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Is combination therapy the next step to overcome resistance and reduce toxicities in melanoma?

Cancer Treatment Reviews, 4, 39, pages 305 - 312

Abstract

In the last few years, several drugs targeting signalling proteins critical for melanoma entered clinical evaluation. In 2011 vemurafenib (Zelboraf®, F. Hoffman-La Roche Ltd.) was approved for BRAF V600-positive melanoma and showed high overall response rates (48–53%). However recent results from a phase II clinical trial also showed that the median duration of response was 6.7 months and median progression free survival was 6.8 months with tumour relapse. Resistance to targeted agents is quite common and understanding of the underlying molecular mechanisms might predict response or failure. The knowledge of the mechanisms involved in intrinsic and acquired resistance to mutated BRAF is increasing swiftly. Subsequently the elucidation of these mechanisms resulted in the development of rational combination therapies to overcome toxicity and resistance. These combination therapies will be discussed.

Keywords: Vemurafenib, Melanoma, Drug resistance, Toxicity, Squamous cell carcinoma, Combination therapy.

Introduction

Vemurafenib (Zelboraf®, F. Hoffman-La Roche Ltd.) has recently been approved as monotherapy for BRAF V600E mutation-positive metastatic melanoma. For a long time dacarbazine was the standard of care with an overall response rate (ORR) of only 15–20% and no improvement in overall survival. 1 New treatment is clearly needed since the median survival of these patients is less than 1 year. 2 After the discovery of activating mutations in the serine/threonine kinase BRAF, found in approximately 50–70%3 and 4 of all melanomas, the possibilities for targeted therapy were investigated. This led to the approval of BRAF inhibitor (BRAFi) vemurafenib by the FDA in 2011. 5 All clinical trials confirmed high overall response rates (48–53%)5, 6, and 7 and in a phase III clinical trial vemurafenib showed, as compared to dacarbazine, both improved progression-free (PFS) and overall survival (OS) in patients with metastatic melanoma with the BRAF V600E mutation. 5 However recent results from a phase II trial showed that after a response duration of 6.7 months some patients show tumour relapse due to mechanisms that are not fully understood. 7 In addition approximately 25% of the patients develop hyperproliferative skin lesions and some even cutaneous squamous cell carcinoma (CSCC). Now investigators focus on the diverse pathways of vemurafenib resistance (and toxicity), because this might lead to new strategies to overcome or delay resistance and prolong responses.

In this paper we discuss the recent developments concerning BRAF signalling in melanoma pathogenesis and the development of possible combination therapies to overcome resistance and to reduce toxicity.

Vemurafenib has shown to benefit patients with BRAF V600E activating mutation in clinical trials as monotherapy. During the escalation phase of a phase I clinical trial 6 the recommended phase II dose was determined at 960 mg twice-daily (BID). In the extension phase, treatment with vemurafenib resulted in complete or partial tumour regression in the majority of patients (81%, 26 patients). In a phase II study 7 , vemurafenib treatment was effective (ORR: 53%) and no severe or life threatening toxic effects occurred. However a large number of patients (26%) developed CSCC or keratoacanthoma (KA). Dose reductions were needed in 45% of the patients and dose interruptions were needed in 64% of the patients. The median OS was 15.9 months. In a phase III clinical trial 5 , comparing vemurafenib to dacarbazine, vemurafenib therapy was associated with a longer median OS of 13.2 months compared to 9.6 months in the dacarbazine arm. The latest update on this study shows a 12-month OS of 55% for vemurafenib and 43% for dacarbazine. 8 Even though the responses are high, the duration of response has been limited due to development of resistance. The development of tumour resistance to single-targeted agents appears inevitable and given the high responses it is of pivotal importance to identify alternative therapies that overcome this problem.

Targeting BRAF: mechanism of action of vemurafenib

To understand the pathways that underlie resistance and toxicity, it is important to understand the mechanism of action of the drug. In 2002 researchers from the Sanger Institute found that a certain RAF kinase in the mitogen-activated protein kinase (MAPK) pathway, BRAF kinase, was mutated in approximately 8% of a cohort of 923 tumours and cancer cell lines. 4 BRAF mutations appeared most common in melanomas (60%), papillary thyroid, low malignant potential ovarian and colorectal cancers. 4 Melanomas that harbour the BRAF V600E mutation constitutively activate the MAPK pathway. Vemurafenib was then developed as a potent kinase inhibitor with specificity for the BRAF V600E mutation within cancer cells.9, 10, and 11

