July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
The Histone Methyltransferase NSD2 is a New Histopathologic Biomarker and Potential Therapeutic Target for Human Retinoblastoma.
Author Affiliations & Notes
  • Rajesh C Rao
    Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, United States
    Pathology, University of Michigan, Michigan, United States
  • Zhenhua Zou
    Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, United States
  • Footnotes
    Commercial Relationships   Rajesh Rao, None; Zhenhua Zou, None
  • Footnotes
    Support  Supported in part by the National Institutes of Health (P30CA046592). R.C.R. is a recipient of an RPB Career Development Award. R.C.R. is supported by Career Development Awards from the National Institutes of Health (K08EY026654) and the A. Alfred Taubman Medical Research Institute, where he is the Leslie and Abigail Wexner Emerging Scholar. R.C.R. received funding from the Leonard G. Miller Ophthalmic Research Fund at the Kellogg Eye Center, Grossman Research Fund, Barbara Dunn Research Fund, Roz Greenspoon Research Fund, Beatrice & Reymont Paul Foundation and March Hoops to Beat Blindness. The authors acknowledge generous support from an unrestricted research grant from RPB to the Department of Ophthalmology and Visual Sciences at the University of Michigan. None of the sponsors participated in the design and conduct of the study, collection, management, analysis and interpretation of the data, preparation, review, approval or submission of the abstract.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 1321. doi:
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      Rajesh C Rao, Zhenhua Zou; The Histone Methyltransferase NSD2 is a New Histopathologic Biomarker and Potential Therapeutic Target for Human Retinoblastoma.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1321.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinoblastoma (RB) is the most common primary intraocular cancer in children. No molecular-targeted therapy for this deadly tumor exists. We recently showed that EZH2, a histone methyltransferase (HMT), is highly expressed in RB and its catalytic activity, via its SET domain, on histone H3 lysine 27 (H3K27me3) is required for RB cell survival (PMID: 26280220). Since EZH2 promotes expression of NSD2 (PMID: 23159737), an HMT for H3K36me, we reasoned that NSD2 might regulate human RB tumorigenesis.

Methods : Expression levels of NSD2 in 43 primary enucleated human retinoblastoma specimens, and two RB (Y79 and Weri-Rb1) cell lines were compared by RT-PCR, immunohistochemistry and Western blotting. shRNA was used to knockdown NSD2, and changes in histone modifications were verified with specific antibodies. The consequences of NSD2 knockdown were detected in ATP-based cell proliferation assay, PI/Annexin V-based apoptosis assay and PI/5’EDU-based cell cycle assay. The functional pathways by NSD2 were identified with CRISPR-edited domain deletions and ChIP-Seq.

Results : NSD2 was enriched in RB cells versus non-tumor retina in primary human specimens. To assess the role of NSD2 in RB cell viability and growth, we used shNSD2 in human RB cell lines and found that NSD2 accelerates S-phase entry in RB cell cycle and inhibits apoptosis. H3K36me2/3 decreased in NSD2-knockdown RB lines. To confirm the functions of NSD2 HMT activities, we used CRISPR gene editing to delete catalytic SET domain of NSD2 and identified the altered downstream target regions of H3K36me2 and NSD2 with ChIP-Seq. Our results showed NSD2 regulates DNA replication through SET-dependent and SET-independent mechanisms.

Conclusions : NSD2 is a histopathologic biomarker for human RB and shows promise as a novel epigenetic target for RB.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Figure. EZH2 and NSD2 expression colocalize in a human RB sample. (A, D, E) Hematoxylin & eosin (purple, H&E) and (brown, B, E, F) EZH2 and NSD2 (brown, C, F, G) staining in corresponding sections of human RB. (A-C) RB (asterisk) fills over half the intraocular space. (D-F) Two EZH2+ and NSD2+ nodules (arrows) form within a strip of detached, non-tumor retina below the main RB mass (asterisk) (20X magnification). (E-G) RB cells within the main mass form EZH2+ (F) and NSD2+ (G) Homer Wright (black arrow) and Flexner-Wintersteiner (white arrow) rosettes (200X magnification).

Figure. EZH2 and NSD2 expression colocalize in a human RB sample. (A, D, E) Hematoxylin & eosin (purple, H&E) and (brown, B, E, F) EZH2 and NSD2 (brown, C, F, G) staining in corresponding sections of human RB. (A-C) RB (asterisk) fills over half the intraocular space. (D-F) Two EZH2+ and NSD2+ nodules (arrows) form within a strip of detached, non-tumor retina below the main RB mass (asterisk) (20X magnification). (E-G) RB cells within the main mass form EZH2+ (F) and NSD2+ (G) Homer Wright (black arrow) and Flexner-Wintersteiner (white arrow) rosettes (200X magnification).

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