Abstract 1006: Combination therapy with MEK inhibitors and a novel anti-neoplastic drug, imipridone ONC212, demonstrates synergy in pancreatic ductal adenocarcinoma cell lines
Authors: Raufi Alexander G. , Parker Casssandra , Zhou Lanlan , Prabhu Varun V. , Allen Josh , El-Deiry Wafik S. 
Published: July 2021

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. Although KRAS is mutated or amplified in nearly 95% of PDAC, no effective KRAS directed therapy has entered clinical practice. There is emerging data suggesting that inhibition of downstream targets of KRAS, such as MEK or ERK, increases autophagic flux and dependence in KRAS-mutated tumors. Autophagy, a catabolic process used by many tumors to maintain viability, can be targeted with various agents such has hydroxychloroquine and chloroquine, and recent preclinical data has shown that the combination of these agents with inhibitors of MEK/ERK can synergistically induce cell death. Using viability assays, we established sensitivity of multiple KRAS-mutated and KRAS wild-type pancreatic cancer cell lines, BxPC3, PANC1, HPAF-II, AsPC-1 to ONC212, trametinib, chloroquine and combinations of these agents. Results were analyzed after 72 hours of incubation using Compusyn and Combenefit. We also performed Western Blotting on cell lysates after treatment for 48 hours with these agents. We found that trametinib exhibited strong synergy with both chloroquine and ONC212 in all cell lines. The synergistic potential appeared to be most profound in those cell lines treated with ONC212 and trametinib. We used MRC5, a normal lung fibroblast cell line, as a control and found that these combinations did not significantly induce cell death. Using Western Blotting, we assessed markers of autophagy, including Beclin-1 and LC3B, as well as key second messenger pathway activation/suppression with p-AKT/AKT and p-ERK/ERK. At increasing doses, trametinib increased conversion of LC3-I to LC3-II, more notably in KRAS mutant cell lines, suggesting increased autophagic flux. ONC212, however, reduced conversion of LC3-I to LC3-II and increased degradation of Beclin-1 suggesting inhibition of autophagy. The combination, did not appear to have as striking of an effect on this pathway. In addition, p-ERK, as expected, was downregulated with MEK inhibition. ONC212 also appeared to reduce p-ERK levels and, interestingly, increased p-AKT. This suggests possible PI3K pathway upregulation, however, this could be influenced by timing and dose of treatments. Western blot analysis also revealed increased cPARP/PARP with combination therapy across cell lines. In conclusion, the combination of trametinib and ONC212 produces a synergistic cytotoxic effect on tumor cells at doses that are non-toxic to normal cells. In KRAS-mutated pancreatic cancer cell lines, we hypothesize that this may, in part, be due to autophagy suppression by ON212. Similar synergistic effects were noted in a KRAS wild-type pancreatic cancer cell lines, suggesting an autophagy-independent mechanism. Thus, this combination may represent a potential therapeutic modality for PDAC.
Citation Format: Alexander G. Raufi, Casssandra Parker, Lanlan Zhou, Varun V. Prabhu, Josh Allen, Wafik S. El-Deiry. Combination therapy with MEK inhibitors and a novel anti-neoplastic drug, imipridone ONC212, demonstrates synergy in pancreatic ductal adenocarcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1006.