BRAF is the second kinase in the cascade consisting of RAS, RAF, MEK (mitogen-activated protein kinase) and ERK (extracellular signalling-regulated kinase [MAPK] kinase) ( Fig. 1 ). It is long known that signal transduction through this pathway is involved in proliferation, invasion and drug resistance of various cancer types. The MAPK pathway is one of the key regulators of cell cycle progression and is commonly activated in human tumours. In normal cells activation of receptor tyrosine kinase (RTK) stimulates phosphorylation of guanine exchange factor (GEF), including SOS1/SOS2, that activate RAS. Activated RAS binds to activated RAF which subsequently leads to phosphorylation of MEK. Finally MEK phosphorylates ERK which enters the nucleus. By inhibiting ERK signalling in a V600 BRAF-selective manner, with a BRAFi such as vemurafenib, cell proliferation is inhibited. 12

gr1

Fig. 1 Mechanism of action of vemurafenib in vemurafenib-sensitive cells. Mutated BRAF V600E causes excessive signalling in the RAS/RAF/MEK/ERK pathway, leading to increased MEK and ERK. (A) Hyperactivation of the pathway leads to excessive proliferation and subsequently to tumour growth. (B) When treated with a BRAF inhibitor such as vemurafenib, the pathway is inhibited, leading to tumour shrinkage. Additionally PTEN normally inhibits the PI3K pathway thus proliferation and cell survival through this pathway is inhibited also. RTK, receptor tyrosine kinase; PDGFRβ, beta-type platelet derived growth factor.

Vemurafenib only inhibits the ERK pathway and cell proliferation in tumours with mutant BRAF. In tumours and normal cells with wild-type BRAF vemurafenib causes paradoxical activation of the pathway which will be discussed in the toxicity and resistance part. However vemurafenib only reactivates the pathway when there is upstream RAS or RTK (receptor tyrosine kinase) activity ( Fig. 2 ). Since vemurafenib is a very specific inhibitor of ERK signalling this underlies its broad therapeutic index in V600E mutated melanoma.

gr2

Fig. 2 Mechanism of the development of cutaneous squamous cell carcinoma in non-mutated BRAF cells. (A) In wild-type BRAF cells, cell growth is regulated by the RAS/RAF/MEK/ERK pathway. (B) In RAS mutated cells with the BRAF wild-type gene that are treated with vemurafenib, BRAF/CRAF heterodimerization induces MEK/ERK signalling. This leads to increased proliferation, which might be the cause of the development of cutaneous squamous cell carcinoma in some patients.

Toxicity and resistance

Mechanisms of toxicity

Vemurafenib has shown remarkable results in clinical trials conducted so far. However approximately 25% of the patients develop hyperproliferative skin lesions and some even CSCC. These cutaneous side effects disappeared after drug withdrawal. An explanation for this can be found in the finding that selective BRAFi can suppress the RAF/MEK/ERK pathway in tumour cells that harbour a BRAF-mutation, but can activate this same pathway in tumour cells with a mutation in the KRAS gene, which possesses a wild type BRAF gene.13, 14, 15, and 16 In normal cells activation of the BRAF/MEK/ERK pathway promotes cell growth, but excessive activation is associated with cancer. Signal activation through RAS enzymes is low when BRAF is mutated, so ERK signalling is predominantly activated by mutated BRAF. This activation may lead to tumour growth ( Fig. 2 ). When BRAFi are given this activation of ERK by the mutated kinase is suppressed, which causes cell death and tumour shrinkage. In RAS mutated cells with the BRAF wild-type gene, BRAF inhibitors block phosphorylation of RAF monomers (BRAF or CRAF), which subsequently form dimeric complexes (BRAF–CRAF). The formation of these complexes causes excessive signalling; a process that is enhanced by the presence of an oncogenic RAS mutation.13, 14, 15, and 16 This activation is also called the paradoxical activation of ERK. This might explain the formation of skin lesions (KA) and CSCC in some patients treated with vemurafenib. Following this finding lesions from patients that were treated with a BRAFi were analysed for oncogenic mutations. Mutations in RAS, and particularly in HRAS, are frequent in CSCC and KA in patients treated with a BRAFi.17, 18, 19, and 20 RAS mutations and phosphoinositide 3-kinase (PI3K) gene mutations have been found as well.17 and 19 In addition the observation of Zimmer et al. (2012) suggest that melanocytes bearing or acquiring oncogenic RAS are at increased risk of developing secondary melanoma. 21 Further investigations are needed in larger groups to fully understand the mechanisms of the potentially melanoma-inducing effect of BRAF inhibitors.

