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.

ACK1; Liquid-liquid phase separation; Lung squamous cell carcinoma; STAT5.