RAS/RAF/MEK Inhibitors in Oncology

1164 Current Medicinal Chemistry, 2012, 19, 1164-1176

0929-8673/12 $58.00+.00 © 2012 Bentham Science Publishers

RAS/RAF/MEK Inhibitors in Oncology

P. Rusconi, E. Caiola and M. Broggini*

Laboratory of Molecular Pharmacology, Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy

Abstract: The RAS/RAF/MEK signaling pathway plays a central role in mediating both proliferation and survival of cancer cells. These

proteins are a group of serine/threonine kinases activated in response to a variety of extracellular stimuli and mediate signal transduction

from the cell surface towards both nuclear and cytosolic targets. In combination with several other signaling pathways, they can

differentially alter phosphorylation status of the transcription factors. A controlled regulation of these cascades is involved in cell

proliferation and differentiation, whereas an unregulated activation of these kinases can result in oncogenesis.

Dysregulation of the RAS/RAF/MEK pathway has been detected in more than 30% of human tumors, however mutations in the MEK1

and MEK2 genes are seldom, so that hyperactivation of MEK1/2 usually results from gain-of-function mutations in RAS and/or

B-RAF.

In addition, alteration of the pathways is often associated with drug resistance in the clinic, such as the case of K-RAS mutant expressing

tumors.

Since RAS protein is a difficult target, alternative ways altering post-translational modifications using farnesyl transferase inhibitors have

been adopted. Drug discovery programs have therefore largely focused on B-RAF and MEK.

In this review we will discuss the most promising strategies developed to target these kinases and the most recent inhibitors facing the

preclinical and clinical setting, also considering their structure-activity relationship (SAR).

Keywords: MAPK inhibitors, RAF, MEK, intracellular signalling, kinases, small molecule inhibitors, ATP competitive, ATP non competitive, allosteric inhibitors, mutant B-RAF.

INTRODUCTION

The RAS/RAF/MEK pathway plays a pivotal role in regulating cell growth and survival. The pathway mediates the signal transduction from the cell surface towards both nuclear and cytosolic targets. It responds to different extracellular stimuli such as the binding of a ligand to its receptor, and is able to cross-talk to several other signal transduction pathways in the cell. It has been demonstrated that this pathway is activated in response to virtually all mitogenic factors [1]. Following activation, transcription of the genes involved in the G1/S transition is promoted in the nucleus [2] and, at the same time, anti-proliferative protein such as Tob1, Foxo3a and p21 are downregulated [3]. The RAS/RAF/MEK pathway also controls many cytosolic factors involved in different cellular functions such as glucose metabolism, apoptosis, protein and nucleotide biosynthesis and cell migration [4]. The ability to control such a wide range of cellular processes is achieved through the interaction with other pathways: one of the most important is the PI3K/AKT/mTOR pathway [5].

The initial response to the extracelullar stimuli consists in activation of RAS in the RAS-GTP bound form, which binds and recruits RAF kinases to the plasma membrane. When anchored to the plasma membrane, RAF is phosphorylated by itself or by other kinases. Activated RAF in turn is able to phosphorylate and activate MEK, which then activates the final downstream effector ERK, that is able to induce the activity of several transcription factors important for cell growth, such as c-myc, c-fos, NFkB and others (Fig. 1).

A controlled regulation of these cascades is fundamental for maintaining cell proliferation and differentiation, whereas an unregulated activation of these kinases can result in oncogenesis.

Dysregulation of the RAS/RAF/MEK pathway has been detected in more than 30% of human tumors. The majority of the tumor-associated mutations are on RAS and RAF genes, while MEK genes are only rarely found mutated in human tumors.

Three distinct RAS proteins (H-RAS, K-RAS and N-RAS), each encoded by different genes have been described. They share high sequence homology and have similar functional domains.

*Address correspondence to this author at the Laboratory of Molecular Pharmacology,

Istituto di Ricerche Farmacologiche “Mario Negri”, via G. La Masa 19, 20156 Milan, Italy; Tel: +39 0239014585; Fax: +39 0239014734; E-mail [email protected]

These proteins do not have a transmembrane domain and need to be post translationally modified -prenylated- by farnesyl transferase to reach the membrane. Mutations in these genes are mostly found at codons 12 and 13 in exon 2 (from 90 to 95% of all the mutations), followed by codon 61 in exon 3. Other possibly relevant, although quantitatively rare, mutations have been found in exon 4. Substitution of glycines at codons 12 and 13 induces a constitutively active form of RAS. K-RAS is predominantly found mutated in colon, lung and pancreatic cancer and is the one - among the RAS family genes - most frequently mutated.

K-RAS mutations have a role either in tumor initiation or in tumor progression. Mutations in K-RAS gene are in fact sufficient to induce tumor formation in mice. At clinical level the presence of K-RAS mutation is associated with a less favorable prognosis, mostly due to resistance to pharmacological treatments.

The inhibition of RAS activity has been obtained targeting the post translational modification of RAS necessary, as already pointed out, for its membrane anchorage.

The farnesyl transferase inhibitors gave interesting preclinical results but failed to get relevant activity in the clinic. It has been reported that this was mainly due to the possibility that geranyl geranyl transferase could act and modify RAS in the absence of farnesyl transferase, hence creating an escape mechanism [6, 7]. The attempts to inhibit both farnesyl and geranylgeranyl transferase ended with an unacceptable toxicity. For this reason the development of RAS inhibitors is at present a less attractive area, while the inhibition of RAS downstream kinases RAF and MEK seems to be more promising.

Also for RAF, three distinct genes, encoding for A-RAF, B-RAF and C-RAF serine/threonine kinase proteins are present in eukaryotic cells. The three genes share sequence homology: they all have a RAS binding domain, necessary for the membrane recruitment, a serine/threonine rich domain, which is essential for binding to regulatory proteins and for activation, and a protein kinase domain located in the C-terminus. The activation of these kinases involves a complex series of steps including translocation to the plasma membrane and the interaction with active RAS after the dissociation from the RAF kinase inhibitory protein, the formation of homo- and heterodimers, several phosphorylation/dephospho-rylation processes on different sites [8]. At least thirteen residues are known to be phosphorylated on RAF kinases and to modulate

RAS/RAF/MEK Inhibitors Current Medicinal Chemistry, 2012 Vol. 19, No. 8 1165

their activity. Some phosphorylation sites are common for the three isoforms [9], while others are specific: S43, S259 and S621 are phosphorylated on C-RAF, S364 and S428 are phosphorylated by AKT on B-RAF. Phosphorylation on S338, Y340 and Y341 is responsible for the activation of C-RAF and A-RAF, whereas in B-RAF only the corresponding residue of S338 (S455) is conserved and constitutively phosphorylated. The tyrosines occupying the other two sites are replaced by aspartic acid (D492 and D493) conferring to the N-terminal region a constant negative charge and an elevated basal kinase activity [10]. Mutations are almost exclusively found in the B-RAF gene: approximately 65 different mutant variants have been found in human tumors, but 90% of the mutations are localized at residue V600. The mutation of valine in this position leads to a constitutive active state.

The principal effectors of RAF kinases are MEK1 and MEK2. They act as dual Serine/Threonine and Tyrosine kinases, that phosphorylate and activate the ERK/MAP kinases ERK -1 and ERK -2. MEK1 and MEK2 are encoded by two distinct genes and differ in their binding to ERKs and in their activation profiles. MEK1 and MEK2 have low sequence homology at the amino-terminal ends. This amino-terminal domain is one of two extended regions of non homology between MEK1 and MEK2 and could be a region involved in differential interaction of MEKs with specific MEK activators or substrates, or subcellular localization. MEK1 and MEK2 are positively regulated by RAF isoforms phospho-rylation on S218 and S222 in their activation loop [11]; it has been reported that, among RAF kinases, B-RAF has the strongest kinase activity on MEK isoforms [8]. The activity of MEK1/2 is regulated by additional phosphorylation/dephosphorylation events as well, as the phosphorylation of Ser386 on MEK1 by ERK, which can inhibit ERK activity [11].

Activating mutations in MEK genes are rarely found in lung carcinomas, melanomas and colon carcinomas with a frequency of about 1% [12]. Despite such a low mutation rate, both in vitro and

in vivo experiments have extensively demonstrated that inhibition of MEK1/2 is a valid approach to suppress proliferation of various leukemic and carcinoma cell lines [13, 14] and to induce significant tumor regression in mouse xenograft models [15].

The next tier in the cascade is represented by ERK1 and ERK2; they are activated by dual phosphorylation, exclusively mediated by MEK1/2 kinases, of a -Thr-Glu-Tyr- motif present in the activation loop. Upon phosphorylation, ERK1/2 activate several transcription factors, including p53, Elk1, c-Jun, c-Fos, Ets1/2, involved in the regulation of proliferation and oncogenic transformation [11].

RAF INHIBITORS

Several anti-cancer agents targeting RAF proteins have been developed and are in various phases of clinical evaluation. Two classes of RAF-inhibitors can be distinguished on the basis of their inhibitory capability: class I and class II [16].

First Generation RAF Inhibitors

Class II kinase inhibitors block RAF with major affinity for its inactive state, thereby preventing kinase activation. Class II compounds are indirect ATP-competitors and are able to inhibit different other kinases (VEGFR, c-KIT, PDGFR).

Sorafenib (Nexavar, BAY43-9006; Bayer, Table 1) is a diphenylurea discovered in 1999 by Bayer and Onyx from a high throughput screening initiated in 1995 on 200,000 compounds with RAF inhibitory activity [17], together with combinatorial chemistry support. From a bis-aryl urea library, a lead compound, a 3-amino isoxazole analogue, was discovered with a C-RAF IC50 of 230 nM. The inhibitory potency of this molecule was then five fold increased by replacing the distal phenyl ring with a 4-pyridyl moiety, which conferred more hydrophilicity to the compound. Other structure-activity relationship (SAR) studies led to the

Fig. (1). Schematic representation of RAS-RAF-MEK signalling cascade.

pRas

R f

GTP

pp

Raf

Mek 1/2

p

Erk 1/2

p

p

Elkcell cycle progression

Erk 1/2p

JunFos AP1

cell survival, protein synthesis

vescicle trafficking

1166 Current Medicinal Chemistry, 2012 Vol. 19, No. 8 Rusconi et al.

replacement of 3-amino-isoxazole group with a phenyl ring and the modification of the distal pyridine ring leading to sorafenib, showing an IC50 of 6 nM in biochemical assays against C-RAF [18]. Initially developed as C-RAF inhibitor, this compound blocks also A-RAF and B-RAF and, less potently than the wild type isoform, the oncogenic V600E B-RAF, as revealed by biochemical and mechanistic cellular assays [19]. Wan et al., showed in their crystallographic study that the distal pyridil group of sorafenib directly interacts with three aminoacids that form the ATP binding region of RAF and that the compound interacts through several hydrogen and hydrophobic bonds with the allosteric site formed by the ‘out’ conformation of the activation loop, thus promoting the shift to the inactive form of RAF kinases [20]. Besides RAF family, sorafenib inhibits also the autophosphorylation of several tyrosine kinase receptors involved in processes like angiogenesis and tumor progression (VEGFR family, PDGFR, Flt-3 and c-KIT) [21].

