Abstract 1679: Combined efficacy of Cediranib and Quinacrine in glioma is enhanced by hypoxia and is associated with autophagic vacuole accumulation.
Authors: Lobo Merryl , Kurre Peter , Schabel Matthias , Gillespie Yancey , Woltjer Randall , Pike Martin 
Published: August 2014

Despite robust vascularity of malignant gliomas, anti-angiogenic therapy largely fails to induce durable responses. We have previously reported that efficacy with Cediranib (Ced), a VEGF/PDGF receptor tyrosine kinase inhibitor, is synergistically enhanced via combination with late-stage autophagy inhibitor quinacrine (Quin), in intracranial 4C8 mouse glioma, resulting in decreased tumor vascularization and growth, increased tumor necrosis and improved mouse survival (#1905, AACR 2012). Our present study was aimed at investigating the role of autophagy (a cellular catabolic pathway that promotes tumor cell survival under hypoxic/nutrient stress), in this synergistic efficacy. MTS assays revealed dose-dependent reductions in cell viability for Ced and Quin under normal (Nrm) and hypoxic (0.5%O2, Hyp) conditions: IC50s(μM) under Nrm and Hyp were 2.7±0.1 and 2.4±0.2 for Ced, and 3.2±0.2 and 2.4±0.04 for Quin, respectively. Greater than additive combined efficacy for Ced+Quin occurred only under Hyp (cell viability reductions for 1μM C + 2.5μM Q: 78±7%(Hyp) vs. 31±3%(Nrm), p&lt;0.05). Western blotting for cleaved caspase 3 also indicated a marked increase in apoptosis with Ced+Quin/Hyp versus all other groups. Western blotting for autophagic vacuole (AV) associated LC3-II protein, which increases with increased autophagic flux and/or late stage autophagic inhibition, not only indicated increases with Quin, but also with Ced, suggesting that Ced may increase autophagic flux. Hypoxia potentiated LC3-II increases in the presence of either Quin or Ced, with the largest increases occurring with Ced+Quin/Hyp, suggesting that hypoxia-induced autophagic flux stimulation combined with late stage autophagic inhibition can trigger AV accumulation and cell death. Bafilomycin A1 (Baf), another late-stage autophagy inhibitor, also decreased cell viability. However, the efficacy of combined Baf and Ced was only additive and lacked substantial potentiation by Hyp (cell viability reductions for 1μM Ced/5ηM Baf: 54±8% (Hyp) vs 37±11% (Nrm)). LC3-II accumulation with Baf was substantially lower than with Quin, either with Hyp or Nrm, suggesting that in addition to inhibiting late-stage autophagy, Quin may also stimulate autophagic flux, consistent with its reported inhibition of PI3K/Akt/mTOR signaling. Thus, a key role for AV accumulation is consistent with its increase with Quin versus Baf in tandem with the improved efficacy of Quin over Baf, in combination with Ced. Our results suggest that the unique cytotoxic efficacy of Ced+Quin we previously reported in vivo, could be associated with increased AV accumulation within hypoxic tumor cells, induced by Ced and possibly Quin, in combination with late-stage autophagic inhibition by Quin. These findings provide a rationale for a careful evaluation of a Ced+Quin combination therapy in patients with malignant gliomas.

Citation Format: Merryl Lobo, Peter Kurre, Matthias Schabel, Yancey Gillespie, Randall Woltjer, Martin Pike. Combined efficacy of Cediranib and Quinacrine in glioma is enhanced by hypoxia and is associated with autophagic vacuole accumulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1679. doi:10.1158/1538-7445.AM2013-1679