RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.

BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

The EGF receptor Hokey-Cokey.

In cancer, the epidermal growth factor (EGF) receptor (EGFR) can be activated by mutations that disrupt the inactive conformation and allow the active conformation to predominate. Structural studies have elucidated the molecular events that lead to EGFR activation and shown that small-molecule anti-EGFR drugs can bind to either the inactive or the active conformation of the kinase domain. In this issue of Cancer Cell, Yun et al. present 12 crystal structures of the wild-type or mutant forms of the EGFR kinase domain bound to four different ligands. This study will prove invaluable to those developing novel anti-EGFR drugs.

Potent BRAF kinase inhibitors based on 2,4,5-trisubstituted imidazole with naphthyl and benzothiophene 4-substituents.

The RAS-RAF-MEK-ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine-threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-{2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl} benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC(50)=190 nM and with cellular GI(50)=2100 nM, and 6-{2-[4-(2-dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-naphthalen-1-ol (1q) with IC(50)=9 nM and GI(50)=220 nM.

Small molecule inhibitors of BRAF in clinical trials.

Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically.

Toward the Scale-Up of a Bicyclic Homopiperazine via Schmidt Rearrangement and Photochemical Oxaziridine Rearrangement in Continuous-Flow.

The scale-up of a chiral bicyclic homopiperazine of pharmaceutical interest was investigated. The outcome and safety profile of a key batch ring-expansion step via Schmidt rearrangement was improved using continuous-flow chemistry. The selectivity of nitrogen insertion for the ring expansion was improved via an alternative photochemical oxaziridine rearrangement under mild conditions, which when converted to continuous-flow in a simple and efficient flow reactor allowed the first photochemical scale-up of a homopiperazine.

Novel tricyclic pyrazole BRAF inhibitors with imidazole or furan central scaffolds.

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.

2-Aminomethylene-5-sulfonylthiazole Inhibitors of Lysyl Oxidase (LOX) and LOXL2 Show Significant Efficacy in Delaying Tumor Growth.

The lysyl oxidase (LOX) family of extracellular proteins plays a vital role in catalyzing the formation of cross-links in fibrillar elastin and collagens leading to extracellular matrix (ECM) stabilization. These enzymes have also been implicated in tumor progression and metastatic disease and have thus become an attractive therapeutic target for many types of invasive cancers. Following our recently published work on the discovery of aminomethylenethiophenes (AMTs) as potent, orally bioavailable LOX/LOXL2 inhibitors, we report herein the discovery of a series of dual LOX/LOXL2 inhibitors, as well as a subseries of LOXL2-selective inhibitors, bearing an aminomethylenethiazole (AMTz) scaffold. Incorporation of a thiazole core leads to improved potency toward LOXL2 inhibition via an irreversible binding mode of inhibition. SAR studies have enabled the discovery of a predictive 3DQSAR model. Lead AMTz inhibitors exhibit improved pharmacokinetic properties and excellent antitumor efficacy, with significantly reduced tumor growth in a spontaneous breast cancer genetically engineered mouse model.

Author Correction: Lysyl oxidase drives tumour progression by trapping EGF receptors at the cell surface.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anti-metastatic Inhibitors of Lysyl Oxidase (LOX): Design and Structure-Activity Relationships.

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase that cross-links collagens and elastin in the extracellular matrix and is a critical mediator of tumor growth and metastatic spread. LOX is a target for cancer therapy, and thus the search for therapeutic agents against LOX has been widely sought. We report herein the medicinal chemistry discovery of a series of LOX inhibitors bearing an aminomethylenethiophene (AMT) scaffold. High-throughput screening provided the initial hits. Structure-activity relationship (SAR) studies led to the discovery of AMT inhibitors with sub-micromolar half-maximal inhibitory concentrations (IC50) in a LOX enzyme activity assay. Further SAR optimization yielded the orally bioavailable LOX inhibitor CCT365623 with good anti-LOX potency, selectivity, pharmacokinetic properties, as well as anti-metastatic efficacy.

Novel inhibitors of the v-raf murine sarcoma viral oncogene homologue B1 (BRAF) based on a 2,6-disubstituted pyrazine scaffold.

