Famed brain cancer drug turns out to hit a metabolic enzyme, not its intended target

A drug hailed as one of the best chemical tools for studying a family of epigenetic enzymes has been caught in an act of molecular deception. QC6352, long considered the gold-standard inhibitor of the KDM4 histone demethylases and celebrated for killing glioblastoma stem cells in laboratory dishes and in mice, does not actually owe its anticancer power to blocking those enzymes at all. Instead, a comprehensive chemical biology investigation published in Nature Chemical Biology reveals that the compound’s true target is dihydroorotate dehydrogenase, or DHODH, a mitochondrial enzyme that sits at the heart of pyrimidine biosynthesis. The finding rewrites the mechanism of a widely used research probe and has immediate implications for zavondemstat, a clinical analog of QC6352 currently being tested in patients with advanced cancers.

The story begins with glioblastoma, the most aggressive form of adult brain cancer. Despite surgery, radiation and chemotherapy, median survival remains roughly 14.6 months, and a major reason is the persistence of glioblastoma stem cells, which drive tumor initiation, progression and resistance to therapy. Because aberrant epigenetic programs help these cells maintain their plasticity, the researchers had previously screened a library of epigenetic chemical probes and found that QC6352 suppressed glioblastoma stem cell viability at nanomolar concentrations. In the new study, the team validated that potency across six patient-derived glioblastoma stem cell lines, including RKI1, MMK1, JK2, FPW1, HW1 and GBM6, with growth-inhibition potency values ranging from 3 to 133 nanomolar. Live-cell imaging showed that nearly 80 percent of treated cells arrested in the G0/G1 phase of the cell cycle, and the drug left normal human astrocytes unharmed up to 1 micromolar, a tenfold therapeutic window.

The cellular phenotype was striking. Treated cells ramped up expression of senescence and apoptosis genes, accumulated senescence-associated beta-galactosidase and Annexin V staining, and lost expression of stemness markers such as NES, SOX2 and SOX9, shifting toward a nonproliferative, differentiated state. In mice bearing orthotopic GBM6 xenografts, QC6352 at 100 milligrams per kilogram slowed tumor growth and extended median survival from 28.5 to 34.5 days without affecting body weight or blood counts. On the surface, everything pointed to a spectacular epigenetic vulnerability. But the team, led by Lenka Munoz of the University of Sydney together with Paul Workman, applied a discipline that is recommended far more often than it is practiced: testing multiple, structurally distinct inhibitors against the presumed target.

That discipline exposed the crack. Alongside QC6352, the researchers evaluated the orthogonal KDM4 inhibitor ML324, which they confirmed inhibits KDM4A with essentially the same biochemical potency, an IC50 of 326 nanomolar versus 355 nanomolar for QC6352. Yet the two compounds behaved nothing alike in cells. While QC6352 at 100 nanomolar completely repressed glioblastoma growth and triggered apoptosis, ML324 at 500 nanomolar had no impact whatsoever, and even at 5 micromolar it failed to block colony formation. Both compounds raised the levels of the histone marks H3K9me3 and H3K36me3 that KDM4 enzymes normally remove, but the team cautioned that such changes can also reflect broader heterochromatin and senescence responses, so they went further, directly testing whether either drug actually engaged KDM4 proteins inside living cells.

It did not. Cellular thermal shift assays detected no stabilization of overexpressed KDM4A even at 100 micromolar of either compound, and single-molecule tracking of SNAP-tagged KDM4A in live HeLa cells revealed no change in the mobility or chromatin-binding behavior of the enzyme after drug treatment. Expression analysis deepened the skepticism: KDM4A and KDM4B transcripts were present at low levels in only some glioblastoma stem cell lines, KDM4C and KDM4D were largely undetectable across twelve lines, and single-cell RNA sequencing of biopsies from 62 patients showed consistently low KDM4 expression in malignant cells. Proteomics found no KDM4 protein at all in A172 glioblastoma cells. Crucially, knocking out KDM4A, KDM4B or KDM4C individually with CRISPR-Cas9 left cells just as sensitive to QC6352 as before, and genome-wide CRISPR dropout screens using the 71,090-guide TKOv3 library identified 3,334 fitness genes in RKI1 cells without a single KDM4 family member among them.

The chemogenomic screens added another layer. Treating TKOv3-transduced cells with QC6352 at its 90 percent effective concentration of 70 nanomolar and at its 20 percent effective concentration of 25 nanomolar revealed 609 sensitizing and 278 resistance genes, with RNA splicing and mRNA and tRNA processing emerging as key biological processes. Again, no KDM4 enzyme met the threshold. The evidence was converging on an uncomfortable conclusion: the celebrated KDM4 probe was doing something else entirely in glioblastoma cells.

