Abstract 2923: Targeting the polycomb repressive complex-2 related proteins in nasopharyngeal carcinoma
Authors: ZHU Junyu , Li Lili , Hui Connie W. , Tong Joanna H. , Chan Raymond , Wong Chi Hang , Ai Qiyong , Hui Edwin P. , Chan Anthony T. , Lo Kwok Wai , Tao Qian , Ma Brigette B. 
Published: September 2020

Aberrant hypermethylation of tumor suppressor genes is common in nasopharyngeal carcinoma (NPC), which is also enriched in denovo methylation at PCR2-related gene loci. PRC2 complex has three core subunits: embryonic ectoderm development (EED), enhancer of Zeste 1/2 (EZH1/2) and suppressor of Zeste 12 (SUZ12), which can mediate gene silencing. This study investigated the pharmacological effect of targeting EED and EZH2 in NPC cell lines (C666-1, C17C, NPC43 and HK1) and an immortalized normal nasopharyngeal epithelial cell line (NP69), either alone or concomitantly with other drugs. The relevance and prognostic significance of EED, SUZ12, EZH2 and H3K27me3 expression were determined in 100 NPC samples. EZH2 and EED was strongly expressed in EBV-positive NPC cell lines (C666-1, C17C and NPC43), but weakly in EBV negative cell line HK1 and NP69. The effect of EED inhibitor (EED226) and EZH2 inhibitor (EPZ-6438) on cell growth was tested in C666-1, NPC43 and HK1. Treatment with EED226 reduced the level of H3K27me3, with only minimal/ modest inhibition on NPC cell growth at 72hr. Drug combinations of PRC2-targeting agents with other epigenetic modulators or chemotherapy drugs were evaluated at, and near their IC50 concentrations in C666-1 and NPC43. Three combinations indicated synergistic effect: (i) EPZ-6438 and HDAC inhibitor (TSA) (synergy score, SS = 12.636); (ii) EED226 and gemcitabine (SS = 10.285); (iii) DNMT inhibitor (AZA) and EED226 (SS = 7.336). RNA-seq analysis were done after treatment with 5M of EED226 for 3, 7 and 14 days. GO enrichment analysis, KEGG pathway enrichment analysis and Artificial Neural Network (ANN) were used for bioinformatics analysis. EED226 at low dose mainly affected the cell-adherence related genes at 3-7 days after treatment, and MHC protein complex and cell cycle related genes were significantly changed after longer drug exposure (7-14 days) in NPC cells. ANN predicted a combination therapy of EED226 and LEE011 (CDK4/6 inhibitor) and we demonstrated a synergistic inhibitory effect on cell growth when EED226 was combined with LEE011 in C666-1 and HK1 cells at 72hr, with the respective combination index (CI) of ED50 &lt; 0.7. In conclusion, EED226 can significantly down- regulate the level of H3K27me3 at low dose, but with minimal growth inhibitory effect as monotherapy. EED and EZH2 inhibitor have synergistic effect when combined with chemotherapy and other epigenetic modulators and CDK4/6 inhibitors in vitro. Validation in vivo model of NPC is warranted. Data on the prognostic of PRC2-related protein significance in advanced NPC will be presented.

Acknowledgement of grant: Direct grant for research, The Chinese University of Hong Kong (#2018.012).

Citation Format: Junyu ZHU, Lili Li, Connie W. Hui, Joanna H. Tong, Raymond Chan, Chi Hang Wong, Qiyong Ai, Edwin P. Hui, Anthony T. Chan, Kwok Wai Lo, Qian Tao, Brigette B. Ma. Targeting the polycomb repressive complex-2 related proteins in nasopharyngeal carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2923.