Review

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Type III or allosteric kinase inhibitors for the treatment of non-small cell lung cancer 1.

Introduction

2.

MEK inhibitors

3.

AKT inhibitors

4.

Conclusion

5.

Expert opinion

Morena Fasano, Carminia Maria Della Corte, Raffaele Califano, Annalisa Capuano, Teresa Troiani, Erika Martinelli, Fortunato Ciardiello & Floriana Morgillo† †

Second University of Naples, Medical Oncology, Department of Experimental and Internal Medicine “ F. Magrassi e A. Lanzara” , Napoli, Italia

Introduction: In recent times, there has been much interest in the development of pharmacological kinase inhibitors that treat NSCLC. Furthermore, treatment options have been guided by the development of a wide panel of synthetic small molecule kinase inhibitors. Most of the molecules developed belong to the type I class of inhibitors that target the ATP-binding site in its active conformation. The high sequence similarity in the ATP-binding site among members of the kinase families often results in low selectivity and additional toxicities. Also, second mutations in the ATP-binding site, such as threonine to methionine at position 790, have been described as a mechanism of resistance to ATP-competitive kinase inhibitors. For these reasons, alternative drug development approaches targeting sites other than the ATP cleft are being pursued. The class III or allosteric inhibitors, which bind outside the ATP-binding site, have been shown to negatively modulate kinase activity. Areas covered: In this review, the authors discuss the most well-characterised allosteric inhibitors that have reached clinical development in NSCLC. Expert opinion: Great progress has made in developing inhibitors with entirely new modes of action. That being said, it is important to highlight that despite their apparent simplicity, biochemical assays will remain at the core of drug discovery activities to better explore these new opportunities. Keywords: AKT, MAPK kinase, non-small cell lung cancer, type III tyrosine kinase inhibitor Expert Opin. Investig. Drugs (2014) 23(6):809-821

1.

Introduction

NSCLC is the most common cause of cancer-related death in both males and females [1]. Standard therapies, including platinum-based chemotherapy and radiotherapy, provide a marginal benefit in survival with associate toxicity. Advances in the understanding of NSCLC biology have enabled the identification of specific cellular kinases as new potential therapeutic targets [2]. Protein kinases play an important role in the control of cellular physiology, including proliferation and apoptosis and their deregulation is intimately involved in the processes of tumour development. Among several cellular kinases, there are kinases with transforming capacity and with oncogenic potential whose constitutive activation is essential for survival of tumour cells, which are therefore, ‘oncogene addicted’ [3]. An example of this class is represented by the tyrosine kinase of the EGFR, which activating mutations in exon 19 or 21 drive lung tumourigenesis. A second class of kinases is not oncogenic, but they are required for cancer cell survival and/or proliferation and are located in key signalling pathways downstream of transforming oncogenes (mitogen-activated protein kinase [MAPK] 10.1517/13543784.2014.902934 © 2014 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 All rights reserved: reproduction in whole or in part not permitted

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Protein kinases play an important role in the control of cellular physiology, including proliferation and apoptosis, and their deregulation is intimately involved in the processes of tumour development. Class III of kinase inhibitors, also called allosteric inhibitors, binds outside the ATP-binding site and negatively modulates kinase activity. This class of inhibitors exploits the allosteric properties of kinases, where allostery is the re-distribution of protein conformational ensembles leading to different dynamic and functional effects. The most well-characterised allosteric inhibitors, which have reached clinical development in NSCLC, are inhibitors of MAPK kinase 1 and 2 (MEK1/2) and Akt. Despite a solid preclinical rationale, results from early clinical studies are controversial showing a non-clear antitumour activity. Successful MEK/AKT-directed treatment of NSCLC potentially depends on two factors: identification of biomarkers to predict sensitivity and selection of optimally beneficial drug combinations. To explore the opportunity to develop protein kinase drugs with different pharmacological properties, such as higher specificity, it is important to utilise assays that allow the probing of different conformations adapted by the target protein.

This box summarises key points contained in the article.

mitogen-activated protein kinase kinase1 [MEK1], MEK2, mammalian target of rapamycin [mTOR], cyclin-dependent kinases, etc.) [4]. The inhibition of these kinases can result in a synthetic lethality when paired with another nonlethal mutation or with the inhibition of other kinases. A third class of kinases is expressed in tumour and surrounding tissues and are important for tumour maintenance in human host (vascular endotheial growth factor, neurotrophic tyrosine receptor kinase kinase, metalloproteinases etc.) [5]. This has generated great interest in the development of pharmacological kinase inhibitors for cancer treatment and a wide panel of synthetic small molecule kinase inhibitors has been developed in past years for NSCLC therapy. Most of them belong to the type I class of inhibitors and target the ATP-binding site in its active conformation. Erlotinib and gefitinib are two type I kinase inhibitors approved for firstline therapy of advanced NSCLC patients whose tumours harbour EGFR-activating mutations and for second- and third-line treatments of EGFR wild-type (WT) patients (erlotinib). Recently, afatinib, an irreversible ErbB family inhibitor, was approved in tyrosine kinase inhibitor (TKI)naive patients with NSCLC harbouring EGFR-activating mutations. Type I inhibitors activity is restricted by the high-sequence similarity in the ATP-binding site among the members of the kinase families, which results in low selectivity and in additional toxicities. In addition, second mutations in the ATP-binding site have been described as mechanism of 810

