l-2-Hydroxyglutarate impairs neuronal differentiation through epigenetic activation of MYC expression
Wen Gu, Xun Wang, Ashley Solmonson, Ling Cai, Yi Xiao, Alpaslan Tasdogan, Jordan Franklin, Yuannyu Zhang, Hua Zhang, Aundrea K. Westfall, Ashley Rowe, Hetali Trivedi, Brandon Faubert, Zheng Wu, Jessica Sudderth, Lauren G. Zacharias, Bushra Afroze, Ilya Bezprozvanny, Sunil Sudarshan, Feng Cai, Samuel K. McBrayer, Thomas P. Mathews, Ralph J. DeBerardinis
Wen Gu, Xun Wang, Ashley Solmonson, Ling Cai, Yi Xiao, Alpaslan Tasdogan, Jordan Franklin, Yuannyu Zhang, Hua Zhang, Aundrea K. Westfall, Ashley Rowe, Hetali Trivedi, Brandon Faubert, Zheng Wu, Jessica Sudderth, Lauren G. Zacharias, Bushra Afroze, Ilya Bezprozvanny, Sunil Sudarshan, Feng Cai, Samuel K. McBrayer, Thomas P. Mathews, Ralph J. DeBerardinis
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Research Article Clinical Research Development Metabolism

l-2-Hydroxyglutarate impairs neuronal differentiation through epigenetic activation of MYC expression

Abstract

High levels of l- and d-2-hydroxyglutarate (2HG), the reduced forms of α-ketoglutarate (αKG), are implicated in neurodevelopmental disorders and cancer by modulating αKG-dependent dioxygenases involved in histone, DNA, and RNA demethylation. L-2HG dehydrogenase (L2HGDH) deficiency, a rare autosomal recessive inborn error of metabolism associated with systemic L-2HG elevation, causes progressive neurological disability and increased brain tumor risk of unclear mechanism. Using an isogenic, patient-derived induced pluripotent stem cell system, we examined the impact of L2HGDH deficiency on neural progenitor cell (NPC) function and neuronal differentiation. L2HGDH deficiency caused L-2HG accumulation, NPC hyperproliferation, increased clonogenicity, and defective neuronal differentiation in 2D cultures and cortical spheroids. Editing the L2HGDH locus to WT reversed these effects. Inhibiting glutaminase reduced L-2HG levels and induced neuronal differentiation. L-2HG–dependent inhibition of KDM5 histone demethylases led to widespread retention of H3K4me2/3, markers of active gene expression, with prominent enrichment at the MYC locus and elevated MYC expression across multiple neural cell types. Despite broadly altered histone methylation, genetically or pharmacologically normalizing MYC completely restored neuronal differentiation. These data indicated that a primary metabolic disturbance activated MYC to favor self-renewal and suppress neuronal lineage commitment.

Authors

Wen Gu, Xun Wang, Ashley Solmonson, Ling Cai, Yi Xiao, Alpaslan Tasdogan, Jordan Franklin, Yuannyu Zhang, Hua Zhang, Aundrea K. Westfall, Ashley Rowe, Hetali Trivedi, Brandon Faubert, Zheng Wu, Jessica Sudderth, Lauren G. Zacharias, Bushra Afroze, Ilya Bezprozvanny, Sunil Sudarshan, Feng Cai, Samuel K. McBrayer, Thomas P. Mathews, Ralph J. DeBerardinis

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Figure 4

Increased MYC expression in L2HGDH-deficient NPCs and cortical spheroids.

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Increased MYC expression in L2HGDH-deficient NPCs and cortical spheroids...
(A) GSEA mountain plot showing enrichment of DANG_MYC_TARGETS_UP gene set in unedited versus corrected patient 1 NPCs. (B) Quantification of MYC mRNA in control H9 NPCs, unedited patient 1 NPCs, and corrected patient 1 NPCs. mRNA levels were obtained from RNA-seq of biological triplicates and expressed as transcripts per kilobase million (TPM). (C) Immunoblot analysis of nuclear c-MYC and Neurogenin-2 in unedited and corrected patient 1 NPCs. TBP was used as a loading control for nuclear lysates. (D) UMAP heatmaps displaying MYC expression in scRNA-seq of unedited and corrected day 45 cortical spheroids. Color intensity (purple) indicates MYC transcript levels in individual cells. (E) Quantification of NEUROG2 mRNA in control H9 NPCs, unedited patient 1 NPCs, and corrected patient 1 NPCs. mRNA levels were obtained from RNA-seq of biological triplicates and expressed as TPM. (F) Immunoblot analysis of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 1 μM CB-839 (GLSi) for 4 or 7 days. TBP served as a loading control for nuclear lysates. Significance was determined based on log-transformed TPM values using 1-way ANOVA followed by Tukey’s HSD test for B and E. Data are shown as mean ± 1 SEM of 3 biological replicates.