Abstract LB-C08: Imipridone ONC212 induces apoptosis, suppresses autophagy and synergizes upon GRP132 knockdown or exposure to lactic acid in preclinical studies of the pancreatic cancer microenvironment

Abstract

Abstract Pancreatic cancer is almost invariably fatal, often diagnosed in advanced stage and carrying a five-year survival rate of less than 5%. Cell-intrinsic oncogenic signaling in a supportive microenvironment renders neoplastic cells resistant to standard chemotherapy or immunotherapy, raising the urgent need for novel agents. ONC212, a fluorinated imipridone, is cytotoxic at nM doses against a variety of cancer preclinical models. ONC212 engages the seven-passage transmembrane receptor GPR132, which is considered a pH sensor and one of the lactic acid receptors. We previously demonstrated that ONC212 inhibits growth of several pancreatic cancer xenografts, some of which underwent apoptosis. We hypothesized that GPR132 modulates ONC212 anti-tumor response in the acidic tumor microenvironment in pancreatic cancer. We studied the growth inhibitory effects of ONC212 in the presence of lactic acid, an essential metabolite within the pancreatic tumor microenvironment resulting from the Warburg effect. We investigated a panel of human pancreatic cancer cell lines (AsPC1, HPAFII, BxPC3, CAPAN2 and PANC1) and two normal fibroblast cell lines (MRC-5 and WI-38) in vitro by CellTiter-Glo assays to assess cell viability and western blots to assess intracellular signaling upon ONC212 treatment. ONC212 inhibited cancer cell growth with a GI50 ranging from 0.09 to 0.47 μM, whereas fibroblasts were resistant. PKC became transiently phosphorylated, as would be expected in response to GPR132 engagement, by 15-30 minutes after ONC212 exposure of AsPC1 cells. Pro-growth and survival Akt and ERK phosphorylation, constitutively detected in most pancreatic cancer cells, was suppressed by 1 hour after ONC212 treatment and this was also observed at 48 hours. After 48 hours, we observed PARP cleavage in the most sensitive pancreatic cancer cell lines such as AsPC1 and HPAFII, whereas the anti-apoptotic protein Mcl-1 decreased in all the cell lines treated with ONC212. Interestingly, LC3II and Beclin 1, two well-recognized autophagic markers, were suppressed by ONC212 in cell lines undergoing apoptosis but not in the others, pointing to autophagy as a possible escape mechanism from ONC212. GPR132 was constitutively expressed in pancreatic cancer cells and decreased upon 48-hour ONC212 treatment. Importantly, GPR132 knockdown reduced cell survival while synergizing with ONC212 in pancreatic cancer cells, suggesting its cytotoxic effect may be mediated by engagement and subsequent abrogation of pro-survival GPR132 signaling. The combination of ONC212 and lactic acid, sharing GPR132 as surface receptor, resulted in strong synergism in inhibiting cancer cell growth. This was evident in fibroblast cell lines too, hinting that stromal tumor-supportive cells residing in cancer acidic microenvironment may be inhibited by treatment. ONC212 shows pre-clinical activity against pancreatic cancer through apoptosis induction and autophagy blockade. Suppression of GPR132 may contribute to ONC212 cytotoxicity. ONC212 is a novel therapeutic active against the acidic tumor microenvironment of pancreatic cancer through suppression of pH sensor and pro-survival GPR132. Citation Format: Isacco Ferrarini, Aakash Jhaveri, Young Lee, Howard Safran, Lanlan Zhou, Wafik S El-Deiry. Imipridone ONC212 induces apoptosis, suppresses autophagy and synergizes upon GRP132 knockdown or exposure to lactic acid in preclinical studies of the pancreatic cancer microenvironment [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 LB-C08. doi:10.1158/1535-7163.TARG-19-LB-C08