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

Extracellular-regulated protein kinase 5 (ERK5) is an emerging therapeutic target in cancer, and small-molecule ERK5 inhibitors have been widely employed to define its role in tumour biology. Here, we show that the commonly used ERK5 inhibitors XMD8-92 and JWG-045 suppress RSL3-induced ferroptosis in breast cancer cells, in contrast to the next-generation ERK5 inhibitors JWG-071 and BAY-885, and the MEK5 inhibitor BIX02189. Using CRISPR-mediated gene editing, we generated ERK5-deficient breast cancer cells and found that XMD8-92 and JWG-045 retained their anti-ferroptotic activity against RSL3 in the absence of ERK5 expression, indicating clear off-target effects. Pathway-level analysis of bulk RNA-sequencing data using FerrDb-curated gene sets revealed no global alteration in ferroptotic activity in BT474 cells following XMD8-92 treatment. Interestingly, XMD8-92 did not inhibit RSL3-induced lipid peroxidation and preserved cell viability even after RSL3-induced ferroptosis initiation. Based on these observations, we propose that XMD8-92 confers transient resistance to ferroptotic cell death by maintaining plasma membrane integrity, potentially through enhanced membrane repair mechanisms. Collectively, these findings reveal a previously unrecognised off-target anti-ferroptotic activity of XMD8-92 and JWG-045, further highlighting the limitation of these compounds for ERK5-specific mechanistic studies.