PLX4032: does it keep its promise for metastatic melanoma treatment?
Elisabeth Livingstone, Lisa Zimmer, Sarah Piel & Dirk Schadendorf†
University Hospital Essen, Department of Dermatology and Skin Cancer Center, Germany

Importance of the field: Activating mutations in the BRAF kinase gene have been identified in 50% of all melanomas. PLX4032, a selective and potent inhibitor of BRAF V600E mutant tumor cells, has shown inhibition of tumor growth in cell lines harboring BRAF V600E mutations. Data from early clinical trials showed promising results in the treatment of patients with metastatic melanoma.
Areas covered in this review: An extensive literature search was conducted that included published articles and abstracts on PLX4032 to evaluate the existing data in both preclinical and Phase I–II studies.
What the reader will gain: The review comprises the rationale for choosing a
selective BRAF inhibitor for certain types of melanoma, its mode of action, associated toxicities and potential pitfalls.
Take home message: Despite the convincing response rates in Phase I trials, duration of tumor response is limited in some patients, and a cure cannot be expected. Intrinsic and acquired PLX4032 resistance still has to be investi- gated; signaling pathway switching is probably the most important factor for development of resistance. Combination therapy with simultaneous inhi- bition of different pathways might be more effective and warrants further investigation. The toxicity profile of PLX4032 is considerably low, and special attention is needed to address the development of keratoacanthomas and cutaneous squamous cell carcinomas.

Keywords: BRAF inhibitor, cutaneous squamous cell carcinoma, MAPK pathway, melanoma, PLX4032, RAS/RAF/MEK/ERK pathway, targeted therapy

Expert Opin. Investig. Drugs (2010) 19(11):1439-1449

⦁ Introduction

Melanoma is a very aggressive, therapy-resistant malignancy that derives from the pigment-producing cells. Although the skin is the most common localization of melanoma, it can generally develop wherever melanocytes are present including the mucosa, uvea and leptomeninges. Melanoma occurs in patients of all skin types; people with a fair complexion, however, are at higher risk of melanoma develop- ment. According to data from the WHO ~ 132,000 cases of melanoma are diag-
nosed globally each year [1]. The age-adjusted incidence rate of melanoma was
calculated to be at 3.1% a year between 1992 and 2004 in the US and doubled over a 10-year period [2]. For 2010, 68,130 new melanoma cases are estimated for the US with 8700 melanoma related deaths [3].
Whereas early-stage lesions with a tumor thickness of < 1 mm can generally be cured by surgery, melanoma treatment -- once metastasized beyond locoregional sites -- remains a challenge with 1-year survival rates of only 33% for advanced metastatic disease (stage M1c) [4]. In the past decades, various chemotherapies and biological agents have been used either as single agents or in combination therapies

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Box 1. Drug summary.
Drug name (generic) PLX4032 (also known as RG7204 or RO5185426) Phase (for indication under discussion) Phase I--III clinical studies
Indication (specific to discussion) Metastatic melanoma

Pharmacology description/mechanism of action
Selective inhibition of mutant BRAF (V600E/K)

Route of administration Oral administration

Chemical structure
A.

CI O

N N

F

O
F N S O
B.

W531

D594

F595

E501

c-helix
C. shift K507 E600

F595

R509
F595

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PLX4032-bound protomer Apo protomer

D.

K507

E600

3D structure of PLX4032 binding to BRAF (V600E) (Bollag et al. [49])
Pivotal trial(s) Phase I trial of PLX4032: Proof of concept for V600E BRAF mutation as a therapeutic target in human cancer [15].
Early efficacy signal demonstrated in advanced melanoma in a Phase I trial of the oncogenic BRAF-selective inhibitor PLX4032 [64].
An open-label multi-center study on the efficacy of continuous oral dosing of RO5185426 on tumor response in previously treated patients with metastatic melanoma.
Clinical Phase II trial, active, not recruiting
(see http://clinicaltrials.gov/ct2/show/NCT00949702?term = plx4032&rank = 2).
A randomized, open-label, controlled, multi-center, global study on progression-free and overall survival in previously untreated patients with unresectable stage IIIC or stage IV melanoma with V600E BRAF mutation receiving RO5185426 or dacarbazine.
Clinical Phase III trial, active, recruiting
(see http://clinicaltrials.gov/ct2/show/NCT01006980?term = Brim3&rank = 1).

