Sequential use of protein kinase inhibitors potentiates their toxicity to melanoma cells: a rationale to combine targeted drugs based on protein expression inhibition profiles.

Targeted therapy has shown high efficacy in the treatment of metastatic melanoma with impressive response rates. However, resistance appears after a few months, underlining the need for simultaneous multiple signalling pathway inhibition to provide a durable benefit. The aim of our study was to evaluate the possible synergistic effect of various protein kinase inhibitor combinations targeting SRC, MEK, PI3K or JAK on the survival of representative melanoma cell lines with WTNRAS/WTBRAF and harbouring the most frequent mutations (Q61LNRAS/WTBRAF or WTNRAS/V600EBRAF). By comparing IC50s and protein inhibition profiles, cell exposure to a single inhibitor for 3 days (condition 1) showed that both WTBRAF lines were at least 15-fold more sensitive to SRC inhibition while V600EBRAF cells were 30-fold more sensitive to MEK inhibition, confirming that the latter cells are largely dependent on the MAPK pathway for growth. Concomitant treatment for 3 days (condition 2) revealed an antagonistic effect between SRC and JAK inhibitors as compared to treatment by each inhibitor alone in all 3 lines, supporting that both SRC and JAK stimulate the STAT pathway. Finally, sequential cell exposure to inhibitors by pre-treatment with a single effector at non-toxic but effective on target inhibition concentrations for 7 days followed by the addition of each of the other inhibitors for 3 days (condition 3) showed that MEK, PI3K or JAK inhibitor acted in synergy with the SRC inhibitor in both wild-type and Q61LNRAS cells, suggesting that the first inhibitor could activate the SRC/STAT compensatory signalling pathway. In conclusion, a treatment strategy consisting in a sequential use of targeted inhibitors to first render melanoma cells more dependent on alternative compensatory pathways that should subsequently be inhibited, may enhance efficacy. By contrast, concomitant exposure to various combinations of inhibitors at different concentrations failed to produce such effect, further supporting the importance of both the duration of cell exposure to inhibitors and their sequential use.