ACK1 condensates promote STAT5 signaling in lung squamous cell carcinoma.

BACKGROUND: ACK1, a non-receptor tyrosine kinase, phosphorylates various substrates involved in cancer progression. Its oncogenic activity is driven by gene amplification, mutations, and post-translational modifications. However, additional regulatory mechanisms that govern ACK1 activity remain to be fully understood. Liquid-liquid phase separation (LLPS) has emerged as a key mechanism of cellular compartmentalization, controlling the spatiotemporal dynamics of signaling pathways. METHODS: Expression plasmids and corresponding mutants were generated using molecular cloning techniques. Protein expression and localization were assessed through western blotting, immunofluorescence, and confocal microscopy. LLPS properties were evaluated using time-lapse imaging, photobleaching, optoDroplet assays, and in vitro droplet formation assays. Cellular functions were examined through colony formation and wound-healing assays. STAT5 signaling activation was assessed by western blotting, co-immunoprecipitation (Co-IP), immunofluorescence, RNA sequencing (RNA-Seq), and Gene Set Enrichment Analysis (GSEA). RESULTS: We demonstrate that ACK1 is frequently amplified and overexpressed in lung squamous cell carcinoma (LUSC). In LUSC cells, ACK1 undergoes LLPS, a process that depends on the intrinsically disordered region (IDR, 96-156 aa) but is independent of its kinase activity. We identify that the IDR induces droplet formation, with the 143-156 aa segment being essential for this activity. Furthermore, our data reveal that ACK1 phosphorylates STAT5 in LUSC cells. ACK1 condensates recruit the non-catalytic adaptors NCK1 and NCK2 and enhance STAT5 signaling. These condensates promote STAT5 nuclear localization and transcriptional activity, thereby facilitating LUSC cell growth and migration. CONCLUSIONS: Our findings highlight the crucial role of ACK1 condensates in oncogenic STAT5 signaling and suggest that targeting the formation of ACK1 condensates could serve as a potential therapeutic strategy for LUSC.