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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: 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.

Pregnancy is a common immunization event, but the molecular mechanisms and immunological consequences provoked by pregnancy remain largely unknown. We used mouse models and human transplant registry data to reveal that pregnancy induced exhausted CD8 T cells (Preg-TEX), which associated with prolonged allograft survival. Maternal CD8 T cells shared features of exhaustion with CD8 T cells from cancer and chronic infection, including transcriptional down-regulation of ribosomal proteins and up-regulation of TOX and inhibitory receptors. Similar to other models of T cell exhaustion, NFAT-dependent elements of the exhaustion program were induced by fetal antigen in pregnancy, whereas NFAT-independent elements did not require fetal antigen. Despite using conserved molecular circuitry, Preg-TEX cells differed from TEX cells in chronic viral infection with respect to magnitude and dependency of T cell hypofunction on NFAT-independent signals. Altogether, these data reveal the molecular mechanisms and clinical consequences of maternal CD8 T cell hypofunction and identify pregnancy as a previously unappreciated context in which T cell exhaustion may occur.

BACKGROUND: Genomic and transcriptomic studies have elucidated new insights into the landscape of diffuse intrinsic pontine glioma (DIPG). However, the role of long non-coding RNAs (lncRNAs) has not been explored at depth in these tumors, and there have not been studies focused on how lncRNAs interact with the K27M histone mutation. In a recent analysis of nearly 200 DIPGs and pediatric high-grade gliomas (pHGG), we previously detected a novel, recurring structural variant in the lncRNA CCDC26. This rearrangement occurs in nearly 10% of all DIPGs, and we have furthermore identified alterations in more than 100 lncRNAs in DIPG. METHODS: To identify lncRNAs required for proliferation of patient-derived DIPG cancer cells, we designed two custom genome-scale lncRNA libraries. We generated a genome-scale lncRNA CRISPR-Cas9 knockout pooled library, consisting of 45,766 single guide RNAs (sgRNAs). Additionally, we generated a genome-wide CRISPR interference pooled library consisting of 45,608 sgRNAs, targeting lncRNA transcription start sites (TSS). RESULTS: We utilized in vitro histone-mutant pHGG models as well as edited clones of these models with the K27M mutant corrected in order to compare lncRNA dependencies in these two contexts. We have successfully performed genome-scale CRISPR-Cas9 knockout and CRISPR interference screens targeting lncRNAs in these cell lines, revealing lncRNA dependencies. Candidate dependencies in our CRISPR-Cas9 knockout screen include LOC100507412, LOC105379524, and LINC02193. CONCLUSION: Genome-wide lncRNA CRISPR knock-out and CRISPR interference screens are a novel approach for the unbiased identification of lncRNAs that are required for pediatric high-grade glioma proliferation. Further validation of specific lncRNAs is required, and these lncRNA dependencies represent potential novel therapeutic targets.

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naïve samples–the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them.

Calcific aortic valve disease (CAVD) is the leading heart valve disorder in the US. It is characterized by an active accumulation of calcium nodules on the aortic valve leaflets which lead to stiffening and remodeling of the valve leaflets causing valve dysfunction, cardiac failure and increased stroke risk. Inflammation and mechanical stresses contribute to CAVD pathogenesis. However, the mechanisms driving the fibrocalcific remodeling of the aortic valve are currently ill-defined. Multiple studies have revealed that the catalytic subunit of telomerase reverse transcriptase (TERT) can induce gene transcription and its overexpression primes mesenchymal stem cells to differentiate into osteoblasts, suggesting that TERT has a role in the activation of osteogenic transcriptional programs. We hypothesized that TERT contributes to early events leading to calcification of the valve leaflet. In human calcified valve tissue, we found that TERT protein is highly expressed in areas of calcification compared to control valve tissue, with no effect on telomere length. Alpha-SMA, a VIC activation marker, and RUNX2, a key transcription factor involved in the osteogenic differentiation of osteoblast, were also elevated in CAVD tissue. Under osteogenic differentiation conditions, human valve interstitial cells (VICs) upregulated TERT, RUNX2, and alpha-SMA protein levels and calcified, while CAVD VICs calcified de novo. Inflammatory stimuli intensified in vitro calcification, and induced TERT, RUNX2, and alpha-SMA protein expression. PLA and ChIP analysis showed that TERT interacts with interacted with Signal Transducer and Activator of Transcription 5A/B (STAT5) and together bind to RUNX2 promoter, respectively. shRNA-mediated TERT downregulation reduced expression of RUNX2 and alpha-SMA and genetic deletion of Tert in murine mesenchymal stem cells and vascular smooth muscle cells prevented calcification. These data provide evidence that TERT is required for calcification, regulates the transition of quiescent VICs into calcifying VICs, and that STAT5 functions as a TERT-interacting partner for DNA binding.

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naïve samples–the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them.

Structural variants (SVs) within the cancer genome reflect defects in DNA damage repair, impact gene regulation, and alter the genomic landscape. SVs impact a larger fraction of the cancer genome than any other genetic aberration, yet their effects on modulating therapeutic response have been largely uncharacterized. Patterns of rearrangements, otherwise known as signatures, vary across tumor and tissue type, and can indicate underlying biological mechanisms responsible for cancer etiology. We have extracted SV signatures from whole genome sequencing data from hundreds of cancer cell lines. Using datasets indicating the cell lines’ response to over a thousand small molecule inhibitors, we have identified associations between SV signatures and response to therapeutics. These results will enhance our understanding of mechanisms underlying rearrangement signatures and nominate new biomarkers of drug response. Citation Format: Simona Dalin, Frank Dubois, Shu Zhang, Alexander Crane, Jeremie Kalfon, Javad Noorbakhsh, Rameen Beroukhim. Associations between structural variant signatures and drug sensitivity in cell lines [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 2710.

BACKGROUND Pediatric High-Grade Gliomas (pHGGs) show recurrent high-level amplifications around the oncogenes MET, MYCN and EGFR. However what drives expression of the oncogenes from these amplicons remains unclear. We aim to discover enhancers on these amplicons that are responsible for oncogene expressions and the core regulatory transcription factors (TFs) they bind. METHOD Using RNA-seq from 12 pHGG cell lines, we identified groups of high and low-expressing pHGG lines for MET, MYCN and EGFR. We then compared the H3K27Ac ChIP-seq between the two groups using diffbind. This allowed us to identify statistically significant peaks that are differentially activated in the oncogene-high v.s. oncogene-low expressing groups. Additionally, we overlapped the positions of these candidate oncogene enhancers with the regions that are recurrently incorporated into high-level amplicons based on published whole genome sequencing data. Using a previously defined set of core regulatory TFs we determined which TF binds the amplified oncogene enhancers and could be driving oncogenic expressions of MET, MYCN and EGFR in pHGGs. RESULTS We identify 3 cell lines for both the high- and low-expressing groups for each oncogene. Cell lines with high expression of the oncogene showed distinct enhancers with significant enrichment in H3K27Ac compared to the cell lines with low expression for each oncogene. Of all enhancers with enrichment high oncogene expression groups those with binding sites for known pHGG core regulatory circuit TF were preferentially incorporated into the high-level amplicons of the oncogene. We also identified core TFs that bind enhancers for MYCN, EGFR and MET as well as core TFs that are unique to a single oncogene. CONCLUSION We identified candidate core transcription factor that drives expression of multiple oncogenes in pHGG. These could serve as a potential novel therapeutic target for pHGGs with addiction to MYCN or RTK signaling.