Induction of apoptosis in human melanoma by the BRAF inhibitor PLX4720: The key to therapeutic success?

Abstract

8559 Background: A high proportion of Patients with metastatic melanoma have shown clinical responses when treated with the PLX4032/NP22657 inhibitor against the mutated V600E form of BRAF. The present study examined an analogue of this inhibitor for its ability to inhibit viability and induce apoptosis in a large panel of established and short term cultured melanoma. Methods: The oncocarta mutation panel was used to assess muations in 31 melanoma lines and short-term cultures. Each line was tested for viability in MTS assays and apoptosis by subG1 PI fractions over 72 hours. Western blots were used to measure Bcl-2 family protein expression. Results: There were 7 lines with no detectable mutations, 10 with mutations in NRAS or KRAS,13 with BRAF mutations and one with mutated cKit.Apoptosis induction was highest in the lines with mutated BRAF and correlated inversely with cell viability except for 2 lines with mutated CDK4 (R24c) which had low viability and low apoptosis.Inhibition of caspases indicated that apoptosis was the main mechanism for reduced cell viabilty. Induction of apoptosis correlated with upregulation of the proapoptotic BH3 protein Bim as reported in previous studies on the MEK inhibitor UO126. In particular there was selective upregulation of the short form of Bim and this correlated with induction of apoptosis. There were no significant changes in the the other BH3 only proteins PUMA or NOXA Combination of a MEK inhibitor with the BRAF inhibitor had strong potentiating effects on cell lines that were relatively resistant to the BRAF inhibitor. Conclusions: These studies confirm that the BRAF inhibitor PLX4720 induces apoptosis in human melanoma with mutated BRAF at concentrations achievable in vivo. Melanoma lines with mutated CDK4 or EGRF receptors had lower cell viability. Apoptosis was correlated with the induction of the proapoptotic Bim, particularly Bim short and may be a useful biomarker to assess apoptotic responses and guide drug doses and combinations in the clinic. No significant financial relationships to disclose.