Results Effects of single agent dabrafenib or AKTi on cell growth and cell signaling In this study, a panel of 23 previously described melanoma cell lines harboring BRAFV600 mutations was used to assess the effects of targeting the MAPK pathway and the PI3K AKT signaling pathway. The panel included 19 drug na ve cell lines and four sub lines with acquired resistance to the BRAF inhibitor vemurafe nib developed by continuous in vitro e posure to this drug. The MAPK pathway was inhibited by the BRAF inhibitor dabrafenib and the PI3K AKT pathway was inhib ited by the AKT inhibitor GSK2141795B. By per forming growth assays and arranging cell lines according to their IC50 values a cut off of 100 nM for resistance to dabrafenib as single drug was determined on the basis of the natural gap in the IC50 values.

This divided the cell lines into two groups sensitive and resistant to dabrafe nib. The sensitive group could further be divided into two groups very sensitive and sensitive. In 8 out of the 13 resistant cell lines, the IC50 was not achieved in the tested concentration range. Based on the inhibitory effects of single agent AKTi and according to the calculated IC50 values for this inhibitor, cells lines were divided into three groups sensitive, intermediate resistant and resistant. PTEN is a known negative regulator of the PI3K AKT pathway and lack of e pression or mutations in the protein can cause over activity of this pathway. Interestingly, most of the PTEN null cell lines were among the AKTi sensitive cell lines including M249, M411, M399, M397 and M397AR, indicated with red bars.

However, M233 has homozygous PTEN loss but was less sensitive to AKTi. The only known AKT mutant in this series, M262, was also found in the sensitive group. The efficacy of the drugs in inhibiting the signaling pathways was verified by western blot analysis of phosphor ylated proteins. Dabrafenib caused a clear reduction in p MEK, p ERK and p S6 at a concen Carfilzomib tration as low as 50 nM in the dabrafenib sensitive cell line M411, whereas such reductions were not evident in the dabrafenib resistant cell line M299. AKTi caused a concentration dependent decrease in p S6, p 4E BP 1 and p GSK 3B in the AKTi sensitive cell line M411. On the contrary, in the AKTi resistant cell line M299, AKTi only reduced p GSK 3B. In both cell lines, both drugs induced p AKTs, suggesting activation of feedback mechanisms.

however the induction of p AKTs was more pronounced by AKTi. Combinatorial treatment with dabrafenib and AKTi enhances cell growth inhibition in dabrafenib sensitive and resistant cell lines After evaluating the growth inhibition resulting from treatment with each drug alone, we e plored whether blocking both pathways by the combination of dabrafenib and AKTi would enhance the growth inhibitory effects.