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 5

Increased activating histone methylation marks H3K4me2 and H3K4me3 at the MYC locus in L2HGDH-deficient NPCs.

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Increased activating histone methylation marks H3K4me2 and H3K4me3 at th...
(A) DNA dot blot analysis of 5mC levels in NPCs, including control H9 NPCs, control iPSC-derived NPCs, unedited patient 1 NPCs, unedited patient 2 NPCs, corrected patient 1 NPCs, and corrected patient 2 NPCs. (B) m6A dot blot analysis of total RNA in NPCs, including control H9 NPCs, control iPSC-derived NPCs, unedited patient 1 NPCs, unedited patient 2 NPCs, corrected patient 1 NPCs, and corrected patient 2 NPCs. (C) Immunoblot analysis of activating histone markers H3K4me2 and H3K4me3 levels in unedited and corrected patient 1 NPCs. Histone H3 was used as a loading control for histone lysates. (D and E) ChIP-seq profiles of H3K4me2 (D) and H3K4me3 (E) in unedited and corrected patient 1 NPCs. ChIP-seq signals were plotted over center peaks (±5 kb from peak center) identified in corrected patient 1 NPCs. Sites were sorted by the ChIP-seq signal intensity from corrected patient 1 NPCs. (F and G) Representative ChIP-seq tracks showing H3K4me2 (F) and H3K4me3 (G) enrichment at the MYC locus in unedited and corrected patient 1 NPCs.