Discovering the mechanisms underlying oncogenesis by ZFTA-RELA and pinpointing therapeutic targets.

PROJECT SUMMARY: Ependymoma (EPN) is an aggressive and chemo-resistant pediatric brain tumor, with treatment remaining surgery and radiation. There are no targeted therapies available to EPN patients at diagnosis or relapse. Over 95% of EPN that arise in the cortex are driven by gene fusions of ZFTA, with ZFTA-RELA fusions (denoted ZRFUS) being the most common. We and others have developed mouse models of ZRFUS EPN and found that ZRFUS binds novel genomic loci to activate oncogenic gene expression and drive tumor formation. Despite these advances, key knowledge gaps regarding ZRFUS biology remain, including: 1) The precise mechanisms of oncogenic gene expression, including recruitment of co-regulatory factors, following ZRFUS DNA binding, 2) How the structured domains and intrinsically disordered regions (IDRs) within the ZRFUS protein synergistically contribute to oncogene activation, and 3) How ZRFUS interaction partners contribute to oncogenesis and whether they represent therapeutic vulnerabilities. Emerging evidence from us and others across multiple pediatric cancers has linked fusion oncoprotein (FO) expression to biomolecular condensate formation, possibly through a phase separation (PS) mechanism, as a possible unifying model of oncogenic gene expression. We demonstrate that ZRFUS forms dynamic, nuclear condensates, and that key ZRFUS IDRs are necessary for condensate formation, oncogenic gene expression and tumorigenesis. In addition to identifying several long IDRs within ZRFUS, we have discovered that the ZFTA zinc finger (ZF) domains in ZRFUS dominantly influence DNA binding, condensate formation, and oncogenic gene expression. Based on these preliminary data, we hypothesize that: ZRFUS forms chromatin-associated, liquid-like condensates that are necessary for oncogenic gene expression and tumor development. To address this hypothesis, we propose the following three objectives/aims, to (1) Understand how ZRFUS condensates regulate oncogenic expression and tumor development, (2) Discover the structural basis of ZFTA zinc finger DNA binding and its role in tumorigenesis, and (3) Interrogate ZRFUS interacting proteins as cancer dependency proteins and therapeutic targets. Overall, we anticipate that deciphering the mechanisms underlying oncogenesis by ZRFUS, including the biophysics of condensate formation and functional roles of its condensate interaction partners, may yield novel therapeutic targets for this aggressive disease.