DROSHA regulates mesenchymal gene expression in Wilms tumor.

Wilms tumor, the most common pediatric kidney cancer, resembles embryonic renal progenitors. Currently, there are no ways to therapeutically target Wilms tumor driver mutations, such as in the microRNA processing gene DROSHA. Here we used a "multi-omics" approach to define the effects of DROSHA mutation in Wilms tumor. We categorized Wilms tumor mutations into four mutational subclasses with unique transcriptional effects: microRNA processing, MYCN activation, chromatin remodeling, and kidney developmental factors. In particular, we find that DROSHA mutations are correlated with de-repressing microRNA target genes that regulate differentiation and proliferation and a self-renewing, mesenchymal state. We model these findings by inhibiting DROSHA expression in a Wilms tumor cell line, which led to upregulation of the cell cycle regulator cyclin D2 (CCND2). Furthermore, we observed that DROSHA mutations in Wilms tumor and DROSHA silencing in vitro were associated with a mesenchymal state with aberrations in redox metabolism. Accordingly, we demonstrate that Wilms tumor cells lacking microRNAs are sensitized to ferroptotic cell death through inhibition of glutathione peroxidase 4 (GPX4), the enzyme that detoxifies lipid peroxides. Implications: This study reveals genotype-transcriptome relationships in Wilms tumor and points to ferroptosis as a potentially therapeutic vulnerability in one subset of Wilms tumor.

The neural stem-cell marker CD24 is specifically upregulated in IDH-mutant glioma.

BACKGROUND: Malignant gliomas have disproportionally high morbidity and mortality. Heterozygous mutations in the isocitrate dehydrogenase 1 (IDH1) gene are most common in glioma, resulting in predominantly arginine to histidine substitution at codon 132. Because IDH1R132H requires a wild-type allele to produce (D)-2-hydroxyglutarate for epigenetic reprogramming, loss of IDH1R132H heterozygosity is associated with glioma progression in an IDH1-wildtype-like phenotype. Although previous studies have reported that transgenic IDH1R132H induces the expression of nestin-a neural stem-cell marker, the underlying mechanism remains unclear. Furthermore, this finding seems at odds with better outcome of IDH1R132H glioma because of a negative association of nestin with overall survival. METHODS: Gene expression was compared between IDH1R132H-hemizygous and IDH1R132H-heterozygous glioma cells under adherent and spheroid growth conditions. The results were validated for (D)-2-hydroxyglutarate responsiveness by pharmacologic agents, associations with DNA methylation by bioinformatic analysis, and associations with overall survival. Bisulfite DNA sequencing, chromatin immunoprecipitation, and pharmacological approach were used. FINDINGS: Neural stem-cell marker genes, including CD44, NES, and PROM1, are generally downregulated in IDH-mutant gliomas and IDH1R132H-heterozygous spheroid growth compared respectively with IDH-wildtype gliomas and IDH1R132H-hemizygous spheroid growth, in agreement with their negative associations with patient outcome. In contrast, CD24 is specifically upregulated and apparently associated with better survival. CD24 and NES expression respond differentially to alteration of (D)-2-hydroxyglutarate levels. CD24 upregulation is associated with histone and DNA demethylation as opposed to hypermethylation in the downregulated genes. INTERPRETATION: The better outcome of IDH-mutant glioma is orchestrated exquisitely through epigenetic reprogramming that directs bidirectional expression of neural stem-cell marker genes.

Extracellular glutamate and IDH1R132H inhibitor promote glioma growth by boosting redox potential.

PURPOSE: Somatic mutations of the isocitrate dehydrogenase 1 (IDH1) gene, mostly substituting Arg132 with histidine, are associated with better patient survival, but glioma recurrence and progression are nearly inevitable, resulting in disproportionate morbidity and mortality. Our previous studies demonstrated that in contrast to hemizygous IDH1R132H (loss of wild-type allele), heterozygous IDH1R132H is intrinsically glioma suppressive but its suppression of three-dimensional (3D) growth is negated by extracellular glutamate and reducing equivalent. This study sought to understand the importance of 3D culture in IDH1R132H biology and the underlying mechanism of the glutamate effect. METHODS: RNA sequencing data of IDH1R132H-heterozygous and IDH1R132H-hemizygous glioma cells cultured under two-dimensional (2D) and 3D conditions were subjected to unsupervised hierarchal clustering and gene set enrichment analysis. IDH1R132H-heterozygous and IDH1R132H-hemizygous tumor growth were compared in subcutaneous and intracranial transplantations. Short-hairpin RNA against glutamate dehydrogenase 2 gene (GLUD2) expression was employed to determine the effects of glutamate and the mutant IDH1 inhibitor AGI-5198 on redox potential in IDH1R132H-heterozygous cells. RESULTS: In contrast to IDH1R132H-heterozygous cells, 3D-cultured but not 2D-cultured IDH1R132H-hemizygous cells were clustered with more malignant gliomas, possessed the glioblastoma mesenchymal signature, and exhibited aggressive tumor growth. Although both extracellular glutamate and AGI-5198 stimulated redox potential for 3D growth of IDH1R132H-heterozygous cells, GLUD2 expression was required for glutamate, but not AGI-5198, stimulation. CONCLUSION: 3D culture is more relevant to IDH1R132H glioma biology. The importance of redox homeostasis in IDH1R132H glioma suggests that metabolic pathway(s) can be explored for therapeutic targeting, whereas IDH1R132H inhibitors may have counterproductive consequences in patient treatment.