The activity of this agent was initially tested in vitro by different groups: sorafenib caused the inhibition of MAPK signalling through RAF inhibition in several cell lines, with the exception of KRAS-mutated non small cell lung cancer (NSCLC) cell lines [21]. Garassino et al., reported that different aminoacid substitutions in the same codon of KRAS, which all led to constitutive activation of the small GTPase, had a different impact on H1299 NSCLC cell line sensitivity to sorafenib [22]. McDermott et al., reported that sorafenib did not show a significant reduction on cell proliferation and survival in cell lines bearing B-RAF activating mutations [23].

Other authors observed that sorafenib exerted antiproliferative activity in human colon, breast, ovarian, pancreatic, melanoma and thyroid cancers cell lines [24]. The drug was also described to determine growth inhibition over xenograft tumors derived from these cell lines [21]. Recently it has been showed that this molecule induces cell death also in glioblastoma [25], lymphoma [26] and myeloma cell lines [27].

Sorafenib carries out its antitumoral activity not only by preventing MAPK cascade activation, but also by other mechanisms, most probably exerting potent antiangiogenetic

effects. In fact Wilhelm et al., showed that sorafenib inhibitory activity against Colo-205 xenograft model was not associated with downregulated levels of phosphorylated ERK, but it was rather due to a significant reduction in microvessels area and density [21]. Also, Chang et al., demonstrated that sorafenib inhibited tumor growth in a murine renal carcinoma (Renca) model by disrupting tumor vasculature [28].

Sorafenib was soon established in the clinical setting as a safe and well tolerated drug being mild hand-foot skin reactions, rush and diarrhea the most frequent adverse events (AE) [29]. Since the molecular targets of sorafenib are involved in the etiology of many types of solid tumors, this compound has been evaluated in different advanced malignancies. The most promising results came from trials on renal cell carcinoma patients, in which sorafenib therapy increased the progression free survival (PFS) [30, 31], and studies with patients with hepatocellular carcinoma, in which this agent prolonged the overall survival [32]. Thanks to these evidences, the FDA and the EMEA approved sorafenib for the treatment of these tumors. Unfortunately, sorafenib failed to demonstrate clinical activity as single agent in patients with melanoma [33], where B-RAF has an undisputed role in tumorigenesis. This negative result is due to the drug’s higher affinity for B-RAF inactive conformation. Conversely in melanomas the oncogenic V600E isoform of B-RAF causes a constitutive activation of the kinase.

In these tumors, however, sorafenib could have a chance to show activity in combination with other small molecules or chemotherapy; in fact more than 300 phase II/III clinical trials evaluating sorafenib alone or in combination with other molecules in patients with different solid tumors are ongoing. Overall, the efficacy of sorafenib as cancer therapy seems to be increasingly attributed to its antiangiogenic effects, exerted through the inhibition of VEGFR and PDGFR, rather than its inhibitory activity over the MAPK signaling pathway through RAF inhibition.

Regorafenib (BAY73-4506; Bayer Schering Pharma, Table 1) is a novel oral multikinase inhibitor resulted from a discovery

Table 1. First Generation, Class II RAF Inhibitors: Mode of Action and Status Development

N

OHN

O

NH

HNO

F

FF

Cl

Sorafenib

ATP indirect competitive pan-RAF/ VEGFR/PDGFR/flt-3/c-Kit

inhibitor

Approved by FDA and EMEA for HCC and RCC

N

OHN

O

F

NH

HNO

F

FF

Cl

Regorafenib

ATP indirect competitive pan-RAF/ VEGFR/ PDGFR/ c-Kit/ TIE2/

RET/ FGFR inhibitor

Phase III ongoing

N

N

NH

F

F

F

O

N

N

NH

F

F

F

RAF-265

ATP indirect competitive pan-RAF/ VEGFR/PDGFR /c-Kit inhibitor

Phase II ongoing

Structure not disclosed XL281

pan-RAF allosteric inhibitor

Phase I ongoing


program aimed at optimizing biological properties of the urea class compounds. Its structure differs from that of sorafenib by the addition of a fluorine atom in the phenyl ring; it appears to have a similar but slightly distinct biochemical profile and to be pharmacologically more potent than its precursor [34].

Like sorafenib, regorafenib showed a potent inhibition of kinase activity against the VEGFR family, all RAF isoforms, PDGFR, c-KIT, TIE2, RET and FGFR at nanomolar concentrations in cellular assays. This compound inhibited tumor cell proliferation to various degree, depending on the cell line tested. In GIST 882 and thyroid carcinoma cell lines, where oncogenic activated mutant c-KIT and RET are expressed, regorafenib inhibited cell growth in the nanomolar range [34]. Regorafenib significantly inhibited tumor growth in a wide range of xenograft models, including colorectal (Colo-205), renal cell carcinoma, breast (MDA-MB-231), ovarian and melanoma at doses comparable to the clinical efficacious ones. This compound prevented lung metastasis formation in an orthotopically injected breast cancer model [34].

Similarly to sorafenib the in vivo antitumor activity of this drug is mainly attributed to its antiangiogenetic effects rather than to its inhibition of MAPK signaling; indeed RAF blockade was observed only in a breast cancer model [34].

A phase I clinical trial in patients with colorectal cancer revealed a good tolerability profile and promising clinical results were observed in a phase II study in untreated subjects with metastatic or unresectable melanoma. Regorafenib is now included in 19 phase II clinical trials with patients affected by several malignancies and in 3 phase III trials involving metastatic colorectal cancer and GIST patients.

RAF-265 (CHIR-265; Novartis Pharmaceuticals, Table 1) is an orally bioavailable small molecule derivative of benzazoles that inhibits with high potency and selectivity all RAF isoforms, included V600E B-RAF, and VEGFR-2, c-KIT and PDGFR [35].

In preclinical setting RAF-265 showed 50 to 100-fold more pharmacologic activity than sorafenib against B-RAF mutant melanoma cell lines [36]. RAF-265 demonstrated efficacy in terms of tumor growth inhibition in xenograft models of colorectal cancers and melanoma, where sorafenib resulted only in a stabilization of the disease [37].

The encouraging data obtained in vitro and in vivo gave rise to phase I/II clinical trials: clinical efficacy is also being assessed in combination with ARRY162 - a second generation MEK inhibitor - in adult patients with advanced solid tumors harboring RAS or B-RAF V600E mutations (NCT01352273 clinical trial).

Table 2. Second Generation, Class I RAF Inhibitors: Mode of Action and Status Development

S

O

O

HN

F

FO

N

NH

Cl

Vemurafenib

ATP direct competitive specific V600E B-RAF inhibitor

Approved by FDA for B-RAF V600E-positive inoperable or

metastatic melanoma

S

O

O

HN

F

FO

N

NH

Cl

PLX 4720

ATP direct competitive specific V600E B-RAF inhibitor

Preclinical ongoing

S

O

OHN

F

F

FSN

N

N

NH2

Dabrafenib

ATP competitive specific V600E

B-RAF/ inhibitor

Phase III ongoing

P

OOH

HO

O O

ON

N

N

N

HN

NS

O

O

N

N

ARQ 736

B-RAF allosteric inhibitor

Phase I ongoing


XL281 (BMS-908662; Exelixis, Table 1) is an oral highly selective pan-RAF allosteric inhibitor. This compound exhibited a strong antitumor activity through inhibition of RAF kinase and inactivation of MAPK signaling also in oncogenic RAS-RAF driven models [35].

A phase I clinical trial with 29 patients with different solid tumors revealed that XL281 is generally well tolerated. Currently, a larger phase I study is aimed at determining safety, tolerability, and maximum tolerated dose (MTD) of daily oral administration of XL281 in adult patients with different solid tumors (NCT00451880). The NCT01086267 clinical trial is ongoing to identify a safe and tolerable dose of XL281 in combination with cetuximab. It will also evaluate the tumor response to XL281 when administered alone or in combination with cetuximab in KRAS or B-RAF mutation-positive advanced or metastatic colorectal cancer subjects (www.clinicaltrials.gov).

Second Generation RAF Inhibitors

Recently a second generation RAF inhibitors was developed with the aim of identifying compounds with enhanced selectivity against RAF kinases, in particular the oncogenic form of B-RAF. Class I RAF inhibitors show high affinity for RAF isoforms in their active conformation so they do not require the activation loop in the Asp-Phe-Gly (DFG) ‘out’ conformation. Consequently they are more specific for the V600E oncogenic B-RAF isoform [38], hence more active as anti-cancer agents in tumors driven by this mutation.

Vemurafenib (PLX4032, RO5185426, RG7204; Plexxikon and Roche Pharmaceuticals, Table 2), disclosed by using a scaffold- and stucture-based discovery approach, belongs to 7-azaindole class and contains a difluoro-phenyl-sulfonamide substructural motif. This compound is an orally administered, ATP-competitive RAF inhibitor and it showed the highest selectivity for the V600E B-RAF mutant isoform among more than 70 kinases tested in in vitro and in vivo screenings [29].

Preclinical studies demonstrated that vemurafenib specifically inhibits cell proliferation at nanomolar concentrations, with a concomitant dose-dependent block of MEK and ERK phosphorylation. It also induces cell cycle arrest and apoptosis in cell lines (melanoma, thyroid cancer, colorectal cancer) bearing the V600E B-RAF mutation, but not in wild type B-RAF cell lines and in the healthy cells [39].

In addition, this molecule showed potent antitumor activity in xenograft models that harbored the V600E mutation, where first generation drugs as sorafenib failed to demonstrate any efficacy. In mice implanted with human melanoma cells, complete tumor regression was observed with vemurafenib [40]. In colorectal cancer xenograft models vemurafenib monotherapy was found to be more efficacious than single treatment with capecitabine or bevacizumab: significant tumor growth delay and tumor growth inhibition were described after treatment with vemurafenib in a V600E B-RAF colorectal xenograft model.

Toxicology studies revealed a wide safety margin which allowed the use of crystalline formulation for phase I clinical trials. Overall, vemurafenib resulted well tolerated and 89% of all adverse events were of mild gravity. In addition, the efficacy data were particularly encouraging thus providing the basis for other clinical trials with patients affected by V600E mutation driven melanoma [41].

