AbstractTargeted agents have emerged as promising molecules for cancer treatment, but most of them fail to achieve complete tumor regression or attain durable remissions due to tumor adaptations. We used dynamic BH3 profiling to identify targeted agents effectiveness and anti-apoptotic adaptations upon targeted treatment in rhabdomyosarcoma. We focused on studying the use of BH3 mimetics to specifically inhibit pro-survival BCL-2 family proteins, overwhelm resistance to therapy and prevent relapse. We observed that the MEK1/2 inhibitor trametinib rapidly depleted the pro-apoptotic protein NOXA, thus increasing MCL-1 availability. Indeed, we found that the MCL-1 inhibitor S63845 synergistically enhanced trametinib cytotoxicity in rhabdomyosarcoma cells in vitro and in vivo. In conclusion, our findings indicate that the combination of a BH3 mimetic targeting MCL-1 with trametinib improves efficiency on rhabdomyosarcoma by blocking tumor adaptation to treatment.