ASCL1 regulates neurodevelopmental transcription factors and cell cycle genes in brain tumors of glioma mouse models

Abstract Glioblastomas (GBMs) are incurable brain tumors with a high degree of cellular heterogeneity and genetic mutations. Transcription factors that normally regulate neural progenitors and glial development are aberrantly coexpressed in GBM, conferring cancer stem‐like properties to drive tumor progression and therapeutic resistance. However, the functional role of individual transcription factors in GBMs in vivo remains elusive. Here, we demonstrate that the basic‐helix–loop–helix transcription factor ASCL1 regulates transcriptional targets that are central to GBM development, including neural stem cell and glial transcription factors, oncogenic signaling molecules, chromatin modifying genes, and cell cycle and mitotic genes. We also show that the loss of ASCL1 significantly reduces the proliferation of GBMs induced in the brain of a genetically relevant glioma mouse model, resulting in extended survival times. RNA‐seq analysis of mouse GBM tumors reveal that the loss of ASCL1 is associated with downregulation of cell cycle genes, illustrating an important role for ASCL1 in controlling the proliferation of GBM.


| INTRODUCTION
Glioblastomas (GBMs) are incurable brain tumors most commonly found in adults. Despite significant advances in imaging and surgical resection techniques combined with aggressive radiotherapy and chemotherapy, the median survival for GBM patients remains stagnated between 14 and 16 months, with greater than 90% of patients succumbing to their disease within 5 years of diagnosis (Ostrom et al., 2016). A major reason for this poor prognosis is due to the high degree of heterogeneity and plasticity of these neoplasms, and the lack of mechanistic insights into the pan-therapeutic resistance of GBM tumor cells (Babu et al., 2016;Brennan et al., 2013;Lathia, Heddleston, Venere, & Rich, 2011).
In this study, we focus on ASCL1, a class II basic-helix-loop-helix (bHLH) transcription factor that forms a heterodimer with class I bHLH E-proteins (such as E47/TCF3) to activate specific target genes (Kageyama, Ohtsuka, Hatakeyama, & Ohsawa, 2005). During embryogenesis, ASCL1 is expressed in specific populations of neural progenitor domains and glial precursor cells throughout the neural tube from the spinal cord to the brain (Helms et al., 2005;Parras et al., 2004Parras et al., , 2007Sugimori et al., 2007Sugimori et al., , 2008Vue, Kim, Parras, Guillemot, & Johnson, 2014), including in neurogenic regions of the adult brain (Kim, Ables, Dickel, Eisch, & Johnson, 2011;Kim, Leung, Reed, & Johnson, 2007). Recently, ASCL1 was shown to be capable of reorganizing and promoting the accessibility of closed chromatin in embryonic stem cells, neural progenitors, as well as glioma cell lines (Casey, Kollipara, Pozo, & Johnson, 2018;Park et al., 2017;Raposo et al., 2015). Not surprisingly, genome wide profiling revealed a critical role for ASCL1 in interacting with both Wnt and Notch signaling pathways to control the tumorigenicity of glioma cells in culture (Park et al., 2017;Rajakulendran et al., 2019;Rheinbay et al., 2013). To date however, whether ASCL1 is absolutely required for glioma tumor development in the brain as it has been shown for a mouse model of small cell lung carcinoma (SCLC) (Borromeo et al., 2016) remains to be determined. Here, we sought to identify the direct in vivo role and transcriptional targets of ASCL1 in brain tumors of previously characterized patient-derived xenograft (PDX)-GBM (Marian et al., 2010;Marin-Valencia et al., 2012) and a genetically engineered glioma mouse model (Lin et al., 2004;Zhu et al., 2001).

| Mouse breeding and tamoxifen administration
The appearance of a vaginal plug was considered embryonic day (E) 0.5, and the day of birth was noted as postnatal day (P) 0. To induce tumor formation in the brains of Glast CreERT2/+ ;Nf1 F/F ;Trp53 F/F mice, tamoxifen (Sigma T5648, dissolved in 10% ethanol/90% sunflower oil) was administered intraperitoneally (62.5 mg/kg body weight) to pregnant females at E14.5. Due to the effects of tamoxifen on birth complications, cesarean section was performed and pups were carefully introduced and raised by a foster female.
Brains were submerged in 30% sucrose/PBS at 4 C, and embedded in O.C.T. for cryosectioning. H&E staining of tumors was done by the UT Southwestern Histopathology Core. Grading of brain tumors was determined by a board certified neuropathologist.
