MYCN and HIF-1 directly regulate TET1 expression to control 5-hmC gains and enhance neuroblastoma cell migration in hypoxia

ABSTRACT

Ten-Eleven-Translocation 5-methylcytosine dioxygenases 1–3 (TET1-3) convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), using oxygen as a co-substrate. Contrary to expectations, hypoxia induces 5-hmC gains in MYCN-amplified neuroblastoma (NB) cells via upregulation of TET1. Here, we show that MYCN directly controls TET1 expression in normoxia, and in hypoxia, HIF-1 augments TET1 expression and TET1 protein stability. Through gene-editing, we identify two MYCN and HIF-1 binding sites within TET1 that regulate gene expression. Bioinformatic analyses of 5-hmC distribution and RNA-sequencing data from hypoxic cells implicate hypoxia-regulated genes important for cell migration, including CXCR4. We show that hypoxic cells lacking the two MYCN/HIF-1 binding sites within TET1 migrate slower than controls. Treatment of MYCN-amplified NB cells with a CXCR4 antagonist results in slower migration under hypoxic conditions, suggesting that inclusion of a CXCR4 antagonist into NB treatment regimens could be beneficial for children with MYCN-amplified NBs.

Key policy highlights

• In MYCN-amplified neuroblastoma cell lines, MYCN directly controls TET1 expression in normoxia.

• In MYCN-amplified neuroblastoma cell lines exposed to hypoxia, HIF-1 augments TET1 expression and TET1 protein stability.

• Hypoxic MYCN-amplified neuroblastoma cell lines have increased cell migration, mediated by genes including CXCR4 that gain 5-hydroxymethylcytosine density.

• Treatment of MYCN-amplified NB cells with a CXCR4 antagonist slows hypoxia-associated migration, suggesting a CXCR4 antagonist could be beneficial in treatment regimens for children with MYCN-amplified neuroblastomas.

KEYWORDS:

• MYCN
• hypoxia
• TET1
• neuroblastoma

Author contributions

A.E.H. designed and performed experiments, analysed data, and wrote the manuscript. S.U. performed experiments. J.Z.C. analysed the data and helped perform experiments. M.A.A. analysed and contributed sequencing data. H.R.S. helped design experiments. L.A.G. and S.L.C. conceived the study, analysed the data, and edited the manuscript.

Acknowledgments

We thank Dr. Paul Geeleher for performing statistical analysis of wound-healing assay data sets. We would also like to thank Dr. Marsha Rosner and Dr. Chilong Nguyen for their invaluable instruction of several protein biochemistry techniques. Finally, we would like to thank Dr. Julie Losman for her CRISPR plasmids targeting HIF1A.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Datasets generated for this study can be found at the Gene Expression Omnibus under the SuperSeries accession number GSE167478.

GEO accession numbers for publicly available ChIP-seq datasets used in this study are GSE80151 (39), GSE138315 (46), and GSE71399 (51). Sequencing data for NB cell lines can be found on GEO databank (GSE89413) and sequencing for NB tumours can be accessed through R2: Genomics Analysis and Visualization Platform (https://r2.amc.nl/) (37,38). NB cell line names can be found in Table S2. RNA-seq data from JQ1 experiments can also be accessed under GSE80154 (39). RNA-seq data from MLN8237 studies can be available under GSE57810 (40).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592294.2022.2106078

Additional information

Funding

This work was supported by Alex’s Lemonade Stand and a Northwestern Mutual Young Investigator Grant. A.E.H. was supported by The Molecular and Cellular Biology Training Grant, and the HHMI Med-into-Grad Translational Training Program. M.A.A. was supported by the National Institutes of Health, K08CA226237.