LAUSR

Background: Cancer cells activate autophagy, a catabolic process to resupply nutrients and recycle damaged organelles, in order to survive stresses such as limited nutrients and hypoxia, or chemotherapy treatments. RAS mutant cancers, in particular, have been found to require autophagy for tumor growth and survival.1,2 Treating RAS mutant tumors with inhibitors of the downstream MAPK pathway has been largely unsuccessful, as these drugs have been shown to further stimulate autophagy, allowing for tumor cell survival.3,4 Inhibiting autophagy in combination with MAPK pathway inhibition may represent a possible new treatment paradigm for RAS mutant cancers. Proof-of-concept for this strategy was obtained in cancer models and in a RAS mutant pancreatic cancer patient by blocking autophagy with derivatives of chloroquine, in combination with MAPK inhibitors.3,4 Chloroquines indirectly block autophagy via disruption of lysosomal function, which may also affect important normal cellular processes. Chloroquines accumulate in tissues, notably the brain, where autophagy may be vital for neuronal health. The potential exists to more selectively inhibit autophagy by targeting specific components of the autophagy pathway. ULK1/2 kinases initiate autophagy and provide the potential for a targeted approach for selectively inhibiting autophagy in RAS mutant cancers. Herein, we describe preclinical studies with the ULK inhibitor DCC-3116, designed as a potential inhibitor of autophagy in RAS mutant cancers. Methods: In vitro kinase assays were performed using cellular levels of ATP (1 mM) and a peptide substrate. In cell assays, ULK activity was assessed using an ELISA for phosphorylated ATG13. Autophagosome formation was measured using the dye, Cyto-ID. Autophagic flux was assessed using cells expressing the autophagy protein LC3 fused to luciferase. The synergy of DCC-3116 in combination with MAPK inhibitors was assessed in 2D or 3D cell growth assays. Xenograft models were used to assess pharmacokinetics (PK) and pharmacodynamics (PD), as well as efficacy in vivo. Results: DCC-3116 is a potent and selective inhibitor of ULK1/2, inhibiting no other kinases within 30-fold of ULK potency, and only 5 kinases within 100-fold. DCC-3116 inhibited phosphorylation of the ULK substrate ATG13 in cancer cell assays. DCC-3116 inhibited autophagosome formation, as well as degradation of the autophagy marker LC3. DCC-3116 exhibited synergy in vitro in combination with MAPK pathway inhibitors in inhibiting cancer cell growth. In PK/PD models, oral doses of DCC-3116 led to sustained inhibition of ATG13 phosphorylation. DCC-3116, in combination with MAPK inhibitors, exhibited additivity or synergy in inhibiting tumor growth in xenograft models. DCC-3116 exhibited low brain penetration in rats, minimizing inhibition of CNS autophagy. Conclusion: Selectively blocking autophagy via inhibition of ULK kinases, in combination with MAPK pathway inhibition, is a promising therapeutic approach for RAS mutant cancers. DCC-3116 warrants further study as an inhibitor of autophagy, and has been selected as a candidate for potential development in the treatment of RAS mutant cancers. Guo et al., Genes and Dev. 2011; 25: 460 Yang et al., Genes and Dev. 2011; 25: 717 Bryant et al., Nature Med. 2019; 25: 628 Kinsey et al., Nature Med. 2019; 25: 620 Citation Format: Bryan D Smith, Lakshminarayana Vogeti, Anu Gupta, Jarnail Singh, Gada Al-Ani, Stacie L Bulfer, Timothy M Caldwell, Mary J Timson, Subha Vogeti, Yu Mi Ahn, Hikmat Al-Hashimi, Chase K Crawley, Cale L Heiniger, Cynthia B Leary, Justin T Proto, Quanrong Shen, Hanumaiah Telikepalli, Karen Yates, Wei-Ping Lu, Daniel L Flynn. Preclinical studies with DCC-3116, an ULK kinase inhibitor designed to inhibit autophagy as a potential strategy to address mutant RAS cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B129. doi:10.1158/1535-7163.TARG-19-B129