Dynamic low-complexity domain interactions of PAX3::FOXO1 mediate endogenous pathological transcriptional hub formation in alveolar rhabdomyosarcoma

Rhabdomyosarcoma (RMS), a cancer of skeletal muscle tissue, is the most common pediatric soft tissue sarcoma with 5-year survival rate less than 30% in high-risk RMS, despite advances in prognosis and treatment over the past few decades. PAX3::FOXO1 (P3F1) is a fusion oncoprotein found in 60% cases of alveolar RMS (aRMS), the most aggressive subtype of RMS, and has been thought to drive oncogenic transcription in aRMS. However, the molecular mechanisms by which P3F1 regulates transcription remain poorly understood, and the extent to which these transcriptional changes contribute to tumorigenesis is unclear. In the current study, we first demonstrated exogenously expressed P3F1 exhibited neomorphic hub formation propensity which is not observed for its parental proteins. Using CRISPR/Cas9-based gene editing method, we endogenously labeled P3F1 with a fluorogenic HaloTag in patient-derived aRMS cells, allowing us, for the first time, to visualize P3F1 at its native pathological environment. We discovered that endogenous P3F1 forms transcriptional hubs at its target genes, and the average hub size is ~117 nm characterized by photoactivatable localization microscopy. Single particle tracking experiment showed that P3F1 molecules have longer chromatin bound residence time in the hubs compared to those outside the hubs, suggesting an important role of P3F1 hubs in target gene transcription. We found disrupting the intrinsically disordered low complexity domains (LCDs) of P3F1 impaired its hub formation propensity, target gene transcription and its ability to recruit coactivator p300. Using a phase-separation-induced interactome detection (PhaseID) method, we mapped unique interacting proteins that P3F1 hubs recruit and utilize to influence oncogenic transcription. In summary, our results uncover a neomorphic hub formation behavior of pathological P3F1 and provide a strong rationale of targeting P3F1 hub formation as new therapeutics for aRMS.