In particular, the phase III BRIM3 trial (NCT01006980) of vemurafenib for the treatment of V600E B-RAF mutant-positive melanoma was crucial for the approval of this drug in the US. Initiated in January 2010, the study enrolled 675 treatment-naive patients and compared vemurafenib with decarbazine to investigate the co-primary endpoints of overall survival (OS) and PFS (www.clinicaltrials.gov). Interim results highlighted that the trial

met both primary endpoints: vemurafenib reduced the risk of death by 63% and reduced the risk of disease progression of 74% compared to decarbazine [42].

These surprising efficacy data led vemurafenib to be approved by the FDA for the treatment of V600E B-RAF mutation-positive inoperable or metastatic melanoma in August 2011. Other clinical trials of this compound in patients affected by malignant metastatic melanoma are ongoing, in order to test its clinical efficacy in combination with chemotherapeutic drugs or other target-specific small molecules.

Beside melanoma, vemurafenib is being tested for the treatment of thyroid and colorectal malignancies which harbour the oncogenic B-RAF isoform. An open-label, multi-center phase II study involving vemurafenib treatment in patients with metastatic or unresectable papillary thyroid cancer (PTC) positive for the V600 B-RAF mutation and resistant to radioactive iodine is ongoing (NCT01286753); In addition, as vemurafenib is able to cross the blood-brain barrier, two phase II clinical trials in patients with malignant melanoma with brain metastases - who have very few treatment options - are ongoing (www.clinicaltrials.gov).

PLX4720 (Plexxikon, Table 2) is a PLX4032 analog obtained by the replacement of the chloro-phenyl group on the 7-azaindole group with a chlorine atom. PLX4720 demonstrated excellent potency for oncogenic B-RAF, because it preferentially binds to the ‘DGF-in’ (active) conformation of the kinase: in enzymatic assay, it inhibited V600E B-RAF with an IC50 of 13 nM, that is 10-fold lower than the concentrations needed to block wild type B-RAF activity [43]. The selectivity towards the oncogenic form of B-RAF exceeded 100-fold in different cellular systems obtained from different types of solid tumors, as reported by Tsai et al., [43]; furthermore, in cells bearing the V600E mutation, ERK phosphorylation was found to be potently inhibited, whereas it was unaffected in wild type B-RAF cell lines. PLX4720 had an antiproliferative effect on cell lines that express the V600E B-RAF kinase, whereas cell lines expressing wild type B-RAF grew at similar rates compared to the nontreated controls. This agent caused cell cycle arrest and apoptosis after 24h of treatment in highly malignant 1205Lu melanoma cell lines, inherently resistant to pharmacological intervention. Xenograft studies confirmed the results obtained in vitro: PLX4720 caused a nearly complete tumor regression in SCID mice implanted with Colo-205 (colorectal), and 1205Lu and C8161 melanoma cell lines. These results were supported by MAPK signaling inhibition detected by immunohistochemical analysis [43]. Currently, clinical trials using PLX4720 have not been designed yet.

Dabrafenib (SB-590885, GSK2118436; GlaxoSmithKline, Table 2) is a triarylimidazole derivative, extremely selective second generation inhibitor of oncogenic B-RAF kinase. In biochemical assays dabrafenib was able to inhibit all RAF kinases at nanomolar range and proved to be selective for RAF family showing approximately 400-fold higher selectivity for B-RAF, compared to the 91% of all the other kinases tested [44]. Cellular assays demonstrated that dabrafenib selectively inhibits proliferation and ERK phosphorylation only in cell lines that bear the mutation V600E for B-RAF. King et al., showed that dabrafenib caused cell cycle arrest in G1 phase and consequent apoptosis in human colorectal and melanoma cell lines that harbored the oncogenic B-RAF mutation. They demonstrated that this compound inhibits tumorigenesis and anchorage-independent growth in melanoma cells. It also blocks tumor growth of V600E B-RAF melanoma (A375P) and colon cancer (Colo205) human xenografts subcutaneously growing in immuno-compromised mice [44]. Preclinical data demonstrated that this compound could represent an additional small molecule targeting B-RAF, especially in oncogenic B-RAF driven tumors. Indeed several clinical trials have been initiated; overall, phase I studies revealed a good tolerability


profile of the drug, with mild adverse events like headache, nausea, fatigue and vomiting [45]. Currently, dabrafenib is tested for the treatment of malignant melanoma bearing V600E mutation in phase III trials in comparison with standard therapy for this malignancy.

A very interesting phase II trial with dabrafenib started in February 2011 (BREAK MB) to assess its efficacy in melanoma patients with brain metastases (www.clinicaltrials.gov); preliminary but encouraging results, hopefully confirmed by the BREAK MB study, came from a subgroup analysis of a phase I/II trial involving dabrafenib-treated patients bearing solid tumors. Out of ten subjects with brain metastases, nine had reductions in overall tumor size ranging from 20-100% of all brain lesions greater than or equal to 3 mm in diameter before treatment. So in the future this compound could become a therapeutic option for these patients who currently have a very poor prognosis.

ARQ 736 (ArQule, Table 2) (chemical name: (S)- [3- [5- [2- [1- (1-Metyl-1H-pyrazol-3-ylsulfonyl) piperidin-3-ylamino] pyrimidin-4-yl] imidazo [2,1-b] oxazol-6-yl] phenoxy] methyl dihydrogen phosphate) is a potent and selective inhibitor of B-RAF kinase activity and targets tumors harboring B-RAF activating mutation. Unlike other small molecule inhibitors, ARQ 736 acts as an allosteric inhibitor of B-RAF. This represents a potential advantage since allosteric inhibitors usually show higher specificity than competitive inhibitors. Biochemical assays revealed that the compound is able to inhibit all RAF isoform activity at nanomolar concentrations; however it was found to be inactive against VEGFR2. ARQ 736 (and its active metabolite ARQ 680) selectively inhibited cell proliferation in V600E B-RAF positive cells at concentrations ranging from 200 to 300 nM, sparing cell lines with wild type B-RAF or KRAS mutations (IC50 values from 4μM to greater than 10 μM). In preclinical murine models, ARQ

736 treatment determined a 54% growth inhibition of A375 melanoma xenograft following daily administration for 13 days and a 77% tumor growth inhibition following constant administration.

Pharmacodynamics analysis revealed that the drug inhibits the MAPK pathway both in vitro and in vivo, and induces a significant reduction of VEGF secretion, which can contribute to the antitumor activity of this compound [46, 47]. A phase I dose escalation study of ARQ 736 in adult subjects with advanced solid tumors harboring B-RAF and/or NRAS mutations is ongoing in order to assess safety and tolerability as primary endpoints (NCT01225536) (www. clinicaltrials.gov).

MEK INHIBITORS

As already discussed, MEK1 and MEK2 genes are rarely mutated in human tumors. Nevertheless, inhibition of MEK1/2 has been proved to be an efficient antitumor strategy at preclinical level and several compounds are under clinical investigation. Furthermore, ERK1/2 are the only known substrates of MEK1 and MEK2, so targeting MEK1/2 should avoid any off-target effect due to interference with other pathways.

First Generation of MEK Inhibitors

PD98059 (Table 3) was the first MEK1/2 inhibitor to be synthesized in 1995 [48]. This molecule specifically inhibits both MEK1 and MEK2 through a non-competitive mechanism, without altering the activity of a panel of other Ser/Thr kinases [49].

Few years later, cell based assays allowed the identification of other two potent MEK1/2 non-competitive inhibitors: U0126 [50] and Ro 09-2210 [51] (Table 3). All these compounds specifically

Table 3. First Generation of MEK Inhibitors: Mode of Action and Development Status

O

O

O

H2N

PD98059

Allosteric non-competitive MEK inhibitor

Preclinical discontinued

S

H2NS NH2

N

N

NH2

NH2

U0126



Structure not disclosed Ro 09-2210



O

NH

F

FO

HN

ICl

CI-1040


Phase II discontinued


target MEK1 and MEK2 but also show activity against MEK5 and the ERK5 MAP kinase pathway at higher doses. These compounds were extensively used in vitro to characterize the MAPK transduction pathway, but none of them entered clinical trials because of their poor pharmacological features.

CI-1040 (PD184352) was developed starting from the compounds identified during the screening that led to the identification of PD98059 (Table 3). Thanks to its improved selectivity and potency, CI-1040 was the first MEK inhibitor to proceed to clinical testing [14]. Crystallographic analysis established that the non-competitive inhibition of CI-1040 over MEK1/2 is due to the binding of the drug to an allosteric inhibitory cleft, close to the ATP binding site. This binding induces a conformational change in unphosphorylated MEK1/2 that locks the kinase into a catalytically inactive form. The binding pocket was found to be exclusively shared by MEK1 and MEK2 and this accounts for the high degree of specificity found for all MEK inhibitors. Actually MEK5 display a 83% aminoacid identity in the allosteric site with MEK1/2, and this explains why the first inhibitors showed unspecific effects also on this MEK isoform [52].

The high affinity between CI-1040 and the allosteric site is due to the hydrophobic interaction between the cleft and the diphenylamine core of CI-1040. The in vitro IC50 was determined to be 17 nM and the compound also demonstrated to reduce human colon cancer in xenograft models, proving for the first time the anticancer activity for this class of drugs [14].

CI-1040 was tested in phase I trial over 77 patients with metastatic or locally advanced solid tumors. The drug resulted to be

well tolerated, since 60% of patients experienced grade 1 or 2 adverse effects, only 2% were grade 3 and no one grade 4. The most common drug-related toxicities comprised rash, asthenia, diarrhea, nausea and vomiting. Six patients also reported transient visual changes including blurred vision and altered light perception. This Phase I trial also displayed encouraging anticancer properties for CI-1040, indeed one patient had partial response (pancreatic cancer) and 28% of patients achieved stable disease with a median duration of 5.5 months [53]. Based on this result the compound passed on to phase II testing in a multicenter, open-label trial in patients with metastatic or inoperable breast cancer, colon cancer, NSCLC or pancreatic cancer. No patients showed either a complete or partial response and stabilization of the disease (median 4.4 months) was observed in only 8 patients. The reduced antitumor activity, together with other problems related to poor solubility and low bioavailability of the compound determined the discontinuation of the clinical development of CI-1040 [54].

Second Generation of MEK Inhibitors

The diphenylamine core and the hydroxamate side chain were modified to improve the negative pharmacokinetic profile of CI-1040 and many second generation MEK inhibitors were synthesized based on these structural improvements.

