Pro-Apoptotic Activity of MCL-1 Inhibitor in Trametinib-Resistant Melanoma Cells Depends on Their Phenotypes and Is Modulated by Reversible Alterations Induced by Trametinib Withdrawal

SIMPLE SUMMARY: Skin melanoma is fully curable by surgery if diagnosed at an early stage. Targeted drugs against BRAF(V600) and MEK1/2 were the first therapeutic breakthrough for patients with advanced melanoma. However, acquired drug resistance resulting in disease relapse interrupts the treatment after a few months. Therefore, new treatment strategies are imperative to address this problem. The multifactorial nature of melanoma drug resistance involves genetic alterations but also an extraordinary capability of melanoma to adapt to changes in the microenvironment including drug holiday and rechallenge. The alterations in the levels of proteins impacting the MCL-1 activity and stability, such as p-ERK1/2, BIM and NOXA, could explain the differences in pro-apoptotic response to MCL-1 inhibitors between trametinib-resistant melanoma cells grown with and without trametinib. Our results underline the importance of a more detailed analysis of resistant melanoma phenotypes in preclinical studies and in stratifying relapsed patients for clinical trials evaluating novel treatment strategies. ABSTRACT: Background: Although BRAF(V600)/MEK inhibitors improved the treatment of melanoma patients, resistance is acquired almost inevitably. Methods: Trametinib withdrawal/rechallenge and MCL-1 inhibition in trametinib-resistance models displaying distinct p-ERK1/2 levels were investigated. Results: Trametinib withdrawal/rechallenge caused reversible changes in ERK1/2 activity impacting the balance between pro-survival and pro-apoptotic proteins. Reversible alterations were found in MCL-1 levels and MCL-1 inhibitors, BIM and NOXA. Taking advantage of melanoma cell dependency on MCL-1 for survival, we used S63845. While it was designed to inhibit MCL-1 activity, we showed that it also significantly reduced NOXA levels. S63845-induced apoptosis was detected as the enhancement of Annexin V-positivity, caspase-3/7 activation and histone H2AX phosphorylation. Percentages of Annexin V-positive cells were increased most efficiently in trametinib-resistant melanoma cells displaying the p-ERK1/2(low)/MCL-1(low)/BIM(high)/NOXA(low) phenotype with EC(50) values at concentrations as low as 0.1 μM. Higher ERK1/2 activity associated with increased MCL-1 level and reduced BIM level limited pro-apoptotic activity of S63845 further influenced by a NOXA level. Conclusions: Our study supports the notion that the efficiency of an agent designed to target a single protein can largely depend on the phenotype of cancer cells. Thus, it is important to define appropriate phenotype determinants to stratify the patients for the novel therapy.