resistance to ATP-competitive kinase inhibitors. Indeed, in approximately half of the cases, NSCLC tumour cells with EGFR-activating mutation acquire a secondary mutation in exon 20 of the kinase domain consisting in a single aminoacid substitution from threonine to methionine at position 790 (T790M), which leads to drug resistance [6]. As a consequence, the ability of reversible TKIs, gefitinib and erlotinib, to compete effectively with the ATP-binding site is reduced because of the presence of the T790M mutation, which increases the ATP affinity of the oncogenic mutant receptor of about five times [6]. For such reasons, alternative approaches for development of inhibitors targeting sites other than the ATP cleft are being pursued [7]. Generally, the non-ATP competitive inhibitors act by inducing a conformational shift in the target enzyme in order to block the kinase function. These inhibitors are classified as type II and III inhibitors. Type II inhibitors interact reversibly with both the ATP pocket and the hydrophobic site adjacent to the ATP cleft blocking the kinase in inactive status [8]. The biological activity depends on a molecular framework common to all type II inhibitors that consist of a conserved set of hydrogen bonds responsible for the binding with the hydrophobic pocket. Examples of type II inhibitors include the abelson murine leukemia viral oncogene, KIT and platelet-derived growth factor receptor (PDGFR) inhibitors; imatinib and nilotinib; and the KIT, PDGFR and Raf inhibitor sorafenib. The third class of kinase inhibitors, also called allosteric inhibitors, bind outside the ATP-binding site and negatively modulates kinase activity. This class of inhibitors exploits the allosteric properties of kinases, where allostery is the redistribution of protein conformational ensembles leading to different dynamic and functional effects. In particular, the conformational variability of the DGF-motif has been utilised to design this class of drugs. The DGF-motif is a sequence of three residues, Asp-Phe-Gly and is located before the activation loop. This motif can adopt a different conformation in which the DFG aspartate and phenylalanine side chains change positions in opposite directions. This rearrangement creates a new hydrophobic binding pocket, adjacent to the ATP-binding site, representing an attractive target for many inhibitors. The amino acids around this new site are less conserved than those of the ATP cleft and therefore, represent unique targets for a particular kinase, thus providing the highest selectivity for this class of drugs [9]. One of the main problems with this class of inhibitors is their poor solubility. Despite very good in vitro activity, many compounds show lower activity in vivo and poor bioavailability. Many efforts are ongoing to improve the pharmacokinetic (PK) properties of these drugs. The most well-characterised allosteric inhibitors, which have reached clinical development in NSCLC, are inhibitors of MEK1/2 and Akt. In this review, we analysed preclinical in vitro and in vivo data and clinical trials on type III or allosteric kinase

Expert Opin. Investig. Drugs (2014) 23(6)

Type III or allosteric kinase inhibitors for the treatment of non-small cell lung cancer

Table 1. Type III TKI in advanced clinical development for NSCLC treatment. Class Drugs

CI-1040 PD0325901

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Selumetinib

Trametinib

Phase of clinical development

Total enrolled pts

Pts with NSCLC

I [16] II [17] I [20] II [21]

77 67 66 34

I [28] II [29]

57 pts 2 pts 40 pts in 84 pts selumetinib group and 44 pts in pem group

II [30]

44 pts in selumetinib + doce vs 43 pts in selumetinib + placebo

87 pts

I [42]

46 pts

46 pts (24 WT and 22 mutKRAS)

I [43]

42 pts

42 pts (19 pts WT KRAS, 23 pts mutKRAS)

II [44]

134 pts (129 pts with mutKRAS NSCLC)

89 pts with trametinib, 45 pts with doce ND

pts pts pts pts

9 pts 18 pts 5 pts 34 pts

MEK-I

Refametinib (RDEA119, BAY 869766) Pimasertib (AS703026, MSC1936369B) RO4987655 (CH4987655) TAK-733 MEK162 (ARRY 438162) WX-554

AKT

Cobimetinib (GDC-0973; XL518) MK2206

I (NCT01764828) Refametinib alone and + Gemin Asian pts Second Phase I 5 pts I [32] 45 pts Pimasertib + ND I [34] SAR245409 I [37]

49 pts

51 pts 17 Japanese pts (NCT01859026) MEK162 + erlotinib (NCT01581060) ND (NCT01581060) ND: WX-554 alone and +WX-037 13 pts I [52]

I I I I I

[45] [48]

I [61,62] I [64]

33 pts MK-2206 + selumetinib 72 pts MK-2206 + or CBDCA and pacli, doce, or erlotinib

3 pts with unknown RAS mutational status ND 4 pts ND ND ND

Best response in pts with NSCLC

1 SD 3 SD 2 SD 7 SD mPFS 1.8 months (95% CI: 1.5 -- 1.9) mOS 7.8 months (95% CI: 4.5 -- 13.9) SD (ND in NSCLC) 2 PR with selumetinib, 1 CR and 1 PR with pem. mPFS 90 vs 67 days (HR 1.08, two-sided 80% CI: 0.75 -- 1.54; p = 0.79) mPFS 5.3 vs 2.1 months (HR) (for progression 0.58; 80% CI: 0.42 -- 0.79; p = 0.014); mOS 9.4 months in the selumetinib group and vs 5.2 months in the placebo group hazard ratio (HR) for(HR 0.80, 80% CI: 0.56 -- 1.14; p = 0.21) mutKRAS pts: 3 PR and 8 SD, ORR: 17%, DCR: 61%. WT pts: 5 PR and 11 SD, ORR: 21%, DCR: 67% All pts: 7 PR and 22 SD, ORR: 17%, DCR: 69%. mPFS: 5.1 months (95%CI: 2.8 -- 7.1 months); WT KRAS pts: 3 PR and 11 SD, ORR: 16%, DCR: 74%. mPFS: 4 months (95% CI: 1.3 -- 8.4 months); mutKRAS pts: 4 PR and 11 SD, ORR: 17%, DCR: 65%. mPFS: 5.8 months (95%CI: 2.8 -- 7.1 months) mPFS: 11.7 vs 11.4 wk for trametinib vs doce (95% CI: 0.75 -- 1.75; p = 0.5197) ORR : 12% for both arms NA SD with an average duration of 8 months 2 SD (with mutKRAS gene)

SD SD NA NA ND ND

ND

SD for 7 months and continues on treatment

NA

ND

13 pts

1 PR lasting 6 months 1 SD lasting 7 months

CBDCA: Carboplatino; CR: Complete response; DCR: Disease control rate; doce: Docetaxel; mPFS: Median progression-free survival; mutKRAS: Mutated KRAS; NA: Not available; ND: Not defined; ORR: Objective response rate; pacli: Paclitaxel; Pem: Pemetrexed; PR: Partial response; Pts: Patients; SD: Stable disease; WT KRAS: Wild-type KRAS.

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Table 2. Most important toxicities of allosteric inhibitors in Phase II study.

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Class

Drugs

Phase of clinical development

Drug dose

CI-1040

II [17]

800 mg b.i.d.