PD0325901 was developed from CI-1040, substituting chlorine with fluorine on the diphenylamine core and replacing the cyclopropane substituent on the hydroxamate side chain with a dihydroxypropoxyl group (Table 4). These modifications determined a significant increase in potency, solubility and

Table 4. Second Generation of MEK Inhibitors: Mode of Action and Development Status

O

NH

HN

IF

F

FO

OH

OH

PD0325901


Phase II discontinued

N

N

O

F

HN

Cl

Br

HN

O

OH

AZD6244


Phase II ongoing

SO O

HN

HOOH

O

HN

I

F

F

F

BAY869766


Phase I /II ongoing


bioavailability compared to CI-1040 [55]. The PD0325901 IC50 value was determined to be around the nanomolar range in biochemical assays against purified MEK1/2, and it lowers to subnanomolar concentrations in cell proliferation assays in different tumor lines [15]. In vivo studies showed tumor regression in 70% of mice with colon tumor xenograft treated with maximum tolerated dose (MTD) of 25 mg/kg/day for 14 days; the remaining animals underwent partial regression [56].

PD0325901 moved to clinical trial in 2004 entering a phase I – II design. Phase I portion enrolled 30 patients with advanced cancer, including breast, colon, NSCLC and melanoma, employing a dose-escalating design [57]. The MTD was found to be 30 mg b.i.d (bis in die) and with doses 2 mg b.i.d. ERK1/2 phosphorylation was demonstrated to be effectively suppressed in tumor biopsies. Two partial responses were observed in melanoma patients and other 8 patients achieved stable disease lasting 3-7 months. Toxicities were generally acceptable with 50% of patients showing rash and diarrhea and one third reporting blurred vision.

Phase II portion involved NSCLC patients treated with a dose of 20 mg b.i.d.. However dosing schedule was reduced twice during the trial because of several significant adverse events, especially those involving ocular function and neurological toxicity. Finally the clinical development of PD0325901 was stopped due to safety concern at doses at 10 mg and higher and because of lacking of antitumor activity. Indeed only a 25% reduction in tumor volume was observed in one patient [58].

Array Biopharma developed AZD6244 (Selumetinib/ARRY-142886) and then licensed it to AstraZeneca. The diphenylamine core of CI-1040 was modified with the introduction of a benzymidazole group and a hydroxyethoxyl group was added to the hydroxamate side chain (Table 4). The resulting molecule is a potent, non-competitive MEK1/2 inhibitor. In enzymatic assays AZD6244 exhibits an IC50 of 14 nM against MEK1, without affecting the activity of other 40 kinases when tested up to 10 M. The compound IC50 ranges between 50 and 200 nM in cell proliferation assays employing cell lines with mutated B-RAF, and similar values were obtained in other cell lines with activated K-RAS (200-500 nM) [59]. AZD6244 demonstrated dose dependent antitumor activity in different mouse xenograft models, including colon rectal, pancreatic, liver, skin and lung cancer. Also, tumor regression was shown to correlate with reduction of phospho-ERK1/2 levels in tumor biopsies [60].

Such encouraging results promoted the entrance of AZD6244 in phase I clinical trial in 2004. 57 patients with advanced solid tumors were enrolled and MTD was established at 100 mg b.i.d. None of the patients reported tumor regression and only 9 patients showed disease stabilization for at least 5 months. The compound was generally well tolerated, being grade 1 or 2 rash and diarrhea the most common adverse events (AE). Nevertheless 7 patients suffered blurred vision, similarly to what described for CI-1040 and PD0325901 [61].

So far 48 clinical trials involving this molecule have been initiated, completed or are ongoing. The activity and tolerability of the compound were compared to classical chemotherapeutic drugs in different phase II trials but they all failed to demonstrate the superiority of AZD6244. As regards advanced melanoma, patients were randomized 1:1 to AZD6244 or temozolomide: the two treatment arms did not show any difference neither in progression free survival, nor in overall survival. Anyway 5 out of 6 partial responses reported in the AZD6244 arm were obtained in patients positive for B-RAF mutation, while this correlation between B-RAF mutational status and response was not seen in the temozolomide-treated patients, since only 3 out of 12 partial responses were observed in B-RAF mutated patients. These results are in accordance to what obtained in vitro, indicating that MEK inhibition seems to be more effective on a mutated B-RAF

background [62]. Another phase II trial compared AZD6244 and pemetrexed in NSCLC patients but no difference was observed in the primary disease progression endpoint [63]. Similarly, comparable efficacy was observed for AZD6244 and capecitabine as second/third line treatment in patients with metastatic colon rectal cancer [64]. The best clinical results for AZD6244 were obtained from a preliminary phase II trial recruiting patients with advanced biliary cancer. Out of 29 patients, 3 were responders (one complete response and two partial responses), 14 patients had stable disease and 5 patients had progressive disease. Significantly B-RAF and K-RAS mutations are often found in this type of cancer [65]. Other phase II trials are currently ongoing in a variety of tumor types.

Valeant Pharmaceuticals International synthesized BAY869766 (previously called RDEA119), another non-competitive, CI-1040 derivative MEK inhibitor. The molecule development was undertaken by Ardea biosciences that licensed the compound to Bayer for post-phase I/II development and commercialization. The classic diphenylamine core presents a major modification consisting in a dihydroxypropyl-cyclopropanesulphonamide substituent (Table 4).

BAY869766 is a potent and specific MEK1/2 inhibitor since it showed an IC50 of 19 nM for MEK1 and 47 nM for MEK2 in enzymatic assays and no significant inhibitory activity was seen in other 205 kinases tested. In vitro assays indicated that BAY869766 is highly effective in suppressing cell growth and ERK phosphorylation levels in B-RAF mutated cancer cell lines, displaying IC50 values in the nanomolar range [66]. Good antitumor activity was described for the compound in different mouse xenograft models, being the best result obtained the complete suppression of A375 melanoma tumors when dosed at 50 mg/kg/day. Also, unlike PD0325901, BAY869766 demonstrated to preferentially partition to tumor tissue and not to brain in mouse models [66]. A strong synergistic action is described for BAY869766 and sorafenib in some cell lines; sorafenib targets RAF, c-KIT, PDGFR, VEGFR2 and VEGFR3 [23] but it was demonstrated that its specific activity against RAF is the one critical for the synergistic activity with BAY869766. Indeed BAY869766 has been described to act synergistically with other RAF inhibitors.

So far one phase I clinical trial in patients with advanced solid cancer was completed in the US, reporting no confirmed objective responses; anyway 10 out of 69 patients achieved stable disease with an average duration of 8 months. The MTD was found to be 100 mg b.i.d. and the most common AEs were skin rash, digestive tract disturbances and central nervous system toxicities. Several additional phase I/II trials are ongoing, assessing BAY869766 as single agent or its combination with sorafenib or gemcitabine in different types of cancer.

GSK1120212 (Trametinib), is a structurally novel MEK1/2 inhibitor. The diphenylamine core has been modified by introducing a pyrido-pyrimidin structure with a N-phenylacetamide group as major substituent (Table 5). This compound acts as a non-competitive inhibitor since it was demonstrated to bind to the same allosteric site bound also by the other MEK inhibitors [67].

In vitro and in vivo data proved that GSK1120212 is a potent and highly specific MEK1/2 inhibitor, as the IC50 was shown to be 10 nM in enzymatic assays against MEK1 and around 50 nM in cell proliferation assays over B-RAF or KRAS mutated cell lines. Similarly to other MEK inhibitors the IC50 was higher for cell lines wild type for these signaling proteins. This behavior is due to the fact that cell lines mutated in the RAS-RAF-MEK-ERK pathway are dependent on these genes for tumor proliferation (“oncogene addiction”) and so they are more sensitive to inhibition of the pathway [68]. In vivo results confirmed the efficacy of the compound, as a potent anti-proliferative activity was reported on B-


RAF mutated xenograft models (both melanoma and colorectal cancer).

What distinguishes this compound from all other MEK1/2 inhibitors are the good results obtained in phase I clinical trial. Indeed, durable clinical activity was observed in patients with advanced solid tumors and lymphomas. 84 patients were enrolled in a dose-escalating phase I trial. Out of the 20 evaluable patients with melanoma and known B-RAF mutational status, five partial responses were observed, all with 50% tumor reduction. 11 out of these 20 patients were B-RAF mutated, and this group reported 3 partial responses, 5 stable diseases and 3 progressive diseases. The 9 patients left were wild type for B-RAF and 2 partial responses and 3 stable disease were observed in this group. The MTD was found to be 3 mg/day and no dose limiting effects nor grade 2 toxicities were described [69]. Based on these encouraging results GSK1120212 moved to phase II trial, where its effectiveness is currently being assessed on pancreatic cancer and relapsed or refractory leukemia. Moreover, clinical development of the compound has already reached phase III for malignant melanoma in patients with mutated B-RAF.

Two other MEK1/2 inhibitors were developed starting from the PD0325901 structure: TAK733 (Takeda) and RO4987655 (previously called CH4987655, Chugai/Roche) (Table 5). Both compounds were modified to overcome the metabolic instability of the hydroxamate group, that pharmacokinetic studies showed to be easily hydrolyzed by the metabolic enzymes [70].

The Takeda approach consisted in incorporating the amide into a fused bicyclic core, generating a pyridopyrimidindione system, similar to that of GSK1120212. Thus, the amide group is protected

from hydrolysis and the bicyclic structure provides the molecule an improved conformational rigidity. On the other hand the 2-fluoro-4-iodoaniline substituent, part of the former diphenylamine core, ensures the hydrophobic interaction with the allosteric site on MEK1/2. Also, the carbonyl group on the pyrido ring has the potential to form an H-bond to S212. Further molecular refinements led to the introduction of a dihydroxypropane side chain, allowing additional interaction with L97 and the ATP phosphate [71]. The resulting compound is a highly selective inhibitor, exhibiting an IC50 of 3.2 nM against MEK1/2 in enzymatic assays, without targeting any other kinase, receptor or ion channel with concentrations up to 10 M. The pharmacokinetics of TAK733 was assessed in different preclinical models giving excellent results in all species, since low clearance and high oral bioavailability were always observed. Also the compound revealed good antitumor activity in many mouse xenograft models, including melanoma, colon rectal, NSCLC, pancreatic and breast cancer [71].

TAK733 is currently in phase I clinical trial in patients with advanced solid tumors. The trial started in 2009 to evaluate safety, pharmacokinetics, pharmacodynamics and MTD of the compound, and it is expected to be completed by the first half of 2012.

