Piperlongumine targeting of the NSUN2-ALYREF onco-epitranscriptomic signaling cascade in retinoblastoma

Abstract

Purpose : The signaling cascade of 5-methylcytosine (m5C), a form of RNA modification, involves NOP/Sun (NSUN) RNA methyltransferases as “writers”, and the mRNA export adaptor protein ALYREF/THOC4 as the “reader”. In the current study, we assessed the potential inhibitory effects of piperlongumine (PPL), a plant-derived compound, on the m5c signaling cascade in retinoblastoma (RB).

Methods : Immunohistochemistry (IHC) was used to detect the expression of ALYREF, and NSUN2 in human RB tumors. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA levels for NSUN1-7, ALYREF, and PFAS (an NSUN2 target gene responsible for purine biogenesis) in RB cell lines RB116, WERI-RB1, and Y79, including changes of mRNA in response to PPL treatment. Western blot analyses were used to study ALYREF and NSUN2 protein levels in RB cell lines. Anti-tumor activities were studied with WST-1 cell proliferation assay and CellEVENT Casp3/7 green detection dye.

Results : Overexpression of ALYREF and NSUN2 was seen in primary RB tumors by IHC. RB cell lines (RB116, WERI-RB1, and Y79) showed significantly higher levels of mRNA for ALYREF, NSUN2/4/5/6/7, and PFAS (phosphoribosylformylglycinamidine synthase), compared to ARPE-19 cells (p<0.05). Western blot showed ALYREF and NSUN2 proteins are overexpressed in these cell lines. RB cell lines treated with PPL (0 to 20 mM) for 24 h showed significant downregulation of mRNAs for NSUN2/4/5/6/7, ALYREF, and PFAS, compared to DMSO-treated controls (p<0.05). Additionally, PPL-treated RB cell lines showed significant cell growth inhibition and caspase-activated cell death compared to DMSO controls, at all doses (p<0.05).

Conclusions : In summary, ALYREF and NSUN2 were overexpressed by primary RB tumor cells suggesting that they may represent potential novel therapeutic targets. RB cell lines overexpressed ALYREF, NSUN2/4/5/6/7, and PFAS, and their expression was inhibited by PPL. The PPL-treated cells also showed growth inhibition and caspase-activated cell death. The inhibition of the RNA “writer” NSUN2 and the RNA “reader” ALYREF, suggest that PPL may be involved in inhibiting m5c methylation, and this is associated with PPL-induced anti-tumor activities of cell growth inhibition and caspase-activated cell death. Drug-mediated modulation of the NSUN-ALYREF signaling cascade may provide a framework for future therapeutic approaches to RB.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.