Zhou J, Toh SH, Tan TK, Balan K, Lim JQ, Tan TZ, Xiong S, Jia Y, Ng SB, Peng Y, Jeyasekharan AD, Fan S, Lim ST, Ong CJ, Ong CK, Sanda T, Chng WJ.
Affiliations
- 1Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Centre for Translational Medicine, Singapore, 117599, Singapore.
- 2Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore.
- 3NUS Centre for Cancer Research (N2CR), 14 Medical Drive, Centre for Translational Medicine, Singapore, 117599, Singapore.
- 4Division of Cellular and Molecular Research, Lymphoma Genomic Translational Research Laboratory, National Cancer Centre Singapore, 11 Hospital Drive, Singapore, 169610, Singapore.
- 5Duke-NUS Medical School, Singapore, 169857, Singapore.
- 6Genomics and Data Analytics Core (GeDaC), Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore, 117599, Singapore.
- 7Department of Medical Oncology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
- 8Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore.
- 9Director’s office, National Cancer Centre, Singapore, 168583, Singapore.
- 10Office of Education, Duke-NUS Medical School, Singapore, 169857, Singapore.
- 11Department of Sarcoma, Peritoneal and Rare Tumours (SPRinT), Division of Surgery and Surgical Oncology, National Cancer Centre, Singapore, 168583, Singapore.
- 12Department of Sarcoma, Peritoneal and Rare Tumours (SPRinT), Division of Surgery and Surgical Oncology, Singapore General Hospital, Singapore, 168583, Singapore.
- 13Laboratory of Applied Human Genetics, Division of Medical Sciences, National Cancer Centre, Singapore, 168583, Singapore.
- 14SingHealth Duke-NUS Oncology Academic Clinical Program, Duke-NUS Medical School, Singapore, 169857, Singapore.
- 15SingHealth Duke-NUS Surgery Academic Clinical Program, Duke-NUS Medical School, Singapore, 169857, Singapore.
- 16Institute of Molecular and Cell Biology, A*STAR Research Entities, Singapore, 138673, Singapore.
- 17Division of Cellular and Molecular Research, Lymphoma Genomic Translational Research Laboratory, National Cancer Centre Singapore, 11 Hospital Drive, Singapore, 169610, Singapore. gmsock@nus.edu.sg.
- 18Cancer and Stem Cell Biology, Duke-NUS Medical School, 8 College Road, Singapore, 169857, Singapore. gmsock@nus.edu.sg.
- 19Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Centre for Translational Medicine, Singapore, 117599, Singapore. csitakao@nus.edu.sg.
- 20Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore. csitakao@nus.edu.sg.
- 21Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Centre for Translational Medicine, Singapore, 117599, Singapore. mdccwj@nus.edu.sg.
- 22Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore. mdccwj@nus.edu.sg.
- 23NUS Centre for Cancer Research (N2CR), 14 Medical Drive, Centre for Translational Medicine, Singapore, 117599, Singapore. mdccwj@nus.edu.sg.
- 24Department of Hematology-Oncology, National University Cancer Institute of Singapore (NCIS), National University Health System (NUHS), 1E, Kent Ridge Road, Singapore, 119228, Singapore. mdccwj@nus.edu.sg.
Abstract
Background: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL.
Methods: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo.
Results: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis.
Conclusions: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.
PMID: 37032358 PMCID: PMC10084643 DOI: 10.1186/s12943-023-01767-1