A 150 bp region around the peak summit was used to identify the primary binding motif and other potential DNA-binding co-Lanfactor motifs.
To analyze mouse tumor RNA-seq data (GSE152401), sequenced reads were aligned to the mouse mm10 genome using TopHat 2.1.0 (Kim et al., 2013). Default settings were used, with the exception of -G, specifying assembly to the mm10 genome, −-library-type fr -first strand, and -no-novel-juncs, which disregards noncanonical splice junctions when defining alignments. DESeq2 (Love, Huber, & Anders, 2014) was used to incorporate RNA-seq data from the five biological replicates for Ascl1 WT and Ascl1 CKO tumor samples, and differentially expressed genes were identified using default parameters.
To investigate the similarity/difference between Ascl1 WT and Ascl1 CKO tumors in comparison to each other and to CNS cell types, multidimensional scaling (MDS) was performed using the plotMDS function available in edgeR package (Robinson, McCarthy, & Smyth, 2010). Finally, to identify enrichment of gene signature sets in rank ordered gene lists obtained from Ascl1 WT and Ascl1 CKO tumor samples, gene set enrichment analysis (GSEA) (Subramanian et al., 2005) was performed and the signal-to-noise ratio metric was used to rank the genes.

| GBM subtype classification and heatmap clustering analyses
The GBM subtype signatures defined by Verhaak et al. (2010) were used for hierarchical clustering for 164 GBM patient samples and five normal brains from TCGA for which RNA-seq data was available (Brennan et al., 2009(Brennan et al., , 2013TCGA, 2008;Verhaak et al., 2010). Spearman rank order correlation and ward.D2 clustering method were applied to identify the various GBM subtypes. Heatmaps were generated using absolute expression values (RPKM) for the selected list of genes or significantly changed genes, and hierarchical clustering was performed using the correlation distance metric and the ward.D2 method using the heatmap.2 function available in the gplots R package.

| Gene targets and pathway enrichment analysis
To identify ASCL1 putative targets, genes associated with the ASCL1 ChIP-seq peaks were annotated using GREAT v3.0.0 (http://great. Quantifications were performed on at least three images taken from different areas per tumor for each marker (N = 4).
To determine the expression of ASCL1, OLIG2, and SOX2 in human GBMs, RNA-seq of 164 TCGA primary GBM and five normal brain samples (Brennan et al., 2013) were analyzed and categorized into the various subtypes using the 840 GBM Subtype Signature Genes (Verhaak et al., 2010 (Gangemi et al., 2009;Ligon et al., 2007;Lu et al., 2016;Park et al., 2017;Rheinbay et al., 2013;Singh et al., 2017;Somasundaram et al., 2005). However, the extent to which these factors are coexpressed in GBM tumors in vivo remains unclear. Using We next sought to determine the expression level of ASCL1, OLIG2, and SOX2 across primary GBMs exhibiting a variety of genomic alterations. Leveraging RNA-seq data of 164 TCGA primary GBMs, along with five normal control brain samples (Brennan et al., 2013), we first classified these primary GBMs into the four GBM subtypes (proneural, neural, classical, mesenchymal) as previously defined using an 840 gene list (Verhaak et al., 2010). Notably, while 107 samples can be classified into one of the four GBM subtypes, the remaining 57 samples expressed signatures of more than one subtype, which we referred collectively to as mixed GBMs ( Figure S1). This finding echoes previous reports demonstrating the presence of multiple GBM subtype identities in different regions or cells of the same GBM tumors Sottoriva et al., 2013). Expression of ASCL1, OLIG2, and SOX2 across these GBM subtypes showed that they were highest in the proneural and classical subtypes, intermediate in the neural and mixed subtypes, but were extremely low in the mesenchymal subtype, even in comparison to normal brain (Figure 1p-r). Collectively, these findings illustrate that ASCL1, OLIG2, and SOX2 are coexpressed at relatively high levels in the majority of primary GBMs with the exception of the mesenchymal subtype.

| ASCL1 binds to genes encoding neurodevelopmental and glial transcription factors, oncogene signaling molecules, and factors involved in cell cycle control and chromatin organization
Chromatin immunoprecipitation followed by deep sequencing (ChIPseq) has previously been performed for ASCL1 in glioma cell lines in culture, and a dual role for ASCL1 was proposed to either promote or attenuate tumorigenicity depending on context (Park et al., 2017;Rheinbay et al., 2013). We performed ChIP-seq for ASCL1 in the  Table S1 for ASCL1 binding peaks and coordinates).
