GPR68-ATF4 Signaling in Glioblastoma Multiforme (GBM)
Executive Summary
This research investigates the role of GPR68, an acid-sensing G protein-coupled receptor (GPCR), in glioblastoma multiforme (GBM), an aggressive and treatment-resistant brain cancer. The study identifies a novel small molecule inhibitor of GPR68, named Ogremorphin (OGM), and demonstrates that GPR68 promotes GBM cell survival by suppressing the transcription factor ATF4. Inhibition of GPR68 with OGM or through genetic means leads to increased ATF4 expression, which subsequently induces ferroptosis, an iron-mediated form of cell death, in GBM cells. This suggests GPR68 is a potential therapeutic target for GBM, especially in combination with existing treatments like temozolomide (TMZ) and radiation
Background and Problem
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GBM Challenge: GBM remains a significant challenge in oncology due to its aggressive nature, limited treatment options, and poor survival rates. "Glioblastoma multiforme (GBM) stands as the most prevalent and lethal primary brain tumor among adults, with a grim median survival of only 14 months despite aggressive standard management strategies."
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Acidic Tumor Microenvironment (TME): A hallmark of GBM is its acidic TME, resulting from the Warburg effect (altered tumor metabolism favoring aerobic glycolysis). This acidification is thought to activate pro-tumorigenic pathways. "Despite variations in cell states and genetic makeup, a distinctive feature of glioblastoma is its acidic extracellular tumor microenvironment (TME), a result of the Warburg effect..."
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GPR68 and Cancer: GPR68 is an acid-sensing GPCR upregulated in radioresistant GBM. It is activated by subtle extracellular acidification (active at pH 6.4, inactive at pH 7.4). Use of Lorazepam, which has off-target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. "GPR68, also known as ovarian cancer G-coupled protein receptor 1 (OGR-1), is a member of the proton sensing GPCR family which is activated in response to subtle extracellular acidification (inactive at pH 7.4 and fully active at pH 6.4)...Alarmingly, the use of anxiolytic Lorazepam...has recently been associated with a 3.8-fold higher rate of pancreatic cancer progression and related death..."
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Unclear Mechanism: The exact mechanism by which cancer cells sense and respond to extracellular pH changes is not fully understood.
Key Findings and Ideas:
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OGM Discovery: The researchers identified Ogremorphin (OGM) through a zebrafish embryonic development screen as a novel, highly specific inhibitor of GPR68. "Here, we describe the discovery of a novel class of small molecule GPR68/OGR-1 inhibitors named ogremorphins..."
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GPR68 Activation by Acidification: GBM cells acidify their microenvironment, and this acidification activates GPR68. Using a pHluorin2 probe, they demonstrated how glioblastoma cells acidify their microenvironment to activate GPR68. "We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68."
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GPR68 Inhibition Induces Cell Death: Blocking GPR68 signaling with OGM or genetic means (siRNA, CRISPRi) results in robust cell death in GBM cell lines, irrespective of genetic/phenotypic heterogeneity or resistance to TMZ. "Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested..."
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Ferroptosis Mechanism: The cell death induced by GPR68 inhibition occurs through ferroptosis, an iron-mediated cell death pathway characterized by lipid peroxidation. RNA-seq analysis and subsequent experiments (Western blotting, qRT-PCR, electron microscopy, Liperfluo staining) confirmed the induction of ferroptosis markers and characteristics. "Moreover, we show that genetic and pharmacological disruption of GPR68 signaling in glioblastoma cells induces ferroptosis, an iron-mediated cell death program, across a diverse set of GBM lines."
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ATF4-Dependent Pathway: GPR68 suppresses the transcription factor ATF4. Inhibition of GPR68 increases ATF4 expression, which then leads to the induction of CHAC1, glutathione depletion, and ultimately, ferroptosis. "In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death."
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Selectivity: OGM was not acutely toxic to zebrafish and its inhibitory effects spared non-malignant neural cells, suggesting it acts specifically on Glioblastoma cells with no overt toxic effects on normal or neuronal tissues, across species and subtypes. This highlights its potential therapeutic selectivity. "Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells."
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Synergistic Effects: OGM showed synergistic killing of GBM cells when combined with TMZ and radiation, suggesting it could improve the efficacy of existing treatments. "Interestingly, OGM and TMZ demonstrated strong synergistic killing of PDX 08-387 cells with a coefficient of drug interaction (CDI) < 0.7..." and "OGM and ionizing radiation demonstrated exceptionally strong synergistic induction of lipid peroxidation..."
Implications
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Therapeutic Potential: GPR68 is a promising therapeutic target for GBM.
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OGM as a Lead Compound: Ogremorphins represent a novel class of compounds with potential for GBM treatment.
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Ferroptosis Induction: Inducing ferroptosis is a viable strategy for targeting GBM, especially given the resistance to apoptosis in many GBM cases.
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Combination Therapy: OGM may be most effective in combination with existing GBM therapies like TMZ and radiation.