Mechanisms of resistance

Even though the responses that are seen are profound, they are also temporary. There are cases known where patients show initial response after 2 months of treatment and then show disease progression another 2 months later. 22 On the other hand, there are also patients who have responses that persisted for 18 months and who remain on therapy. 23 Approximately 20% of patients with BRAF mutated melanoma tumours are not responsive at all due to intrinsic resistance. 24 It is important to know which mechanisms underlie the emergence of resistance following initial response to develop new rational therapeutic strategies to prevent or overcome resistance. So far, there is no evidence of gatekeeper resistance in BRAF inhibitor resistance. 25

Cell proliferation pathways are often complex and overlap each other. Thus, there may be different genetic alterations in cell proliferation pathways that can bypass BRAF inhibition. Mutations in the PI3K/AKT pathway and PTEN loss can affect responses to BRAFi ( Fig. 3 ).26 and 27 Moreover, overexpression of cyclin D1 in combination with CDK4 (cyclin-dependent kinase 4), may contribute to resistance to BRAF-specific inhibitors in BRAF mutated melanoma. 28 The overlap between mutations in cyclin D and CDK4 and BRAF mutations in melanoma is approximately 40%. 29 Additionally deletion of retinoblastoma protein (Rb) has also been reported to mediate intrinsic resistance. 27

gr3

Fig. 3 Mechanisms of intrinsic and acquired resistance in the RAS/RAF/MEK/ERK pathway. Intrinsic resistance for BRAF inhibitors can be mediated by alterations in the PI3K pathway. Due to decreased activity of PTEN, caused by PTEN loss, PTEN is unable to inhibit the PI3K pathway which leads to activation of PI3K. There are several mechanisms known for acquired resistance. Increases of IGF-1R and in AKT activity also leads to activation of the pathway and thus to increased proliferation. Activation of RAS due to RAS mutations or upstream activation of PDGFRβ or another tyrosine kinase receptor promotes the formation of RAF dimers. When a BRAF inhibitor (vemurafenib/dabrafenib) binds to one member of the dimers, the other one is activated. In these cells vemurafenib does not inhibit the pathway, leading to resistance. Additionally overexpression of COT results in activation of MEK and ERK in a RAF-independent manner and thus in resistance. When BRAF is mutated LKB1 (tumour supressor) is inhibited by p90rsk and ERK and subsequently AMPK is unable to inhibited cell growth.

So far there several mechanisms have been discovered that describe acquired resistance ( Fig. 3 ). One of these mechanisms involves the formation of RAF protein dimers. As described above, vemurafenib only inhibits RAF activation in cells with a BRAF V600 mutation. In cells with normal RAS, RAF proteins form dimers, and the binding of the drug to one RAF in the dimer activates the other.13, 14, and 16 In cells with a BRAF V600E mutation, however the levels of activated RAS are too low to promote adequate formation of RAF dimers and vemurafenib inhibits RAF activity and ERK signalling. If mutant RAS is introduced, cells with mutant BRAF become resistant to vemurafenib. 16 When RAS is highly active, due to RAS mutation or upstream activation of a receptor tyrosine kinase (RTK), RAS promotes formation of RAF dimers ( Fig. 3 ). Vemurafenib inhibits only one member of the dimer and the other is still activated, so the pathway is not inhibited. 16 Nazarian et al. (2010) confirmed this theory in a patient in whom resistance to vemurafenib developed by a mutation in NRAS. 15 They also suggested that platelet derived growth factor receptor β (PDGFRβ) is overexpressed in cellular models for BRAF-inhibitor resistance. PDGFRβ overexpression was associated with resistance despite continued inhibition of ERK signalling in the presence of the drug. 15 Other RTKs may also be involved in mechanisms of resistance. Villanueva et al. (2010) found that under conditions of chronic BRAF inhibition, IGF-1R is increased in post-relapse human tumour samples. 30 IGF-1R promotes cell proliferation and survival through the PI3K/AKT/mTOR pathway.