To validate the quality and efficiency of our ChIP-seq, we next analyzed ASCL1 binding at known canonical targets (DLL1, DLL3, NOTCH1, HES5, HES6, and INSM1) which have previously been shown to be directly regulated by ASCL1 in numerous contexts (Borromeo et al., 2014(Borromeo et al., , 2016Castro et al., 2011;Jacob et al., 2009;Park et al., 2017;Somasundaram et al., 2005;Ueno et al., 2012;Vias et al., 2008). As expected, ChIP-seq tracks revealed the presence of strong ASCL1 binding peaks at loci of all the canonical target genes examined (asterisk, Figure 2b). Moreover, ASCL1 is known to preferentially bind to degenerate CANNTG E-box motifs to regulate gene expression (Borromeo et al., 2014(Borromeo et al., , 2016Casey et al., 2018;Castro et al., 2011). Using de novo motif analysis (Heinz et al., 2010), we identified the bHLH CAGCTG E-box motif as being highly enriched directly beneath 74% of the 13,457 ASCL1 combined peaks called, further confirming the quality of the ChIP-seq. Interestingly, we found that SOX and FOXO motifs were also significantly enriched within ASCL1 binding peaks (Figure 2c), suggesting that ASCL1 may function in combination with these transcription factor families to regulate gene expression in GBMs.
To identify putative-targets of ASCL1 in GBMs, we then used GREAT (McLean et al., 2010) to associate nearby genes that were upstream or downstream of the 13,457 ASCL1 binding peaks. From this analysis, we uncovered a total of 8,791 genes (red oval, Figure 2d) (see Table S2 for list of ASCL1 target genes). We reasoned that if these genes are regulated by ASCL1 then they should also be expressed in a manner correlated with ASCL1 expression in GBMs. By applying Spearman's rank-ordered correlation (>0.4) to RNA-seq of the 164 TCGA GBM samples, we then identified the top 10% of genes that showed a positive correlation with ASCL1 expression across these tumor samples. We found 2,136 genes that are positively correlated with ASCL1 expression (green oval, Figure 2d) (see Table S3 for list of ASCL1 correlated genes in GBMs).
When we cross referenced these 2,136 genes with the 8,791 genes identified from the ASCL1 ChIP-seq, there was an overlap of 1,106 genes, which we define as ASCL1 putative target genes in GBM (yellow area, Figure 2d). Supporting the validity of this approach, all ASCL1 canonical targets examined were included in this 1,106 putative-target gene list (Figure 2d) (see Table S4 for list of ASCL1 putative targets).  Ki67+,OLIG2+,or SOX2+ (n). N = 4 PDX-GBM. (p-r) Box whisker plot of RNA-seq data from 164 TCGA Primary GBMs and 5 normal brain samples (Brennan et al., 2013) demonstrating that ASCL1 (p), OLIG2 (q), and SOX2 (r) are highly expressed in the majority of GBM subtypes but are low in MS subtype and normal brain (Br). GBM subtype was determined using the 840 GBM Subtype Signature Genes (Verhaak et al., 2010). CL, classical; MS, mesenchymal; NE, neural; PN, proneural. Mixed GBM subtype express multiple subtype signatures. Scale bar is 1 mm for (b) and 50 μm for (c-m), and 12.5 μm for all insets in (d-m) [Color figure can be viewed at wileyonlinelibrary.com] By evaluating the ASCL1 putative-target gene list (Table S4) We next wanted to know how the expression of the 1,106 ASCL1 putative-target genes sort across the various GBM subtypes using RNA-seq of the 164 primary GBMs. Heatmap and dendrogram analysis revealed that, similar to ASCL1, the 1,106 putative-target genes were highly expressed in the proneural and classical GBM subtypes, in the majority of neural and mixed GBM subtypes, but was mostly absent in the mesenchymal GBM subtype (Figure 2i). In all, 109 of the TCGA GBM samples were positive for the ASCL1 putative-target genes, while the remaining 55 samples express very little or low levels of the ASCL1 putative-targets.
