(A, B) CALU1 cells were treated with DMSO or 50 nM trametinib and the indicated concentration of N-acetyl cysteine (NAC) for 16 days. (A) ROS was measured by DCFDA assay. Error bars represent SD of two replicates. (B) Population doublings of trametinib-treated cells compared to DMSO-treated cells. Error bars represent SD of two replicates. (C) KEAP1-KO reduces trametinib- and BSO-induced ROS in CALU1 cells. Cells were treated for 72 hr, and ROS was measured by DCFDA assay. Error bars represent SD of two replicates. (D) KEAP1-KO increases cell viability in CALU1 cells treated with trametinib and BSO. 20,000 CALU1 cells were treated with DMSO plus BSO for 7 days or trametinib plus BSO for 12 days. Error bars represent SD of triplicate wells. (E) Model of how KEAP1-KO confers resistance to trametinib. MAPK pathway inhibitors turn off MAPK signaling and also induce ROS, leading to low level activation of NRF2. Upon KEAP1 loss, NRF2 activity further increases, altering expression of genes involved in the antioxidant response and metabolism, allowing for proliferation in the absence of MAPK signaling.