LAUSR

Metastasis poses a major challenge in cancer management, including EML4‐ALK‐rearranged non‐small cell lung cancer (NSCLC). As cell migration is a critical step during metastasis, we assessed the anti‐migratory activities of several clinical ALK inhibitors in NSCLC cells and observed differential anti‐migratory capabilities despite similar ALK inhibition, with brigatinib displaying superior anti‐migratory effects over other ALK inhibitors. Applying an unbiased in situ mass spectrometry‐based chemoproteomics approach, we determined the proteome‐wide target profile of brigatinib in EML4‐ALK+ NSCLC cells. Dose‐dependent and cross‐competitive chemoproteomics suggested MARK2 and MARK3 as relevant brigatinib kinase targets. Functional validation showed that combined pharmacological inhibition or genetic modulation of MARK2/3 inhibited cell migration. Consistently, brigatinib treatment induced inhibitory YAP1 phosphorylation downstream of MARK2/3. Collectively, our data suggest that brigatinib exhibits unusual cross‐phenotype polypharmacology as, despite similar efficacy for inhibiting EML4‐ALK‐dependent cell proliferation as other ALK inhibitors, it more effectively prevented migration of NSCLC cells due to co‐targeting of MARK2/3.