In order to protect the amide group a different strategy was pursued with RO4987655. Indeed the introduction of a 3-oxo-[1,2]oxazinan-2-ylmetil group at the 5-position of the diphenylamine core was found to strongly improve the metabolic stability of the compound, thanks to the ability of this substituent to perturb the interaction with metabolizing enzymes. Also, the ring structure of this newly introduced side chain develops novel interactions with residues located in the allosteric cleft of MEK1/2,

Table 5. Second Generation of MEK Inhibitors (Continued): Mode of Action and Development Status

O

HN N

N

N

O

OO

HN

I

F

GSK1120212


Phase II ongoing

N

N

N

O

O

NHI

F

F

HO

OH

TAK733


Phase I ongoing

O

HN

O

N

O

O

OH

NH

I

F

F F

RO04987655


Phase I ongoing

Structure not disclosed ARRY162


Phase II ongoing


significantly increasing the binding affinity of RO4987655 compared to PD0325901. Similarly to TAK733, this molecule shows high specificity for MEK (IC50 = 5.2 nM) without affecting the activity of other 400 kinases, and antitumor activity was shown with B-RAF mutated tumor cell lines with IC50 values in the low nanomolar range [70]. In vivo data demonstrated a strong tumor regression in many mouse xenograft models and efficacy was even improved when RO4987655 was combined with mTOR inhibitors [72]. Further in vivo studies reported a good pharmacokinetic profile, indicating higher metabolic stability than PD0325901 and no pERK inhibitory activity in the brain [70]. Preliminary results from an ongoing phase I trial on healthy volunteers confirmed the favorable pharmacokinetic profile [73].

Another relevant MEK1/2 inhibitor that reached clinical development is ARRY162 (MEK162, structure not disclosed). Initially the compound was developed by Array BioPharma and in 2010 they entered into an agreement with Novartis for collaboration in the clinical development of the molecule. ARRY162 is an allosteric inhibitor of MEK1/2 that showed tumor growth inhibition in xenograft models regardless of RAF and RAS mutational status. Thanks to its anti-inflammatory properties (inhibition of pro-inflammatory cytokines production – such as TNF and IL-1) the molecule reached phase II clinical trial for the treatment of rheumatoid arthritis but its development was discontinued after failing to reach the primary endpoint. Nevertheless ARRY162 entered phase I development for solid tumors in the US (both monotherapy and combination therapy) and reached phase II trial in advanced malignant melanoma for patients with either B-RAF or N-RAS mutations.

CONCLUSIONS

In the last few years highly potent small molecules RAF inhibitors have been developed and are now showing great promise as novel therapeutic strategies for melanomas and other solid tumors harboring the activating V600E B-RAF mutation, which for many years was assumed to induce resistance to all forms of therapeutic intervention; these new drugs showed a good initial response in several clinical trial, but it is predictable that acquired resistance will be a major factor limiting the clinical benefit of selective B-RAF inhibitors. Indeed it has been recently reported that progression free survival of patients treated with second generation B-RAF inhibitors is about 7 months [41]. Preclinical models of acquired resistance have in part elucidated the possible mechanisms responsible for the lack of response in patients bearing the V600E B-RAF mutation. These models will be a useful tool to develop effective therapeutic strategies to overcome or prevent resistance. For instance, a recent preclinical study showed that the presence of a second mutation in one of the so-called ‘gatekeeper’ residues in B-RAF kinase is critical for drug resistance. These residues are situated in the ATP binding region and the consequent aminoacid changes prevent the drug to occupy the hydrophobic pocket of the kinase. Whittaker et al., [74] identified a B-RAF ‘gatekeeper‘ site in T259 residue and demonstrated that the mutation occurred after vemurafenib treatment in a cellular model. Unfortunately, this secondary substitution has never been found in biopsies of patients resistant to B-RAF inhibitors therapy. Montagut et al., proposed that ERK reactivation after B-RAF specific inhibition in V600E B-RAF positive tumors is associated with A-RAF and C-RAF increased expression [75]. Additional mechanisms of resistance to B-RAF inhibitors are emerging. The understanding of these processes will help in designing new analogues with chemical structures able to overcome resistance. Among the mechanisms of resistance so far described in the literature are the overexpression of the COT/Tpl2 kinase [76], the acquired mutations in NRAS (Q61N) [77], or in MEK1 (C121S) [78] and the loss of PTEN [79]; anyway such mechanisms seem to be very rare events in the clinic.

Other potential problems deal with the use of inhibitors in B-RAF wild type tumors: indeed it has been shown that B-RAF inhibitors are effective against the mutated V600E B-RAF isoform and actually inhibit the MAPK signaling pathway; on the contrary, in B-RAF wild type tumors, these inhibitors can induce dimerization of B-RAF and activation of the RAS/MEK/ERK pathway, enhancing tumor growth [80]. It has also been observed that drugs like vemurafenib and dabrafenib could enhance tumor progression and cause skin lesion formations like keratocanthomas and, less frequently, squamous cell carcinomas, fortunately easily spotted and removable [41].

In conclusion, mutant B-RAF inhibitors have been a very important discovery, especially for melanomas and other types of tumors bearing oncogenic B-RAF mutation, for which conventional treatments were ineffective. Nevertheless, resistance occurs quickly and there is a urgent clinical need for therapeutic strategies that could restore sensitivity to these agents. Corcoran et al., proposed to treat patients who relapse on B-RAF inhibitor with the combination of a B-RAF and a MEK inhibitor, and for tumors driven by RTKs overexpression or constitutive activation, to add a PI3K inhibitor to B-RAF or MEK inhibitor monotherapy [81]. Indeed, clinical trials testing these therapeutic interventions are already ongoing (NCT01072175, NCT01337765) (www.clinical-trials.gov).

As regards MEK1/2 kinases, they occupy a critical position within the MAPK cascade, as they represent a converging point of transducted signals originating from many growth factor receptors located on the cell surface and moving towards the nucleus. The upstream elements of the pathway – RAS and RAF - gather all these signals and route them to MEK1/2 that are monogamous kinases. So MEK1/2 focus the signal on ERK1/2, the MAP kinases, that in turn represent the diverging point of the pathway, since they are polygamous kinases, phosphorylating many different and tissue-specific substrates. Based on these assumptions, MEK1/2 results an attractive target because of its crucial location within the pathway and because its inhibition should avoid any off-target effect related to interference with other cellular pathways. Furthermore MEK inhibitors, since the development of the first generation compounds, have always been characterized by a high specificity, thanks to their binding to an allosteric site, close to the ATP site, that is conserved only in MEK kinases [82].

On the other hand the validity of the MEK targeted therapy, supported by a plethora of in vitro and in vivo data, has not already been proved at therapeutic level. Despite encouraging pre-clinical data, most of the compounds developed so far exhibited disappointing results in the clinical phases, with poor therapeutic activity and some serious adverse events such as ocular and neurological toxicities (PD0325901, AZD6244, RDEA119). These side effects are due to the involvement of the MAPK pathway in many different physiological processes, so its inhibition can lead to alteration of the homeostasis in different tissues [83]. More recent compounds, such as GSK1120212 and RO4987655 demonstrated to avoid side effects in the central nervous system of pre-clinical models, but these data still need to be confirmed in clinical trials .

In vivo and in vitro data showed a recurrent feature for MEK1/2 inhibitors, as their activity seems to be strongly influenced by the mutational status of other members of the MAPK pathway. Indeed pre-clinical studies suggest that patients harboring activating mutations in RAS or B-RAF genes are better candidates for treatment with these kinase inhibitors. So patient enrolment for clinical trials should carefully consider such genetic lesions as these will probably be critical for the outcome of the trial itself. Preliminary data from the trials are in accordance with pre-clinical studies and seem to indicate that the use of MEK inhibitors as monotherapy will likely be limited to a subset of cancer patients with specific gene mutations, such as B-RAF mutations in melanoma and thyroid tumor [68].


Nevertheless combination therapies with conventional chemotherapeutic drugs or targeted agents have shown encouraging results in pre-clinical studies. This approach can prevent the emergence of resistance due to new mutated elements in the MAPK pathway or in other pathways regulating cell proliferation, such as the PI3K/AKT/mTOR pathway. In fact activating mutations in PI3K gene or PTEN loss of function were shown to significantly decrease the response of KRAS mutant cancer cells to MEK inhibitors [84]. The concomitant inhibition of the ERK and PI3K pathway was found to exert a synergistic effect on tumor regression [85]. According to these evidences, many clinical trial are now assessing the efficacy of different MEK inhibitors combined with chemotherapy or targeted agents; for instance GSK1120212 is undergoing phase I and II trials in combination with GSK2118436 (another MEK inhibitor), everolimus (mTORC1 inhibitor), erlotinib (EGFR inhibitor), gemcitabine, taxanes, carboplatin and pemetrexed in different types of cancer.

All these data seem to outline a picture in which future therapeutic employment of MEK1/2 inhibitors will be based on personalized treatments. The identification of predictive biomarkers coupled with the characterization of the patient’s mutational profile will allow to define what mono or combination therapy is the most suitable for every single patient.

In conclusion the inhibition of the signal transduction pathway involving RAS-RAF and MEK remains an attractive possibility for cancer therapy. The level of inhibition of the available small molecule inhibitors of both RAF and MEK can be considered satisfactory, particularly for MEK inhibitors. There is space, probably, for the modification of the chemical structures with the aim of reducing toxicity, yet preserving high potency against the target.

CONFLICT OF INTEREST

None declared.

ACKNOWLEDGEMENT

Dr Paolo Rusconi is a fellow of the Monzino Foundation, Milan, Italy.

REFERENCES

[1] Jones, S.M.; Kazlauskas, A. Growth-factor-dependent mitogenesis requires

two distinct phases of signalling. Nat. Cell Biol., 2001, 3, 165-172. [2] Sears, R.; Nuckolls, F.; Haura, E.; Taya, Y.; Tamai, K.; Nevins, J.R. Multiple

Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev., 2000, 14, 2501-2514.

[3] Hwang, C.Y.; Lee, C.; Kwon, K.S. Extracellular signal-regulated kinase 2-dependent phosphorylation induces cytoplasmic localization and degradation

of p21Cip1. Mol. Cell. Biol., 2009, 29, 3379-3389.

[4] Friday, B.B.; Adjei, A.A. Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein kinase cascade with MEK inhibitors for cancer

therapy. Clin. Cancer Res., 2008, 14, 342-346. [5] Ramjaun, A.R.; Downward, J. Ras and phosphoinositide 3-kinase: partners in

development and tumorigenesis. Cell Cycle, 2007, 6, 2902-2905. [6] Berndt, N.; Hamilton, A.D.; Sebti, S.d.M. Targeting protein prenylation for

cancer therapy. Nat. Rev. Cancer, 2011, 11, 775-791. [7] Sousa, S.F.; Fernandes, P.A.; Ramos, M.J. Farnesyltransferase inhibitors: a

detailed chemical view on an elusive biological problem. Curr. Med. Chem.,

2008, 15, 1478-1492.