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with only modest response rates, low complete response rates and little impact on overall survival [5-7].
In the US, dacarbazine, an alkylating agent, and IL-2 have been approved as standard therapies for first-line treatment in metastatic melanoma. In the EU, only dacarbazine obtained approval for the untreated metastatic melanoma patients
> 30 years ago. With reported response rates of only 5 — 10% in unselected patients and no survival benefit [8], dacarbazine cannot be considered the gold standard for mela- noma treatment. To date, no polychemotherapeutic regimens or combination regimens with added immunotherapy have shown to significantly prolong survival in controlled clinical trials. Recently, ipilimumab, a T-cell stimulating human mAb directed against CTLA-4, was shown to provide a sur- vival benefit in advanced stage disease for the first time [9]; response rates, however, are low with consistently reported ranges of only 10 — 15%. Due to these dismal therapeutic options, international guidelines for melanoma recommend the treatment of patients with metastatic disease within clini- cal trials investigating promising new agents as the first choice option [10-14].
One of these promising new targeted agents is PLX4032 (also known as RG7204 or RO5185426), an oral drug with high affinity for the mutated BRAF, which has shown impres- sive response rates in melanoma patients in a Phase I study (Box 1) [15]. This review summarizes available preclinical and clinical data regarding PLX4032 in cancer therapy with a special emphasis on its potential in patients with metastatic melanoma.

⦁ Ras/Raf/MEK/ERK pathway and melanoma

The substantial progress that has been made in understanding the molecular changes associated with the development of melanoma has led the way to the identification of new targets for therapeutical drugs. One of the most promising target pathways in melanoma is the upregulated Ras/Raf/MEK/ ERK pathway (Figure 1) [16,17].
Approximately a third of all cancers harbor genetic altera- tions that aberrantly upregulate MAPK-dependent signal transduction [18]. The MAPK cascade is activated by binding of a ligand to the membrane-bound receptor tyrosine kinase, which subsequently leads to activation of RAS. GTP-bound RAS, in turn, promotes the activation of RAF family proteins (ARAF, BRAF, CRAF) and then, through MEK 1/2 and ERK 1/2, the signal reaches the nucleus and a cellular response to the initial stimulus is delivered [19]. Activation of this path- way is related to cell proliferation [20] and resistance to apopto- sis [21]. Apart from endogenous receptor–ligand interactions, the pathway can be activated through mutations in NRAS or BRAF in melanoma. Other RAS isoforms and receptors (e.g., GPCRs) activate the pathway in other cancers, and the restriction to NRAS and BRAF only applies to melanoma.
In a milestone publication in 2002, Davies et al. reported that BRAF is mutated in ~ 8% of all human tumors [22] and
occurs in 50 — 70% of melanoma cell lines and tumors [22,23] but has also been observed in papillary thyroid (30 — 70%), ovarian (15 — 30%) and colorectal cancers (5 — 20%). Partic- ularly, melanoma of sun-exposed sites show BRAF altera- tions [24] and mutations persist from the primary lesion through later stages of vertical growth phase and to metastatic disease [19]. In roughly 90% of cases, a single glutamic acid for valine substitution at residue 600 (V600E) within the activa- tion segment of the kinase domain is present [22,25] which leads to a 500-fold increase in activity compared to the wild-type protein kinase [26]. In melanoma cells, BRAF V600E causes deregulated proliferation by overcoming the G1 restriction point [27,28] and causing D1 production in mid-G1 [28]. BRAF silencing has shown to induce regression in melanoma xenografts [29], indicating the essential role of BRAF for cell survival.

