Gene knockout or selective inhibition of AKT-3, but not AKT-1 or AKT-2 isoform, enhances apoptosis in CD133+ melanoma cancer stem cells treated with the MEK inhibitor trametinib

CD133-expressing melanoma-initiating stem cells are associated with tumorigenesis, metastasis, and drug resistance in malignant human melanoma. CD133 knockout (KO) or doxycycline (Dox)-inducible expression shows that CD133 activates an alternate survival pathway, PI3K/AKT, bypassing the MAPK pathway, resulting in apoptosis inhibition, increased melanoma cell survival, and resistance to the MEK-inhibitor trametinib. Combinational inhibition of the MAPK and PI3K/AKT pathways with trametinib and the pan-AKT inhibitor capivasertib synergistically induces melanoma cell apoptosis in vitro and inhibits tumor growth in vivo . To determine the relative contributions of each of three AKT isoforms, AKT-1, -2, and -3, to apoptosis inhibition, we used CRISPR-Cas9 KO or pharmacological inhibitors of each isoform, in combination with trametinib in a Dox-inducible NRAS-mutant melanoma cell line BAKP. Immunoblot analysis with anti-AKT-1, -2, or -3 and DNA sequence analysis confirmed gene knockout. BAKP control cells and cells with single or triple KO of each or all of the AKT isoforms were then exposed to trametinib, alone or in combination with capivasertib, and subjected to Annexin V apoptosis assays and immunoblot analysis with antibodies to apoptosis markers. Single KO of AKT-2 or -3, but not AKT-1, sensitized BAKP cells exposed to trametinib alone, partially obviating the requirement for inhibition of AKT by capivasertib. Triple KO of all three isoforms further sensitized cells to trametinib alone. We next investigated apoptosis induction by selective inhibitors of each of the AKT isoforms; afuresertib for AKT-1, CCT128930 for AKT-2, or uprosertib for AKT-3, as a monotherapy or in combination with trametinib. The combination of uprosertib and trametinib proved to be the most effective in synergistically inducing apoptosis in melanoma cells, even targeting the CD133-expressing melanoma stem cells. Together, this indicates that AKT-3 plays an essential role in apoptosis inhibition, consistent with reports that implicate AKT-3 in drug resistance in melanoma and glioblastoma. Cells treated with trametinib, alone or combined with capivasertib or uprosertib, exhibited loss of mitochondrial membrane potential, suggesting a mitochondrial pathway of apoptosis. Future studies will focus on optimizing the effect of selective inhibitors of AKT-3 in dose response experiments, and test its effects in in vivo mouse xenograft studies. Simultaneously targeting the AKT and MAPK survival pathways with trametinib and uprosertib underscores the importance of combination therapies to eliminate recalcitrant melanoma stem cells.