Abstract A049: <i>ZFTA::NCOA2</i>-dependent DNA binding and transcriptional regulation: Comparison to <i>ZFTA::RELA</i> and other common non-<i>RELA ZFTA</i> fusions

Abstract Introduction ZFTA::NCOA2 (ZN2) has been identified as a rare but recurrent alternative fusion to the more common ZFTA::RELA (ZR) in ependymoma. In non-RELA ZFTA fusions, ZFTA typically fuses with partner genes at mid-exon 5, preserving all four zinc finger domains of ZFTA in fusion oncoproteins. Recent studies, including findings in our patient cohort, have revealed "new-anatomy" ZFTA::NCOA1/2 (ZN) fusions with ZFTA exon 2 joining with partner genes, resulting in the preservation of only one zinc finger domain of ZFTA. While methylome and transcriptome profiling of ZN fusions have been reported, molecular characterization of the "new-anatomy" ZN fusion remains underrepresented in previous studies. We developed an in vitro model to study ZN2-dependent transcriptional regulation. Methods Human primary astrocytes (HPAs) were transduced with inducible expression constructs for ZFTA, NCOA2 and ZN2 (exon 2::exon 13), respectively. Stably transduced HPAs were subjected to Chromatin Immunoprecipitation Sequencing (ChIP-seq) and RNA-seq analyses. To investigate the functional impact of ZN2 expression, we comprehensively analyzed its DNA binding properties and transcriptional effects by integrating ChIP-seq data with gene expression profiles. GSEA and KEGG pathway enrichment analysis were performed to classify functional roles of genes dysregulated by ZN2. Results Genome-wide chromatin binding analysis identified 6,669 ZN2 binding peaks, 77.4% of which overlapped with the 40,460 peaks of wild-type ZFTA. Both shared and ZN2-only binding regions were significantly enriched for previously reported ZR binding motifs. Despite their shared DNA-binding feature, ZN2 preferentially bound to active-enhancer chromatin regions, whereas ZFTA was enriched at promoter regions. The integrated ChIP-seq and RNA-seq analysis demonstrated that the ZN2 fusion protein preferentially bound to regulatory region of genes targeted by the wild-type ZFTA and activated the expression of genes transcriptionally repressed by ZFTA. Comparing ZN2- to ZFTA-expressing cells, we identified 1,295 upregulated genes, including 1,211 genes directly targeted by both ZN2 and ZFTA, and 84 with ZN2-only binding peaks. Notable, 61 out of 93 (65.6%) previously defined ZFTA-fusion signature genes are present among the 1211 shared targets. KEGG pathway analysis revealed that these 1,211 genes were enriched in MAPK, Wnt, and Rap1 signaling pathways, consistent with findings from prior studies of ZR fusion. In contrast, the 84 ZN2-specific target genes were enriched in pathways related to cell adhesion molecules and other distinct biological processes. Conclusions As the first study to investigate ZN2 in human astrocytes, our work uncovered both shared and distinct transcriptional programs compared to ZR fusions, providing new insights into the molecular mechanisms underlying ZN2-driven oncogenesis. Functional classification of genes directly upregulated by ZN2 revealed similarities to those activated by ZR, alongside unique pathways specific to ZN2. Citation Format: Xuefei Ma, Rebecca Voss, Wojciech Rosikiewicz, Ajay Palagani, Wenqing Qi, Zhaohong Yin, Lu Wang. ZFTA::NCOA2-dependent DNA binding and transcriptional regulation: Comparison to ZFTA::RELA and other common non-RELA ZFTA fusions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A049.