Knight Cancer Pilot Project Awardee: Martin Pike, Ph.D.

Martin Pike, Ph.D., Associate Scientist, Advanced Imaging Research Center

Helikson Memorial Award for Brain Cancer Research

Title: "Anti-Glioma Efficacy with Combined PP2A Activation and Autophagy Inhibition"

Despite robust vascularity of malignant gliomas, anti-angiogenic therapy largely fails to induce durable responses. Autophagy, a cytoprotective cellular degradation pathway, can promote drug resistance and its late-stage inhibition can induce tumor cell death. We hypothesized that efficacy with Cediranib, a VEGF/PDGF receptor tyrosine kinase inhibitor, can be enhanced via combination with the late-stage autophagy inhibitor Quinacrine. To investigate this, we exposed 4C8 glioma cells to Cediranib, Quinacrine or the combination, under normoxic and hypoxic (0.5% O2) conditions. Cell viability assays revealed dose-dependent cytotoxic effects of each agent under both conditions but a greater than additive combined efficacy under hypoxic conditions. Western blotting for autophagic vacuole (AV) marker LC3-II, indicated increases with Quinacrine as well as Cediranib, suggesting that Cediranib stimulates autophagic flux. Maximal LC3-II accumulation and Caspase 3 cleavage were observed with Cediranib+Quinacrine/Hypoxia. This effect was abrogated with 3-Methyladenine, an early-stage autophagy inhibitor which prevents AV formation. Employing a dual-bolus perfusion MRI approach, we assessed relative cerebral blood flow and volume (rCBF, rCBV), and vascular permeability (Ktrans) in intracranial 4C8 mouse glioma. Cediranib or quinacrine monotherapy did not alter tumor growth, while combined Cediranib/quinacrine reduced it by over 2-fold (p<0.05). Cediranib or quinacrine monotherapy did not significantly alter mean tumor rCBF or Ktrans compared to untreated while combined Cediranib/quinacrine substantially reduced both (p<0.05), indicating potent tumor devascularization. Tumor necrosis and mean vessel density (MVD), assessed  immunohistologically, were unchanged by Cediranib or quinacrine monotherapy. In contrast, MVD was reduced by nearly 2-fold (p<0.01), and necrosis increased by 3-fold (p<0.05 one-tailed) in Cediranib/quinacrine treated versus untreated. In conclusion, hypoxia-potentiated autophagic flux stimulation by Cediranib, combined with late stage autophagic inhibition by Quinacrine, induces AV accumulation and glioma cell death. Combined Cediranib/quinacrine treatment synergistically increased anti-vascular/anti-tumor efficacy in intracranial 4C8 mouse glioma, suggesting a promising and facile treatment strategy for malignant glioma.

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