Inhibition of GPR68 induces ferroptosis in diffuse intrinsic pontine gliomas

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive pediatric brainstem tumor with limited treatment options and dismal prognosis. Previously, we showed that Ogremorphin (OGM), a specific GPR68 inhibitor, induced ferroptosis in glioblastoma multiforme (GBM) cells. Given their shared originating cell type, we investigated OGM's efficacy in inducing ferroptosis in DIPG cells, using normal human astrocytes (NHAs) for comparison.

DIPG cell lines (SF7761, DIPG36, DIPG007) and NHAs were evaluated. OGM sensitivity was assessed via CellTiter-Glo viability assays. Ferroptosis markers including glutathione (GSH) levels, lipid peroxidation (BODIPY C11), and qRT-PCR of ATF4 and SLC7A11 were measured. Rescue experiments used liproxstatin-1 for ferroptosis and Z-VAD-FMK for apoptosis. Genetic validation used shRNA knockdown and overexpression of GPR68/ATF4, plus an ATF4 luciferase reporter assay.

OGM treatment in N5 media revealed that NHAs were more resistant (LD₅₀ = 3.84 µM) than DIPG cells (LD₅₀ ≤ 0.79 µM). DIPG sensitivity to OGM was comparable to GBM. Knockdown of GPR68 via shRNA confirmed that GPR68 inhibition induces cell death in DIPG cells. OGM robustly triggered ferroptosis marked by elevated lipid radicals, reduced GSH, and increased ATF4 and SLC7A11 expression in DIPG and NHA cells, except SF7761, which lacked SLC7A11 induction. Furthermore, liproxstatin-1 rescued the loss of cell viability, whereas Z-VAD-FMK had no effect. ATF4 overexpression caused cell death, while its knockdown rescued OGM-mediated effects.

These findings suggest that GPR68 inhibition is an attractive therapeutic target for DIPG. Notably, GPR68 inhibition mirrors the effects observed in GBM, inducing ATF4-dependent ferroptotic cell death.