DHODH identified as main driver of QC6352 activity in glioblastoma

QC6352, widely used as a best-in-class inhibitor of KDM4 histone demethylases, showed potent activity against patient-derived glioblastoma stem cell models, but its growth-suppressive effects were largely independent of KDM4 inhibition. The compound reduced proliferation across multiple glioblastoma lines and extended survival in an orthotopic mouse model to 34.5 days versus 28.5 days without notable toxicity signals.

Orthogonal testing weakened the case for KDM4 as the relevant cellular target. ML324, a structurally distinct KDM4 inhibitor with comparable KDM4A inhibition in biochemical assays, did not reproduce QC6352’s antiproliferative effects. CRISPR screens and individual knockout experiments also found no major fitness role for KDM4A, KDM4B or KDM4C in the tested glioblastoma cells.

Mechanistic experiments instead identified DHODH, an enzyme in de novo pyrimidine biosynthesis, as the functional target. QC6352 inhibited recombinant DHODH with IC50 = 0.3 nM, caused metabolic changes consistent with DHODH blockade and lost its growth-inhibitory activity when cells were supplied with uridine or expressed an inhibitor-resistant DHODH A58T mutant.

Zavondemstat, a clinical analog of QC6352, also potently inhibited DHODH, suggesting its activity in glioblastoma models is driven mainly by pyrimidine biosynthesis disruption. The results highlight the need for orthogonal chemical probes when assigning drug mechanisms in cells.