Combined PKC and MEK inhibition for treating metastatic uveal melanoma

Abstract

Uveal melanoma (UM) is the most common primary intraocular malignancy and the second most common form of melanoma. UM has a strong tendency for metastatic disease, and no effective treatments have yet been identified. Activating oncogenic mutations are commonly found in GNAQ and GNA11 in UM, and inhibiting key downstream effectors of the GNAQ/11 signaling pathway represents a rational therapeutic approach for treating metastatic UM. Chen et al., doi:10.1038/onc.2013.418, now confirm activation of the MAPK and PKC pathways as a result of GNAQ and GNA11 activating mutations in melanocytes, and they demonstrate that MAPK activation occurs downstream of PKC activation. PKC inhibitors disrupt MAPK signaling and block proliferation of GNAQ/11 mutant UM cell lines and slow the in vivo growth of xenografted UM tumors without inducing their shrinkage. However, a combination of PKC and MEK inhibition led to sustained MAPK pathway inhibition and tumor regression in vivo. Hence, the authors concluded that MEK and PKC inhibition is synergistic, with superior efficacy to treatment of GNAQ/GNA11 mutant UMs with either drug alone.

Uveal melanoma (UM) is the most common primary intraocular malignancy and the second most common form of melanoma.¹ The primary intraocular tumor is usually treated by radiotherapy or enucleation (eye removal). Despite successful treatment of the eye in the vast majority of cases, up to one-half of patients are at risk for fatal metastatic disease. Unfortunately, there are no systemic treatments with proven efficacy for metastatic UM. As early as 2005, the RAS-RAF-MEK-ERK (extracellular signal-regulated kinase) or mitogen-activated protein kinase (MAPK) pathway was shown to be constitutively activated in UM despite an absence of BRAF or RAS mutations.² Hence, it was concluded that activation of the MAPK pathway in UM occurs through a mechanism different to that of cutaneous melanoma.² Activating mutations at either codon 209 or 183 in the Gq alpha subunits GNAQ and GNA11 are now known to represent early, mutually exclusive events that constitutively activate the MAPK pathway in the development of UM.^3–5 Other genetic drivers of UM include mutations in the BRCA1-associated protein-1 (BAP1), which are found in ~84% of metastasizing class 2 UMs,⁶ and in splicing factor 3B subunit 1 (SF3B1), which are found in ~15% of UMs and are associated with a more favorable outcome.⁷ Alterations in Eukaryotic translation initiation factor 1 A, X linked (EIF1AX) are present in ~8% of tumors with favorable outcome that lack SF3B1 mutations.⁸ Genetic discoveries in UM have led to new clinical trials to assess several classes of compounds, including MEK, protein kinase C and histone deacetylase inhibitors.⁹ Now, Chen et al.¹⁰ discuss the use of a combination of PKC and MEK inhibitors for the treatment of GNAQ/GNA11-mutant metastastic UM.

GNAQ/11 SIGNALING AND EARLIER INHIBITORS

Along with the MAPK pathway, protein kinase C (PKC) is a target of GNAQ/11 signaling that ultimately leads to ERK1/2 (MAPK3/MAPK1) activation. In early studies, it was suggested that PKC inhibition might provide therapeutic benefit for GNAQ-mutant UM.¹¹ UM cells were treated with the PKC inhibitors enzastaurin or AEB071 (sotrastaurin), and their effect on proliferation, apoptosis and signaling events were evaluated. Both drugs led to the downregulation of several PKC isoforms, including PKC-betaII, PKC-theta, PKC-epsilon and/or their phosphorylation in GNAQ-mutated cells. These PKC inhibitors also inhibited the growth of GNAQ mutant, but not GNAQ wild type, UM cells through induction of G1 arrest and apoptosis. This led to inhibition of ERK 1/2 and nuclear factor of kappa B (NF-kB) through decreased phosphorylation, decreased expression of cyclin D1, survivin, Bcl-xL and XIAP, and increased expression of cyclin-dependent kinase inhibitor p27(Kip1). Inhibitors of ERK1/2 and NF-kB pathways were also shown to reduce viability of UM cells.