IDH1R132H is intrinsically tumor-suppressive but functionally attenuated by the glutamate-rich cerebral environment.

Recurrent heterozygous mutation of isocitrate dehydrogenase 1 gene (IDH1), predominantly resulting in histidine substitution at arginine 132, was first identified in glioma. The biological significance of IDH1R132H, however, has been controversial, and its prevalent association with glioma remains enigmatic. Although recent studies indicate that IDH1R132H is nonessential to tumor growth or even anti-tumor growth, whether IDH1R132H initiates gliomagenesis remains obscure. In this study, we report that IDH1R132H is intrinsically tumor-suppressive but the activity can be attenuated by glutamate-the cerebral neurotransmitter. We observed that IDH1R132H was highly suppressive of subcutaneous tumor growth driven by platelet-derived growth factor B (PDGFB), but IDH1R132H tumor growth and glioma penetrance were virtually indistinguishable from those of IDH1-wildtype tumors in orthotopic models. In vitro, addition of glutamate compromised IDH1R132H inhibition of neurosphere genesis, indicating glutamate promotion of oncogenic dominance. Furthermore, we observed that IDH1R132H expression was markedly decreased in tumors but became more permissible upon the deletion of tumor-suppressor gene Cdkn2a. To provide direct evidence for the opposing effect of IDH1R132H on PDGFB-driven glioma development, we explored tandem expression of the two molecules from a single transcript to preclude selection against IDH1R132H expression. Our results demonstrate that when juxtaposed with oncogenic PDGFB, IDH1R132H overrides the oncogenic activity and obliterates neurosphere genesis and gliomagenesis even in the glutamate-rich microenvironment. We propose therefore that IDH1R132H is intrinsically suppressive of glioma initiation and growth but such tumor-suppressive activity is compromised by the glutamate-rich cerebral cortex, thereby offering a unifying hypothesis for the perplexing role of IDH1R132H in glioma initiation and growth.

In Vivo Manipulation of HIF-1α Expression During Glioma Genesis.

Hypoxia has long been recognized as a driving force of tumor progression and therapeutic resistance, and the transcription factor HIF-1α is believed to play a crucial role in these processes. Here we describe an efficient RCAS/Nes-TVA system that allows for in vivo manipulation of HIF-1α expression in the mouse neural progenitor cells. Simple production of the recombinant avian virus RCAS enables quick delivery of gene of interest through injection into the neural progenitors of transgenic mice expressing the viral cognate receptor TVA under the nestin promoter. By crossing with various commercially available genetically engineered mouse strains, a repertoire of mouse models can be created to study gene-specific effects on glioma genesis. This chapter provides details of plasmid construction, viral production, and intracranial delivery of transgenes, a methodology that can be easily adapted to a specific purpose.

Functional requirement of a wild-type allele for mutant IDH1 to suppress anchorage-independent growth through redox homeostasis.

Mutations of isocitrate dehydrogenase 1 (IDH1) gene are most common in glioma, arguably preceding all known genetic alterations during tumor development. IDH1 mutations nearly invariably target the enzymatic active site Arg132, giving rise to the predominant IDH1R132H. Cells harboring IDH1 R132H -heterozygous mutation produce 2-hydroxyglutarate (2-HG), which results in histone and DNA hypermethylation. Although exogenous IDH1 R132H transduction has been shown to promote anchorage-independent growth, the biological role of IDH1R132H in glioma remains debatable. In this study, we demonstrate that heterozygous IDH1 R132H suppresses but hemizygous IDH1 R132H promotes anchorage-independent growth. Whereas genetic deletion of the wild-type allele in IDH1 R132H -heterozygous cells resulted in a pronounced increase in neurosphere genesis, restoration of IDH1 expression in IDH1 R132H -hemizygous cells led to the contrary. Conversely, anchorage-independent growth was antagonistic to the mutant IDH1 function by inhibiting gene expression and 2-HG production. Furthermore, we identified that in contrast to IDH1 R132H -hemizygous neurosphere, IDH1 R132H -heterozygous cells maintained a low level of reducing power to suppress neurosphere genesis, which could be bypassed, however, by the addition of reducing agent. Taken together, these results underscore the functional importance of IDH1 mutation heterozygosity in glioma biology and indicate functional loss of mutant IDH1 as an escape mechanism underlying glioma progression and the pathway of redox homeostasis as potential therapeutic targets.