To find the real mechanism, the team turned QC6352’s transcriptional fingerprint into a search query against the Connectivity Map, comparing its signature with more than 20,000 pharmacologic perturbations across 71 cell lines. The top matches were brequinar, a known DHODH inhibitor, and floxuridine and raltitrexed, both inhibitors of thymidylate synthase. All three converge on a single metabolic pathway: de novo pyrimidine synthesis, the assembly line that produces the nucleotide building blocks of DNA, RNA and lipids. The logic of the pathway also explained a curious secondary hit, the cardiac glycosides digoxin, strophanthidin and bufalin, which indirectly block the uridine salvage route by suppressing sodium-dependent nucleoside uptake. When the researchers simply added uridine to the culture medium, it completely rescued glioblastoma cells from QC6352-induced growth arrest, the classic signature of a blocked pyrimidine supply.

Biochemistry then delivered the decisive numbers. In a recombinant enzyme assay, QC6352 inhibited human DHODH with an IC50 of 0.3 nanomolar, making it more potent than the established DHODH inhibitors BAY2402234 at 1.1 nanomolar and brequinar at 2.6 nanomolar. ML324, by contrast, showed no DHODH inhibition at all. Metabolomics sealed the case: within 24 hours of treatment, both QC6352 and BAY2402234 drove a roughly 10,000-fold accumulation of the upstream DHODH substrates dihydroorotate and carbamoylaspartate while depleting the downstream nucleotides UMP, UDP and UTP. A 1.95-angstrom cocrystal structure of QC6352 bound to human DHODH showed the compound occupying the hydrophobic channel leading to the active site, blocking ubiquinone cofactor binding, with its isonicotinic acid head forming polar contacts with R136, Q47 and Y356 and its tail packing into a hydrophobic tunnel. The same scaffold, in the KDM4A structure, chelates the catalytic nickel ion, explaining how one molecule can moonlight in two entirely different enzyme families.

The clinical implications came into sharp focus with zavondemstat, a structural analog of QC6352 now in trials for advanced and metastatic cancers. The R-enantiomer inhibited DHODH at 0.6 nanomolar, outperforming brequinar, farudodstat and BAY2402234, while its S-enantiomer was 40-fold weaker against DHODH, a chirality-dependent potency gap that tracked far better with the compounds’ cellular efficacy than their modest twofold difference in KDM4A inhibition. Uridine fully reversed the effects of both enantiomers, and expressing an inhibitor-resistant DHODH A58T mutant rescued cells from QC6352, zavondemstat and BAY2402234 alike, while uridine could not rescue the unrelated drugs GSK-J4 or doxorubicin. Guided by the crystal structures, the team even designed three new isonicotinic acid analogs that retain potent KDM4A inhibition, with IC50 values between 41 and 208 nanomolar, while showing no DHODH activity above 20 micromolar, providing the field with genuine KDM4-selective probes for the first time in this chemical series.

Beyond glioblastoma, the study is a cautionary tale for chemical biology as a whole. Despite long-standing guidelines urging the use of at least two structurally distinct probes plus inactive controls, reliance on a single compound remains common, and QC6352 shows exactly how that shortcut can produce confident but wrong conclusions about drug mechanism. DHODH itself is a validated therapeutic target, with the inhibitors leflunomide and teriflunomide already approved for autoimmune disease, though no DHODH inhibitor has yet been approved for cancer. The authors note that zavondemstat’s KDM4-independent mechanism does not diminish its clinical promise, and may even explain it, but they urge that ongoing trials be benchmarked against prior DHODH inhibitor studies. They also flag that DHODH should now be considered a frequent hidden target in phenotypic drug discovery, since multiple unrelated chemotypes keep landing on it. For glioblastoma, a disease desperate for options, the message is paradoxically hopeful: a drug already in the clinic may work through a metabolic vulnerability that is increasingly recognized as one of cancer’s most druggable Achilles heels.

Subject of Research: Target deconvolution of the KDM4 inhibitor QC6352 revealing DHODH as the driver of antiglioblastoma activity

Article Title: DHODH is the key target of the KDM4 inhibitor QC6352 in glioblastoma stem cells

Article References: Sterling, J. R., Holtsmark, J. R., Joun, G. L., Graus, M. S., Du, T. Y., Kuchibhotla, M., Johns, T. G., deWeck, A., Loo, L., Neely, G. G., Tran, K., Shard, C., Gomez, G. A., Patil, A., Shi, D. D., Losman, J.-A., Saxton, A. J., Baker, J. R., Lei, Z., … Munoz, L. (2026). DHODH is the key target of the KDM4 inhibitor QC6352 in glioblastoma stem cells. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02306-x

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02306-x

Keywords: glioblastoma, DHODH, QC6352, zavondemstat, KDM4, pyrimidine biosynthesis, chemical probes, target deconvolution, epigenetics, drug mechanism, cancer metabolism, CRISPR screens