Synergistic Effect of EZH2 Inhibitor Dznep and Flavonoid GL-V9 in AML through Inhibition Hells and Induction of DNA Damage

Background

Epigenetic alterations play critical roles in the oncogenesis of acute myeloid leukemia (AML). EZH2 is a chromatin-remodeling enzyme, responsible for trimethylation of K27 sites on H3 (H3K27me3). Alteration of chromatin architecture can greatly modulate cellular sensitivity to DNA damage (Gamze Kuser-Abali,2018). Recent evidence has implicated that EZH2 is recruited to the site of DNA damage, implicating a crosstalk between epigenetics and DNA repair (Lisa B. Caruso,2018). The newly synthesized flavonoid GL-V9 derivatized from wogonin with elegant effects against several solid tumors (Qinglong Guo, Hui Hui,2023), while the effect of GL-V9 in AML remains unknown. Here we report the synergistic promising impact of EZH2 inhibitor DZNeP and flavonoid GL-V9 that kills AML cells by triggering gross DNA damage response.

Methods

Cell proliferation was measured by Cell Counting Kit-8(CCK-8) in AML cells (U937, THP-1, MV4-11, and KG-1) treated with vehicle, DZNeP, GL-V9, and combination (DZNeP+GL-V9). Cell cycle and apoptosis were examined by flow cytometry. The colony-forming assay was conducted to assess the colony-forming ability following drug withdrawal. RNA-seq was performed in U937 cells treated with 2μM DZNeP, MV4-11 cells treated with 4μM GL-V9, and vehicle for 48 hours. The comet assay was used for measuring DNA strand breaks (DSB). Immunofluorescence staining was visualized on a confocal microscope. Expression of target genes was detected by western blot and RT-qPCR. The R2 database and GEPIA 2 were utilized to analyze the mRNA expression level and overall survival.

Results

Both DZNeP and GL-V9 had a dose-dependent and time-dependent effect on growth inhibition in U937, THP-1, MV4-11, and KG-1 cells. Co-treatment of GL-V9 with DZNeP exerted synergistic effects on reduced cell viability and clone formation versus single-drug in U937 and MV4-11 cells (p<0.001). The total apoptosis rate increased significantly in the GL-V9+DZNeP group compared to single-drug controls (p<0.001). DZNeP induced G0/G1 arrest; and GL-V9 induced G2/M arrest, whereas the combination significantly decreased the number of cells in the S-phase. Comet assay showed that the combination treatment increased comet tails. Immunofluorescence staining indicated GL-V9 triggers micro-, multi-nucleation, and cytoskeleton abnormalities. The loose chromatin was observed in DZNeP treatment cells, while an extensive nuclear fragmentation was noted in the combined treatment indicating an increase in apoptotic cells. Consistently, the combination dramatically up-regulated apoptotic effectors PARP, cleaved Caspase-3, p53, and DNA damage marker γH2AX versus single-drug. Collectively, our data indicates that the synergistic anti-tumor effect of DZNeP combined with GL-V9 is mediated by inducing DNA damage.

To understand the molecule mechanism underlying the synergistic effect, the transcriptomes were analyzed by RNA-seq, and 242 different expression genes (DEGs) were consistently regulated by DZNeP and GL-V9. The overlapped DEGs mainly enriched in the pathways concerning DNA damage repair. Particularly, the DNA damage repair genes, HELLS, MCM4, MCM6, CDC6, POLE2, ERCC6L, XRCC2, FANCI, PCLAF, GINS4, GINS2, CLSPN, MSH6, PRKDC, POLA1, CCNE2, CCNE1, FEN1 are simultaneously downregulated by DZNeP and GL-V9. Accumulation of DNA damage and deficiency of DNA damage repair pathway could disturb cellular homeostasis or cause cell death. Also, protein-protein interaction network analysis showed HELLS (Lymphoid-specific helicase) acts as a hub gene. HELLS is a multifunctional chromatin remodeling protein related to SNF2/helicase family members, participates in the DNA damage response, and plays a critical role in maintaining chromatin structure (Annalisa Tameni,2024). RT-qPCR showed that combination treatment significantly decreased the mRNA level of HELLS compared to single agents and vehicle control. Database analysis shows that HELLS is overexpressed in AML patients and the high level of HELLS is related to poor clinical outcomes.

Conclusions

Our results demonstrated the DZNeP sensitizes the effect of the new flavonoid compound GL-V9 by promoting gross DNA damage of AML cells via targeting HELLS and eventually promoting p53-mediated cell death. Our study reveals a new potential combinational strategy of EZH2 inhibitor with a natural flavonoid compound for AML.