PD0325901

II [21]

Start with 15 mg b.i.d. After Phase I study results: 15 mg b.i.d. for 5 days a week, 3 weeks on, 1 week off

MEK-I Selumetinib

Trametinib

II [29] II [30]

II [44]

100 mg b.i.d. 75 mg b.i.d.

2 mg/day

Principal toxicities

Diarrhoea, nausea, rash, oedema and abdominal pain Anorexia fatigue and vomiting Anaemia, nausea and reversible visual disturbances Diarrhoea and rash Nausea, anaemia,reversible visual disturbances, pulmonary embolism and one case of congestive heart failure

Diarrhoea and fatigue Diarrhoea and skin rash Diarrhoea, stomatitis, anorexia, alopecia, fatigue and pyrexia and rush Nausea, vomiting, asthenia and febrile neutropenia Asthenia, oedema and nausea Haematological toxicities

Grade of toxicities (G) with percentage of patients (%) G1-2: 17 -- 50% of pts G3-4: 6 -- 17% of pts G1-2: 38 -- 46% of pts G3: 8% of pts G1-2: 5 -- 48% of pts

G3-4: 5 -- 10% of pts G1-2: 30 -- 43% of pts G1-2: 23 -- 73% of pts G3-4: 2 -- 9% of pts G1-2: 50% of pts G3-4: 26% of pts

b.i.d.: Bis in die (twice a day); Pts: Patients.

inhibitors, such as MEK and Akt inhibitors, for the treatment of NSCLC (Tables 1 and 2). 2.

MEK inhibitors

MAPKs are serine/threonine and tyrosine protein kinases that respond to extracellular signals and regulate cell proliferation, apoptosis, differentiation and gene expression. Alteration of MAPKs may result in carcinogenesis and in maintenance of the malignant phenotype in various types of tumours. Three major subfamilies of mammalian MAPKs are known to play important biological functions; they are the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase and p38, which phosphorylate a substrate containing the minimal consensus sequence Ser/Thr-Pro. The most important MAPK pathway in the tumourigenesis is the Ras-Raf-MEKERK pathway, which is deregulated in a significant percentage of solid tumours, including approximately 20 -- 35% on NSCLCs. In particular, activating KRAS mutations are highly frequent in NSCLC and till now, targeted therapy developed for KRAS-mutant NSCLC failed to demonstrate clinical activity. Despite decades of effort, KRAS itself remains undruggable, and now attention has recently focused on inhibition of the KRAS-downstream signalling. Mutations of KRAS and RAF lead to a sustained activation of MEK1 and MEK2, which in turn phosphorylate ERK1 and ERK2, thus predicting response to MEK inhibitors in laboratory models [10-12]. In addition, recent work from our group demonstrated the role of MEK1/2 activation as an important mechanism of 812

acquired resistance to ATP-competitive inhibitors, gefitinib and erlotinib, in advanced NSCLC [13]. Indeed, the aberrant activation of the Ras-Raf-MEK-ERK pathway is responsible of the acquisition of a mesenchymal phenotype, in the context of an epithelial-mesenchymal transition (EMT), and the inhibition of MEK1/2 can revert the resistance to EGFR inhibitors [13]. Several MEK inhibitors are in preclinical and clinical development for the treatment of NSCLC. We discussed those inhibitors that reached most advanced clinical development. CI-1040 (PD184352) The most studied allosteric kinase inhibitor is CI-1040 (PD184352), a highly selective oral noncompetitive inhibitor of MEK-1/2. In vitro experiments demonstrated antitumour activity of CI-1040 in several types of cancer along with a dosedependent G1 arrest. Significant antitumour activity was demonstrated also in vivo, in a wide panel of human xenograft models, including tumours of the colon, breast, pancreas, lung and kidney [14]. In particular, as paclitaxel has been demonstrated to activate the MAPK pathway in tumour cells, a combined treatment with CI-1040 and paclitaxel has been tested in mice xenografted with NSCLC cell lines or heterotransplanted by fragments of tumours from NSCLC patients. The combination treatment reduced both growth and angiogenesis of tumours. Delay in tumour growth was associated with the suppression of pERK ‡ 50% and was significantly 2.1

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Type III or allosteric kinase inhibitors for the treatment of non-small cell lung cancer

greater when pERK was suppressed by ‡ 90%. The inhibition of phosphorylation of ERK has been therefore proposed as a potential marker of in vivo activity of CI-1040 [15]. Supported by these strong preclinical evidences, CI-1040 was studied in a Phase I dose-escalation trial to assess the toxicity, PKs, pharmacodynamics (PD), maximum tolerated dose (MTD) and clinical activity of the drug. Seventy-seven metastatic patients affected by different tumours (colorectal, NSCLC, pancreas, kidney cancer and melanoma) resistant to conventional therapy were enrolled. The MTD was identified at 800 mg bis in die (twice a day [b.i.d.]) and was based on 2 out of 7 patients who developed grade 3 asthenia during their first cycle. The most common drug-related adverse events were generally grade 1/2 and consisted of diarrhoea, asthenia, rash, nausea and vomiting. Sixty-six patients were evaluable for response and only one patient with pancreatic cancer demonstrated a partial response (PR) and a significant improvement of symptoms. Nineteen patients (28%) achieved stable disease (SD) with median duration of 5.5 months (range: 4 -- 17 months). Of the nine enrolled patients with NSCLC, only one obtained SD [16]. PD of CI-1040 was evaluated by measurement of pERK levels on peripheral blood mononuclear cells (PBMCs) or tumour biopsy tissues. Inhibition of tumour pERK (median: 73%; range: 46 -- 100%) was demonstrated in all evaluable patients. Assessment of pERK inhibition in PBMCs demonstrated an association between CI-1040 concentration and target suppression and was achieved at drug plasma concentrations exceeding 100 ng/ml. Based on the good tolerability, favourable PK and biomarker response, the 800 mg bid dose was selected as the recommended Phase II study dose (RP2D). This dose was tested in a multi-centre, open-label, Phase II study whose objective was to evaluate the antitumour activity and safety of CI-1040 in pretreated patients with different types of cancer in advanced stage (colon, NSCLC, breast or pancreas cancer). Of 67 patients enrolled, 18 had advanced NSCLC, of which only one in first-line treatment. No complete or partial responses were observed, and three patients with NSCLC achieved SD. Treatment was well tolerated, with 81% of patients with grade 1 -- 2 toxicity. Similarly to the Phase I trial, most common adverse events were diarrhoea, nausea, fatigue, rash, oedema, vomiting, abdominal pain and anorexia. The immunohistochemical evaluation for activated ERK (pERK) of archived tumour samples revealed a mild association (p 0.055) between baseline pERK expression and SD [17]. Although the insufficient antitumour activity of CI-1040 limited further single-agent development, the efficacy in targeting MAPK pathway encouraged the clinical development of a second-generation MEK inhibitor, PD0325901, more potent against MEK and with better oral bioavailability. PD0325901 PD0325901 has been obtained from structural changes of CI-1040. It is a potent inhibitor of MEK1/2 and shows 2.2