To gain insights into the collective significance of the 1,106 ASCL1 putative targets, we then performed gene set over-representation analysis to annotate their function using ConsensusPathDB, a comprehensive collection of molecular interaction databases integrated from multiple public repositories (Herwig et al., 2016). Interestingly, the top most enriched pathway identified was cell cycle (Figure 2j). This is con- Cancer were also enriched for ASCL1 targets (Figure 2j) (see Table S5 for list ASCL1 target enriched biological pathways). Taken together, these findings suggest that ASCL1 is a transcriptional regulator at the epicenter of multiple biological processes that are fundamental to cancer development.
3.3 | ASCL1, OLIG2, and SOX2 are coexpressed in early and terminal stage tumors of a glioma mouse model To functionally test ASCL1's role in gliomagenesis in vivo, we began by characterizing the temporal expression pattern of ASCL1 along with OLIG2, SOX2, and glial lineage markers in brain tumors induced in a mouse model carrying floxed alleles of the tumor suppressor genes Neurofibromin 1 (Nf1) and tumor protein 53 (Tp53) (Nf1 F/F ; Tp53 F/F ) (Lin et al., 2004;Zhu et al., 2001). NF1 and TP53 are two of the most highly mutated genes in human GBM (Brennan et al., 2013;Verhaak et al., 2010), and Cre-recombinase deletion of these two tumor suppressor genes (Nf1;Tp53 CKO ) in neural progenitors or glial precursor cells have previously been shown to be fully penetrant in producing glioma tumors in the brains of mice (Alcantara Llaguno et al., 2009;Alcantara Llaguno et al., 2015;Zhu et al., 2005). When mice carrying a Glast CreERT2/+ knock-in allele (Mori et al., 2006) was reporter line (Madisen et al., 2010), we found that tdTomato fluorescence was restricted in the brain of neonatal pups if tamoxifen was administered at E14.5 (Figure 3a-c), making Glast CreERT2/+ ideal to combine with the Nf1 F/F ;Tp53 F/F alleles to induce brain tumors.
To visualize the tumors as they develop in the brain, a R26R LSL-YFP reporter allele (Srinivas et al., 2001) Information Tables S2-S4. (e-h) ChIP-seq tracks of ASCL1 binding peaks at loci of neurodevelopmental and glial transcription factors (e), cell cycle and mitotic genes (f), chromatin modifying genes (g), and oncogenic signal transduction genes (h). (i) Heatmap and dendrogram illustrating relative expression of 1,106 ASCL1 putative target genes in GBM subtypes using RNA-seq of 164 TCGA primary GBM samples (Brennan et al., 2013). Note that ASCL1 target-positive GBMs include all subtypes except mesenchymal, while ASCL1 target-negative GBMs include all mesenchymal and some neural and mixed GBM subtypes. (j) Gene set over-representation analysis of 1,106 ASCL1 putative-target genes using ConsensusPathDB (cpdb.molgen.mpg.de). Biologically relevant enriched pathways are illustrated. Size of circle indicates the number of genes per pathway, size of edge indicates degree of gene overlaps between the pathways, and color indicates database sources. The number of ASCL1 putative-target genes over-represented in each pathway, and respective p-value are indicated. See Supporting Information Table S5 for  another tumor suppressor, Pten (Lei et al., 2011), and implies that the majority of GFAP+ cells are reactive astrocytes that have infiltrated the YFP+ tumor bulk in our model.
From P60-120, we found that 100% of Nf1;Tp53 CKO mice exhibited neurological symptoms and had tumors that evolved into an expanded mass with high mitotic index and microvascular proliferation resembling that of high-grade gliomas (Figure 4a,b). We termed these Ascl1 WT tumor mice (N = 29, blue line), which exhibited a median survival of 102 days, while CreER-negative littermate controls (N = 19, green line) were tumor-free and healthy (Figure 4p). Over 90% of the tumors were found in the cortex and/or striatum area, while a minority was also found in olfactory bulb, diencephalon, mid-

| Loss of ASCL1 decreases the proliferation of gliomas and increases the survival of tumor bearing mice
Currently, the direct requirement of ASCL1 in brain tumor formation and progression from low-grade gliomas to high-grade GBMs in vivo remains unknown. To address this, we incorporated Ascl1 GFP knock-in (null) and Ascl1 Floxed alleles into the glioma mouse model to generate Glast CreERT2 ;Ascl1 GFP/F ;Nf1 F/F ;Tp53 F/F and Glast CreERT2 ;Ascl1 F/F ;Nf1 F/F ; Tp53 F/F mice, respectively, both of which when administered with tamoxifen at E14.5 will result in triple conditional knock-out of Ascl1 along with Nf1 and Tp53 (Ascl1;Nf1;Tp53 CKO ). To control for the possible effects of genetic background on glioma phenotype observed, we also generated Glast CreERT2 ;Ascl1 GFP/+ ;Nf1 F/F ;Tp53 F/F and Glast CreERT2 ; Ascl1 F/+ ;Nf1 F/F ;Tp53 F/F mice in parallel for comparison, both of which developed tumors that are still heterozygous for Ascl1 when induced with tamoxifen, and are referred to as Ascl1 HET tumor mice.