⦁ MAPK pathway as a therapeutical target

Several drugs have been developed which target at different levels of the Ras/Raf/MEK/ERK pathway. To this date, kinase inhibitors, farnesyl transferase inhibitors, antisense oligonu- cleotides and heat shock protein 90 inhibitors have been tested preclinically and in recent clinical trials. Especially, the pathway kinases are relatively easily druggable targets and attractive for therapy as cancers are often dependent on this class of molecule (‘oncogene addiction’) while normal cells are not [30,31].
Sorafenib, a small molecule multikinase inhibitor, is a pan RAF kinase inhibitor with the most extensive clinical experi- ence to date. Initially developed as a selective CRAF inhibitor, later studies demonstrated its inhibitory effect of several other protein kinases including VEGF receptor 2 and 3, PDGF receptor, Flt-3, c-kit and FGF receptor 1 [32,33]. In vivo, sora- fenib showed to be a relatively weak inhibitor of BRAF [30] and did not meet the expectations as a single agent [33] or in combination with cytotoxic drugs in the treatment of melanoma patients [34]. A Phase III trial of sorafenib in com- bination with carboplatin and paclitaxel in patients with advanced melanoma failed to meet its primary end point of improvement in overall survival [35]. Other small molecule inhibitors of RAF are currently being tested in clinical trials. XL281 (Exelixis) has led to a clinical benefit (partial response (PR) or stable disease) in 43% of patients (13/30) in a Phase I dose-escalation study [36]. RAF265 (Novartis) inhibits all three isoforms of RAF as well as mutant BRAF and has anti- angiogenic activity through inhibition of VEGFR-2 [37,38]. In a BRAF mutant xenograft mouse model, RAF265 (CHIR-265) causes tumor regression and dose-dependent tumor growth inhibition [39], and a Phase I study is currently being conducted. First data of a Phase I–II clinical trial of the selective BRAF inhibitor GSK2118436 (GlaxoSmithKline) in patients with metastatic melanoma and other solid tumors were reported at ASCO 2010 [40]. Response rates of GSK2118436 were comparable to PLX4032 with a > 20%

Figure 1. Simplified depiction of the MAPK and PI3K pathways: extracellular ligands bind to membrane-bound receptor tyrosine kinase which subsequently leads to activation of downstream kinases via phosphorylization. The signal reaches the nucleus resulting in cell proliferation and resistance to apoptosis.

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tumor decrease in 60% of patients with BRAF mutations at first restaging (8 — 9 weeks). Maximum tolerated dose had not been reached by the time of report; adverse events were very similar to those documented for PLX4032. Phase III clinical trials are planned for this substance.
MEK inhibitors were among the first to be tested in clinical trials [41] but so far have not shown to display sufficient anti- neoplastic activity as a single agent despite the fact that they selectively kill melanoma cells that carry BRAF V600E muta- tions in preclinical studies [42]. Ongoing studies are now test- ing the efficacy of the MEK1/MEK2 inhibitor AZD6244 in trials in which study entry is restricted to patients with activat- ing mutations in BRAF and/or RAS. Monotherapy with AZD6244 led to a wide range of mutations in MEK quickly causing resistance to treatment in those patients, which leads to PLX4720 resistance as well [43]. Furthermore, a number of new papers have appeared showing that PLX4032 resistant melanoma cell lines are dependent on MEK signaling [44,45] making combinations of agents targeting mutant BRAF and MEK1/MEK2 of high interest [46].

⦁ Pharmacokinetic data and preclinical characteristics of PLX4032

PLX4032 (RG7204 or RO5185426) is co-developed by Plexxikon, Inc. and Hoffmann-La Roche Pharmaceuticals under their 2006 license and collaboration agreement. The chemical structure of PLX4032 was published only very recently [47] and previous reports were somewhat misleading. The discovery and development of PLX4720, an analog of
PLX4032, are described in a publication by Bollag et al. [47]
and by Tsai et al. [48].
PLX4032 is an orally available and highly selective inhibi- tor of BRAF kinase activity with an IC50 of 31 nM against V600E-mutant BRAF and an IC50 of 100 nM for wild- type BRAF [49]. As PLX4032 showed a high potency against the growth of the BRAF-mutated A375 melanoma cell line (IC50 = 310 nM) and good selectivity versus other kinases, it was selected for further development [50]. Its selectivity of inhibition of mutated BRAF protein over the wild-type kinase as well as its selectivity against a panel of 70 other kinases that cover all branches of the kinome was demonstrated at the American AACR 2006 [51].
The cytotoxic effect exerted by PLX4032 was confirmed to be BRAF V600E specific in in vitro and in vivo melanoma models [52-56] leading to the inhibition of phospho-ERK, G1-phase cell cycle arrest and apoptosis [57]. Studies by Hersey et al. confirmed that the BRAF inhibitor PLX4720 induced apoptosis in human melanoma with mutated BRAF at concentrations achievable in vivo [55]. Sensitivity of the drug was found to broadly correlate with duration of the MAPK inhibitory effect and the reduction of cyclin D1 expression in a melanoma mouse xenograft [57]. The same study group demonstrated that recovery of MAPK signaling following PLX4032 treatment can occur but can be overcome by MEK inhibitor treatment [57]. Thus, it is clear that mutant BRAF is a viable therapeutic target. Treatment with selective BRAF inhibitors such as PLX4032 may rapidly induce hyper- activation of the MEK/ERK1/2 pathway in mutant N-RAS melanoma cells as recently described [58]. Furthermore, these