higher anticancer activity and better oral bioavailability than CI-1040. PD0325901 has been tested in genetically engineered mouse models of NSCLC with KRASG12D or BRAFV600E mutation. Oral administration of PD0325901 induced tumour regression and prevented expansion of new tumour lesions, which increased again soon after treatment interruption [18]. PD and PK profile of PD0325901 has also been investigated in male Sprague-Dawley rats treated with oral (p.o.) and intravenous (i.v.) doses at 10, 30 and 100 mg/kg and 60,180 and 600 mg/mq, respectively. Inhibition on pMAPK in lung tissue was quantised by western blot and by measurement of plasma levels. Both p.o. and i.v. administration showed analogue bioavailability, hepatic metabolism and toxicities. The MTD was 100 mg/kg with following adverse events: red staining of the muzzle, diarrhoea, hypo-activity and (when administered orally p.o.) also ataxia [19]. PD, PK and tolerability of PD0325901 has been evaluated in a Phase I study on 66 patients with multiple solid tumours. In the study, 15 mg b.i.d. was the highest dose with an acceptable incidence (< 33%) of DLT. However, it was associated with delayed development of retinal vein occlusion (RVO). Although an alternative dose of 10 mg b.i.d. given 5 days on/2 days off, was tested, one RVO event occurred so further enrollment to this trial was stopped. Most common adverse events were diarrhoea, asthenia, nausea, acneiform rash and transient ocular disorders. Preliminary evaluation of clinical activity on 58 patients showed 3 PR (all melanoma patients) and 23 SD (21 patients with melanoma and 2 patients with NSCLC). Doses of 2 mg b.i.d. consistently caused 60% suppression of phosphorylated ERK in melanoma. Fifteen patients showed significant decreases in Ki-67 [20]. PD0325901 was also evaluated in a multi-centre, single-arm, open-label Phase II study in metastatic NSCLC patients after failure of previous therapy [21]. In this study, two schedules of treatment were used. PD0325901 was initially administered orally at 15 mg b.i.d. with food (3 weeks on, 1 week off). Results of safety profile from the Phase I trial suggested continuing with a different schedule of treatment (15 mg b.i.d. for 5 days a week, 3 weeks on, 1 week off). A total of 34 pretreated NSCLC patients were enrolled, 13 patients in the first schedule and 21 patients in the second one. Overall response rate (ORR), which was the primary endpoint, was not reached: no objective responses were reported; only seven patients presented SD. Median progression-free survival (PFS) was 1.8 months (95% CI: 1.5 -- 1.9) and median overall survival (OS) was 7.8 months (95% CI: 4.5 -- 13.9). The principal adverse events were anaemia, diarrhoea, rash, nausea and reversible visual disturbances. In the group treated with the second schedule, two cases of pulmonary embolism and one case of grade 2 congestive heart failure were reported, all drug-related. We suggest further studies should consider lower doses of PD0325901 with intermittent schedule and should focus on patient’s screening based on BRAF and KRAS mutations. In

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addition, PFS or disease control rate (DCR) may represent better endpoints to evaluate activity of this drug class. The PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are known to interact with each other at several points, and increasing evidences suggest that blockade of both pathways is required to achieve higher anticancer effects; PD0325901 has been studied in combination with PI3K/mTOR inhibitors. In addition, activating mutations of PIK3CA have been described as mechanism of acquired resistance to MEK inhibitors [22]. The combination of PD0325901 and rapamycin (mTOR inhibitor) has been investigated in human lung tumour xenografts or heterotransplants in nude mice. Combined treatment was well tolerated and more effective in tumour regression than single agent [23]. PD0325901 has been also studied in association with PF-04691505 (PF-502), a PI3K/mTOR inhibitor, in orthotopic lung tumour models in mice derived from H460 cell line (with KRAS and PIK3CA mutations) and in GEMMs KRASG12D-LSL. The combination was stronger in inhibition of tumour expansion, tumour invasiveness and development of other NSCLC lesions in both models as compared with single agent [24]. A Phase I trial is ongoing in patients with advanced cancer evaluating the PI3K/mTOR inhibitor PF-05212384 plus PD-0325901 or irinotecan (NCT01347866).

Selumetinib (AZD 6244; ARRY-142886) Preclinical efficacy of selumetinib has been widely studied in several human tumours [25]. In NSCLC models, selumetinib has been demonstrated to block cell proliferation and tumour progression, especially in those lung tumours with acquisition of mesenchymal phenotype at progression after an EGFR inhibitor [13]. The role of selumetinib has also demonstrated strong activity in vitro and in vivo in combination with an mTOR inhibitor (AZD 8055) [26]. PD profile showed inhibition of both MAPK and AKT signalling and activation of apoptosis by combination schedule. Based on the evidence that MAPK activation is one of the possible escape mechanisms of cancer cells to survive to radiation treatment, selumetinib has been also studied in combination with radiation therapy. The combined treatment lead to a doubling time of NSCLC A549 cell line-derived xenografts [27]. Single-agent selumetinib has been tested in Phase I study on 57 patients with advanced cancer. The RP2D was defined as 100 mg b.i.d., with grade 1 -- 2 diarrhoea, skin rash and hypoxia as most frequent adverse events. Less frequently, patients showed nausea, fatigue, peripheral oedema, derangement of liver function tests and blurred vision. Best response was SD reported in 33% of patients. Although the number of patients was too low for a statistical analysis, those with tumours harbouring mutated KRAS or BRAF seemed to have a better response [28]. 2.3