BRAF, a serine/threonine kinase, plays a key role in the development of certain types of cancer, particularly melanoma. 2-(3,4,5-Trimethoxyphenylamino)-6-(3-acetamidophenyl)-pyrazine, 1, was identified as a low micromolar (IC 50 = 3.5 microM) BRAF inhibitor from a high-throughput screen of a library of 23000 compounds. This compound was chosen as the starting point of a program aimed at developing inhibitors of mutant (V600E)BRAF. We have already reported on the optimization of the trimethoxyphenylamino moiety of 1. In this paper, we describe the synthesis of a series of compounds derived from 1 with the purpose of optimization of the pyrazine central core and the phenylacetamido moiety in order to increase the potency against (V600E)BRAF compared to CRAF. The biological activity of the new inhibitors was assessed against mutant (V600E)BRAF in vitro. Several compounds were identified with IC 50s of 300-500 nM for (V600E)BRAF, and all compounds that were assessed showed selectivity for (V600E)BRAF compared to CRAF by 5-->86-fold.

Long functionalized poly(ethylene glycol)s of defined molecular weight: synthesis and application in solid-phase synthesis of conjugates.

A concise synthesis of long-chain poly(ethylene glycol) (PEG) of defined molecular weight up to 29 ethyleneoxy units is described. These PEG diols were converted in a two-step synthesis into Fmoc-protected PEG amino acids, suitable as long linkers and compatible with solid-phase peptide synthesis. Long PEG chains (MW > 1000) can be readily synthesized with this method, which has the advantage of defined single molecular weight products over the comparable commercial polymers. The application of these PEG linkers to the synthesis of peptide-PEG-folate conjugates on a solid support was investigated. A method for the solid support synthesis of the targeting component of the conjugate, folic acid-cysteine, was developed, resulting in improved yields with respect to literature methods. The assembly of the peptide, PEG linker, and targeting group on solid support resulted in the synthesis of a conjugate of defined molecular weight and structure.

Lysyl oxidase drives tumour progression by trapping EGF receptors at the cell surface.

Lysyl oxidase (LOX) remodels the tumour microenvironment by cross-linking the extracellular matrix. LOX overexpression is associated with poor cancer outcomes. Here, we find that LOX regulates the epidermal growth factor receptor (EGFR) to drive tumour progression. We show that LOX regulates EGFR by suppressing TGFß1 signalling through the secreted protease HTRA1. This increases the expression of Matrilin2 (MATN2), an EGF-like domain-containing protein that traps EGFR at the cell surface to facilitate its activation by EGF. We describe a pharmacological inhibitor of LOX, CCT365623, which disrupts EGFR cell surface retention and delays the growth of primary and metastatic tumour cells in vivo. Thus, we show that LOX regulates EGFR cell surface retention to drive tumour progression, and we validate the therapeutic potential of inhibiting this pathway with the small molecule inhibitor CCT365623.

Novel inhibitors of B-RAF based on a disubstituted pyrazine scaffold. Generation of a nanomolar lead.

B-RAF, a serine/threonine kinase, plays an important role in the development of certain classes of cancer, especially melanoma. As a result of high-throughput screening of a 23,000 compound library, 2-(3,4,5-trimethoxyphenylamino)-6-(3-acetamidophenyl)pyrazine, 1, was identified as a low micromolar (IC(50) = 3.5 microM) B-RAF inhibitor. This compound was chosen as the starting point of a program aimed at producing potent inhibitors of B-RAF. We have synthesized a series of 40 novel compounds, which involved extensive modifications to the 2-(3,4,5-trimethoxyphenylamino) moiety (ring A) of 1. Their biological profiles against isolated B-RAF and mutated B-RAF in a cellular assay have been determined. These efforts led to the identification of two compounds exhibiting activities lower than 800 nM against B-RAF.

V599EB-RAF is an oncogene in melanocytes.

The oncogenic version of B-RAF, (V599E)B-RAF, is found in approximately 70% of human melanomas. However, the role that this oncogene plays in melanoma is unclear because (V559E)B-RAF is also found in approximately 80% of benign nevi. We have examined the role of oncogenic B-RAF in the early stages of melanoma by expressing (V599E)B-RAF in cultured melanocytes. In these cells, (V599E)B-RAF induced constitutive mitogen activated ERK-activating kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, 12-O-tetradecanoylphorbol-13-acetate-independent growth, and tumorigenicity in nude mice. Intriguingly, in RAS-transformed melanocytes, B-RAF depletion did not block MEK-ERK signaling or cell cycle progression. Similarly, B-RAF depletion blocked MEK-ERK signaling in human melanoma cells harboring oncogenic B-RAF, but not in melanoma cells harboring oncogenic RAS. Thus, although B-RAF can act as a potent oncogene in the early stages of melanoma by signaling through MEK and ERK, it is not required for this signaling in RAS-transformed melanocytes due to innate redundancy within the pathway. These findings have important implications for future therapeutic strategies.

