Combined Inhibition of Lysine-Specific Demethylase 1 and Kinase Signaling as a Preclinical Treatment Strategy in Glioblastoma

Glioblastoma is the most common primary malignant brain tumor in adults, with limited treatment options resulting in a poor prognosis. Lysine-specific demethylase 1 (LSD1) is overexpressed in glioblastoma and contributes to tumor growth and treatment resistance. Inhibitors of LSD1 have shown preclinical promise but have had limited clinical development for glioblastoma. Given the frequent kinase pathway alterations in glioblastoma, the interplay between LSD1 inhibition and kinase signaling pathways was investigated to identify vulnerabilities that could be exploited through rationally designed combination therapies. We hypothesized that LSD1 inhibitors would activate kinase signaling pathways, such as MAPK, and the addition of kinase inhibition would enhance their efficacy. Glioblastoma stem cell (GSC) lines and normal human astrocytes (NHAs) were treated with catalytic LSD1 inhibitors, NCD38 and bomedemstat, and the LSD1 scaffolding inhibitor, seclidemstat. Cells were treated with LSD1 inhibitors alone and in combination with kinase inhibitors, including osimertinib, afatinib, and ulixertinib. The effect on cell viability, proliferation, and neurosphere formation was assessed, and synergy scores were calculated using Bliss synergy models. Kinase signaling was analyzed via western blot analysis, and in vivo efficacy was evaluated in orthotopic xenograft models. LSD1 knockdown and seclidemstat reduced kinase signaling in the GSC lines, while catalytic LSD1 inhibitors increased kinase activity or had no effect. Catalytic LSD1 inhibitors combined with kinase inhibitors, particularly the combination of NCD38 and osimertinib, synergistically reduced GSC viability and proliferation while sparing NHAs. Combination treatment consistently reduced phospho-S6 ribosomal protein levels in three different GSC lines, and basal phospho-S6 ribosomal protein levels across the GSCs and the NHAs were negatively correlated with a synergistic response. The generation of an NCD38-resistant GSC line showed increased kinase activity and was associated with enhanced osimertinib sensitivity. Finally, the combined treatment with NCD38 and osimertinib in glioblastoma-bearing mice delayed tumor growth and improved survival outcomes. This work provides several key contributions to glioblastoma research and preclinical drug development for LSD1 inhibitors. It reveals the distinct effects of LSD1 inhibition on kinase signaling and demonstrates the therapeutic potential of combining catalytic LSD1 inhibitors with EGFR inhibitors in glioblastoma. These findings provide a rationale for further investigation of combination therapies and dual-targeted inhibitors to overcome resistance and improve treatment outcomes in glioblastoma.