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authors demonstrate that C-RAF is the major RAF isoform involved in this process.
There is some evidence that PLX4032 may be more effec- tive in melanoma cell lines harboring homozygous BRAF mutations than those with heterozygous BRAF muta- tions [52,56,59]. Also, response to PLX4032 seems to depend on the cellular line, as chemical blockade with PLX4032 or BRAF silencing by short hairpin RNA only led to growth arrest and little or no cell death in the anaplastic thyroid carcinoma cell line ARO whereas it induced apoptosis in the melanoma cell line A375 [19], although both are driven by mutant BRAF. There are only limited preclinical studies investigating the effect of a combination treatment with chemotherapeutical agents and PLX4032. In an in vitro cell culture model of colon cancer, PLX4032 enhanced the activity of the cytotoxic drugs 5FU, SN-38 and oxaliplatin [54]. Response to combina- tion of PLX4032 with both capecitabine and bevacizumab was superior to monotherapy with PLX4032, capecitabine
or bevacizumab in a colon cancer xenograft system [60,61].
In the preclinical xenograft studies, PLX4032 was reported to have good bioavailability that allowed for prolonged expo- sure in both rodents and primates (F > 70%, t1/2 = 10 h) [62]. In initial reports from Plexxikon at the 2006 AACR meeting in Washington, no appreciable toxicity and no significant alterations in body weight were noted following once daily administration of PLX4032 over 14 days to mice. No other preclinical animal xenograft studies have reported toxic effects following the administration of either PLX4032 or PLX4720 [48,53,57,62,63].

⦁ PLX4032 in clinical trials

PLX4032 has been tested in a Phase I dose-escalation study in 49 patients with metastatic melanoma, 3 patients with thyroid cancer and 1 patient each with rectal, ovarian and germ cell carcinoma [15]. At the time of first data presentation at ASCO 2009, 9 of the 16 melanoma patients (69% M1C) with the BRAF V600E mutation treated at doses of 240 mg twice a day (b.i.d.) or higher of the increased bioavailability formulation had a PR by RECIST criteria, with tumor regres- sion of up to 83%. Five BRAF wild-type patients had progres- sive disease. The median progression-free survival of the 16 BRAF V600E positive melanoma patients was ~ 6 months.
In addition, three patients with BRAF V600E positive thyroid
cancer demonstrated stable disease or tumor regression.
In the 28 patients receiving the optimized formulation of PLX4032, AUC was dose-proportional and above target plasma levels at 240 mg b.i.d. and higher (AUC0 — 24 h > 240 mg b.i.d.
~ 500 — 1000 µM·h). Dose-limiting toxicity was reached at
1120 mg b.i.d. with three of five patients experiencing grade
3 rash and fatigue and one grade 3 arthralgia; one grade 4 pan- cytopenia was documented at 720 mg b.i.d. Adverse events up to a dose of and including 720 mg b.i.d. were reported to be mild and transient. A summary of adverse events is given in Table 1.
Based on these promising results, 31 exclusively mutation- positive V600E metastatic melanoma patients were subse- quently enrolled in an extension study [64]. Most patients had an advanced disease (stage M1C) with previous systemic therapy for metastatic disease. PLX4032 was well tolerated at 960 mg b.i.d. and set as the maximum tolerated dose. Of the 27 patients evaluable at the time of report, complete response was documented in 1 patient treated for 3 cycles, PRs of > 30% tumor regression by RECIST criteria were observed in 18 patients, with 15 patients showing responses of > 50%. Minor responses in six patients showed tumor regression between 10 and 30%. Figure 2 shows results of tumor response in an exemplary patient. Drug-related adverse events were predominantly mild in severity and included rash, joint pain, photosensitivity and fatigue [64]. An example of phototoxicity is given in Figure 3. Serious adverse events were observed in some patients after chronic treatment, including seven patients with cutaneous squamous cell carcinoma (keratoacanthoma subtype) that were treated by excision, while treatment with PLX4032 was continued.
A Phase II single-arm trial in treatment-naive patients opened at 13 sites in the US and Australia in September 2009. Results of this study are anticipated early 2011. A Phase III multi-center trial with the primary end point of overall survival recruited the first patient in January 2010. In this study, ~ 700 BRAF V600E mutation positive previ-
ously untreated metastatic melanoma patients will be ran-
domized 1:1 with PLX4032 960 mg b.i.d. or dacarbazine. Recruitment is being conducted at ~ 100 sites in the US, Australia and Europe and Canada with enrollment comple- tion anticipated by the end 2010. Results of this study are expected in early 2012.
Although almost 90% of BRAF mutations constitute V600E mutations which are targeted by PLX4032, there is mounting evidence that other V600 mutations also might be susceptible to selective inhibition, for example, melanoma patients carrying the V600K mutation are allowed to enter the clinical trial with GSK2118436 and recently Rubinstein et al. demonstrated that V600K mutations are also sensitive to PLX4032 action [65].
Importantly, as PLX4032 might induce hyperactivation of the MEK/ERK1/2 pathway which promotes resistance to apoptosis in both non-invasive and invasive mutant N-RAS melanoma cells, there is increasing awareness to genotypically stratify melanoma patients before enrollment on a mutant B-RAF inhibitor trial [58].
FDG-PET was demonstrated to be an effective tool in the early assessment of clinical response to PLX4032 treatment in metastatic melanoma patients [66]. Tumor lesions within the same patients responded relatively homogenously. The authors, therefore, speculated that there is little intrapatient molecular heterogeneity of melanoma cells with respect to effects of BRAF inhibition. However, early FDG-PET responses were not correlated with the best overall response rate.

