ORY-1001 inhibits glioblastoma cell growth by downregulating the Notch/HES1 pathway via suppressing lysine-specific demethylase 1 expression

Objective
This study aimed to investigate the inhibitory effect of ORY-1001, a lysine-specific histone demethylase 1 (LSD1) inhibitor, on the growth of glioblastoma (GBM) and explore the underlying molecular mechanisms.

Methods
LSD1 expression levels in GBM and normal brain tissues were analyzed using data from the TCGA and HPA databases. Female BALB/c mice carrying xenografts derived from U87 cells or cells modified with lentivirus-mediated LSD1 silencing or Notch overexpression were treated by oral gavage with either saline or 400 µg/kg ORY-1001 every seven days. Tumor development and the mice’s survival were monitored. The effects of ORY-1001 on GBM cell viability and LSD1 expression were evaluated using Western blotting. Gene pathways associated with LSD1 were identified through bioinformatics analysis. The expression of the Notch/HES1 pathway was assessed through Western blotting and qRT-PCR after LSD1 silencing or ORY-1001 treatment. Chromatin immunoprecipitation assays were conducted to study ORY-1001’s regulatory impact on the Notch/HES1 pathway, and the effect of ORY-1001 on U87 cells with Notch1 silencing or overexpression was also evaluated.

Results
LSD1 was highly expressed in GBM, with higher levels correlating negatively with patient survival (P < 0.001). Both ORY-1001 treatment and LSD1 silencing significantly reduced tumor burden and extended the survival of Iadademstat GBM-bearing mice. ORY-1001 effectively inhibited GBM cell viability and reduced LSD1 expression in a dose-dependent manner, though this inhibitory effect was diminished when LSD1 was silenced. Bioinformatics analysis showed that LSD1-regulated genes were enriched in the Notch pathway. LSD1 silencing and ORY-1001 treatment downregulated Notch/HES1 pathway expression. Additionally, overexpression of Notch1 weakened the anti-tumor effects of ORY-1001. Mechanistically, ORY-1001 disrupted the interaction between LSD1 and several Notch target genes, including *Notch3*, *HES1*, and *CR2*.

Conclusion
ORY-1001 inhibits GBM growth in mice by suppressing LSD1 expression, which in turn downregulates the Notch/HES1 pathway.