Another mechanism involves COT protein kinase (MAP3K8 or Tpl2) 31 , which can activate ERK and MEK in a RAF-independent manner. 32 A correlation has been found between increased COT expression and acquired resistance to BRAFi in tissue from patients with relapsing tumours. 31 ERK can also be activated in a MEK-dependent manner in which COT directly activates ERK. This would suggest that ERK inhibition or direct COT inhibition may be needed to intercept this mechanism. 33

Vemurafenib combination therapy for the treatment of melanoma

Patients with BRAF V600E positive melanoma will eventually experience relapse or progression on vemurafenib treatment. As described many mechanisms are now being investigated that may underlie resistance to RAF inhibitors. 15 In addition to resistance, toxicities associated with BRAFi, such as the appearance of CSCC, must be overcome. Therefore there is a strong need for rational combination strategies that target oncogenic pathways along with BRAF. More and more preclinical and clinical studies show encouraging results towards the future of melanoma treatment. But to answer the question whether or not the suggested combination regimens can overcome resistance and toxicities the proposed interference mechanisms of these combinatorial drugs need to be evaluated. Below these mechanisms will be discussed ( Fig. 4 ).

gr4

Fig. 4 Targeting the RAS/RAF/MEK/ERK pathway to overcome BRAF inhibitor induced resistance in melanoma. Several targets to overcome resistance due to an active member of RAF dimers are investigated. By simultaneously blocking MEK and mutated BRAF inhibition of cell growth might be enhanced. Due to loss of PTEN the PI3K pathway is stimulated resulting in proliferation and survival. By inhibiting PI3K in combination with inhibition of mutated BRAF or MEK the cross-talk between the RAS/RAF/MEK/ERK and PI3K/AKT pathway is blocked. Additionally activation through AKT is inhibited. Metformin can activate AMPK indirectly by increasing the cellular AMP/ATP ratio. Activated AMPK inhibits cell proliferation and survival.

MEK inhibitors

As discussed before, the development of mutations in genes such as NRAS 15 or MEK 34 seem to potentiate signalling through the RAS–MEK–ERK pathway by reactivation of MEK in tumours that are exposed to BRAFi. This observation led to the suggestion that the combination of BRAF and MEK inhibitors may enhance growth inhibition. While BRAFi only inhibit ERK signalling in cells with mutant BRAF, MEK inhibitors block the ERK pathway in both tumour and normal tissue. Therefore therapeutically acceptable doses of these compounds are limited by the side effects associated with ERK inhibition in normal tissue, such as intolerable skin rashes.23 and 35

The combination of BRAFi GSK2118436 (dabrafenib) and GSK1120212 (trametinib) is currently widely investigated. The oral drug trametinib is a selective inhibitor of MEK 1 and MEK 2 and showed evidence of tumour regression and disease stabilization in patients with V600E and V600K positive melanoma in clinical trials. In a phase III clinical trial 36 PFS was 4.8 months for trametinib and 1.5 months for chemotherapy (dacarbazine or paclitaxel). At 6 months the rate of overall survival (OS) was 81% in the trametinib group and 67% in the chemotherapy group, despite crossover. In 74% of the patients some degree of tumour regression was observed and 22% had a sufficient degree of sustained tumour regression to qualify as a confirmed objective response according to RECIST. This response rate that is associated with trametinib seems to be inferior to that with vemurafenib. The molecular basis for this lesser degree of tumour regression observed with a MEK inhibitor than with a BRAFi is unknown. No cases of CSCC were observed during the course of treatment with trametinib in this trial.

A phase III trial comparing dabrafenib to dacarbazine was recently published.37 and 38 This study shows a median PFS of 5.1 months for dabrafenib and 2.7 for dacarbazine. The confirmed ORR was 53% for dabrafenib and 19% for dacarbazine. This response rate is consistent with the outcome of a phase I clinical trial in patients with melanoma, untreated brain metastasis, and other solid tumours where they found an ORR of 47%. 39 Serious adverse events on the dabrafenib arm included pyrexia (4%), CSCC (6%) and new primary melanomas (2%).37 and 38

