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Protein kinase A regulates ferroptosis by controlling GPX4 m6A modification through phosphorylation of ALKBH5

ABSTRACT: GPX4-dependent ferroptosis has emerged as a therapeutic strategy for cancer treatment. Here, we demonstrated that protein kinase A (PKA) participates in the regulation of ferroptosis by controlling the m6A modification of GPX4 in an ALKBH5-dependent manner. Notably, we identified ALKBH5, an m6A demethylase, as a novel target of PKA, which drives phosphorylation-dependent degradation of ALKBH5 protein. Moreover, the deletion of ALKBH5 represses ferroptotic cell death by maintaining GPX4 m6A modification and stability. Thus, by regulating ALKBH5-dependent GPX4 stability, PKA acts as a key regulator of ferroptosis. Our study unveils the involvement of PKA in m6A modification, which could control GPX4-dependent ferroptosis and tumor progression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE283869 | GEO | 2025/12/09

REPOSITORIES: GEO

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Project description:Numerous neurological diseases involve neuroinflammation, a process in which immune cells, particularly microglia, contribute to neuronal death. Ferroptosis, a recently identified form of regulated cell death, is implicated in various diseases characterized by neuronal injury. Nicotinamide mononucleotide (NMN), a potent NAD+ precursor supplement, has been found to inhibit neuroinflammation and ferroptosis. However, the mechanisms of NMN in both ferroptosis and neuroinflammation remains unclear. The present study aimed to investigate the impact of NMN on neuroinflammation and the susceptibility of microglia to ferroptosis. Ferroptosis markers in microglia exposed to lipopolysaccharide (LPS) were analyzed using CCK8, flow cytometry, ELISA, and RT-qPCR. The effects of NMN on LPS-induced ferroptosis in microglia were evaluated through flow cytometry, western blotting, and immunofluorescence staining. RT-qPCR analysis assessed the inflammatory cytokine production of microglia subjected to ferrostatin-1-regulated ferroptosis. RNA sequencing elucidated the underlying mechanisms of NMN-associated microglia ferroptosis under LPS induction. In BV2 microglia, an inhibitor of Glutathione Peroxidase 4(GPX4), RSL3, was employed to suppress GPX4 expression. Intracerebroventricular injection of LPS was performed to evaluate neuroinflammation and microglia activation in vivo. LPS treatment resulted in decreased cell viability, accompanied by upregulation of ferroptosis markers SLC7A11 and GPX4, and elevated levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and total iron in a dose-dependent manner. NMN effectively rescued LPS-induced ferroptosis and improved cell viability in microglia. Co-administration of NMN and ferrostatin-1 significantly reduced proinflammatory cytokine production in microglia following the introduction of LPS stimuli. Mechanistically, NMN facilitated glutathione (GSH) production, and promoted resistance to lipid peroxidation in a GPX4-dependent manner, repressing cytokine transcription and protecting cells from ferroptosis. RNA sequencing elucidated the underlying mechanism of NMN-associated microglia ferroptosis under LPS induction. Furthermore, simultaneous injection of NMN ameliorated LPS-induced ferroptosis and neuroinflammation in mouse brains. The data from the present study indicated that NMN enhances GPX4-mediated ferroptosis defense against LPS-induced ferroptosis in microglia by recruiting GSH, thereby inhibiting neuroinflammation. Therefore, therapeutic approaches targeting ferroptosis in diseases using NMN should consider both its anti-ferroptosis and anti-inflammatory effects to achieve optimal outcomes, presenting promising strategies for treating neuroinflammation-related diseases or disorders.

Project description:Background In the ongoing battle against BCR-ABL+ leukemia, despite significant advances with tyrosine kinase inhibitors (TKIs), the persistent challenges of drug resistance and the enduring presence of leukemic stem cells (LSCs) remain formidable barriers to achieving a cure. Methods In this study, we demonstrated that Disulfiram (DSF) induces ferroptosis to synergize with TKIs in inhibiting BCR-ABL+ cells, particularly targeting resistant cells and LSCs, using cell models, mouse models, and primary cells from patients. We elucidated the mechanism by which DSF promotes GPX4 degradation to induce ferroptosis through immunofluorescence, co-immunoprecipitation (CO-IP), RNA sequencing, lipid peroxidation assays, and rescue experiments. Results Here, we present compelling evidence elucidating the sensitivity of DSF, an FDA-approved drug for alcohol dependence, towards BCR-ABL+ cells. Our findings underscore DSF's ability to selectively induce a potent cytotoxic effect on BCR-ABL+ cell lines and effectively inhibit primary BCR-ABL+ leukemia cells. Crucially, the combined treatment of DSF with TKIs selectively eradicates TKI-insensitive stem cells and resistant cells. Of particular note is DSF's capacity to disrupt GPX4 stability, elevate the labile iron pool, and intensify lipid peroxidation, ultimately leading to ferroptotic cell death. Our investigation shows that BCR-ABL expression induces alterations in cellular iron metabolism and increases GPX4 expression. Additionally, we demonstrate the indispensability of GPX4 for LSC development and the initiation/maintenance of BCR-ABL+ leukemia. Mechanical analysis further elucidates DSF's ability to circumvent resistance by reducing GPX4 levels through the disruption of its binding with HSPA8, thereby promoting GPX4 ubiquitination and subsequent degradation. Furthermore, the combined treatment of DSF with TKIs effectively targets both BCR-ABL+ blast cells and drug-insensitive LSCs, conferring a significant survival advantage in mouse models. Conclusion In summary, the dual inhibition of GPX4 and BCR-ABL presents a promising therapeutic strategy to synergistically target blast cells and drug-insensitive LSCs in patients, offering potential avenues for advancing leukemia treatment.

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Protein kinase A regulates ferroptosis by controlling GPX4 m6A modification through phosphorylation of ALKBH5

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