Previous reports demonstrate that ASCL1 is essential for the proliferation of GBM cell lines in vitro (Park et al., 2017;Rheinbay et al., 2013). In contrast, in vivo we found that Ascl1;Nf1;Tp53 CKO mice (hence forth referred to as Ascl1 CKO tumor mice, N = 39) still developed high-grade tumors that were phenotypically consistent with high-grade gliomas (Figure 5a). Furthermore, Ascl1 CKO tumor penetrance and location (Figure 5l,m) in the brain was similar to the Ascl1 HET (not shown) and Ascl1 WT tumors (Figure 4o,p). We confirmed that ASCL1 was indeed absent in Ascl1 CKO tumors. As illustrated for a Glast CreERT2 ;Ascl1 GFP/F ;Nf1 F/F ;Tp53 F/F mouse, GFP driven by the endogenous Ascl1 locus marks precisely the tumor cells but ASCL1 was no longer detected (Figure 5b,c). Notably, OLIG2 and SOX2 ( Figure 5d,e,j,k), which we identified as ASCL1 target genes, were still expressed, indicating that expression of these two transcription factors do not depend solely on ASCL1. Similarly, OPC markers such as PDGFRα, the chondroitin sulfate NG2, and SOX10 were still expressed in the Ascl1 CKO tumors (Figure 5d,f,g). As observed in  (Zhang et al., 2014). A multidimensional scaling (MDS) plot shows that both the Ascl1 WT and Ascl1 CKO tumors cluster together and away from the CNS cell types, and therefore are more similar to each other than to neurons or any of the glial lineage cells (Figure 6b). When we further analyzed RNA-seq of Ascl1 WT and Ascl1 CKO tumors using the top 50 signature genes for each CNS cell type, both tumor types more closely resemble that of OPCs rather than the other CNS cell types (Figure 6c). This finding further supports the notion that OPCs, which are highly proliferative and migratory, may be the precursor cell-of-origin for the glioma tumors in this model.
Finally, we sought to identify genes that are differentially expressed between Ascl1 WT and Ascl1 CKO tumors. Analysis of ASCL1 canonical target genes revealed that Dll3, similar to Ascl1, was significantly decreased, while Hes5 and Hes6 were lowered (Figure 6d), but Dll1, Notch1, and Insm1 (not shown) were unchanged in Ascl1 CKO tumors. Interestingly, glial transcription factors Nfia and Nfib, and several mitotic (Aurkb) and chromatin modifying (Hdac5) genes were significantly decreased, whereas Olig1, Olig2, and cell cycle genes (Ccnd2 and Cdk4) were somewhat reduced in Ascl1 CKO tumors (Figure 6e,g).
In contrast, Sox genes were bidirectionally affected by the loss of ASCL1. For instance, although Sox3 and Sox11 were decreased, Sox2 and Sox4 appeared upregulated in the Ascl1 CKO tumors (Figure 6f).
Heatmap and dendrogram analysis of all 1,054 ASCL1 putative targets (converted from a list of 1,106 genes from human GBMs, Table S4), revealed that there were as many genes being upregulated as there were genes being downregulated by the loss of ASCL1 (Figure 6h, Table S7). We also identified about 50 indirect targets of ASCL1 that were either upregulated or downregulated in the Ascl1 CKO tumors ( Figure 6i, Table S8). Finally, in agreement with our earlier finding that tumor cell proliferation is decreased in the absence of ASCL1, geneset-enrichment analysis revealed that cell cycle related genes were highly enriched in the downregulated genes in the Ascl1 CKO tumors ( Figure 6j). This suggests that a decreased in cell-cycle related gene expression may contribute to the increase in survival of the Ascl1 CKO tumor mice.
In summary, our findings highlight an in vivo role for ASCL1 in modulating the expression of a variety of genes, including neurodevelopmental or glial transcription factors and cell cycle genes, either directly or indirectly, that are crucial for the proliferation of glioma tumors in the brains of genetically relevant mouse models.