Mutations that activate RAS or MEK lead to hyperactive RAS–MEK–ERK pathway which is insufficiently inhibited by BRAF or MEK inhibition alone. In in vitro models Greger et al. were able to demonstrate that the combination of these inhibitors show profound growth arrest and restoration of transcriptional output. 40 This transcriptional output includes a decrease in genes designated as ERK transcriptional output genes. Conclusions from this data are that the combination of a BRAF and MEK inhibitor, in their case dabrafenib and trametinib, can overcome resistance to BRAFi due to NRAS and/or MEK mutations in vitro. This hypothesis is now tested in clinical phase I/II trials.41, 42, and 43 Recently published data confirmed response rates of 76% (n = 6) when given 150 mg BID dabrafenib and 2 mg once-a-day (QD) trametinib compared with 54% with dabrafenib monotherapy (150 mg BID). 44 Median progression-free survival in the combination group (150 mg BID dabrafenib and 2 mg QD trametinib) was 9.4 months, as compared with 5.8 months in the dabrafenib monotherapy group (150 mg BID). The most common grade 3/4 adverse events were pyrexia, fatique and dehydration. CSCC occurred in 7% of patients that received the combination 150 mg BID dabrafenib and 2 mg QD trametinib and in 19% receiving monotherapy. Pyrexia was more common in the combination group (71%) than in the monotherapy group (26%). 44 These results show that the combination therapy of dabrafenib and trametinib has a lower incidence of BRAFi-induced hyperproliferative skin lesions compared with single treatment. 43 The clinical activity observed in patients with the V600 BRAF mutant melanoma is encouraging and will be investigated in two phase III trials. In the first phase III trial dabrafenib plus trametinib will be compared to vemurafenib monotherapy ( NCT01597908 ). In the second phase III trial dabrafenib in combination with trametinib will be compared to dabrafenib in combination with a placebo in unresectable or metastatic BRAF V600E/K cutaneous melanoma ( NCT01584648 ).

PI3K/mTOR inhibitors

It is known that if BRAF is repressed that melanomas trigger an alternative signalling pathway, thus the tumour can continue to rely on MAPK for maintenance of the malignant phenotype. Villanueva et al. found that under conditions of chronic BRAF inhibition, melanomas rely on IR/IGF-1R-mediated survival pathways. 30 In addition to increased activation of BRAF and MEK, the members of the PI3K pathways are elevated in metastatic melanoma.30 and 45 The enhanced activity of PI3K/AKT suggests the possible existence of a negative crosstalk between these two pathways. The PI3K/AKT and RAS/RAF/MEK/ERK pathway can both be activated by oncogenic RAS and appear to provide some compensatory signalling when one or the other is inhibited. When mTOR, a downstream target of AKT, is inhibited, PI3K can activate MAPK via RAS.30 and 46 Crosstalk between MAPK and PI3K has been reported in other cancer systems, but not much is known in melanoma. Simultaneous MEK and IGF-1R/PI3K/AKT/mTOR inhibition led in preclinical experiments to cytotoxicity in melanomas that are resistant to BRAFi.30, 40, 47, and 48

In in vitro melanoma models inhibition of PI3K/mTOR reduces the growth of tumours. 49 It was also found that the addition of PI3K/mTOR inhibitor GSK2126458 to either dabrafenib or trametinib further reduced cell proliferation. The combination of GSK2126458 (PI3K/mTor inhibitor) and trametinib (MEKi) is more potent and more effective than the combination of GSK2126458 and dabrafenib (BRAFi) in short- and long-term assays. 40 These preclinical studies provided a clear rationale for the investigation of the clinical effect of these combination therapies. Shimizu et al. (2012) evaluated the clinical relevance of the dual-targeting strategy involving PI3K/AKT/mTOR and RAF/MEK/ERK pathways in patients with advanced cancer. 50 One group received a PI3K pathway inhibitor in combination with a MAPK pathway inhibitor and the other group was treated with an inhibitor of either the PI3K or MAPK pathways. They concluded that the inhibition of both pathways may potentially have favourable efficacy compared with the inhibition of either pathway, at the expense of greater toxicity. This approach may be especially important in patients with a coexisting PI3K mutation, but it is uncertain how well tolerated the combination will be. Another phase I/II trial combining a PI3K inhibitor (BKM120) and a BRAFi (vemurafenib) has just started.50 and 51

