Braf Wild-Type Melanoma Survival Hinges Upon Akt Activity And Adaptive Autophagic Reprogramming In Response To Chemotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Strategies for patients whose melanomas are BRAF wild-type are completely lacking. We here report on the mechanisms underlying the long-term responses (>12 months) of 2 BRAF wild-type (WT) melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. A panel of 7 BRAF/NRAS-WT cell lines was leveraged to investigate strategies to overcome chemoresistance. Electron microscopy, western blotting and flow cytometry assays were integrated to simultaneously follow fluctuations in autophagic flux, reactive oxygen species levels and cytotoxicity caused from chronic treatment of cells with chemotherapy, MK-2206, lysomotropic agents and/or anti-oxidants. MK-2206 potently inhibited phospho-AKT signaling, but did not impact melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin induced little apoptosis following 3-day treatment, but led to cytotoxicity in long-term colony formation and 3D spheroid assays. Mechanistically the combination was noted to induce autophagy and disrupt reactive oxygen species homeostasis, as shown by increased LC3-II expression, the formation of LC3 punctae, acridine orange protonation, the accumulation of autophagosomes and increased ROS detection using DHR-123. At earlier time points, autophagy was protective with cytotoxicity enhanced following chloroquine and bafilomycin treatment as well as the increased cytotoxic effects seen in ATG5-/- MEFs. Although prolonged MK-2206/chemotherapy treatment (6-9 days) led to high levels of autophagy induction and a switch to caspase-dependent apoptosis, drug resistant clones still eventually emerged. The importance of autophagy for the escape process was suggested by the lack of ATG5-/- MEF colonies seen following combination therapy treatment. Autophagy inhibition enhanced the cell death response through increased generation of ROS and could be reversed by anti-oxidants. In summary, we demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma. We further show that the administration of an autophagy inhibitor may prevent resistance from emerging to this drug combination. Citation Format: Vito W. Rebecca, Renato Massaro, Inna Fedorenko, Geoffrey T. Gibney, Vernon K. Sondak, Ravi K. Amaravadi, Shengkan Jin, Silvya S. Maria-Engler, Ragini Kudchadkar, Keiran S. M. Smalley. BRAF wild-type melanoma survival hinges upon AKT activity and adaptive autophagic reprogramming in response to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 809. doi:10.1158/1538-7445.AM2014-809