| DISCUSSION
We demonstrate in this study that ASCL1 is highly expressed in the majority of PDX-GBM cells in vivo, with over 90% of ASCL1+ cells coexpressing OLIG2 and SOX2. Interestingly, in addition to OLIG2 and SOX2, we find that expression of a variety of other genes encoding transcription factors such as those for NFI, POU domain, Sal-like, SOX, and homeobox families are also highly correlated with ASCL1 expression in RNA-seq of primary GBMs (Table S3). This finding is similar to that previously reported in GSCs from cultured GBM cell lines (Rheinbay et al., 2013;Suva et al., 2014). Accordingly, we find that these transcription factor encoding genes are also targets of ASCL1 binding (Tables S1 and S2). These findings support a complex transcription factor interaction network in which the coexpression of these transcription factors may be interdependent on each other, and this coexpression is essential for regulating genes crucial for maintaining glioma cells in an aberrant stem-like state of dedifferentiation and proliferation. In agreement with this, it is not surprising that combinatorial overexpression of multiple transcription factors is necessary and sufficient to reprogram differentiated glioma cells or immortalized astrocytes into tumor propagating cells (Singh et al., 2017;Suva et al., 2014).
ChIP-seq for ASCL1 has previously been performed for glioma cell lines in culture revealing that ASCL1 directly interacts with Wnt signaling by binding to genes such as AXIN2, DKK1, FZD5, LGR5, LRP5, TCF7, and TCF7L1. A model was proposed in which ASCL1 functions at least in part by repressing an inhibitor of Wnt signaling, DKK1, resulting in increased signaling through this pathway to maintain the tumorigenicity of glioma cells (Rheinbay et al., 2013). Analysis of microarray data in primary GBM cultures also divided GSCs into ASCL1 high and ASCL1 low subgroups, where the former is closely correlated with the proneural subtype, whereas the latter is associated with the mesenchymal subtype (Park et al., 2017). Notably, dual attenuation of both Wnt and Notch signaling in the ASCL1 high subgroup upregulated a neurogenic program similar to that observed during development, resulting in decreased tumorigenicity of GSCs (Park et al., 2017;Rajakulendran et al., 2019). Together, these studies support the conclusion that ASCL1 levels in gliomas acts in a balance with both Wnt and Notch signaling pathways to regulate the GSC status of tumor cells in culture.
Here we also find that both Wnt and Notch related genes are also directly bound by ASCL1 in PDX-GBMs grown orthotopically in the brains of mice. Additionally, expression of many of these genes is  Table S2). Despite these findings, RNA-seq from a Nf1;Tp53 CKO mouse model of glioma where the tumors also lack ASCL1 revealed that although some Notch related genes (Dll3, Hes5, Hes6) were decreased, expression of many of the Wnt related genes seemed unaffected by the loss of ASCL1. Thus, although ASCL1 binds to and may contribute to the regulation of some of these target genes, particularly in an in vitro setting (Rheinbay et al., 2013), expression of many of these target genes remains in gliomas in vivo in the absence of ASCL1.
Interestingly, ChIP-seq for ASCL1 in PDX-GBMs and RNA-seq of mouse Ascl1 CKO tumors in our study revealed that cell cycle and mitotic genes are major targets of ASCL1 binding (Figure 2f,j), and these targets were most impacted by the loss of ASCL1 (Figure 6g,j).