B-RAF is a therapeutic target in melanoma.

B-RAF is a serine/threonine-specific protein kinase that is mutated in approximately 70% of human melanomas. However, the role of this signalling molecule in cancer is unclear. Here, we show that ERK is constitutively activated in melanoma cells expressing oncogenic B-RAF and that this activity is required for proliferation. B-RAF depletion by siRNA blocks ERK activity, whereas A-RAF and C-RAF depletion do not affect ERK signalling. B-RAF depletion inhibits DNA synthesis and induces apoptosis in three melanoma cell lines and we show that the RAF inhibitor BAY43-9006 also blocks ERK activity, inhibits DNA synthesis and induces cell death in these cells. BAY43-9006 targets B-RAF signalling in vivo and induces a substantial growth delay in melanoma tumour xenografts. Our data demonstrate that oncogenic B-RAF activates ERK signalling, induces proliferation and protects cells from apoptosis, demonstrating that it is an important therapeutic target and thus provides novel strategies for clinical management of melanoma and other cancers.

Paradox-breaking RAF inhibitors that also target SRC are effective in drug-resistant BRAF mutant melanoma.

BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.

Structure-activity relationship in cationic lipid mediated gene transfection.

Non-viral synthetic vectors for gene delivery represent a safer alternative to viral vectors. Their main drawback is the low transfection efficiency, especially in vivo. Among the non-viral vectors currently in use, the cationic liposomes composed of cationic lipids are the most common. This review discusses the physicochemical properties of cationic lipids, the formation, macrostructure and specific parameters of the corresponding formulated liposomes, and the effect of all these parameters on transfection efficiency. The optimisation of liposomal vectors requires both the understanding of the biological variables involved in the transfection process, and the effect of the structural elements of the cationic lipids on these biological variables. The biological barriers relevant for in vitro and in vivo transfection are identified, and solutions to overcome them based on rational design of the cationic lipids are discussed. The review focuses on the relationship between the structure of the cationic lipid and the transfection activity. The structure is analysed in a modular manner. The hydrophobic domain, the cationic head group, the backbone that acts as a scaffold for the other domains, the linkers between backbone, hydrophobic domain and cationic head group, the polyethyleneglycol chains and the targeting moiety are identified as distinct elements of the cationic lipids used in gene therapy. The main chemical functionalities used to built these domains, as well as overall molecular features such as architecture and geometry, are presented. Studies of structure-activity relationships of each cationic lipid domain, including the authors', and the trends identified by these studies, help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery, and therefore the rational design of new improved cationic lipids vectors capable of achieving clinical significance.

Synthesis of novel cationic lipids: effect of structural modification on the efficiency of gene transfer.

A series of novel cationic lipids was designed and synthesized in an effort to understand the importance of the various structural features with respect to transfection efficiency. Particular attention has been paid to the hydrophobic domain and the cationic headgroup. An efficient method of synthesizing asymmetric diether lipids is described, using alkyl chains ranging from C(12) to C(18) and the unsaturated oleyl group. The ternary formulations including the diether lipid 3beta-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol) and dioleoyl phosphatidylethanolamine (DOPE) were up to 10-fold more efficacious in in vitro assays than the DC-Chol/DOPE control. The shorter and most asymmetric diether lipids performed the best. The chemical nature and basicity of the headgroups have been varied by the coupling of the four naturally occurring amino acids with cationic side chains-arginine, histidine, lysine, and tryptophan. Transfection efficiency was highest for arginine/lysine derivatives, with binary formulations containing the amino acid derivative alone and DOPE proving superior.

Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT.

Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2 (CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered CPG2 (stCPG2(Q)3) or control beta-galactosidase, are assessed. The effect of various structural features on stability, kinetics of activation, and biological activity is discussed. The p-methylamino prodrugs are the most stable compounds from this series, with the largest cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-[N-[4'-Bis(2' '-iodoethyl)aminophenyl]-N'-methylcarbamoyloxymethyl]phenylcarbamoyl-l-glutamic acid, compound 39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing beta-galactosidase. An additional six compounds show better cytotoxicity differential than the published N-[4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl]-l-glutamic acid (CMDA) prodrug.

BRAF inhibitors induce metastasis in RAS mutant or inhibitor-resistant melanoma cells by reactivating MEK and ERK signaling.

Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF-ERK (extracellular signal-regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)-ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor-resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.