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BACKGROUND Glioblastoma (GBM) is the most aggressive and lethal type of brain tumor. Activation of PI3K/mTOR pathway along with the loss of its primary negative regulator, phosphatase and tensin homolog (PTEN), occurs in nearly 50% of GBM patients. As PTEN is known to promote DNA damage repair deficiency, here we investigated whether PTEN deficiency presents a vulnerability to a simultaneous induction of DNA damage and suppression of repair mechanisms by combining topoisomerase I (TOP1) and PARP inhibitors. METHODS We used patient-derived GBM cells and stem-like cells to determine response to LMP400 (Indotecan), a novel non-camptothecin TOP1 inhibitor, and the PARP inhibitors Olaparib or Niraparib. Treatment efficacy was also determined using cell viability, cell cycle, DNA damage, repair, and apoptosis assays in a pair of isogenic PTEN-null and PTEN-WT glioma cell lines derived from a genetically engineered mouse GBM model. RNAseq analysis was performed to identify treatment-induced dysregulated pathways. RESULTS PTEN-deficient cells are highly sensitive to LMP400 and PTEN rescue lessens sensitivity to the treatment. Combining LMP400 with PARP inhibitors, Olaparib or Niraparib, leads to synergistic cytotoxicity. LMP400/Niraparib combination induces G2/M cell cycle arrest, DNA damage, suppression of homologous recombination (HR)-related proteins and activation of caspase 3/7 activity significantly more in PTEN-null cells compared to isogenic PTEN-WT cells. Gene set enrichment analysis revealed suppression of cell cycle and DNA damage repair as well as activation of cell death pathways. Finally, CRISPR-Cas9 KO screening suggests that LMP400 is not likely to be a substrate for ABC transporters, suggesting the brain penetration and supporting the use in brain tumor patients. CONCLUSION Combined inhibition of TOP1 and PARP induces synergistic antiglioma effects selectively in PTEN-null glioblastoma cells, providing a strong scientific premise for a clinical trial of combined treatment with LMP400 and Niraparib in a subset of GBM with PTEN deficiency.

BACKGROUND IDH mutations are common in diffuse gliomas and confer an increased dependency on oxidative mitochondrial function. Zotiraciclib, a multi-kinase inhibitor, not only suppresses CDK9-mediated gene transcription process but also causes mitochondrial dysfunction in preclinical models. We investigated the selective activity of zotiraciclib in IDH-mutant gliomas. METHODS Cytotoxicity of zotiraciclib was determined in patient-derived glioma cells with/without IDH mutations. Immunoblotting and flow cytometry were used to evaluate the cell death pathways. Real-time ATP production was measured by Seahorse assay. RESULTS The EC50 of zotiraciclib in IDH-wt cells was twice as high as in IDH-mutant cells. In low-dose zotiraciclib (15nM)-treated IDH-mutant cells, RNA Pol II phosphorylation and expression of antiapoptotic proteins, including XIAP, MCL-1 and survivin were decreased significantly. Cleaved caspase 3/7, 8, 9 and LC3B were also detected in treated IDH-mutant cells but not in IDH-wt cells, suggesting activation of intrinsic and extrinsic cell apoptosis pathways, and subsequent autophagy of IDH-mutant cells. Low-dose treated IDH-mutant, but not IDH-wt cells, induced decreased expression of a panel of mitochondrial respiration genes, and mitochondrial respiration complexes, cellular ATP depletion and increased ROS production, suggesting a greater disruption of mitochondrial function in zotiraciclib-treated IDH-mutant cells. Zotiraciclib treatment prolonged the survival in mice bearing intracranial IDH-mutant gliomas (30 days in DMSO-treated group vs 33 days in zotiraciclib-treated group, p = 0.01), but not in mice with glioma established with the isogenic IDH-wt glioma cells (18 days in DMSO-treated vs 20 days in zotiraciclib-treated group, p = 0.10). CONCLUSIONS There is selective activity of zotiraciclib in IDH-mutated glioma. Low-dose exposure of zotiraciclib induces cell death and suppresses transcriptional process and mitochondrial biogenesis in IDH-mutant glioma, not in IDH-wt tumors. These findings support a clinical trial testing zotiraciclib in IDH-mutated glioma. The treatment efficacy seen even at low concentrations further supports testing it as a single agent, thereby reducing toxicity concerns.