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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

Supplementary Figure from FOXR2 Is an Epigenetically Regulated Pan-Cancer Oncogene That Activates ETS Transcriptional Circuits

Supplementary Table from FOXR2 Is an Epigenetically Regulated Pan-Cancer Oncogene That Activates ETS Transcriptional Circuits

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: Diffuse midline gliomas (DMGs) are a universally fatal brain tumor of childhood. While histone mutations are a critical tumor initiating event, they are insufficient to drive gliomagenesis. Histone mutations co-occur with somatic alterations in other pathways including TP53, MAPK, and MYC signaling. However, the mechanisms through which these pathways are activated have not been fully elucidated. 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 recently found that a subset of DMGs aberrantly express FOXR2, a forkhead transcription factor. FOXR2 is both sufficient to enhance tumor formation, and necessary for FOXR2-expressing DMGs. While FOXR2 indeed enhances MYC protein stability, FOXR2 exerts oncogenesis through MYC-independent functions and specifically hijacks E26-transformation specific (ETS) transcriptional circuits and FOXR2 DNA-binding is highly enriched at ETS motifs. We have performed proteomic and phospho-proteomic analysis of FOXR2-expressing human neural stem cells to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells. Conclusion: Taken together, this study elucidates how FOXR2 interacts with ETS transcription factors to mediate oncogenesis, and further highlights a role for FOXR2 in activating ETS and MAPK signaling. Citation Format: Jessica W. Tsai, Paloma Cejas, Marissa Coppola, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Annarah Charles, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Volker Hovestadt, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Dissecting mechanisms underlying FOXR2-mediated gliomagenesis in diffuse midline gliomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3562.

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.

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) expression has been associated with a subset of cancers including CNS and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation 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 found FOXR2 to be aberrantly upregulated in 70% of all cancer types, and 8% of all individual tumors. We identified genetic and epigenetic mechanisms that induce its expression, including hypomethylation of a novel promoter in the vast majority (78%) of FOXR2-expressing cases. We demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, using both in vitro and in vivo models. Conclusion: Taken together, this study demonstrates the role of FOXR2 as a potent oncogene across human cancers, and highlights a novel mechanism by which its expression is activated. Citation Format: Jessica W. Tsai, Paloma Cejas, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, J'Ya Hunter, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. FOXR2 is an oncogenic driver across adult and pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5730.