The phosphoinositide 3-kinase (PI3K)/AKT pathway is also activated in UM, which led Khalili et al. to evaluate a combination of small molecules inhibiting both MEK and PI3K in UM cells with different GNAQ/11 mutation backgrounds.¹² These authors observed that GNAQ/11 mutation status was not a determinant of whether cells would undergo cell-cycle arrest or growth inhibition to MEK and/or PI3K inhibition. However, a reverse correlation was observed between MAPK and AKT phosphorylation after MEK or PI3K inhibition, respectively. Neither MEK nor PI3K inhibition alone was sufficient to induce apoptosis in the majority of cell lines; however, the combination of MEK +PI3K inhibitor treatment resulted in the marked induction of apoptosis in a GNAQ/11 mutant-dependent manner.¹² Hence, these authors concluded that combined MEK and PI3K inhibition may be an effective combination therapy in UM, given the inherent reciprocal activation of these pathways.

Ho et al.¹³ investigated the impact of dual pathway inhibition upon UM cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. Synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, although apoptosis was preferentially induced in BRAF mutant cells and in a BRAF mutant xenograft model but not GNAQ mutant model.

Chen et al.¹⁰ confirm activation of the MAPK and PKC pathways as a result of GNAQ and GNA11 activating mutations in melanocytes and demonstrate (not unexpectedly) that MAPK activation occurs downstream of PKC activation. This dual activation was not seen in GNAQ/11 wild-type tumors, where they speculate that the MAPK pathway might be activated via another mechanism. Activation of PKC in GNAQ/11 mutant tumors was determined on the basis of increased phosphorylation of the PKC substrate MARCKS, whereas activation of the MAPK pathway was determined on the basis of the presence of p-ERK and pp90RSK. The authors then examined the effect of PKC inhibition on the basis of treatment with two different inhibitors (AEB071 and AHT956). These molecules inhibited pMARCKS and MAPK signaling and proliferation of melanoma cell lines in GNAQ/11 mutant cells, whereas the MEK inhibitor PD0325901 did not inhibit proliferation of these lines. Treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status (Figure 1). The authors interpreted this to be that in the context of GNAQ or GNA11 mutation, MAPK activation can be attributed to activated PKC.

Figure 1.

Simplified view of cellular pathways in UM arising as a consequence of activating mutations in GNAQ or GNA11 and pharmacological agents that disrupt them.

Chen et al.¹⁰ then investigated the effect of these inhibitors upon an allograft model of GNAQ-Q209L-transduced melanocytes. Treatment with the PKC inhibitor AEB071 significantly slowed the growth of tumors in vivo, but did not induce tumor shrinkage. The authors hypothesize that they were unable to suppress MAPK activation due to a compensatory mechanism. They then examined the effect of combined PKC and MEK inhibition and showed that it led to sustained MAPK pathway inhibition and produced a strong synergistic effect in halting proliferation and inducing apoptosis in vitro. Combined PKC and MEK inhibition was also efficacious in vivo, leading to marked tumor regression in a UM xenograft mouse model. The authors conclude that PKC is a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations, and that combined MEK and PKC inhibition provides a synergistic effect with superior efficacy compared with treatment with either approach alone. This therefore leads one to ask whether the PKC and RAS/RAF/MEK pathways leading to MAPK activation are independent in the context of mutant GNAQ/GNA11. This study also leaves several important questions unanswered. What will be the price to be paid for combined MEK and PKC inhibition in terms of increased systemic toxicity? Will inhibition of the GNAQ/11 pathway alone be sufficient for sustained efficacy in human subjects with metastatic UM, when the vast majority of them will have BAP1 mutations that may also require pharmacologic modulation? Combination therapies that target different downstream targets of the GNAQ/11 pathway, such as the approach in this study, undoubtedly represent the next step in treating metastatic UM, but it is likely that combined therapies will ultimately need to target early and late mutational events for maximal efficacy.