Table 1. Summary of adverse events in $ 10% of patients (n = 55) of the clinical Phase I study.
studies above [67-69] are important to mention because the endogenous MAPK signaling in melanoma cells will eventually influence the outcome of the inhibitor treatment, whereas data

Adverse event All related adverse events
Related grade $ 3
from recombinant proteins can provide additional information regarding the protein conformation and drug binding.

Rash 29% 2%
Halaban et al. [56] stated that in their melanoma cell models

Fatigue 24% 2% PLX4032 activated ERK1/2 wild-type BRAF regardless of the
Pruritus 20% 2% status of mutations in NRAS or PTEN. In contrast to other
Photosensitivity reaction 14% 0% reports, the authors assume that the mode of action by which
Nausea 14% 0% PLX4032 activates RAF1 in wild-type BRAF is independent

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Anemia 13% 0%
Cutaneous squamous 11% 11%
cell carcinoma
Alopecia 11% 0%

Adapted from Flaherty et al. [15].

⦁ Potential of selective BRAF inhibitors to activate MAPK pathway in wild-type BRAF

Recent studies showed that selective BRAF inhibitors can lead to the activation of the RAF/MEK/ERK pathway in wild-type BRAF melanoma cells via RAF1 activation.
The selective BRAF inhibitor 885-A was demonstrated to bind to wild-type BRAF in KRAS-mutant cells and promote the formation of BRAF–CRAF complexes, CRAF activation and consequently MEK/ERK signaling [67]. These authors report the formation of endogenous BRAF–CRAF complexes as a result of PLX4720 treatment in NRAS mutant melanoma cells [67]. Interestingly, the authors describe that in contrast to 885-A this complex formation was sensitive to MEK activa- tion, an important observation with regard to the endogenous situation in melanoma cells.
Hatzivassiliou et al. [68], demonstrated that the BRAF- selective inhibitor GDC0879 promotes the formation of dimeric complexes of RAF-type kinases, specifically BRAF– CRAF, BRAF–ARAF and CRAF–CRAF complexes via a con- formational change in the kinase whereas PLX4720 prevented the formation of BRAF–CRAF complexes and was assumed to lead to conformational changes in BRAF which specifically impairs BRAF binding to other RAF kinases. This inhibitory effect of PLX4720 on the formation of BRAF–CRAF com- plexes was not observed in the endogenous situation but by using recombinant (and not full length) proteins [68]. The activation of the RAF/MEK/ERK pathway induced by PLX4720 was suggested to be promoted by the formation of CRAF–CRAF homodimers, a view that was supported by Poulikakos et al. [69]. Poulikakos et al. also demonstrated that MEK/ERK activation does not only occur in RAS- mutant cells but also in cells in which the RAS/RAF/MEK/ ERK pathway is activated by other oncogenes such as HER2. Poulikakos et al. also used recombinant proteins not expressed in melanoma cells to analyze a CRAF–CRAF complex, whereas the authors did not perform this experiment for a BRAF–CRAF complex. These different aspects of the three
of RAS-GTP as the mutant cell line expressing RAF1 R89L
was activated to the same degree as its wild-type counterpart. The findings of these studies underline the importance that BRAF-mutant specific inhibitors should only be used for can- cers caused by BRAF-mutant tumors and avoided especially in
cancers caused by RAS mutations [70].

