Avoid common mistakes on your manuscript.
Long Interspersed Element-1 (LINE-1 or L1) is the only autonomously active human retrotransposable element shown to mobilize in cancers, which can disrupt normal gene function or regulation [6]. However, L1 regulatory elements have not been implicated in human tumorigenesis.
We identified an infant high-grade glioma (HGG, Fig. 1a) showing DNA methylation profiles (Fig. 1b) and FOXR2 overexpression (Fig. 1c) characteristic of FOXR2-activated CNS neuroblastoma (NBL) [1]. However, histology review confirmed typical HGG findings—infiltrating astrocytic tumor cells demonstrated strong and diffuse GFAP expression and were negative for synaptophysin. This suggests that aberrant FOXR2 activation may have driven tumorigenesis and the observed methylome profile.
The tumor’s whole genome sequencing (WGS) data revealed a cluster of soft-clipped (SC) reads containing sub-regions unmapped to the reference genome located within intron 1 of FOXR2. The reads contained a poly-A or L1 5’UTR sequence, indicating an L1 insertion event (Supplementary Fig. 1a, online resource). PCR amplification of the genomic sequence revealed a ~ 3 kb somatic insertion (Supplementary Fig. 1b, online resource). Targeted PacBio sequencing identified a 5’ inverted L1 insertion with a nearly intact L1 5’UTR, which contains an RNA pol-II promoter in the same orientation as FOXR2 but inverted with respect to the remaining truncated L1 sequence, where a partial L1 open reading frame (ORF2) was present, followed by the L1 3’UTR, a 31 bp poly-A tail, a 29 bp transduction sequence, and a 96 bp poly-A tail (Fig. 1d). The insertion site was flanked by a target-site duplication (TSD; 5’-GTTGATATCTTT). The transduction sequence enabled us to trace the full-length 6p24.1 L1 as the source element responsible for the somatic insertion (Supplementary Fig. 1c, online resource) [2, 4], which was also confirmed by shared L1 sequence variants between the 6p24.1 L1 and the FOXR2 L1 (Supplementary Table 1, online resource).
RNA-seq data indicated “donation” of the L1 promoter initiated FOXR2 transcription as we identified a chimeric L1/FOXR2 transcript spanning the first 97 bp of L1 5’UTR from a known L1 splice donor site to the acceptor site of exon 2 of a non-canonical FOXR2 isoform (Fig. 1d and Supplementary Fig. 2b, online resource) [3]. There was no expression of FOXR2 exon 1 nor splice junction reads upstream the L1 insertion (Supplementary Fig. 2a, online resource). To further confirm promoter activity of the FOXR2 L1, we performed bisulfite sequencing on its 5’UTR. We observed hypomethylation of all CpG sites profiled, while the source 6p24.1 L1 5’UTR remained hypermethylated (i.e., inactive) (Fig. 1e). These results support an active L1 promoter driving aberrant FOXR2 transcription in the tumor.
Molecular profiling of serial tumor samples projected the temporal order of mutation acquisition as follows (Fig. 1f): a somatic L1 insertion at the FOXR2 locus led to aberrant oncogenic FOXR2 expression and chimeric L1/FOXR2 transcripts. The insertion was an early tumor-initiating event, as it was the only driver present at diagnosis and, as a founder mutation, persisted through tumor recurrence. While wild-type p53 expression was confirmed in the primary tumor, a clonal TP53 R175H mutation with loss of heterozygosity was acquired in recurrent tumors (Supplementary Fig. 3, online resource).
Our study presents the first example of L1 promoter “donation” as a novel cancer-initiating mechanism, as compared to previously reported L1-mediated disruption of tumor suppressors or oncogene repressors [6]. We screened an additional 183 pediatric HGG samples and 22 CNS tumors [7] and did not observe another L1/FOXR2 fusion, likely due to low L1 activity in CNS tumors [6]. Nevertheless, the findings made in the index HGG broaden oncogenic L1 retrotransposition mechanisms, providing a new direction for investigating genomic drivers in non-coding regions. Optimal treatment strategies for this hybrid histological HGG and molecular CNS NBL FOXR2 tumor demand further investigation which may involve assessing the functional impact of FOXR2 activation, known to stabilize cMYC [5], on global methylome changes in neural progenitor cells.
References
WHO Classification of Tumours Editorial Board (2021) Central nervous system tumours [Internet]. Lyon (France): International Agency for Research on Cancer (WHO classification of tumours series, 5th ed; vol 6). Available from: https://tumourclassification.iarc.who.int/chapters/45. Accessed 9 Mar 2022
Beck CR, Collier P, Macfarlane C, Malig M, Kidd JM, Eichler EE et al (2010) LINE-1 retrotransposition activity in human genomes. Cell 141:1159–1170. https://doi.org/10.1016/j.cell.2010.05.021
Belancio VP, Hedges DJ, Deininger P (2006) LINE-1 RNA splicing and influences on mammalian gene expression. Nucleic Acids Res 34:1512–1521. https://doi.org/10.1093/nar/gkl027
Kidd JM, Cooper GM, Donahue WF, Hayden HS, Sampas N, Graves T et al (2008) Mapping and sequencing of structural variation from eight human genomes. Nature 453:56–64. https://doi.org/10.1038/nature06862
Li X, Wang W, Xi Y, Gao M, Tran M, Aziz KE et al (2016) FOXR2 interacts with MYC to promote its transcriptional activities and tumorigenesis. Cell Rep 16:487–497. https://doi.org/10.1016/j.celrep.2016.06.004
Scott EC, Devine SE (2017) The role of somatic L1 retrotransposition in human cancers. Viruses. https://doi.org/10.3390/v9060131
Sturm D, Orr BA, Toprak UH, Hovestadt V, Jones DTW, Capper D et al (2016) New brain tumor entities emerge from molecular classification of CNS-PNETs. Cell 164:1060–1072. https://doi.org/10.1016/j.cell.2016.01.015
Acknowledgements
This work was supported by National Cancer Institute (NCI) grants to J.C., J.Z. and S.J.B. (P30CA021765 and P01CA096832). Additionally, R01CA216391 funds J.Z. and X.C. The St. Jude 3D Genome Consortium also funds in part J.Z., S.J.B. and J.E. All authors receive support from American Lebanese Syrian Associated Charities (ALSAC). We thank Drs. Kim Stegmaier and Pratiti Bandopadhayay for their shared interest in investigating FOXR2 activation in pediatric HGGs. We thank Drs. Karol Szlachta and Liqing Tian for additional analysis efforts and Emily Plyer and Haseeb Zubair for their technical assistance. Drs. Rick Young and Tom Look provided a critical review of the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
Authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Flasch, D.A., Chen, X., Ju, B. et al. Somatic LINE-1 promoter acquisition drives oncogenic FOXR2 activation in pediatric brain tumor. Acta Neuropathol 143, 605–607 (2022). https://doi.org/10.1007/s00401-022-02420-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00401-022-02420-9