Abstract P084: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models

Abstract

Abstract Background: Activating mutations in EGFR have been reported in ~30% of patients with non-small cell lung cancer (NSCLC). Three generations of small molecule EGFR kinase inhibitors have been approved by the FDA to treat these patients, however, multiple mechanisms of resistance cause cancer progression. In addition to drug-resistant mutations that arise and re-activate EGFR, other signaling pathways can be activated to cause resistance. Although EGFR inhibitors such as osimertinib and afatinib (also a pan-ErbB inhibitor) have demonstrated clear clinical benefits, patients inevitably develop resistance. Herein we demonstrate mutant EGFR NSCLCs activate autophagy upon treatment with EGFR inhibitors as a drug resistance mechanism. Therefore, a combination of an EGFR inhibitor with an autophagy inhibitor has the potential to deepen and prolong responses and improve patient outcomes. Materials and Methods: Human cancer cells with EGFR mutations were cultured using recommended complete medium. Inhibition of ULK1/2 was measured through standard biochemical assays and cellular readouts including NanoBRET and ELISA-based ATG13 phosphorylation assays. Autophagosome formation was measured using the CytoID assay (Enzo Life Sciences). For in vivo studies, NCI-H1975 cells that harbor an EGFR T790M resistance mutation were inoculated into BALB/c nude mice. Statistical analyses for the in vivo studies were performed using the Student’s t-test. Results: Erlotinib, gefitinib, osimertinib, and afatinib activated autophagy 3–4-fold over basal levels in the HCC827 cell line (EGFR exon 19 deletion) as measured by increases in phosphorylated ATG13, a cellular substrate of the autophagy-initiating kinases ULK1/2. DCC-3116, an investigational potent and selective dual inhibitor of ULK1 (IC50 6 nM) and ULK2 (9 nM) in cellular assays, inhibited both EGFR-induced and basal phosphorylation of ATG13 with IC50 values of 61–66 nM. Treatment of the NCI-H1975 EGFR mutated (L858R/T790M) NSCLC cell line with osimertinib or afatinib induced autophagy 3-fold over basal levels. DCC-3116 potently inhibited osimertinib and afatinib induced phosphorylation of ATG13 with IC50 values of 91 nM and 71 nM, respectively, and inhibited the increase in autophagosomes induced by these agents. Importantly, these in vitro effects translated to in vivo efficacy. The combination of DCC-3116 with osimertinib or afatinib resulted in significantly greater tumor responses than single agent treatments in the NCI-H1975 NSCLC xenograft model (combination vs. osimertinib p = 0.0005; combination vs. afatinib p = 0.0001; osimertinib combination vs vehicle p < 0.0001; afatinib combination vs. vehicle p < 0.0001). These data provide a strong rationale to study the combination of the ULK inhibitor DCC-3116 with EGFR inhibitors such as osimertinib and afatinib in cancer patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with a documented RAS or RAF mutation (NCT04892017). Citation Format: Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Yu Zhan, Bryan D. Smith, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P084.