⦁ Significance of therapy-associated development of cutaneous neoplasms

Data from the Phase I dose escalation study and the exten- sion cohort study showed that ~ 20% of patients treated with PLX4032 developed therapy-associated cutaneous neo- plasms [71]. Keratoacanthomas prevailed but squamous cell carcinomas were also found. The neoplasms occurred within
8 — 12 weeks after therapy initiation primarily in sun-exposed areas (head/neck 41%) and partially arose at multiple sites. Histologically, the neoplasms were well-differentiated with a low probability of invasive or metastatic potential.
Development of keratoacanthomas and cutaneous squa- mous cell carcinomas has also been observed in other RAF inhibitors such as sorafenib [72]. Speculation has, therefore, arisen that the occurrence of these cutaneous neoplasms in patients treated with RAF inhibitors is attributable to the acti- vation of ERK signaling [70,73]. This assumption is backed up by the findings that: i) the BRAF inhibitor GDC0879 causes hyperactivation of ERK and hyperproliferation in skin cells in mice [68] and ii) RAS mutations can be found in up to 20% of squamous cell carcinomas and in a subset of actinic keratoses pre-malignant lesions that can precede squamous cell carcinoma [74].
Behind this background, Mateus et al. [75] were the first to prospectively investigate biopsies of normal skin from patients treated with sorafenib or placebo for keratinocyte prolifera- tion and expression of proteins involved in the MAPK and PI3K pathway by immunohistochemistry. They could dem- onstrate that sorafenib stimulates keratinocyte proliferation and significantly activates ERK phosphorylation in normal skin when compared to placebo treated patients.

⦁ Expert opinion and conclusion

The promising results that PLX4032 has shown in clinical tri- als so far gives justifiable hope in the historically frustrating treatment of metastatic melanoma. The selectivity of

⦁ B.

Figure 2. Patient No. 69 (MD Anderson Cancer Center) from Phase I extension study [59] with impressive and early tumor response. A. PET scans at baseline and day 15 after initiation of PLX4032 treatment. B. Response of liver metastases, images of pretreatment phase, cycle 2 and cycle 4.

Figure 3. A. Phototoxicity emerged within 1 week after initiation of PLX4032 in this female patient after sun exposure on both arms. Patients complain of itching and discomfort similar to sunburn and should be advised to refrain from UV exposure. Phototoxicity usually responds to topical steroid treatment. B. Desquamation of skin similar to sunburn 1 week after appearance of phototoxicity reaction.

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PLX4032 for BRAF mutant tumor cells and the possible par- adoxical activation of the RAS/RAF/MEK/ERK pathway in wild-type BRAF emphasize the imperative of pretreatment screening of prospective patients. But, even if an activating BRAF mutation exists, some melanoma will be intrinsically
resistant to PLX4032 as shown previously in the Phase I clin- ical trial and in vitro studies [15,44,45,52]. These cell lines might rely on the co-activation of other signaling pathways includ- ing the PI3K/Akt pathway [52] or might show amplifications or overexpression of key cell cycle and survival components

that may limit the effectiveness of molecularly targeted thera- pies [76]. Further studies investigating the mechanisms of intrinsic resistance against PLX 4032 are warranted.
The duration of the response to PLX4032 is yet unknown but development of resistance has already been demonstrated in vitro [57] and requires investigation. Especially, signaling pathway switching is thought to be responsible for acquired resistance allowing for com- pensatory survival signals when BRAF is inhibited. The simultaneous administration of a MEK inhibitor has demonstrated to overcome resistance in these cell lines in vitro [46,57,58,66]. The dual inhibition of the MAPK and PI3K/Akt pathway is another approach thought to improve effectiveness of melanoma treatment as BRAF mutations are rarely found in the absence of genetic alter- ations of PTEN, Akt or PI3 kinase [77]. Clinical trials investigating combination therapies of PLX4032 with other kinase inhibitors can, therefore, be expected for the future. As normal cells also rely on these signaling path- ways, side effects as seen with the development of keratoacanthomas and cutaneous squamous cell carcinomas
in patients treated with PLX4032 have to be expected and will probably multiply if several pathways are blocked.
The otherwise well tolerable side effects as well as its oral availability make PLX4032 an attractive drug in mela- noma treatment. Whether the median PFS of around 6 — 8 months translates into an overall survival benefit needs confirmation in the currently recruiting clinical Phase III study. However, if this survival benefit is validated, PLX4032 would be a highly valuable drug inducing clinical responses in patients with higher tumor volume and in combination with other investigational agents such as MEK inhibitors and ipilimumab.

