Abstract 443: Therapeutic targeting of GPR68 activated by acidic extracellular microenvironment induces ferroptosis in glioblastoma cells

Abstract

Abstract The Warburg Effect is a common feature of cancer cells characterized by increased glucose uptake and fermentation of glucose to lactate even in the presence of oxygen. While it is commonly accepted that Warburg Effect promotes the growth, survival, proliferation, and long-term maintenance of cancers, its precise function and its downstream mediators remain unclear. A key physiological consequence of the Warburg effect is lactate secretion, which acidifies the tumor milieu, thought to promote oncogenesis and confer tumor resistance to chemotherapy and radiotherapy. Glioblastoma multiforme (GBM) is one of the most aggressive and deadly cancers, characterized by cellular heterogeneity and plasticity, which are thought to drive extreme therapeutic resistance. Despite their heterogeneity, common hallmarks of GBM tumors are high levels of aerobic glycolysis (“Warburg Effect”) and a resultant acidic tumor microenvironment (TME), which promotes tumor progression. In an in vivo zebrafish developmental screen, we identified ogremorphin (OGM), a small molecule inhibitor of GPR68/OGR-1, a G-protein coupled receptor (GPCR) which is activated by extracellular protons. Using ogremorphin and pHluorin2-GPI, a novel sensor of extracellular acidification, we demonstrate that glioblastoma cells acidify their own environment in vitro and activate GPR68, and visualize, for the first time, the establishment of the acidic extracellular microenvironment during the formation of GBM spheroids in vitro. Selective inhibition of GPR68 causes robust cell death in all 12 glioblastoma cell lines tested to date, despite genetic and molecular heterogeneity, without toxicity on healthy cells in whole animals. Mechanistically, GPR68 inhibition activates ferroptosis, a programmed cell death characterized by lipid peroxidation, in an ATF4 (activating transcription factor 4)-dependent manner. Finally, in GBM cells, ogremorphin treatment demonstrates strong synergistic effects with the frontline therapeutics temozolomide and ionizing radiation. Our results indicate that GPR68 activation by extracellular acidification is a key cancer survival pathway downstream of the Warburg Effect, and that GPR68 inhibition, either alone or in combination with temozolomide and radiation therapy, is a promising therapeutic approach to selectively induce ferroptosis in GBM tumors. Citation Format: Charles H. Williams, Leif R. Neitzel, Jessica Cornell, Samantha Rea, Ian Mills, Maya Silver-Isenstadt, Jovanni D. Ahmad, Henry Brem, Betty Tyler, Eli E. Bar, Charles C. Hong. Therapeutic targeting of GPR68 activated by acidic extracellular microenvironment induces ferroptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 443.