814

The efficacy of selumetinib in NSCLC patients was evaluated in two Phase II trials. Hainsworth et al. investigated selumetinib versus pemetrexed as second-/third-line treatment, with PFS as the primary endpoint. Treatments were generally well tolerated, with different toxicity profile: diarrhoea and skin rash for selumetinib versus asthenia, nausea, myelotoxicity for pemetrexed. Two patients showed response in both arms (two PR with selumetinib, one CR and one PR with pemetrexed). Median PFS was not statistically different between two arms (90 vs 67 days, HR 1.08, two-sided 80% CI: 0.75 -- 1.54; p = 0.79, for pemetrexed and selumetinib, respectively). These results suggested that selumetinib was not better than pemetrexed in the unselected NSCLC population [29]. More recently, a prospective, randomised, double-blinded Phase II trial evaluated docetaxel plus selumetinib/placebo in previously treated patients with KRAS-mutant advanced NSCLC [30]. Forty-four patients received selumetinib at 75 mg b.i.d. plus docetaxel at 75 mg/mq on day 1 of a 21-day cycle (selumetinib group) and 43 were treated with placebo and docetaxel (placebo group). OS was the primary endpoint. This was not met but the trial demonstrated a not significant trend for longer OS in the selumetinib arm (9.4 vs 5.2 months; HR 0.80, 80% CI: 0.56 -- 1.14; p = 0.21). Median PFS was significantly longer in the selumetinib arm (5.3 vs 2.1 months, HR 0.58, 80% CI: 0.42 -- 0.79; p = 0.014). In addition, response rate was higher in the selumetinib group (37 vs 0%, p < 0.0001). Grade ‡ 3 adverse events were more frequent in the selumetinib group (82 vs 67%). Notably, the incidence of febrile neutropenia was higher in the selumetinib group (18 vs 0%) and 36 and 55% of patients had a dose reduction and a dose interruption of selumetinib, respectively. Only 10% of patients in the placebo group needed a dose interruption and there were no dose reductions of placebo. Selumetinib plus docetaxel showed promising efficacy, albeit with a higher number of adverse events than with docetaxel alone, in previously treated advanced KRAS-mutant NSCLC. Given the promising results of selumetinib/docetaxel in the KRAS mutant population, a randomised, double-blind, placebo-controlled Phase III trial, SELumetinib Evaluation as Combination Therapy-1 (SELECT-1) is evaluating the safety and efficacy of selumetinib plus docetaxel as a second-line therapy in pretreated KRAS-mutant advanced NSCLC patients (NCT01933932). Patients will be randomised in a ratio of 1:1 to receive either selumetinib (75 mg, p.o. and b.i.d.) or matching placebo in combination with docetaxel (75 mg/mq, on day 1 of every 21-day cycle). PFS is primary endpoint and OS the secondary endpoint. Refametinib (RDEA119, BAY 869766) Several in vivo studies demonstrated a good antitumour activity of refametinib, also known as RDEA119 or BAY 869766, in xenograft models of various human tumours types along with an acceptable PK profile and a good tolerability [31]. 2.4

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Type III or allosteric kinase inhibitors for the treatment of non-small cell lung cancer

In addition, preclinical studies, on several human NSCLC xenograft tumour models, suggest a stronger activity when combined with a PI3K inhibitor (BAY 80 -- 6946) [32]. Two Phase I studies have been conducted to evaluate safety and recommended Phase II dose. One trial has tested refametinib as a single agent and in combination with gemcitabine (NCT01764828 trial) in Asian patients, and results are not yet available. The second Phase I study has been conducted to investigate the MTD of refametinib, its PK and PD and the tolerability profile. The MTD was £ 100 mg once daily (OD) or 50 mg b.i.d. Most common adverse events all G1-G2 were: skin rash, diarrhoea, asthenia, nausea and vomiting [33]. Among the five patients enrolled with advanced NSCLC, the best response was SD, with an average duration of 8 months [12]. Pimasertib (AS703026-MSC1936369B) Pimasertib has demonstrated to strongly inhibit proliferation and progression of NSCLC, in vitro and in vivo [13]. Similarly to other MEK-inhibitors, pimasertib demonstrated synergism when combined with the PI3K inhibitor BEZ235, or with the multitargeted inhibitor sorafenib, in nude mice xenografted with the NSCLC cell line H1975, which harbour KRASNRAS-BRAF WT genes and mutation of PI3KCA and EGFR (T790M and L858R) genes. The efficacy of the combination was superior to pimasertib alone [34]. Currently, only one Phase I dose-escalation trial on the combination treatment of pimasertib plus the PI3K/mTOR inhibitor, SAR245409, has been conducted. MTD was 90 mg OD for pimasertib plus 70 mg of SAR245409 with a RP2D of 60 mg OD for pimasertib and 70 mg OD for SAR245409. Most common adverse events were represented by skin rash, asthenia, diarrhoea, nausea and vomiting. On a total of 45 enrolled patients, 2 PR and 7 SD (lasting > 12 weeks) were observed, which included two NSCLC patients with mutated KRAS gene [35]. 2.5

RO4987655 (CH4987655) RO4987655 (CH4987655) is a highly selective oral MEK inhibitor that showed good antitumoural activity in vivo in various types of tumours (NSCLC, pancreatic and hepatocellular cancer) inducing cancer regression in 70% of cancer xenograft models [36]. A Phase I trial in healthy volunteers demonstrated an excellent PK and a good bioavailability with a single dose of RO4987655 administered orally [37]. Another dose-escalation Phase I trial evaluated safety, PK, PD, MTD, DLT and efficacy of RO4987655 in 49 patients affected by metastatic solid cancer (three patients with NSCLC with unknown KRAS mutational status). RO4987655 was administrated as oral capsules at dose-escalation schedule from 1 to 2.5 mg OD in a first cohort (13 patients) and then increased in doses from 3 to 21 mg b.i.d. (36 patients). MTD was 8.5 mg b.i.d. About 20% of patients showed PR 2.6