[8] McCubrey, J.A.; Steelman, L.S.; Chappell, W.H.; Abrams, S.L.; Wong, E.W.; Chang, F.; Lehmann, B.; Terrian, D.M.; Milella, M.; Tafuri, A.;

Stivala, F.; Libra, M.; Basecke, J.; Evangelisti, C.; Martelli, A.M.; Franklin,

R.A. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim. Biophys. Acta, 2007, 1773,

1263-1284. [9] Steelman, L.S.; Pohnert, S.C.; Shelton, J.G.; Franklin, R.A.; Bertrand, F.E.;

McCubrey, J.A. JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia, 2004, 18, 189-218.

[10] Marais, R.; Light, Y.; Paterson, H.F.; Mason, C.S.; Marshall, C.J. Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and

tyrosine kinases. J. Biol. Chem., 1997, 272, 4378-4383.

[11] Shaul, Y.D.; Seger, R. The MEK/ERK cascade: from signaling specificity to

diverse functions. Biochim. Biophys. Acta, 2007, 1773, 1213-1226. [12] Marks, J.L.; Gong, Y.; Chitale, D.; Golas, B.; McLellan, M.D.; Kasai, Y.;

Ding, L.; Mardis, E.R.; Wilson, R.K.; Solit, D.; Levine, R.; Michel, K.; Thomas, R.K.; Rusch, V.W.; Ladanyi, M.; Pao, W. Novel MEK1 mutation

identified by mutational analysis of epidermal growth factor receptor signaling pathway genes in lung adenocarcinoma. Cancer Res., 2008, 68,

5524-5528.

[13] Milella, M.; Kornblau, S.M.; Estrov, Z.; Carter, B.Z.; Lapillonne, H.; Harris, D.; Konopleva, M.; Zhao, S.; Estey, E.; Andreeff, M. Therapeutic targeting

of the MEK/MAPK signal transduction module in acute myeloid leukemia. J.

Clin. Invest., 2001, 108, 851-859.

[14] Sebolt-Leopold, J.S.; Dudley, D.T.; Herrera, R.; Van Becelaere, K.; Wiland, A.; Gowan, R.C.; Tecle, H.; Barrett, S.D.; Bridges, A.; Przybranowski, S.;

Leopold, W.R.; Saltiel, A.R. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat. Med., 1999, 5, 810-816.

[15] Solit, D.B.; Garraway, L.A.; Pratilas, C.A.; Sawai, A.; Getz, G.; Basso, A.; Ye, Q.; Lobo, J.M.; She, Y.; Osman, I.; Golub, T.R.; Sebolt-Leopold, J.;

Sellers, W.R.; Rosen, N. BRAF mutation predicts sensitivity to MEK inhibition. Nature, 2006, 439, 358-362.

[16] Ribas, A.; Flaherty, K.T. BRAF targeted therapy changes the treatment

paradigm in melanoma. Nat. Rev. Clin. Oncol., 2011, 8, 426-433. [17] Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.; Smith, R.A.;

Schwartz, B.; Simantov, R.; Kelley, S. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat. Rev. Drug Discov.,

2006, 5, 835-844. [18] Lowinger, T.B.; Riedl, B.; Dumas, J.; Smith, R.A. Design and discovery of

small molecules targeting raf-1 kinase. Curr. Pharm. Des., 2002, 8, 2269-2278.

[19] Wellbrock, C.; Hurlstone, A. BRAF as therapeutic target in melanoma. Biochem. Pharmacol., 2010, 80, 561-567.

[20] Wan, P.T.; Garnett, M.J.; Roe, S.M.; Lee, S.; Niculescu-Duvaz, D.; Good, V.M.; Jones, C.M.; Marshall, C.J.; Springer, C.J.; Barford, D.; Marais, R.

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic

mutations of B-RAF. Cell, 2004, 116, 855-867. [21] Wilhelm, S.M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.;

Chen, C.; Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.; Adnane, L.; Lynch, M.; Auclair, D.;

Taylor, I.; Gedrich, R.; Voznesensky, A.; Riedl, B.; Post, L.E.; Bollag, G.; Trail, P.A. BAY 43-9006 exhibits broad spectrum oral antitumor activity and

targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res., 2004, 64, 7099-7109.

[22] Garassino, M.C.; Marabese, M.; Rusconi, P.; Rulli, E.; Martelli, O.; Farina, G.; Scanni, A.; Broggini, M. Different types of K-Ras mutations could affect

drug sensitivity and tumour behaviour in non-small-cell lung cancer. Ann.

Oncol., 2011, 22, 235-237.

[23] McDermott, U.; Sharma, S.V.; Dowell, L.; Greninger, P.; Montagut, C.;

Lamb, J.; Archibald, H.; Raudales, R.; Tam, A.; Lee, D.; Rothenberg, S.M.; Supko, J.G.; Sordella, R.; Ulkus, L.E.; Iafrate, A.J.; Maheswaran, S.; Njauw,

C.N.; Tsao, H.; Drew, L.; Hanke, J.H.; Ma, X.J.; Erlander, M.G.; Gray, N.S.; Haber, D.A.; Settleman, J. Identification of genotype-correlated sensitivity to

selective kinase inhibitors by using high-throughput tumor cell line profiling. Proc. Natl. Acad. Sci .U S A, 2007, 104, 19936-19941.

[24] Kim, S.; Yazici, Y.D.; Calzada, G.; Wang, Z.Y.; Younes, M.N.; Jasser, S.A.; El-Naggar, A.K.; Myers, J.N. Sorafenib inhibits the angiogenesis and growth

of orthotopic anaplastic thyroid carcinoma xenografts in nude mice. Mol.

Cancer Ther., 2007, 6, 1785-1792.

[25] Siegelin, M.D.; Raskett, C.M.; Gilbert, C.A.; Ross, A.H.; Altieri, D.C.

Sorafenib exerts anti-glioma activity in vitro and in vivo. Neurosci. Lett.,

2010, 478, 165-170.

[26] Chapuy, B.; Schuelper, N.; Panse, M.; Dohm, A.; Hand, E.; Schroers, R.; Truemper, L.; Wulf, G.G. Multikinase inhibitor sorafenib exerts cytocidal

efficacy against Non-Hodgkin lymphomas associated with inhibition of MAPK14 and AKT phosphorylation. Br. J. Haematol., 2011, 152, 401-412.

[27] Ramakrishnan, V.; Timm, M.; Haug, J.L.; Kimlinger, T.K.; Wellik, L.E.; Witzig, T.E.; Rajkumar, S.V.; Adjei, A.A.; Kumar, S. Sorafenib, a dual Raf

kinase/vascular endothelial growth factor receptor inhibitor has significant anti-myeloma activity and synergizes with common anti-myeloma drugs.

Oncogene, 2010, 29, 1190-1202. [28] Chang, Y.; Adnane, J.; Trail, P.; Levy, J.; Henderson, A.; Xue, D.; Bortolon,

E.; Ichetovkin, M.; Chen, C.; McNabola, A.; Wilkie, D.; Carter, C.; Taylor,

I.; Lynch, M.; Wilhelm, S. Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC

xenograft models. Cancer Chem. Pharm., 2007, 59, 561-574. [29] Montagut, C.; Settleman, J. Targeting the RAF-MEK-ERK pathway in

cancer therapy. Cancer Lett., 2009, 283, 125-134. [30] Escudier, B.; Eisen, T.; Stadler, W.M.; Szczylik, C.; Oudard, S.; Siebels, M.;

Negrier, S.; Chevreau, C.; Solska, E.; Desai, A.A.; Rolland, F.; Demkow, T.; Hutson, T.E.; Gore, M.; Freeman, S.; Schwartz, B.; Shan, M.; Simantov, R.;

Bukowski, R.M. Sorafenib in advanced clear-cell renal-cell carcinoma. N.

Engl. J. Med., 2007, 356, 125-134.

[31] Ratain, M.J.; Eisen, T.; Stadler, W.M.; Flaherty, K.T.; Kaye, S.B.; Rosner, G.L.; Gore, M.; Desai, A.A.; Patnaik, A.; Xiong, H.Q.; Rowinsky, E.;

Abbruzzese, J.L.; Xia, C.; Simantov, R.; Schwartz, B.; O'Dwyer, P.J. Phase

II placebo-controlled randomized discontinuation trial of sorafenib in


patients with metastatic renal cell carcinoma. J. Clin. Oncol., 2006, 24, 2505-

2512. [32] Llovet, J.M.; Ricci, S.; Mazzaferro, V.; Hilgard, P.; Gane, E.; Blanc, J.F.; de

Oliveira, A.C.; Santoro, A.; Raoul, J.L.; Forner, A.; Schwartz, M.; Porta, C.; Zeuzem, S.; Bolondi, L.; Greten, T.F.; Galle, P.R.; Seitz, J.F.; Borbath, I.;

Haussinger, D.; Giannaris, T.; Shan, M.; Moscovici, M.; Voliotis, D.; Bruix, J. Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med., 2008,

359, 378-390.

[33] Eisen, T.; Ahmad, T.; Flaherty, K.T.; Gore, M.; Kaye, S.; Marais, R.; Gibbens, I.; Hackett, S.; James, M.; Schuchter, L.M.; Nathanson, K.L.; Xia,

C.; Simantov, R.; Schwartz, B.; Poulin-Costello, M.; O'Dwyer, P.J.; Ratain, M.J. Sorafenib in advanced melanoma: a Phase II randomised

discontinuation trial analysis. Br. J. Cancer, 2006, 95, 581-586. [34] Wilhelm, S.M.; Dumas, J.; Adnane, L.; Lynch, M.; Carter, C.A.; Schutz, G.;

Thierauch, K.H.; Zopf, D. Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine

kinases with potent preclinical antitumor activity. Int. J. Cancer, 2011, 129, 245-255.

[35] Wong, K.K. Recent developments in anti-cancer agents targeting the Ras/Raf/MEK/ERK pathway. Recent Pat. Anticancer Drug Discov., 2009, 4,

28-35.

[36] Shepherd, C.; Puzanov, I.; Sosman, J.A. B-RAF inhibitors: an evolving role in the therapy of malignant melanoma. Curr. Oncol. Rep., 2010, 12, 146-152.

[37] Fecher, L.A.; Amaravadi, R.K.; Flaherty, K.T. The MAPK pathway in melanoma. Curr. Opin. Oncol., 2008, 20, 183-189.

[38] Liu, Y.; Gray, N.S. Rational design of inhibitors that bind to inactive kinase conformations. Nat. Chem. Biol., 2006, 2, 358-364.

[39] Sala, E.; Mologni, L.; Truffa, S.; Gaetano, C.; Bollag, G.E.; Gambacorti-Passerini, C. BRAF silencing by short hairpin RNA or chemical blockade by

PLX4032 leads to different responses in melanoma and thyroid carcinoma cells. Mol. Cancer Res., 2008, 6, 751-759.

