The proneural gene ASCL1 governs the transcriptional subgroup affiliation in glioblastoma stem cells by directly repressing the mesenchymal gene NDRG1

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.


Supplementary Fig. 3 -NDRG1 expression associates with high grade staging and IDH1 wildtype mutational status, is not regulated by mTORC2 but is regulated directly by ASCL1 as other known transcriptional targets.
(a-b) NDRG1 expression is significantly increased in high grade gliomas when compared to low grade (astrocytomas, a; oligoastrocytomas, oa; oligodendroglioma, od; glioblastoma, gbm) as well as in IDH1 wild type vs IDH1 mutant gliomas. ASCL1 expression shows the opposite trend. R2 analysis on Tumor Glioma French dataset comprising 284 samples. (c) The pattern of activation of the NDRG1 upstream activator pSGK1 does not correlate with that of pNDRG1. (d) mTORC2 inhibition by long-term treatment with rapamycin in ASCL1-overexpressing GBM CSCs only partially rescues NDRG1 expression. (e) The expression pattern of NDRG1 and EGFR primary transcripts by qPCR mirrors that of the corresponding proteins. (f) Binding of ASCL1 is detected within the promoter and enhancer of DLL3 and DKK1 genes, respectively.

Supplementary Fig. 4 -NDRG1 expression is inversely related to ASCL1 expression in GBM specimens from the TCGA
(a) Pearson correlation analysis on human GBM samples from the TCGA data set highlights the significant anticorrelation of ASCL1 and NDRG1 in terms of gene expression. (b) Heatmaps of microarray data indicate that high expression of ASCL1 correlates with low expression of NDRG1, thus supporting its role of repressor. (c) In tumors with high expression of ASCL1, NDRG1 is on average lowly expressed (upper panel). The opposite is also true, as high expression of NDRG1 correlates with lower expression of ASCL1 (lower panel).

Supplementary Fig. 5 -Modulation of the expression of ASCL1 and NDRG1 in GBM CSCs affects their tumorigenic behavior and subgroup affiliation.
(a) ASCL1-overexpressing L0306 CSCs failed to form tumors even at the latest time point assessed for controls. Human-specific EGFR staining: brown, 20x. (b-c) NDRG1-overexpressing L0605 and L0125 CSC-derived tumors displayed increased MES morphological features, such as the development of areas made up by spindle-shaped cells with large and elongated nuclei (H&E, 400x), as well as increased MES marker immunoreactivity at the expense of the expression of PN markers (all markers stained in brown, 400x).

Supplementary Fig. 6 -Modulation of the expression of ASCL1 and NDRG1 in GBM CSCs affects their tumorigenic behavior and subgroup affiliation.
Efficient NDRG1 silencing in MES CSC lines was obtained by different shRNA clones (Mission, Sigma-Aldrich) Supplementary Table 3 -Gene set enrichment analysis (GSEA) report for the two distinct gene sets generated through the comparison between ASCL1 high /NDRG1 low or ASCL1 high /NDRG1 high CSCs vs GCLs, when tested on ranked lists of proneural and mesenchymal genes.
A significant FDR q-value (> 0.25) has been retrieved for both comparisons (highlighted in bold italic). A lower NES in the proneural subgroup was observed for genes upregulated in comparison 2 as compared to those in comparison 1, in line with a positive trend in the level of enrichment detected in the mesenchymal subgroup.