductive) in all models tested. To elucidate the specific contributions of inhibition of PDGF and VEGF receptors to the in vivo efficacy of SU11248, we employed two selective inhibitors, SU10944 (VEGF receptor inhibitor) and Gleevec (PDGF receptor inhibitor). SU10944 alone induced a tumor growth delay in all models evaluated, consistent with a primarily antiangiogenic mode of action. In contrast, Gleevec resulted in modest growth inhibition in tumor models in which the cancer cells expressed its targets (PDGFRbeta and KIT), but was not efficacious against tumors not driven by these target receptor tyrosine kinases. Strikingly, in all but one tumor model evaluated, the antitumor efficacy of SU10944 combined with Gleevec was similar to that of single-agent SU11248, and was greatly superior to that of each compound alone, indicating that the antitumor potency of SU11248 in these models stems from combined inhibition of both PDGF and VEGF receptors. The one exception was a model driven by an activated mutant of FLT3, in which the activity of SU11248, which targets FLT3, was greater than that of SU10944 plus Gleevec. Moreover, SU10944 combined with Gleevec inhibited tumor neoangiogenesis to an extent comparable to that of SU11248. Thus, the potent efficacy of SU11248 in models representing diverse signaling paradigms results from simultaneous inhibition of individual target receptors expressed both in cancer cells and in the tumor neovasculature, supporting the hypothesis that multitargeted inhibitors have the cumulative antitumor efficacy of combined single-target inhibitors.

Hair depigmentation is a biological readout for pharmacological inhibition of KIT in mice and humans.

Deregulated activation of the KIT receptor tyrosine kinase has been implicated in several human cancers and in inflammation, making it an attractive target for therapeutic intervention. Conversely, deficiencies in KIT signaling have been implicated in human and animal hair pigmentation disorders, reflecting a role for KIT in the development and function of melanocytes. The goal of this study was to explore the potential utility of hair depigmentation as a biological readout for systemic inhibition of KIT by SU11248 5-[5-fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide), an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activity through targeting platelet-derived growth factor receptors, vascular endothelial growth factor receptors, KIT, and FLT3. Oral SU11248 treatment induced dose-dependent depigmentation of newly regrown hair in depilated C57BL/6 mice. Similar effects were seen after administration of a KIT-neutralizing antibody. SU11248-induced hair depigmentation was reversible with cessation of treatment. Histological and immunohistochemical evaluation of mouse skin samples supported these observations and revealed that SU11248 has no effect on levels of KIT-positive melanocytes associated with hair follicles, indicating that the inhibitory effect is at the level of melanocyte function rather than their development/survival. Similar hair depigmentation has been noted in several cancer patients receiving SU11248 in phase I trials. Strikingly, patient scalp hair exhibits bands of depigmentation and pigmentation that correspond, respectively, to periods of treatment and dosing rest periods. These data demonstrate that hair pigmentation can serve as a dose-dependent, dynamic, biological readout for KIT inhibition in mice, and, apparently, in humans.

Src family kinase activity is required for signal tranducer and activator of transcription 3 and focal adhesion kinase phosphorylation and vascular endothelial growth factor signaling in vivo and for anchorage-dependent and -independent growth of human tumor cells.

The Src family kinases (SFKs) Src and Yes are believed to play critical roles in tumor growth, angiogenesis, invasion, and dissemination. Using a panel of highly selective and structurally diverse Src inhibitors, we found that phosphorylation of signal transducer and activator of transcription 3 [STAT3 (Y705)] and focal adhesion kinase [FAK (Y861)] was SFK dependent in cultured human colon, breast, lung, and ovarian tumor cells. These findings were reproduced in vivo in target modulation studies using tumors derived from fibroblasts overexpressing activated Src. Additionally, treatment of mice with multiple Src inhibitors resulted in inhibition of phosphorylation of FAK (Y861) and of a putative Src autophosphorylation epitope (Y419) in HT-29 human colon tumor xenografts. Next we pharmacologically examined the requirement for SFKs in asynchronous proliferation of human tumor cells. At concentrations sufficient to selectively inhibit Src, structurally diverse Src inhibitors inhibited growth of cultured human colon, breast, and lung cells on plastic under low serum conditions. In addition, these compounds inhibited anchorage-independent growth of HT-29 human colon tumor cells in soft agar. The role of SFK activity in vascular endothelial growth factor signaling was also evaluated. Inhibition of SFK signaling using structurally distinct Src inhibitors resulted in complete inhibition of vascular endothelial growth factor-dependent vascular permeability in vivo. These data demonstrate that STAT3 (Y705) and FAK (Y861) phosphoepitopes are SFK-dependent in tumor cells and reveal a requirement for SFK function in tumor cell proliferation and vascular permeability.