or SD lasting > 16 weeks; adverse events recorded were skin toxicity, nausea, vomiting, diarrhoea, stomatitis, ocular disorders and CPK elevation. Eleven patients stopped treatment due to drug-induced toxicity. In this trial, a correlation with mutational status of KRAS and RAF was found and therefore evaluation of other four cohorts of patients was planned: melanoma with or without BRAF V600E mutation, colorectal cancer with KRAS and/or BRAF V600E mutations and NSCLC with KRAS mutation [38]. Trametinib (GSK1120212) Trametinib is another oral, potent and selective allosteric inhibitor of MEK1/2. Data in vivo revealed long circulating half-life and high activity with 75% of tumour growth inhibition [39]. MTD, RP2D, safety, PD, PK and ORR represented the main objectives of a multi-centre, dose-escalation Phase I study performed in 206 patients with metastatic solid cancers. MTD resulted as 3 mg/die, whereas RP2D was 2 mg/die. ORR was 10% on a total of 21 patients. The most important adverse events were: skin rash and diarrhoea; retinopathy was high-dose related [40]. Another Phase Ib study studied safety, tolerability and RP2D of trametinib in association with gemcitabine, which resulted in 2 mg/day of trametinib in combination with gemcitabine. At this dose, the principal adverse events were thrombocytopenia and leucopoenia, skin rash, asthenia, diarrhoea, nausea and vomiting. One CR was reported in a patient affected by breast cancer, four PR were seen in three patients affected by pancreatic cancer and one with salivary gland cancer [41]. Two Phase I trials using trametinib in combination with chemotherapy in patients with NSCLC have been conducted. The first study was a two-part, five-arm, Phase I/Ib, openlabel study designed to evaluate safety, tolerability and efficacy of trametinib in combination with docetaxel, erlotinib, pemetrexed, pemetrexed+carboplatin/cisplatin or nab-paclitaxel in patients with advanced solid tumours (NCT01192165). Part I consisted of a dose-escalation trametinib in combination with chemotherapy; part II was a dose expansion in patients with NSCLC and pancreatic carcinoma. Based on the promising activity from the first part of the study, in part 2, NSCLC patients were treated with trametinib plus docetaxel combination and/or trametinib plus pemetrexed combination and were stratified for mutational status of KRAS gene (KRAS WT or KRAS mutant) [42]. Forty-six NSCLC patients (24 WT and 22 mutated KRAS) were treated at the trametinib plus docetaxel RP2D. The most frequent toxicities were diarrhoea, fatigue, asthenia and nausea. In patients with KRAS mutation, there were three PR and eight SD with an ORR of 17% and a DCR of 61%. In KRASWT patients, 5 PR and 11 SD with an ORR of 21% and a DCR of 67% were observed. The final response and PFS data are not yet available [43]. Forty-two patients were treated with the combination trametinib at the RP2D plus pemetrexed. Reported adverse 2.7

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events were asthenia, oedema, nausea and G3-4 haematological toxicities. Fifteen patients needed a dose reduction for diarrhoea, asthenia or ejection fraction decrement. ORR was 17% with 7 PR and 22 SD, and DCR was 69% for the whole population of NSCLC patients, including patients previously treated with pemetrexed and in patients with squamous histology. PFS was 5.1 months (95% CI: 2.8 -- 7.1 months) for all patients with NSCLC. Among the patients with KRAS WT (n = 19 patients), 3 PR and 11 SD were observed with a DCR of 74% and an ORR of 16%. Median PFS was 4 months (95% CI: 1.3 -- 8.4 months). In patients with KRAS mutation (n = 23 patients) were reported 4 PR and, 11 SD were reported with an ORR of 17% and DCR of 65%, RR of 15% and > 20% of tumour reduction in 15% of patients. In this subgroup, median PFS was 5.8 months (95% CI: 2.8 -- 7.1 months) [44]. In a second study not yet open for recruitment, trametinib will be evaluated in association with docetaxel as second-line treatment for Japanese patients with advanced NSCLC. A Phase II study, NCT01362296, was designed to evaluate the efficacy of trametinib compared with docetaxel in KRASmutant platinum-pretreated NSCLC patients. Primary end point was PFS, and secondary end points were OS, ORR, duration of response and safety. Patients were randomised 2:1 to trametinib (2 mg every day [QD]) or docetaxel (75 mg/mq i.v. every 3 weeks), and crossover at disease progression was allowed. Median PFS was 11.7 weeks versus 11.4 weeks for trametinib versus docetaxel, respectively (95% CI: 0.75 -- 1.75; p = 0.5197). ORR was 12% for both arms. The principal adverse events with trametinib were rash, diarrhoea, nausea, hypertension and dyspnoea [45]. TAK-733 TAK-733 is a small molecule MEK1/2 inhibitor that has demonstrated strong tumour activity in different types of mouse xenograft models such as colorectal, NSCLC, melanoma, pancreatic and breast cancer. It was well tolerated with OD oral dosing in humans. Preliminary results of a Phase I study, dose-escalation of TAK-733 in patients with metastatic solid cancers (NCT00948467), were presented at the ASCO Annual meeting 2013. Fifty-one patients who received dose escalation of TAK-733 were enrolled. The MTD was 16 mg OD and correlated with pERK inhibition in peripheral blood samples. SD was recorded in 15 patients; only 1 patient with melanoma had a PR after 4 cycles of treatment. Forty-five (88%) patients had adverse events < G2, of which the most frequent was acneiform rash (47%), and 20% of patients experienced > G3 drug-related adverse events. Seven patients interrupted treatment due to adverse events [46]. 2.8

MEK162 (ARRY 438162) MEK162 has been demonstrated to inhibit tumour growth in vivo regardless of any RAS/RAF pathway deregulation [47]. MEK162 is now being investigated in Phase I trials alone [48] 2.9