Immunotherapy

One of the best qualities of cellular immunotherapy in cancer responses is that they are extremely long lived, frequently measured in years. However, these durable objective responses are rare and noted in approximately 15–20% of the patients.52 and 53 Begley and Ribas (2008) proposed that the antitumor activity of adequately stimulated tumour antigen, specifically of T cells, is limited by local factors within the tumour environment and that pharmacologic modulation of this milieu may overcome tumour resistance to immunotherapy. 54 They hypothesised that by understanding the mechanisms of cancer cell immune escape, it may be possible to design rational combinatorial approaches of novel therapies. Pharmacological interventions with specific inhibitors of oncogenic events in cancer cells could sensitise cancer cells to immune attack. This has been termed immunosentization. 54 Targeted drugs that block key oncogenic mechanisms in cancer cells, that induce a proapoptotic cancer cell environment and that do not negatively influence the critical lymphocyte functions would be ideal as immune sensitising agent. 54 Vemurafenib seems to meet most of the criteria’s since it selectively inhibits a driver oncogene in cancer cells 55 , which is neither present nor required for the function of lymphocytes. 56 Vemurafenib treatment results in rapid melanoma cell death which is seen in clinical trials by the high frequency of early tumour responses in patients.5, 6, and 7

The combination of immunotherapy and targeted therapy is even more interesting because of the results from clinical trials with immunostimulant ipilimumab. A 2-year survival of more than 30% in patients with stage IV metastatic melanoma was reported.57 and 58 Given the results of BRAFi and ipilimumab the understanding of immune response to melanoma following selective BRAFi treatment is important to develop combination therapies. Wilmott et al. (2011) found that tumour infiltration by CD4+ and CD8+ lymphocytes increased markedly following BRAFi treatment. 59 More important the increase of CD8+ lymphocytes correlated with a decrease in the size and metabolic activity of tumours. The mechanisms that are involved in this increase of tumour infiltrating lymphocytes (TILs) are not yet defined. However prior studies would support the view that this may involve suppression of the release of immunosuppressive factors from the melanoma and increased melanoma antigen expression leading to more effective T-cell recognition. 59

Koya et al. (2012) provided another study which supports the combination of immunotherapy and selective BRAF inhibition. 60 They found that combined therapy with vemurafenib and T cell receptor (TCR) engineered adoptive cell transfer (ACT) immunotherapy, resulted in superior antitumor effects against a fully syngeneic BRAF V600E mutant melanoma cell line. Vemurafenib did not change the cell expansion or distribution of adoptively transferred cells by morphological and molecular imaging studies. However, the lymphocytes that were exposed to vemurafenib had higher pERK, which is a key feature of an activated MAPK signalling pathway. They also noted that intrinsic ability to increase the cytotoxic function of antigen-specific T cells, and TILs from vemurafenib-treated mice had higher functional activation with increased ability to release the immune stimulating cytokine IFN-y upon antigen re-exposure.

These studies all support the combination of selective BRAFi and immunotherapeutic agents such as ipilimumab. Currently the enrolment is ongoing of a phase I/II trial of vemurafenib and ipilimumab in patients with BRAF V600 mutation-positive metastatic melanoma. 61

Metformin

One of the new discoveries is that there is a linkage between BRAF and LKB1/AMPK (AMP-activated protein kinase).50, 51, and 62 The liver kinase B1 (LKB1) is a serine/threonine kinase that functions as a tumour suppressor gene and is inactivated in Peutz–Jeghers syndrome. LBK1 in combination with low energy conditions activates the AMP-activated protein kinase (AMPK), which results in inhibition of cell growth and proliferation. LBK1 can be phosphorylated by ERK and p90RSK, but this compromises its ability to bind and activate AMPK. In BRAF V600E mutant melanoma cells there is an uncoupling of the LKB1–AMPK complex.63 and 64 This suggests that AMPK can no longer be phosphorylated and therefore activated by LKB1, resulting in AMPK being unable to inhibit cell growth, proliferation and survival. This uncoupling of the LKB1–AMPK complex allows BRAF V600E oncogene-driven cancer cells to become resistant to energy stress and avoid apoptosis. 63 AMPK is activated by metabolic stress which results in an increase of the cellular AMP/ATP ratio either by inhibition of ATP synthesis (ischaemia or hypoxia) or accelerating ATP consumption (muscle contraction). 62

Recent studies report direct antitumor effects of modulating AMPK in melanoma tumour lines with a BRAF mutation and another with an NRAS mutation. 65 This study suggests that AMPK may have a role as a negative regulator and suppressor of malignant melanoma cell growth, which promotes the evidence of expanding investigations of the role of AMPK in melanoma.