A selective and oral small molecule inhibitor of vascular epithelial growth factor receptor (VEGFR)-2 and VEGFR-1 inhibits neovascularization and vascular permeability.

Vascular endothelial growth factor (VEGF) is a key driver of the neovascularization and vascular permeability that leads to the loss of visual acuity in diabetic retinopathy and neovascular age-related macular degeneration. Our aim was to identify an orally active, selective small molecule kinase inhibitor of vascular endothelial growth factor receptor (VEGFR)-2 with activity against both VEGF-induced angiogenesis and vascular permeability. We used a biochemical assay to identify 3-[5-methyl-2- (2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-proprionic acid (SU10944), a pyrrole indolinone, which is a potent ATP-competitive inhibitor of VEGFR-2 (Ki of 21 +/- 5 nM). In cellular assays, SU10944 inhibited VEGF-induced receptor autophosphorylation (IC50 of 227 +/- 80 nM) as well as downstream signaling (IC50 of 102 +/- 27 nM). In biochemical assays, SU10944 exhibits potent inhibitory activity against VEGFR-1; weak activity against other related subgroup members, including stem cell factor receptor (SCFR), platelet-derived growth factor receptor beta (PDGFRbeta), and fibroblast growth factor receptor-1 (FGFR-1); and no detectable activity against other protein tyrosine kinases such as epidermal growth factor receptor (EGFR), Src, and hepatocyte growth factor receptor. In cellular assays, the selectivity for SU10944 to inhibit VEGFR is maintained compared with other tyrosine kinases (IC50 for SCFR of 1.6 +/- 0.3 microM, for PDGFRbeta of 30.6 +/- 13.3 microM, for FGFR-1 of >50 microM, and for EGFR of >50 microM). Upon oral administration, SU10944 gave a clear dose response in the corneal micropocket model with an ED50 value for inhibition of neovascularization of approximately 30 mg/kg and a maximum inhibition of 95% at 300 mg/kg. Similarly, upon oral administration in the Miles assay, SU10944 potently inhibited VEGF-induced vascular permeability. Our data indicate that small molecule inhibitors of VEGFR signaling have the potential to ameliorate VEGF-induced neovascularization as well as vascular permeability.

In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship.

One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU11248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU11248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.

SU6668 inhibits Flk-1/KDR and PDGFRbeta in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice.

SU6668 is a small molecule inhibitor of the angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRbeta, and FGFR1. In mice, SU6668 treatment resulted in regression or growth arrest of all large established human tumor xenografts examined associated with loss of tumor cellularity. The events underlying loss of tumor cellularity were elucidated in detail in several tumor models. SU6668 treatment induced apoptosis in tumor microvessels within 6 h of the initiation of treatment. Dose-dependent decreases in tumor microvessel density were observed within 3 days of the first treatment. These changes were accompanied by decreased tumor cell proliferation and increased tumor cell apoptosis. Rapid increases in VEGF transcript levels were seen, consistent with the induction of tumor hypoxia. Using Western blot analyses, we determined that these in vivo antiangiogenic and proapoptotic effects of SU6668 occur at doses comparable to those required to inhibit Flk-1/KDR and PDGFRbeta phosphorylation in tumors. Potent, dose-dependent inhibition of Flk-1/KDR activity in vivo was independently demonstrated using vascular permeability as a readout. These data demonstrate that SU6668-induced inhibition of angiogenic receptor tyrosine kinase activity in vivo is associated with rapid vessel killing in tumors, leading to broad and potent antitumor effects.