Abstract 612: ONC212 affects ClpXP complex, impairs mitochondrial bioenergetics and synergizes with glycolysis inhibition in pancreatic cancer

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human cancers and still represents an unmet clinical need. Survival, proliferation and drug-resistance of pancreatic cancer cells are driven by KRAS mutation, which supports both mitochondrial and glycolytic bioenergetics. ONC212 is a fluorinated imipridone with cytotoxic activity against pancreatic cancer cell lines and patient-derived xenografts. It has been reported to bind to and hyperactivate ClpXP, a mitochondrial protease composed by the regulatory subunit ClpX and the catalytic chamber ClpP, which is involved in the maintenance of respiratory chain function. Here we evaluated the effect of ONC212 on ClpXP expression in a panel of pancreatic cancer cell lines and investigated the metabolic changes occurring in vitro upon ONC212 exposure. Western blot analysis showed that ONC212 destabilized ClpXP complex through the suppression of its regulatory subunit ClpX, while leaving ClpP relatively unaffected. NDUFA12, SDH-A and SDH-B, three ClpP substrates belonging to the respiratory chain complexes I and II, decreased after treatment with ONC212. Moreover, ClpP transient knockdown partially protected cancer cells from ONC212 cytotoxicity. Seahorse XF ATP assay revealed that mitochondrial ATP production was impaired by ONC212, while glycolytic ATP proportionally increased. Accordingly, ONC212 caused a dose-dependent accumulation of lactate in culture supernatant along with the restoration of NAD+, an essential coenzyme fueling glycolytic flux. Interestingly, there was an inverse correlation between basal mitoATP% and ONC212 GI50. Furthermore, AsPC1, one of the most sensitive cell lines, was far less efficient in upregulating glycolysis as compared to the others and suffered a profound decline in ATP production. As a consequence of bioenergy imbalance, AMPK became phosphorylated at Thr172 upon ONC212 treatment in most cell lines. Culturing PDAC cells in galactose-containing medium, favoring mitochondrial metabolism over glycolysis, sensitized them to ONC212. Conversely, hypoxia, which forces cancer cells to generate ATP through glycolysis, rendered them more resistant to the drug. The combination of ONC212 plus 2-Deoxy-d-Glucose, an inhibitor of the first steps of the glycolytic pathway, resulted in highly synergistic loss of cell viability. In addition, the combination treatment, but not ONC212 alone, inhibited ERK1/2 phosphorylation and induced PARP cleavage in PANC1 and BxPC3 cells. Overall, ONC212 targets ClpXP and compromises mitochondrial bioenergetics in pancreatic cancer. Enhancement of glycolytic flux may represent an escape mechanism active in most PDAC cells. Switching metabolic dependence towards mitochondria or combining ONC212 with a glycolytic inhibitor may significantly increase its antineoplastic potential in this highly aggressive malignancy. Citation Format: Isacco Ferrarini, Lanlan Zhou, Wafik S. El-Deiry. ONC212 affects ClpXP complex, impairs mitochondrial bioenergetics and synergizes with glycolysis inhibition in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 612.