PLX4032 and melanoma: resistance, expectations and uncertainty

Keywords::

• melanoma
• PLX4032
• resistance

Figure 1. The short and long roads to PLX4032 resistance.

(A) In cells expressing mutant BRAF, overexpression of RAF1 or activation of RAS due to N-RAS mutation results in the formation of BRAF–RAF1 heterodimers and/or RAF1–RAF1 homodimers, causing resistance to PLX4032. Alternatively, overexpression of COT results in RAF-independent activation of MEK and ERK, and thus resistance to PLX4032. In such cells, therefore, PLX4032 resistance is mediated by reactivation of the MAPK–ERK signaling pathway. (B) Another possibility is that activation of upstream RTKs such as PDGFRβ makes MEK activity redundant by triggering downstream effectors of cell transformation through parallel signaling pathways.

PDGFRβ: PDGF receptor-β

Reproduced from [4,5], with permission from Nature Publishing Group.

Last year, the results of a Phase I/II study demonstrated an 80% response rate to PLX4032 (Plexxikon; RG7204, Roche Pharmaceuticals) among genotype-selected metastatic melanoma patients with V600E mutation in the BRAF gene [1]. The PLX4032 drug targeting BRAF mutations inhibits the RAF–MEK–ERK signaling pathway and regresses cancer cells containing BRAF mutations [2,3]. Given that almost all patients develop resistance [1], more recent studies have been designed to explore the mechanisms that result in treatment failure. Understanding the molecular basis of resistance may result in the development of both biomarkers for predicting response, as well as new drugs to overcome this primary or acquired resistance [4,5]. Awaiting the results of a Phase III trial that is currently underway, how optimistic can we be for the efficacy of PLX4032 to improve the overall survival of patients with melanoma?

The statistically significant increase in the incidence of melanoma over the last decades, and the poor survival outcome in more advanced stages in the USA and worldwide, have suggested the need for more accurate diagnosis at an early stage as well as improved treatment strategies. In 2010, 68,130 newly diagnosed cases of invasive melanoma and 46,770 cases of in situ melanoma were expected in the USA, and approximately 8700 people were predicted to die from melanoma [6]. The importance of early diagnosis is revealed by the 5-year survival rate, which is over 90% in early-stage (localized) cancer but falls to less than 65% for regional spread, and less than 15% when there is evidence of distant metastases at diagnosis [6]. Incidence and death rates of advanced or metastatic melanoma continue to rise faster than the rates observed with most cancers [6]. The median survival of patients with melanoma who have distant metastases (American Joint Committee on Cancer stage IV) is less than 1 year [7].

The two therapies approved by the US FDA, high-dose IL-2 and dacarbazine, are associated with response rates of only 10–20% and a small percentage of complete responses; neither is thought to improve overall survival [8]. In randomized trials, the median survival among patients treated with dacarbazine was less than 8 months [9]. These disappointing results have shifted translational research to the search for new therapeutics with RAF and MEK inhibitors, which has recently attracted major interest by research and industry.

BRAF mutation

Among patients with melanoma, 40–60% carry an activating mutation in the gene encoding the serine–threonine protein kinase BRAF[10]. In total, 90% of reported BRAF mutations result in a substitution of glutamic acid for valine at amino acid 600 (the V600E mutation). This BRAF mutation constitutively activates BRAF and downstream signal transduction in the MAPK pathway. This identification offers an opportunity to test oncogene-targeted therapy for the treatment of melanoma.

PLX4032

PLX4032 is an orally available potent inhibitor of BRAF genes carrying the V600E mutation. Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF–MEK–ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts [11].

Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase I clinical trials using a crystalline formulation of PLX4032 [1].

In their clinical trial recently published in the New England Journal of Medicine, Flaherty and colleagues conducted a Phase I/II trial to test the safety and efficacy of PLX4032 [1]. The study was sponsored by Plexxikon and Roche Pharmaceuticals. In the Phase I dose–escalation trial, 55 patients (49 of whom had melanoma) were enrolled to assess the maximum dose that could be administered without adverse effects. In the subsequent Phase II trial, 32 additional patients with metastatic melanoma who carried BRAF with the V600E mutation received the recommended Phase II dose of 960 mg twice daily. Among these 32 patients in the extension cohort, 24 patients displayed a partial response and two had a complete response. With an overall response rate of 80%, the estimated median progression-free survival among all patients was greater than 7 months. Despite this impressive high response rate, all the patients had primary or acquired resistance to PLX4032. The authors conclude that it is still not known whether treatment with PLX4032 will improve overall survival; an ongoing Phase III trial (ClinicalTrials.gov no.: NCT01006980) is addressing that question [1].

