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Abstract Background: Pediatric brain tumors are associated with the highest rates of morbidity and mortality of all childhood cancers. Diffuse midline gliomas (DMGs) are universally fatal pediatric tumors with a median survival of less than one year. DMGs are thought to arise from stalled developmental programs in the context of histone mutations (H3 K27M) which are thought to be a critical initiating event. However, these histone mutations alone are insufficient for gliomagenesis and cooperate with other alterations, including mutations in the TP53 pathway (TP53, PPM1D), growth factor receptor pathway activation (PDGFRA, EGFR, PIK3CA), and MYC amplifications (MYC, MYC-N) to induce tumor formation. There is a high degree of genetic heterogeneity amongst DMGs, and other alterations are also critical to DMG pathogenesis. This work is focused on Forkhead Box R2 (FOXR2), a forkhead family TF that we have found to be aberrantly expressed in many cancers, including DMGs. Expression of FOXR2 is sufficient to enhance glioma formation, and we have shown that regulation of FOXR2 occurs through a previously unrecognized epigenetic mechanism. Expression of FOXR2 is regulated by novel promoters, and we have demonstrated that these promoters are necessary for FOXR2 expression in several cancer lineages using CRISPR interference technology, including DMGs, melanoma, neuroblastoma, and non-small cell lung cancers. Methods: We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. Results: We have found that FOXR2 is activated across multiple cancer lineages, including DMGs. FOXR2 is required for proliferation in vitro, and it is sufficient to enhance gliomagenesis using an in utero electroporation in vivo mouse model. FOXR2 enhances MYC protein stability, even in the presence of cycloheximide. However, FOXR2 also exerts its oncogenic effects through MYC independent functions. FOXR2 is highly enriched at E26-transformation (ETS) motifs and specifically activates ETS transcriptional gene sets. To determine protein-protein interactions of FOXR2, we have performed immunoprecipitation and mass spectrometry to identify FOXR2 protein interactors. Moreover, we have performed both proteomic and phospho-proteomic analyses of FOXR2-expressing human neural stem cells. These proteomic studies have allowed us to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells, and that could be potential therapeutic targets in FOXR2-expressing cancers. Conclusion: Taken together, this study elucidates how FOXR2 protein interactors mediate oncogenesis in FOXR2-expressing diffuse midline gliomas. These studies proposed here have broad applicability across a range of cancers as FOXR2 is aberrantly expressed in 8% of all cancers. Citation Format: Jessica W. Tsai, Marissa Coppola, Phonepasong Arounleut, Jared B. Collins, Patrick Hart, Hasmik Keshishian, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Delineating mediators of oncogenesis in FOXR2-expressing cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2854.
Tsai et al. (Fri,) studied this question.