Declaration of interest

D Schadendorf has served on the Advisory boards for Schering-Plough, Roche, GlaxoSmithKline, Bristol-Myers Squibb and Plexxikon and has received fees for clinical study participation from Schering-Plough. The other authors declare no conflict of interest and have received no payment in preparation of this manuscript.

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7 epub
.. An outstanding paper describing the development of PLX4032.
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B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci USA 2008;105(8):3041-6
. An interesting paper giving insight into the development of selective BRAF inhibitors.
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⦁ Expert Opin. Investig. Drugs Downloaded from informahealthcare.com by York University Libraries on 01/01/15 For personal use only.
⦁ Smalley KS. PLX-4032, a small-molecule B-Raf inhibitor for the potential treatment of malignant melanoma.
Curr Opin Investig Drugs 2010;11(6):699-706
⦁ A concise and informative paper giving a comprising overview on clinical and preclinical data on PLX4032.
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J Transl Med 2010;8:39
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AACR-NCI-EORTC Int Congress 2007;23:abstract 252
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Am Assoc Cancer Res Ann Meeting 2008;99th:abstract 3437
⦁ Kaplan FM, Shao Y, Mayberry MM, Aplin AE. Hyperactivation of
MEK–ERK1/2 signaling and resistance to apoptosis induced by the oncogenic B-RAF inhibitor, PLX4720, in mutant N-RAS melanoma cells. Oncogene,
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.. An outstanding paper describing the first clinical results in melanoma using PLX4032.
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et al. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med 2010;8:67
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et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 2010;140(2):209-21
⦁ A very interesting paper describing possible mechanisms for tumor progression in non-mutant BRAF.
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activate the MAPK pathway and enhance growth. Nature 2010;464(7287):431-5
⦁ A very interesting paper describing possible mechanisms for tumor progression in non-mutant BRAF.
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wild-type BRAF. Nature 2010;464(7287):427-31
⦁ A very interesting paper describing possible mechanisms for tumor progression in non-mutant BRAF.
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2010;28(7 Suppl):abstract 8592
.. The first abstract giving further insight into the development of keratoacanthomas and squamous cell carcinomas in patients treated
with PLX4032.
⦁ Arnault JP, Wechsler J, Escudier B, et al. Keratoacanthomas and squamous cell carcinomas in patients receiving sorafenib. J Clin Oncol
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⦁ A well-written paper giving an overview on currently investigated inhibitors of the MAPK pathway.
⦁ Spencer JM, Kahn SM, Jiang W, et al. Activated ras genes occur in human actinic keratoses, premalignant precursors to squamous cell carcinomas.
Arch Dermatol 1995;131(7):796-800
⦁ Mateus C, Arnault J, Tomasic G, et al. Activation of the MAP-kinase pathway in the skin of patients treated with sorafenib: clinical and molecular characterization of skin tumors associated with the multikinase inhibitor sorafenib. J Clin Oncol
2010;28(7 Suppl):abstract 9130
⦁ Further insight into the development of keratoacanthomas and squamous cell carcinomas in patients with PLX4032 treatment.

Expert Opin. Investig. Drugs Downloaded from informahealthcare.com by York University Libraries on 01/01/15 For personal use only.

⦁ Smalley KS, Nathanson KL,
Flaherty KT. Genetic subgrouping of melanoma reveals new opportunities for targeted therapy. Cancer Res 2009;69(8):3241-4
⦁ Flaherty KT, Smalley KS. Preclinical and clinical development of targeted therapy in melanoma: attention to schedule. Pigment Cell Melanoma Res 2009;22(5):529-31

Affiliation
Elisabeth Livingstone MD, Lisa Zimmer MD, Sarah Piel MD & Dirk Schadendorf† MD
†Author for correspondence University Hospital Essen, Department of Dermatology, Hufelandstr 55,
45122 Essen, Germany
Tel: +49 201 723 4342; Fax: +49 201 723 5935;
E-mail: [email protected]