PAX3::FOXO1 (P3F1) is a fusion oncoprotein that drives fusion-positive alveolar rhabdomyosarcoma (aRMS), a highly lethal pediatric cancer with <30% 5-year survival in high-risk patients. Targeted therapy development has been limited by P3F1’s largely disordered structure and poorly understood interaction mechanisms. Recent work has shown that short peptides targeting intrinsically disordered regions (IDRs) can disrupt transcriptional hubs and impair tumor growth, but this strategy requires identifying key interaction partners that often regulate other signaling pathways. We previously discovered that P3F1 demonstrates a neomorphic hub formation propensity that corresponds with its transcriptional activity, suggesting that disrupting these hubs can hinder said activity.
\n\n\n\nTo design short peptides modelled from the P3F1 IDR itself that disrupt P3F1 hubs and suppress P3F1-mediated transcription and aRMS cell proliferation. Experimental Procedures and Results: Using the LacO array assay, an established in-cellulo method that quantifies IDR interaction strength, we discovered that truncating the P3F1 392-510 region significantly decreased IDR interaction strength. Further, the same truncation significantly altered its interaction strength with p300, a key transcriptional coactivator known to promote P3F1’s function. Finally, dual luciferase assay results showed that a 392-510 deletion significantly decreased P3F1’s transactivation ability. Taken together, this suggested that P3F1 392-510 is a key interaction hotspot that influences its transcriptional capability. Sequence analysis revealed that P3F1 392-510 is enriched in cationic and aromatic residues, consistent with cation-π and π-π interactions common in IDRs. FINCHES interaction mapping showed that mutating either all cationic or all aromatic residues in this region reduces P3F1 homotypic interactions, with aromatic-to-alanine mutations producing the stronger effect. Ongoing experiments utilizing the LacO array assay are probing these interaction modes to guide the design of optimized P3F1 392-510 peptide mimics.
\n\n\n\nP3F1 392-510 is critical for P3F1’s IDR-mediated interactions and transcriptional activity. Its cationic and aromatic composition suggests cation-π and/or π-π interactions as key interaction modes. Future work will experimentally dissect these interaction modes and develop peptide inhibitors informed by this mechanistic insight.
", "doi": "10.1158/1538-7445.am2026-623", "issn": "0008-5472", "publisher": "American Association for Cancer Research (AACR)", "publication": "Cancer Research", "publication_date": "2026-04-03", "series_number": "7_Supplement", "volume": "86", "issue": "7_Supplement", "pages": "623-623" }, { "id": "authors:hwz3s-98150", "collection": "authors", "collection_id": "hwz3s-98150", "cite_using_url": "https://authors.library.caltech.edu/records/hwz3s-98150", "type": "article", "title": "Abstract 5950: Dynamic low-complexity domain interactions of PAX3::FOXO1 mediate endogenous pathological transcriptional hub formation in alveolar rhabdomyosarcoma", "author": [ { "family_name": "Zhong", "given_name": "Yanghao", "orcid": "0000-0002-0747-3041", "clpid": "Zhong-Yanghao" }, { "family_name": "Di Martino", "given_name": "Michael", "clpid": "Di-Martino-Michael-T" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" } ], "abstract": "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.
", "doi": "10.1158/1538-7445.am2026-5950", "issn": "0008-5472", "publisher": "American Association for Cancer Research (AACR)", "publication": "Cancer Research", "publication_date": "2026-04-03", "series_number": "7_Supplement", "volume": "86", "issue": "7_Supplement", "pages": "5950-5950" } ]