[40] Yang, H.; Higgins, B.; Kolinsky, K.; Packman, K.; Go, Z.; Iyer, R.; Kolis, S.; Zhao, S.; Lee, R.; Grippo, J.F.; Schostack, K.; Simcox, M.E.; Heimbrook,

D.; Bollag, G.; Su, F. RG7204 (PLX4032), a selective BRAFV600E

inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res., 2010, 70, 5518-5527.

[41] Flaherty, K.T.; Puzanov, I.; Kim, K.B.; Ribas, A.; McArthur, G.A.; Sosman, J.A.; O'Dwyer, P.J.; Lee, R.J.; Grippo, J.F.; Nolop, K.; Chapman, P.B.

Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J.

Med., 2010, 363, 809-819.

[42] Chapman, P.B.; Hauschild, A.; Robert, C.; Larkin, J.M.G.; Haanen, J.B.A.G.; Ribas, A.; Hogg, D.; O'Day, S.; Ascierto, P.A.; Testori, A.;

Lorigan, P.; Dummer, R.; Sosman, J.A.; Garbe, C.; Lee, R.J.; Nolop, K.B.; Nelson, B.; Hou, J.; Flaherty, K.T.; McArthur, G.A. Phase III randomized,

open-label, multicenter trial (BRIM3) comparing BRAF inhibitor vemurafenib with dacarbazine (DTIC) in patients with V600EBRAF-mutated

melanoma. ASCO Meeting Abstracts, 2011, 29, LBA4.

[43] Tsai, J.; Lee, J.T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.; Kong, J.; Haass, N.K.; Sproesser, K.; Li, L.; Smalley, K.S.M.;

Fong, D.; Zhu, Y.-L.; Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu, C.; Settachatgul, C.; Shellooe, R.; Cantwell, J.;

Kim, S.-H.; Schlessinger, J.; Zhang, K.Y.J.; West, B.L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P.N.; Hirth, P.; Artis, D.R.; Herlyn, M.; Bollag, G.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad. Sci .U S A, 2008, 105, 3041-3046.

[44] King, A.J.; Patrick, D.R.; Batorsky, R.S.; Ho, M.L.; Do, H.T.; Zhang, S.Y.; Kumar, R.; Rusnak, D.W.; Takle, A.K.; Wilson, D.M.; Hugger, E.; Wang,

L.; Karreth, F.; Lougheed, J.C.; Lee, J.; Chau, D.; Stout, T.J.; May, E.W.;

Rominger, C.M.; Schaber, M.D.; Luo, L.; Lakdawala, A.S.; Adams, J.L.; Contractor, R.G.; Smalley, K.S.; Herlyn, M.; Morrissey, M.M.; Tuveson,

D.A.; Huang, P.S. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer

Res., 2006, 66, 11100-11105. [45] Kefford, R.; Arkenau, H.; Brown, M.P.; Millward, M.; Infante, J.R.; Long,

G.V.; Ouellet, D.; Curtis, M.; Lebowitz, P.F.; Falchook, G.S. Phase I/II study of GSK2118436, a selective inhibitor of oncogenic mutant BRAF kinase, in

patients with metastatic melanoma and other solid tumors. ASCO Meeting

Abstracts, 2010, 28, 8503.

[46] Chen, C.-R.; Lapierre, J.-M.; Szwaya, J.D.; Liu, Y.; Namdev, N.; Lowe, D.; Savage, R.E.; Bull, C.O.; Chan, T.C.K.; Ashwell, M.A.; France, D.S.

Abstract 4501: Development and biological annotation of a novel RAF

inhibitor amenable for clinical evaluation against BRAF (V600E)-harboring human tumors. Cancer Res., 2010, 70, 4501.

[47] Yu, Y.; Zhao, X.; Gu, X.; Chang, E.; Cousens, L.; Chiesa, E.; Lowe, D.; Liu, Y.; Bull, C.O.; Waghorne, C.G.; Chan, T.C.K. Abstract 2517: Pharmaco-

dynamic biomarkers for ARQ 736, a small molecule BRAF inhibitor. Cancer

Res., 2010, 70, 2517.

[48] Dudley, D.T.; Pang, L.; Decker, S.J.; Bridges, A.J.; Saltiel, A.R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad.

Sci .U S A, 1995, 92, 7686-7689. [49] Alessi, D.R.; Cuenda, A.; Cohen, P.; Dudley, D.T.; Saltiel, A.R. PD 098059

is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem., 1995, 270, 27489-27494.

[50] Favata, M.F.; Horiuchi, K.Y.; Manos, E.J.; Daulerio, A.J.; Stradley, D.A.;

Feeser, W.S.; Van Dyk, D.E.; Pitts, W.J.; Earl, R.A.; Hobbs, F.; Copeland,

R.A.; Magolda, R.L.; Scherle, P.A.; Trzaskos, J.M. Identification of a novel

inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem., 1998, 273, 18623-18632.

[51] Williams, D.H.; Wilkinson, S.E.; Purton, T.; Lamont, A.; Flotow, H.; Murray, E.J. Ro 09-2210 exhibits potent anti-proliferative effects on

activated T cells by selectively blocking MKK activity. Biochemistry, 1998, 37, 9579-9585.

[52] Ohren, J.F.; Chen, H.; Pavlovsky, A.; Whitehead, C.; Zhang, E.; Kuffa, P.;

Yan, C.; McConnell, P.; Spessard, C.; Banotai, C.; Mueller, W.T.; Delaney, A.; Omer, C.; Sebolt-Leopold, J.; Dudley, D.T.; Leung, I.K.; Flamme, C.;

Warmus, J.; Kaufman, M.; Barrett, S.; Tecle, H.; Hasemann, C.A. Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel

noncompetitive kinase inhibition. Nat. Struct. Mol. Biol., 2004, 11, 1192-1197.

[53] LoRusso, P.M.; Adjei, A.A.; Varterasian, M.; Gadgeel, S.; Reid, J.; Mitchell, D.Y.; Hanson, L.; DeLuca, P.; Bruzek, L.; Piens, J.; Asbury, P.; Van

Becelaere, K.; Herrera, R.; Sebolt-Leopold, J.; Meyer, M.B. Phase I and pharmacodynamic study of the oral MEK inhibitor CI-1040 in patients with

advanced malignancies. J. Clin. Oncol., 2005, 23, 5281-5293. [54] Rinehart, J.; Adjei, A.A.; Lorusso, P.M.; Waterhouse, D.; Hecht, J.R.;

Natale, R.B.; Hamid, O.; Varterasian, M.; Asbury, P.; Kaldjian, E.P.; Gulyas,

S.; Mitchell, D.Y.; Herrera, R.; Sebolt-Leopold, J.S.; Meyer, M.B. Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients

with advanced non-small-cell lung, breast, colon, and pancreatic cancer. J.

Clin. Oncol., 2004, 22, 4456-4462.

[55] Dummer, R.; Robert, C.; Chapman, P.B.; Sosman, J.A.; Middleton, M.; Bastholt, L.; Kemsley, K.; Cantarini, M.V.; Morris, C.; Kirkwood, J.M.

AZD6244 (ARRY-142886) vs temozolomide (TMZ) in patients (pts) with advanced melanoma: An open-label, randomized, multicenter, phase II study.

ASCO Meeting Abstracts, 2008, 26, 9033. [56] Barrett, S.D.; Bridges, A.J.; Dudley, D.T.; Saltiel, A.R.; Fergus, J.H.;

Flamme, C.M.; Delaney, A.M.; Kaufman, M.; LePage, S.; Leopold, W.R.; Przybranowski, S.A.; Sebolt-Leopold, J.; Van Becelaere, K.; Doherty, A.M.;

Kennedy, R.M.; Marston, D.; Howard Jr, W.A.; Smith, Y.; Warmus, J.S.;

Tecle, H. The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901. Bioorg. Med. Chem. Lett., 2008, 18, 6501-6504.

[57] Menon, S.S.; Whitfield, L.R.; Sadis, S.; Meyer, M.B.; Leopold, J.; Lorusso, P.M.; Krishnamurthi, S.; Rinehart, J.R.; Nabell, L.; Croghan, G.

Pharmacokinetics (PK) and pharmacodynamics (PD) of PD 0325901, a second generation MEK inhibitor after multiple oral doses of PD 0325901 to

advanced cancer patients. ASCO Meeting Abstracts, 2005, 23, 3066. [58] Haura, E.B.; Ricart, A.D.; Larson, T.G.; Stella, P.J.; Bazhenova, L.; Miller,

V.A.; Cohen, R.B.; Eisenberg, P.D.; Selaru, P.; Wilner, K.D.; Gadgeel, S.M. A phase II study of PD-0325901, an oral MEK inhibitor, in previously

treated patients with advanced non-small cell lung cancer. Clin. Cancer Res.,

2010, 16, 2450-2457.

[59] Yeh, T.C.; Marsh, V.; Bernat, B.A.; Ballard, J.; Colwell, H.; Evans, R.J.;

Parry, J.; Smith, D.; Brandhuber, B.J.; Gross, S.; Marlow, A.; Hurley, B.; Lyssikatos, J.; Lee, P.A.; Winkler, J.D.; Koch, K.; Wallace, E. Biological

characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin. Cancer Res.,

2007, 13, 1576-1583. [60] Davies, B.R.; Logie, A.; McKay, J.S.; Martin, P.; Steele, S.; Jenkins, R.;

Cockerill, M.; Cartlidge, S.; Smith, P.D. AZD6244 (ARRY-142886), a potent inhibitor of mitogen-activated protein kinase/extracellular signal-

regulated kinase kinase 1/2 kinases: mechanism of action in vivo, pharmacokinetic/pharmacodynamic relationship, and potential for

combination in preclinical models. Mol. Cancer Ther., 2007, 6, 2209-2219.

[61] Adjei, A.A.; Cohen, R.B.; Franklin, W.; Morris, C.; Wilson, D.; Molina, J.R.; Hanson, L.J.; Gore, L.; Chow, L.; Leong, S.; Maloney, L.; Gordon, G.;

Simmons, H.; Marlow, A.; Litwiler, K.; Brown, S.; Poch, G.; Kane, K.; Haney, J.; Eckhardt, S.G. Phase I pharmacokinetic and pharmacodynamic

study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers. J.