816

and in association with the PI3K inhibitor, BYL719, in advanced solid cancers (NCT01449058 trial). Recently, preliminary results of a multi-centre Phase I trial (NCT01469130) of MEK162 in Japanese pretreated patients with metastatic solid tumours was published. Primary objective was MTD and/or RP2D of MEK162. The study consisted of a dose-escalation part in patients with advanced solid tumours and of an expansion part in patients with BRAF/KRAS-mutant tumours. Seventeen patients were treated (4 patients with NSCLC), 14 in the dose-escalation and 3 in the expansion part. Main adverse events were rash, increased lipase, acneiform dermatitis, decreased appetite and stomatitis. Four patients discontinued treatment and 16 patients required a dose reduction/discontinuation due to adverse events. In the dose-escalation part, the MTD of MEK162 was identified at 45 mg b.i.d. [49]. Another Phase I/IB trial of MEK162 in combination with erlotinib in NSCLC harbouring KRAS or EGFR mutation is ongoing (NCT01859026 trial). In the Phase I doseescalation study, MEK162 is tested at 30 mg b.i.d. dose plus 100 -- 150 mg of erlotinib daily. WX-554 The Phase I trial was designed to determine safety, tolerance and the optimal biological dose for the inhibition of the MEK system by WX-554 in 25 healthy volunteers [50]. In addition to safety and tolerance, the PD and PK properties were also investigated. The study tested five increasing dose levels, each administered once by a 15-min infusion of WX-554 in five healthy male volunteers. WX-554 showed very good bioavailability and inhibition of the MEK signal transduction pathway in a dose-dependent manner achieving long-lasting inhibition at 100 mg. WX-554 was also safe and well tolerated. One subject at the highest dose level obtained a skin rash. A Phase Ib/II study of an oral formulation of WX-554 in patients with advanced cancer was started in April 2012 (NCT01581060), and a Phase I study with WX-554 alone and in combination with WX-037 in solid tumours is ongoing (NCT 01859351). 2.10

Cobimetinib (GDC-0973; XL518) Cobimetinib (GDC-0973; XL518) is an orally bioavailable allosteric inhibitor designed to selectively target MEK and showed encouraging preclinical in vitro and in vivo results [51]. A Phase I dose-escalating study of GDC-0973 was initiated in subjects with solid tumours. Preliminary results from 13 patients indicate that GDC-0973 is well tolerated with no drug-related serious adverse events being reported. One patient with NSCLC had SD for 7 months and continues on treatment [52]. 2.11

3.

AKT inhibitors

Many of the mutations discovered in NSCLC affect the phosphatidylinositol 3-kinase/AKT/mTOR (PI3K/AKT/mTOR)

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pathway, which is a key regulator of many cellular processes, including cell survival, proliferation and differentiation. The PI3K pathway activation, which occurs in 50 -- 70% of NSCLCs [53], confers a poor prognosis [54]. Activation of the pathway can result from upstream activation (e.g., EGFR, ALK, MET), alterations throughout the pathway itself (PTEN deletions; PI3K, AKT mutations) or parallel pathways [55,56]. For example, KRAS activation of the RAS/RAF/ MEK pathway can lead to cross talk through PI3K [57]. For these reasons, the development of serine/threoninespecific protein kinase AKT inhibitors could represent a new possibility to treat patients with acquired resistance to RTKs inhibitors, or with PI3K/AKT/mTOR pathway mutations or with parallel pathway activation. The high degree of homology in the ATP-binding pocket between AKT, protein kinase A and protein kinase C have initially hindered the development of AKT inhibitors. However, novel agents acting elsewhere within the protein have recently been developed. For example, MK-2206 target regions near the pleckstrin homology domain of AKT, which prevents AKT translocation to the plasma membrane. This keeps AKT inactive and renders it unable to activate the downstream targets that drive cell growth, metabolism, survival and proliferation. MK2206 MK-2206 is a first-in-class allosteric AKT1/2/3 inhibitor with evidence of preclinical activity. Preclincal studies demonstrated synergistic activity in combination with cytotoxic agents (doxorubicin, gemcitabine, docetaxel and carboplatin) in lung NCI-H460 cells. Furthermore, MK-2206 suppresses AKT phosphorylation induced by carboplatin and gemcitabine, thus improving efficacy of this agents by suppressing activation of this cell-survival pathway [58]. MK-2206 has also been shown to enhance erlotinib activity in erlotinibsensitive and -resistant NSCLC cell lines and to re-sensitise cells rendered resistant through c-MET activation by hepatocyte growth factor [59]. In a recent study, Lida et al. demonstrated that the activation of AKT is an important pathway in acquired resistance to cetuximab. The combination treatment with MK-2206 and cetuximab, in an in vitro resistance model of NCI-H226 NSCLC cell line, reduced the activity of both AKT and MAPK, thus highlighting the importance of simultaneous pathway inhibition to overcome acquired resistance to cetuximab [60]. In a Phase I study, MK-2206 caused tumour regressions in a patient with pancreatic cancer with documented loss of PTEN expression and caused minor tumour regression in one patient with melanoma and in one patient with a neuroendocrine tumour [61]. Common reversible drug-related toxicities included rash, nausea, fatigue and hyperglycaemia. In addition, on the basis of the preclinical rationale, the combination of MK-2206 and the MEK inhibitor, selumetinib, has been investigated and has shown encouraging activity, notably in NSCLC. 3.1

The objectives of the Phase I study were to evaluate safety, PK, PD and preliminary antitumour activity of the MK-2206 in combination with selumetinib (NCT01021748). Thirtythree patients with advanced solid tumours were treated with MK-2206 either every other day (QOD) or once weekly (QW), in combination with selumetinib administered continuously either OD or b.i.d. In the MK-2206 QOD-dosing schedule, MTD was 45 mg QOD with selumetinib 75 mg OD. For QW MK-2206, MTD was 90 mg QW with selumetinib 75 mg OD. Responses were reported in a KRASmutated low-grade ovarian cancer with a 64% Ca125 fall and in lung adenocarcinoma (KRAS status awaited) [62,63]. Another multi-arm, open-label, dose-escalation Phase I trial (NCT00848718) was designed to assess the MTD, DLTs, PK and efficacy of MK-2206 in combination with either carboplatin (AUC 6.0) and paclitaxel 200 mg/mq (arm 1), docetaxel 75 mg/mq (arm 2) or erlotinib 100 or 150 mg daily (arm 3). MTD of MK-2206 was 45 mg QOD or 200 mg Q3W (arm 1); MTD was 200 mg Q3W (arm 2) and 135 mg QW (arm 3). Main adverse events were skin rash, febrile neutropenia, tinnitus and stomatitis. In arm 2, a female patient with NSCLC progressed to two prior lines of platinum-based chemotherapy and erlotinib demonstrated a PR lasting 6 months. The patient withdrew from the study before documentation of progressive disease because of docetaxel-related toxicities. No ORR was observed in arm 3; the best response recorded in a male patient with NSCLC was SD lasting 7 months [64]. Two Phase I trials for metastatic NSCLC are active: one trial aims to establish dose of MK2206 in combination with gefitinib in patients with activating EGFR mutations who have failed chemotherapy and EGFR-TKI treatment (NCT01147211) and a second trial proposing testing MK2206 plus erlotinib to evaluate objective response and DCR in patients who have progressed on erlotinib treatment (NCT01294306). 4.