Metformin is a well known guanidine derivative and is used for over 50 years in the treatment of type-2 diabetes. Both metformin and its analogue phenformin enter the cell by the organic cation transporter-1 (OCT-1). There it inhibits mitochondrial ATP production and can thus activate AMPK indirectly by increasing the cellular AMP/ATP ratio. 66 Deletion of LKB1 in mice diminished the effect of metformin on AMPK activity and blood glucose levels. This establishes the role of LKB1 as the main kinase that mediates AMPK activation upon exposure to metformin. 67

In a preclinical study Niehr et al. (2011) tested if the combination of BRAF oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone. 62 They found that metformin monotherapy inhibited proliferation in 12 out of 19 cell lines. The combination of vemurafenib (BRAFi) and metformin showed synergistic anti-proliferative effects on BRAF V600E mutant cell lines. However this combination did not reverse natural or acquired resistance to vemurafenib in the BRAF V600E mutant cell lines. More details about the understanding of these effects should be generated before vemurafenib and metformin should be considered in the clinic. 62

CKD4 inhibition

Smalley et al. (2008) found that increased expression of cyclin D1 in combination with CDK4 can mediate BRAFi resistance in BRAF V600E mutated melanoma. 28 Currently the only potential pharmacologic target is CDK4 and CDK4 inhibitors have recently entered early clinical development. In patients with mantle cell lymphoma, in which a translocation leads to increased cyclin D1 expression and CDK4 activity, the CDK4/6 inhibitor PD0332991 inhibited phosphorylation of markers of proliferation such as Rb. 68 In the future it may be possible to combine CDK4 inhibitors with a BRAFi in patients with a translocation resulting in enhanced CDK4 activity and cyclin D1 expression and a BRAF mutation.

HSP90 inhibition

Thus far many diverse mechanisms of resistance for BRAFi are discussed. The likelihood that others exist can complicate the design for combination therapies and future clinical trials. Therefore Paraiso et al. (2012) hypothesised that resistance may be best managed by targeting strategies that inhibit multiple pathways simultaneously. 69 The heat shock protein 90 (HSP90) regulates many functions of many RTKs and kinases that are required for oncogenic transformation.70 and 71 Mutated BRAF, CRAF, IGF-1R, cyclin D1, CDK4 and AKT are all clients of HSP90 which is required for melanoma initiation and progression.72 and 73

There are currently more than 13 HSP90 inhibitors at various stages of preclinical and clinical development. 71 Thus far they have shown limited single-agent activity, but more promising clinical efficacy has been seen when combined with other drugs.74, 75, 76, and 77 Paraiso et al. (2012) investigated the potential use of the HSP90 inhibitor (XL888) in different cell models of vemurafenib resistance (xenografts). 69 They found that XL888 inhibited tumour growth and induced apoptosis in vemurafenib-resistant melanoma cell lines. HSP90 inhibition showed to be more effective in restoring BIM (apoptosis inducer) and down regulating Mcl-1 (prosurvival protein) than combined MEK/PI3K inhibitor therapy. HSP90 inhibition may be a highly effective in the treatment of the diverse pathways of BRAFi resistance.

Conclusions

When reviewing the possibilities for vemurafenib combination therapy one is required to enlist all mechanisms of resistance because they are inextricably linked. These possible opportunities for resistance highlight the continuous adaptive switch of cancer cells to different signalling pathways as a survival strategy and the advantages and (future) successes using sequential and combination treatment. The ease in which tumour cells adapt to survive is important to keep in mind when trying to answer the question if combination therapy can overcome toxicities and resistance.

In conclusion, the combinations discussed are definitely a promising approach to overcome unresponsiveness of melanoma. The combination of BRAF and MEK inhibitors for instance, shows a decrease in CSCC cases and so far an increased PFS of 7.4 months. 43 However head-to-head comparison of a BRAF inhibitor and the combination of a BRAF inhibitor and a MEK inhibitor should be postured to unravel whether the combination approach has added therapeutic value. Other combinations are worth investigating clinically, as in some cases only preclinical results are available. Additionally, the elucidation of pathways underlying toxicities and resistance is important as a basis to develop effective strategies to reverse unresponsiveness.

Conflict of interest statement

The authors have no conflict of interest.

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Footnotes

a Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands

b Division of Clinical Pharmacology, Department of Medical Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

c Division of Pharmacoepidemiology and Clinical Pharmacology, Faculty of Science, Department of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

d Division of Immunology and Medical Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

lowast Corresponding author. Tel.: +31 20 512 5008; fax: +31 20 512 4753.

1 Tel.: +31 20 512 2570.

2 Tel.: +31 20 512 2446.

3 Tel.: +31 20 512 4342; fax: +31 20 512 4753.