Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma.

Neel M Fofaria,Ryan J Sullivan,Dennie T Frederick,Keith T Flaherty,Sanjay K Srivastava

doi:10.18632/oncotarget.5755

Abstract

Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. Here, we establish a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival and apoptosis. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression and induction of apoptosis by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors.

Highlights

Melanoma, a malignant transformation of melanocytes accounts for the highest number of skin cancer related deaths with a 5-year survival probability of less than 5% [1]
Our results showed a significant upregulation of Myeloid cell leukemia – 1 (Mcl-1) expression upon vemurafenib treatment in both the cell lines (Fig. 1C)
Vemurafenib treatment increased the expression of Mcl-1 by 4.7, 5.2 and 4.5 fold in A375 cells and by 10, 11 and 14 fold in SK-MEL-28 cells at 0.1, 0.2 and 0.4 μM respectively (Fig. 1C)

Summary

Introduction

A malignant transformation of melanocytes accounts for the highest number of skin cancer related deaths with a 5-year survival probability of less than 5% [1]. BRAF mutation is observed in almost 60% melanomas [2, 3]. BRAFV600E have shown significant clinical activity in the patients with late stage metastatic melanoma amongst which vemurafenib has been recently approved by US-FDA [6,7,8]. In spite of promising initial response, there have been several recent reports of acquired resistance within 6–9 months of treatment with BRAF inhibitors in most of the patients [8, 9]. About 20–30% patients develop squamous cell carcinoma and more develop tumor recurrence limiting vemurafenib therapy as well as other BRAF targeted therapies [10, 11]

Methods

Results

Conclusion