XMD8-92 and JWG-045 exhibit anti-ferroptotic activities, independently of inhibiting ERK5

Extracellular-regulated protein kinase 5 (ERK5) is a promising target in cancer therapy, and several ERK5 inhibitors have been developed. In this study, we observed that the inhibitors XMD8-92 and JWG-045 exhibited n anti-ferroptotic activity in breast cancer cells, which was not detected with two other ERK5 inhibitors JWG-071, BAY-885, or the MEK5 inhibitor BIX02189. Using CRISPR-mediated gene editing, we generated ERK5- eficient breast cancer cells and found that XMD8-92 and JWG-045 protected these cells from ferroptosis, indicating clear off-target effects of these inhibitors. RNA sequencing revealed that XMD8-92 might influence erroptosis through the regulation of the oxidative stress-related genes KEAP1 and GCH1. Collectively, these results demonstrate that XMD8-92 and JWG-045 can act independently of ERK5 inhibition, underscoring the mportance of using more specific inhibitors, such as JWG-071, BAY-885, or BIX02189, to evaluate the role of ERK5 signalling in cancer. Furthermore, our data emphasise the critical need for combining bioinformatics nd experimental validation to correctly assign functional roles to specific proteins in biological processes. Overall design: BT474 cells were treated with 5 µM XMD8-92, an ERK5 inhibitor, for 0, 2, 4, and 6 hours. For each time point, three biological replicates were collected, resulting in 12 samples. Additionally, DMSO-treated control samples (one replicate each) were included for the 2-, 4-, and 6-hour time points, bringing the total number of samples to 15.