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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)
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.
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.
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.
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).
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