Understanding resistance mechanisms

Understanding the resistance mechanism can provide clues both for developing new drugs and for identifying new biomarkers for predicting resistance and sensitivity, and also for guiding the selection of appropriate drug combinations for therapy. The paradigm of secondary mutations responsible for imatinib resistance and treatment failure of patients with chronic myeloid leukemia might guide the development of more active drugs than PLX4032 in melanoma patients. Indeed, the discovery that drug-resistant tumor cells often contain secondary mutations in the target kinase that prevent the drug from binding while retaining the kinase’s full oncogenic activity of resistance in chronic myeloid leukemia, accelerated the development of two next-generation inhibitors (dasatinib and nilotinib), which are likely to become front-line therapies in the near future [12,13].

Surprisingly, however, Nazarian et al. show that this is not the case [5]. Using next-generation sequencing technology, the authors examined the genomes of tumor samples from 12 patients with acquired resistance to PLX4032, and found no secondary BRAF mutations.

Exploring treatment failure to PLX4032, Johannessen et al.[4] and Nazarian et al.[5] suggest at least three different potential mechanisms of resistance, all of which share the common theme of ‘oncogene bypass’.

Following the EGF receptor/MET precedent, Johannessen et al. obtained two compelling hits: RAF1 and COT/TPL2, which, like BRAF and RAF1, functions upstream of the kinase MEK (BRAF functions through the MAPK–ERK signaling pathway). In both cases, resistance seems to be due to restored MEK activation in the face of PLX4032 treatment – that is, bypass of BRAF(Figure 1A).

In the second paper, Nazarian and colleagues focused on changes in signaling pathways in 12 matched pairs of drug-sensitive and drug-resistant tumor samples obtained from patients with BRAF-mutant melanoma who were treated with PLX4032 [5]. The data of this study from one patient’s sample suggest that any perturbation that restores MEK activation (RAS mutation, RAF1 or COT overexpression, and perhaps other perturbations) has the potential to cause resistance to PLX4032. Moreover, Nazarian et al. when examining the samples from five patients in relapse, revealed a different mechanism. They documented increased activation of the RTK PDGF receptor-β (PDGFRβ) compared with baseline. Like increased expression of COT and mutant N-RAS, overexpression of PDGFRβ was sufficient to confer resistance to PLX4032, albeit independently of MEK activation (Figure 1B). In the remaining six of the 12 patients the authors studied, the resistance mechanism remains unexplained.

Evaluating these two works [4,5], Solit and Sawyers attempted to address the crucial question of whether these findings will have a clinical impact [14]. They conclude that it is too early to tell, given the limited number of patients examined and the heterogeneity of potential resistance mechanisms. However, the fact that restored MEK activation is sufficient to confer resistance in models of BRAF mutant melanoma begs the question of whether, at least in some patients, resistance to PLX4032 can be overcome using MEK inhibitors. Solit and Sawyers propose that if MEK activation is commonly found at relapse, one could make a strong argument for combined therapy using PLX4032 and a MEK inhibitor to either prevent or delay resistance [14].

Future perspective

Both studies are focused on the RAF–MEK–EPK pathway for understanding PLX4032 resistance, without considering interconnections with other signaling pathways or a more global approach to understand the perturbation of signaling pathways networks as a whole signal-transduction circuit. Despite this limitation, the findings are a crucial step towards determining how to overcome PLX4032 resistance. Pharmaceutical firms have already started clinical trials to test PLX4032 in combination with experimental drugs that inhibit MEK; it may be necessary to combine four or more drugs in these studies to create a potent cancer-fighting cocktail. PLX4032 is a good place to start, because it’s highly selective targeting of BRAF means it is likely to have fewer unwanted side effects [15].

Complex interactions

Interconnections between the components of RAS–RAF–MAPK pathways with PI3K–AKT–PTEN–mTOR (the other main downstream pathway of EGF receptor), as well as other signaling pathways, such as the Wnt/β-catenin or TGF pathways, may be crucial for understanding resistance.

Currently, next-generation sequencing technologies and other latest high-throughput techniques have revolutionized basic and translational biomedical research, providing us with deeper insights into animal models and the human genome. Beyond the structural basis of the genome, we are now beginning to understand how functional regulatory systems operate, and how perturbation of molecular networks can result in cancer or other major diseases [16]. Scientific evidence for the importance of regulatory networks has more recently been provided by the Model Organism Encyclopedia of DNA (modENCODE) project [17,18]. Such exciting basic research discovering the crucial functional regulatory role of transcription factors, epigenetic modifications and microRNAs is now being translated into clinical strategic approaches towards a next-generation of biomarkers and agents targeting cancer networks, rather than single components of biological systems [19–21].

Conclusion

Positive results of a Phase I/II trial have recently been reported by treating patients with metastatic melanoma and BRAF mutations with PLX4032. Whether the progression-free survival benefit with this agent will be translated into an overall survival benefit is still unknown, and will hopefully be revealed by a Phase III randomized trial currently underway. Several resistance mechanisms to the treatment with PLX4032 are being explored in a manner that may lead to new drug targets. However more rational hope, than other research directions, have now provided us with the latest advances in understanding mechanisms underlying cancer networks and how deregulated signaling pathways networks and regulatory systems in cancer could be restored.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.