Targeting of the MAPK and AKT pathways in conjunctival melanoma shows potential synergy

Abstract

PurposeConjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in BRAF and NRAS. We studied whether CM as well as conjunctival benign and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth in vitro.Methods131 conjunctival lesions obtained from 129 patients were collected. The presence of BRAF V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206).ResultsThe BRAF V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both BRAF mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines.ConclusionERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma.

Evaluation of (fli:GFP) Casper Zebrafish Embryos as a Model for Human Conjunctival Melanoma

“…44 It has been used to target metastases and a primary CM. 45 Vemurafenib was previously shown to have a selective effect on CM cell lines in vitro 46 and we used that information to validate the usability of the zebrafish CM model. In our experiments, the effects of the vemurafenib in the treatment of engrafted embryos were the same as those observed in vitro showing that the zebrafish embryo model can be used in drug screens against human CM.…”

Systemic BRAF/MEK Inhibitors as a Potential Treatment Option in Metastatic Conjunctival Melanoma

“…In up to 50% of CM, activating BRAF mutations can be found [11,16,23]. Another notable mutation that is common in CM is hyperactivated NRAS (in up to 18%) [18]. BRAF and NRAS mutations are considered to be mutually exclusive [51].…”

“…Additionally, Cao et al [18] examined the prevalence of activating mutations in the MAPK and AKT (protein kinase B) pathways in benign conjunctival melanocytic lesions, premalignant conjunctival melanocytic lesions, and CM in a total of 129 patients. The study showed that the BRAF V600E mutation could be detected in 19% of the nevi and in 26% of the CM but not in the PAM.…”

“…In contrast, nuclear and cytoplasmic phosphorylated (p)-ERK as well as nuclear and cytoplasmic p-AKT could be detected immunohistochemically in every group. In order to determine the therapeutic potential of either BRAF, MEK, or AKT inhibition in the treatment of CM, Cao et al [18] then studied the effects of vemurafenib, dabrafenib, a MEK inhibitor (MEK162), and an AKT inhibitor (MK2206) on 3 CM cell lines. Of these cell lines, 2 displayed a BRAF mutation (CRMM1 and CM2005.1) and 1 had a BRAF wild type (CRMM2).…”

“…Similar to the melanoma of the skin, CM has been shown to harbor activating mutations of BRAF (rapidly accelerated fibrosarcoma), an oncogenic serine/threonine kinase in the mitogen-activated protein kinase (MAPK) pathway [18]. In skin melanoma, BRAF mutations have been described to occur in 32-60% of cases [17,19,20,21,22].…”