Clin. Oncol., 2008, 26, 2139-2146. [62] Hersey, P.; Bastholt, L.; Chiarion-Sileni, V.; Cinat, G.; Dummer, R.;

Eggermont, A.M.M.; Espinosa, E.; Hauschild, A.; Quirt, I.; Robert, C.; Schadendorf, D. Small molecules and targeted therapies in distant metastatic

disease. Ann. Oncol., 2009, 20, vi35-vi40. [63] Tzekova, V.; Cebotaru, C.; Ciuleanu, T.E.; Damjanov, D.; Ganchev, H.;

Kanarev, V.; Stella, P.J.; Sanders, N.; Pover, G.; Hainsworth, J.D. Efficacy

and safety of AZD6244 (ARRY-142886) as second/third-line treatment of patients (pts) with advanced non-small cell lung cancer (NSCLC). ASCO

Meeting Abstracts, 2008, 26, 8029. [64] Lang, I.; Adenis, A.; Boer, K.; Escudero, P.; Kim, T.; Valladares, M.;

Sanders, N.; Pover, G.; Douillard, J. AZD6244 (ARRY-142886) versus capecitabine (CAP) in patients (pts) with metastatic colorectal cancer

(mCRC) who have failed prior chemotherapy. ASCO Meeting Abstracts,

2008, 26, 4114.

[65] Bekaii-Saab, T.; Phelps, M.; Li, X.; Saji, M.; Kosuri, K.; Goff, L.; Kauh, J.; O'Neil, B.; South, C.; Thomas, J.; Balsom, S.; Chattah, N.; Balint, C.;

Liersemann, R.; Vasko, V.; Marsh, W.; Doyle, L.A.; Ellison, G.; Ringel, M.; Villalona Calero, M. Abstract #LB-129: * A Multi-institutional Study of

AZD6244 (ARRY-142886) in Patients with Advanced Biliary Cancers.

AACR Meeting Abstracts, 2009, 2009, LB-129-.


[66] Iverson, C.; Larson, G.; Lai, C.; Yeh, L.T.; Dadson, C.; Weingarten, P.;

Appleby, T.; Vo, T.; Maderna, A.; Vernier, J.M.; Hamatake, R.; Miner, J.N.; Quart, B. RDEA119/BAY 869766: A potent, selective, allosteric inhibitor of

MEK1/2 for the treatment of cancer. Cancer Res., 2009, 69, 6839-6847. [67] Gilmartin, A.G.; Bleam, M.R.; Groy, A.; Moss, K.G.; Minthorn, E.A.;

Kulkarni, S.G.; Rominger, C.M.; Erskine, S.; Fisher, K.E.; Yang, J.; Zappacosta, F.; Annan, R.; Sutton, D.; Laquerre, S.G. GSK1120212 (JTP-

74057) is an inhibitor of MEK activity and activation with favorable

pharmacokinetic properties for sustained in vivo pathway inhibition. Clin.

Cancer Res., 2011, 17, 989-1000.

[68] Chapman, M.S.; Miner, J.N. Novel mitogen-activated protein kinase kinase inhibitors. Expert Opin. Investig. Drugs, 2011, 20, 209-220.

[69] Infante, J.R.; Fecher, L.A.; Nallapareddy, S.; Gordon, M.S.; Flaherty, K.T.; Cox, D.S.; DeMarini, D.J.; Morris, S.R.; Burris, H.A.; Messersmith, W.A.

Safety and efficacy results from the first-in-human study of the oral MEK 1/2 inhibitor GSK1120212. ASCO Meeting Abstracts, 2010, 28, 2503.

[70] Isshiki, Y.; Kohchi, Y.; Iikura, H.; Matsubara, Y.; Asoh, K.; Murata, T.; Kohchi, M.; Mizuguchi, E.; Tsujii, S.; Hattori, K.; Miura, T.; Yoshimura, Y.;

Aida, S.; Miwa, M.; Saitoh, R.; Murao, N.; Okabe, H.; Belunis, C.; Janson, C.; Lukacs, C.; SchÃ ck, V.; Shimma, N. Design and synthesis of novel

allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

Bioorg. Med. Chem. Lett., 2011, 21, 1795-1801. [71] Dong, Q.; Dougan, D.R.; Gong, X.; Halkowycz, P.; Jin, B.; Kanouni, T.;

O'Connell, S.M.; Scorah, N.; Shi, L.; Wallace, M.B.; Zhou, F. Discovery of TAK-733, a potent and selective MEK allosteric site inhibitor for the

treatment of cancer. Bioorg. Med. Chem. Lett., 2011, 21, 1315-1319. [72] Aida, S.; Yoshimura, Y.; Ishii, N.; Sakamoto, H.; Isshiki, Y.; Ishikawa, Y.;

Miwa, M.; Shimma, N.; Okabe, H.; Aoki, Y. Abstract #3701: CH4987655 (RO4987655), a selective orally bioavailable MEK inhibitor, shows highly

potent and broad antitumor activity as a monotherapy and in combination with other antitumor agents. AACR Meeting Abstracts, 2009, 2009, 3701-.

[73] Lee, L.; Niu, H.; Rueger, R.; Igawa, Y.; Deutsch, J.; Ishii, N.; Mu, S.; Sakamoto, Y.; Busse-Reid, R.; Gimmi, C.; Goelzer, P.; De Schepper, S.;

Yoshimura, Y.; Barrett, J.; Ishikawa, Y.; Weissgerber, G.; Peck, R. The

safety, tolerability, pharmacokinetics, and pharmacodynamics of single oral doses of CH4987655 in healthy volunteers: target suppression using a

biomarker. Clin. Cancer Res., 2009, 15, 7368-7374. [74] Whittaker, S.; Kirk, R.; Hayward, R.; Zambon, A.; Viros, A.; Cantarino, N.;

Affolter, A.; Nourry, A.; Niculescu-Duvaz, D.; Springer, C.; Marais, R. Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci.

Transl. Med., 2010, 2, 35ra41. [75] Montagut, C.; Sharma, S.V.; Shioda, T.; McDermott, U.; Ulman, M.; Ulkus,

L.E.; Dias-Santagata, D.; Stubbs, H.; Lee, D.Y.; Singh, A.; Drew, L.; Haber, D.A.; Settleman, J. Elevated CRAF as a potential mechanism of acquired

resistance to BRAF inhibition in melanoma. Cancer Res., 2008, 68, 4853-4861.

[76] Johannessen, C.M.; Boehm, J.S.; Kim, S.Y.; Thomas, S.R.; Wardwell, L.;

Johnson, L.A.; Emery, C.M.; Stransky, N.; Cogdill, A.P.; Barretina, J.;

Caponigro, G.; Hieronymus, H.; Murray, R.R.; Salehi-Ashtiani, K.; Hill,

D.E.; Vidal, M.; Zhao, J.J.; Yang, X.; Alkan, O.; Kim, S.; Harris, J.L.; Wilson, C.J.; Myer, V.E.; Finan, P.M.; Root, D.E.; Roberts, T.M.; Golub, T.;

Flaherty, K.T.; Dummer, R.; Weber, B.L.; Sellers, W.R.; Schlegel, R.; Wargo, J.A.; Hahn, W.C.; Garraway, L.A. COT drives resistance to RAF

inhibition through MAP kinase pathway reactivation. Nature, 2010, 468, 968-972.

[77] Nazarian, R.; Shi, H.; Wang, Q.; Kong, X.; Koya, R.C.; Lee, H.; Chen, Z.;

Lee, M.K.; Attar, N.; Sazegar, H.; Chodon, T.; Nelson, S.F.; McArthur, G.; Sosman, J.A.; Ribas, A.; Lo, R.S. Melanomas acquire resistance to B-

RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature, 2010, 468, 973-977.

[78] Wagle, N.; Emery, C.; Berger, M.F.; Davis, M.J.; Sawyer, A.; Pochanard, P.; Kehoe, S.M.; Johannessen, C.M.; Macconaill, L.E.; Hahn, W.C.; Meyerson,

M.; Garraway, L.A. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol., 2011, 29, 3085-3096.

[79] Villanueva, J.; Vultur, A.; Lee, J.T.; Somasundaram, R.; Fukunaga-Kalabis, M.; Cipolla, A.K.; Wubbenhorst, B.; Xu, X.; Gimotty, P.A.; Kee, D.;

Santiago-Walker, A.E.; Letrero, R.; D'Andrea, K.; Pushparajan, A.; Hayden, J.E.; Brown, K.D.; Laquerre, S.; McArthur, G.A.; Sosman, J.A.; Nathanson,

K.L.; Herlyn, M. Acquired resistance to BRAF inhibitors mediated by a RAF

kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell, 2010, 18, 683-695.

[80] Hatzivassiliou, G.; Song, K.; Yen, I.; Brandhuber, B.J.; Anderson, D.J.; Alvarado, R.; Ludlam, M.J.C.; Stokoe, D.; Gloor, S.L.; Vigers, G.; Morales,

T.; Aliagas, I.; Liu, B.; Sideris, S.; Hoeflich, K.P.; Jaiswal, B.S.; Seshagiri, S.; Koeppen, H.; Belvin, M.; Friedman, L.S.; Malek, S. RAF inhibitors prime

wild-type RAF to activate the MAPK pathway and enhance growth. Nature,

2010, 464, 431-435.

[81] Corcoran, R.B.; Settleman, J.; Engelman, J.A. Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF

mutant cancers. Oncotarget, 2011, 2, 336-346. [82] Fremin, C.; Meloche, S. From basic research to clinical development of

MEK1/2 inhibitors for cancer therapy. J. Hematol. Oncol., 2010, 3.

[83] Pearson, G.; Robinson, F.; Beers Gibson, T.; Xu, B.E.; Karandikar, M.; Berman, K.; Cobb, M.H. Mitogen-activated protein (MAP) kinase pathways:

regulation and physiological functions. Endocr. Rev., 2001, 22, 153-183. [84] Wee, S.; Jagani, Z.; Xiang, K.X.; Loo, A.; Dorsch, M.; Yao, Y.M.; Sellers,

W.R.; Lengauer, C.; Stegmeier, F. PI3K pathway activation mediates resistance to MEK inhibitors in KRAS mutant cancers. Cancer Res., 2009,

69, 4286-4293. [85] Engelman, J.A.; Chen, L.; Tan, X.; Crosby, K.; Guimaraes, A.R.; Upadhyay,

R.; Maira, M.; McNamara, K.; Perera, S.A.; Song, Y.; Chirieac, L.R.; Kaur, R.; Lightbown, A.; Simendinger, J.; Li, T.; Padera, R.F.; Garcia-Echeverria,

C.; Weissleder, R.; Mahmood, U.; Cantley, L.C.; Wong, K.K. Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA

H1047R murine lung cancers. Nat. Med., 2008, 14, 1351-1356.

Received: October 14, 2011 Revised: January 04, 2012 Accepted: January 06, 2012

RAS/RAF/MEK Inhibitors in Oncology

Documents

Transcript of RAS/RAF/MEK Inhibitors in Oncology