Paper Report: The Role of MDH2 in Epitranscriptomic Metabolic Regulation of Glioblastoma Stem Cells

Introduction and Background

Glioblastoma (GBM) is the most common and lethal primary brain malignant tumor in adults. GBM cells utilize a metabolic pathway known as the Warburg effect, which involves increased glucose uptake through aerobic glycolysis to produce lactate for sustaining growth. This process involves reprogramming of the tricarboxylic acid (TCA) cycle to generate tumor metabolites that promote tumor formation and maintenance. Additionally, glioblastoma stem cells (GSCs) are a unique subpopulation at the apex of the tumor cell hierarchy, characterized by self-renewal, persistent proliferation, and recurrence capabilities. Previous studies have shown that GSCs exhibit metabolic characteristics distinct from differentiated GBM cells (DGCs), and their metabolic regulation plays a crucial role in promoting GSC maintenance and aggressiveness. However, the relationship between metabolism and RNA epitranscriptomic regulation remains to be further explored.

To address this, scientists from various research institutions (Lv Deguan, Deobrat Dixit, Andrea F. Cruz, etc.) published a study on November 5, 2024, in “Cell Metabolism,” exploring the role of the key enzyme Malate Dehydrogenase 2 (MDH2) in the TCA cycle, analyzing how metabolic regulation of GSCs affects their epitranscriptome, thereby regulating tumor stemness and treatment resistance.

Research Objectives and Methods

This study aims to elucidate the regulatory role of MDH2 on GSC metabolism and epitranscriptome and explore how targeting MDH2 can weaken the proliferation ability and stemness of GSCs, thus providing potential therapeutic targets. Research methods include: 1. Metabolic Profiling: Using liquid chromatography-mass spectrometry (LC-MS) for metabolic analysis of GSCs and DGCs. 2. Gene Expression and Epigenetic Analysis: Integrating metabolic data with gene expression data to detect genes related to GSC metabolic states, including the expression status of genes from the Malate-Aspartate Shuttle (MAS) pathway. 3. In Vitro and In Vivo Experiments: Using short hairpin RNA (shRNA) to knock down MDH2 expression to observe its effects on GSC proliferation and stemness; simultaneously applying small molecule inhibitors and combination drug therapies to analyze the therapeutic effects of MDH2 inhibition on GBM cells.

Research Process and Results

1. GSC Metabolic Reprogramming and Preference for the MAS Pathway

Metabolic profiling shows that MAS activity in GSCs is significantly higher than in DGCs. MAS transfers electrons generated by glycolysis from the cytoplasm to the mitochondrial inner membrane to sustain energy production. Experiments show an increase in intermediate metabolites such as malate and fumarate in GSCs, further proving enhanced glucose utilization and TCA cycle metabolic activity in GSCs. Moreover, MAS-related genes such as MDH2, Glast, and OGC are upregulated in GSCs, indicating a pivotal role of the MAS pathway in GSCs.

2. Regulation of GSC Proliferation and Stemness by MDH2

Inhibition of MDH2 expression through shRNA significantly reduces GSC proliferation and sphere formation capability, and MDH2 inhibition suppresses GSC cell viability causing apoptosis. In contrast, DGCs and neural stem cells (NSCs) are less sensitive to MDH2 inhibition, indicating that MDH2 is a specific target for maintaining GSC growth and stemness. Additionally, MDH2 inhibition leads to the accumulation of multiple upstream TCA cycle metabolites (such as malate, fumarate, succinate), whereas downstream metabolites (such as citrate and aspartate) decrease, further showing the key role of MDH2 in GSC metabolic reprogramming.

3. Influence of MDH2 on m6A RNA Methylation

To reveal the role of MDH2 in the epitranscriptome, the study further analyzed the effect of MDH2 on m6A RNA methylation. m6A methylation is a widespread mRNA modification primarily regulated by a “writer-eraser-reader” mechanism. The study found that MDH2 knockdown increased key metabolite α-ketoglutarate (AKG) in GSCs, which is a co-factor for m6A demethylases such as ALKBH5. With rising AKG levels, the demethylase activity of ALKBH5 is enhanced, resulting in decreased m6A methylation levels in GSCs. This transcriptomic regulation further influences GSC proliferation and stemness.

4. Epitranscriptomic Regulation of PDGFRB Gene and Therapeutic Target Research

m6A methylation sequencing results showed that MDH2 knockdown significantly reduced m6A methylation levels of PDGFRB mRNA in GSCs, further affecting PDGFRB stability and protein expression. PDGFRB is essential for GSC growth and stemness maintenance, and the reduction of its methylation level leads to decreased GSC proliferation ability. Furthermore, overexpression of PDGFRB can partially reverse cell death caused by MDH2 inhibition, indicating that MDH2-regulated m6A methylation plays a crucial role in PDGFRB stability and function.

5. Development of MDH2 Inhibitors and Combination Drug Therapy

Researchers also explored the effects of combining MDH2 inhibitors with a known multi-kinase inhibitor, Dasatinib. Dasatinib is an oral multi-target kinase inhibitor considered to have therapeutic potential in GSCs. However, experiments indicate that MDH2 inhibition can enhance Dasatinib’s anti-cancer effects. In xenograft mouse models, combination therapy groups exhibited significantly slowed tumor growth and extended overall survival.

Research Conclusions

This study, through systematic metabolic and epitranscriptomic analyses, revealed the critical role of MDH2 in GSC maintenance. MDH2 not only plays a key role in metabolic reprogramming but also affects GSC stemness and proliferation by regulating m6A RNA methylation. Targeting MDH2 could serve as a potential GBM therapeutic strategy, particularly when used in combination with multi-kinase inhibitors, showing significant synergistic therapeutic effects. This provides a new direction for developing novel targeted therapies for GBM.

Research Highlights and Significance

1. Integration of Metabolic Reprogramming and Epitranscriptome Analysis: This study first systematically reveals how MDH2 regulates GSC metabolism and the epitranscriptome through the MAS pathway, thereby maintaining its stemness.
2. MDH2 as a Therapeutic Target: MDH2 has specific effects on GSCs, and its knockdown significantly inhibits GSC growth, providing a new paradigm for targeted GBM therapy.
3. New Mechanism of m6A RNA Methylation Regulation: The study discovered that changes in metabolite AKG influence m6A methylation in GSCs by affecting the demethylation activity of ALKBH5, revealing a novel link between metabolism and epitranscriptomic regulation.
4. Potential of Combination Drug Therapy: The combination of MDH2 inhibitors with Dasatinib showed significant synergistic effects both in vivo and in vitro, providing experimental evidence for combined GBM therapy.

Summary

MDH2 plays a crucial role in the metabolic and epitranscriptomic regulation of glioblastoma stem cells. Targeting its metabolic function or combining with existing multi-kinase inhibitors could potentially delay tumor growth and recurrence. This study not only provides new molecular targets for GBM treatment but also reveals the complex relationship between tumor metabolic regulation and the epitranscriptome, offering important directions for future research.