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Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis. Significance: This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers. See related commentary by Liu and Northcott, p. 2977

BACKGROUND: Understanding how aberrant transcription factors (TFs) hijack normal development to induce oncogenesis is a critical question in oncology. Forkhead box (FOX) proteins are a superfamily of transcriptional regulators characterized by a forkhead DNA-binding domain. Within this family, Forkhead Box R2 (FOXR2) has been identified as a candidate structural variant (SV) driver in a subset of pediatric cancers including CNS embryonal tumors and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation, other non-SV mechanisms of activating aberrant expression, and the true extent of its role as an oncogene across all cancers have not been systematically evaluated. METHODS: We applied an integrative approach using transcriptomics, epigenetics, in vitro cancer models, and in vivo mouse models to systematically evaluate the mechanisms by which FOXR2 is activated across human cancers. RESULTS: We performed a pan-cancer analysis of FOXR2 activation across over 10,000 adult and pediatric cancer samples, and surprisingly found FOXR2 to be aberrantly upregulated in 70% of all cancer types (including diffuse midline gliomas), and 8% of all individual tumors. FOXR2 expression occurred predominantly in the absence of rearrangement/fusions, single nucleotide variants, or copy number aberrations at the DNA level. Transcriptomic and epigenomic analyses show the vast majority of tumors (78%) aberrantly express FOXR2 through a previously undescribed epigenetic mechanism via hypomethylation of a novel promoter. Using both in vitro and in vivo models, we demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, including DMGs. CONCLUSION: Taken together, this study demonstrates that FOXR2 is a novel and potent oncogene across pediatric and adult cancers, and highlights a new epigenetic mechanism by which its expression is activated.