Conclusion

Protein kinases represent an attractive target for drug discovery. Considering the high frequency of mutation of the RAS/RAF/MEK/MAPK and PI3K/AKT/mTOR pathways in NSCLC, the development of MEK and AKT inhibitors has implemented the armamentarium of drugs available for such disease. Despite a solid preclinical rationale, results from early clinical studies are controversial showing a nonclear antitumour activity. From these studies, we have learned that successful MEK/AKT-directed treatment of NSCLC potentially depends on two factors: . .

identification of biomarkers to predict sensitivity selection of optimally beneficial drug combinations.

At this time, KRAS mutation may be the most reliable predictor of a cancer cell’s dependence on these pathways

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but other techniques have been evaluated to refine further selection. Concurrent treatment with cytotoxic agents, as pemetrexed or gemcitabine, has shown early promise in Phase II trials. Dual targeting of MEK with inhibition of other kinases in the same pathway (such as EGFR) or with inhibition of a parallel pathway (such as the PI3K/Akt pathway) are also promising directions for ongoing trials. According to the development of new kinase inhibitors for cancer treatment, there is a need to improve the knowledge about efficacy and safety of such drugs during pre- and postmarketing experience. However, premarketing drug development studies are lacking in information about a safe drugs use. Therefore, postmarketing surveillance activities, such as the intensive monitoring drug studies, are important to allow the early detection of unexpected and/or serious adverse reactions, which have a considerable negative impact on both health and healthcare costs. For such reasons, further specific studies are needed in order to evaluate the risk-benefit profile of kinase inhibitors [65,66]. 5.

Expert opinion

In last decades, novel therapeutics that specifically target proteins involved in signalling pathways have been developed to improve prognosis of patients with NSCLC, who experienced poor response and important toxicity with conventional treatments (platinum-based chemotherapy and radiotherapy). Erlotinib, gefitinib and afatinib, known as tyrosine kinase type 1 inhibitors, have received clinical approval in first-line setting of NSCLC patients with activating EGFR mutations, and in subsequent treatment lines also in EGFR WT tumours (only for erlotinib), representing the first step in developing a personalised cancer therapy. The employment of these drugs is limited by patient characteristics, poor selectivity and, especially, by early onset of resistance mechanisms. These include the appearance of EGFR T790M mutation, MET gene amplification, EGFR amplification, the acquisition of mesenchymal phenotype (EMT) and mutations in the PIK3CA gene. Thus, new approaches for the development of potent inhibitors with high selectivity are focused on the study of the signal downstream of EGFR. As protein kinases play an important part in human diseases, many efforts are undertaken to develop new stronger protein kinase inhibitors. Based on the central role in mediating transmission of growth-promoting, survival, differentiation and antiapoptotic signals from different growth factor receptors, the RAS/RAF/ MEK/ERK and PI3K/AKT/mTOR cascades provide main potential therapeutic targets in oncology. Mutations of such pathways (PIK3CA mutated in 5% of both adenocarcinomas and squamous cell carcinomas,

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AKT1 mutated in 5% squamous cell carcinomas, BRAF mutated in 5% adenocarcinomas, KRAS mutated in 15% adenocarcinomas and 5% squamous cell carcinomas, MAP2K1 mutated in 5% adenocarcinomas) are described in NSCLC and represent a potential new target for biological agents. Over the past few years, structural biology and medicinal chemistry have shown that protein kinases are very flexible and can adapt a variety of conformations, revealing new ‘druggable’ pockets other than the ATP-binding site. This offers the possibility of developing protein kinase drugs with different pharmacological properties, such as higher specificity. To explore these opportunities, it is important to utilise assays that allow the probing of different conformations adapted by the target protein. Biochemical assays can be utilised for this purpose, although the identification and profiling of conformation-specific inhibitors needs a careful design of the biochemical assays. These assays were previously carried out to identify classical ATP-competitive inhibitors. To identify inhibitors with new binding modes, new assays will require the integration of in vivo regulation of protein kinases, which will be helpful for the design of the biochemical assay. The protein status (length of the construct, phosphorylation levels, etc.), the nature of the substrate (synthetic peptide vs protein) and the presence of cofactors (additional proteins, etc.) are key parameters influencing the output of an assay and consequently the type of inhibitors identified. Most probably, it will be necessary to develop more than one assay to target different conformational states of the same protein. Furthermore, the complete characterisation of inhibitors may require the use of biophysical methods, especially if the compounds bind to a catalytically inactive conformation of the enzyme. In summary, the protein kinase field has made great progress with the result that many opportunities exist today for developing inhibitors with entirely new modes of action. Despite their apparent simplicity, biochemical assays will remain at the core of drug discovery activities to explore these new opportunities.

Declaration of interest This work has been supported by Associazione Italiana Per La Ricerca Sul Cancro (AIRC)-Project MFAG 2013-N.14392. The authors have no other relevant affiliations or financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Affiliation Morena Fasano1, Carminia Maria Della Corte1, Raffaele Califano2,3, Annalisa Capuano4, Teresa Troiani1, Erika Martinelli1, Fortunato Ciardiello1 & Floriana Morgillo†1 † Author for correspondence 1 Second University of Naples, Medical Oncology, Department of Experimental and Internal Medicine “F. Magrassi e A. Lanzara” , Via S. Pansini 5, 80131 Napoli, Italia Tel: +39 081 5666745; Fax: +39 081 5666732; E-mail: [email protected] 2 Cancer Research UK Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, M20 4BX, UK 3 University Hospital of South Manchester, Department of Medical Oncology, Manchester, M23 9LT, UK 4 Second University of Naples, Regional Centre of Pharmacovigilance and Pharmacoepidemiology. Department of Experimental Medicine “L. Donatelli”, Via De Crecchio, 7, 80138 Naples, Italy

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