In agreement with this, the percentage of Ki67+ cells was significantly decreased in Ascl1 CKO relative to Ascl1 WT tumors of the mouse model  (Zhang et al., 2014). Ascl1 WT and Ascl1 CKO tumors are more similar to each other than to any of the CNS cell types. AS, astrocytes; OPC, oligodendrocyte precursor cells; NFO, newly formed oligodendrocytes; MO, myelinating oligodendrocytes; WC, whole cortex. See Table S6 for normalized gene expression (RPKM) in Ascl1 WT and Ascl1 CKO tumors. (c) Heatmap and dendrograms using the top 50 CNS cell lineage signature genes for each cell type (Zhang et al., 2014). Dendrograms on top show that Ascl1 WT and Ascl1 CKO tumors express signature genes that are more similar to OPCs than to the other CNS cell types. (d-g) Box and whisker plots of ASCL1 putative-target genes in Ascl1 WT and Ascl1 CKO tumors. Canonical targets of ASCL1 (d), glial transcription factors (e), and mitotic, chromatin modifying, and cell cycle genes (g) are expressed at lower level while neural stem cell/Sox genes (f) are bidirectionally affected in Ascl1 CKO compared to Ascl1 WT tumors. Asterisks indicate target genes significantly altered (p < .05, Wilcox test). (h-i) Heatmap and dendrograms of differentially expressed genes (DEGs) in Ascl1 WT and Ascl1 CKO GBMs. DEGs consist of ASCL1 putative direct targets (h) (see Table S7) and indirect target DEGs (FDR < 0.05) (i) (see Table S8). (j) Gene-set-enrichment-analysis (GSEA) showing that cell cycle genes are enriched in the downregulated genes in Ascl1 CKO compared to Ascl1 WT tumors [Color figure can be viewed at wileyonlinelibrary.com] the Nf1;Tp53 CKO glioma mouse model is to drive tumor cell proliferation. This function of ASCL1 is similar to what has been observed in neural progenitor cells during development as well as in the adult brain. In particular, although overexpression of ASCL1 is predominantly known to promote cell cycle exit, cell fate specification, and neuronal differentiation, a prominent phenotype associated with Ascl1 mutants is a loss in the overall progenitor pool as a result of a decrease in proliferation and a disruption in the ASCL1/NOTCH balance, which is essential to maintain the progenitor pool (Borromeo et al., 2014;Casarosa, Fode, & Guillemot, 1999;Castro et al., 2011;Horton, Meredith, Richardson, & Johnson, 1999;Nakada, Hunsaker, Henke, & Johnson, 2004). As a validation of ASCL1's direct role in progenitor cell proliferation, ChIP-seq of mouse embryonic telencephalon, adult neural progenitors, and neural stem cells in cultures also found that ASCL1 directly binds to a large number of genes involved in cell cycle progression (Andersen et al., 2014;Castro et al., 2011).
These target genes include positive cell cycle regulators and oncogenic transcription factors, some of which are also identified here in our ChIP-seq of ASCL1 in the PDX-GBMs (Table S2). Similarly, within regions of adult neurogenesis such as the subventricular zone (SVZ) of the lateral ventricle and subgranular zone (SGZ) of the hippocampal dentate gyrus, ASCL1 is expressed at very low level or is undetectable in quiescent neural stem cells which exhibit radial glial-like morphology and express GFAP and Nestin. In contrast, ASCL1 expression is highest in transiently amplifying progenitors (TAPs), which are highly proliferative (Kim et al., 2011). Conditional knock-out studies have shown that ASCL1 is responsible for promoting neural stem cells from a quiescent into an activated state in the SGZ, and the number of proliferating progenitors is significantly decreased with the loss of ASCL1 (Andersen et al., 2014;Kim et al., 2011;Urbán et al., 2016). Using a similar conditional knock-out approach, we also demonstrated that ASCL1 is specifically required for the proliferation of OPCs in both embryonic and adult spinal cord (Kelenis et al., 2018). Indeed, the level of ASCL1 is positively correlated with the rate of OPC proliferation (Kelenis et al., 2018;Nakatani et al., 2013), and the decrease in proliferation of OPCs in the absence of ASCL1 is similar to that observed for Ascl1 CKO tumors.
In addition to gliomas, ASCL1 is highly expressed in cancers with neuroendocrine characteristics from multiple tissues including SCLC, prostate cancer, and thyroid medullary carcinoma (Chen, Kunnimalaiyaan, & Van Gompel, 2005;Rapa et al., 2013;Zhang et al., 2018). Previously, we reported that ASCL1 is required for tumor formation in a mouse model of SCLC (Borromeo et al., 2016). This finding reflects the requirement for ASCL1 in the generation and survival of pulmonary neuroendocrine cells (PNECs), a presumptive cell-oforigin for SCLC. In contrast, we find that ASCL1 is not required for glioma formation in the brain of the mouse model, although disease progression is altered and the animals have extended survival. Based on cell lineage markers in the glioma mouse model used here, our finding implicates OPCs as the presumptive cell-of-origin for the tumors.
OPC specification and generation in the CNS is dependent upon OLIG2 (Lu et al., 2002;Zhou, Choi, & Anderson, 2001); however, ASCL1 also plays an important role to regulate the number and proliferation of OPCs (Kelenis et al., 2018;Nakatani et al., 2013;Parras et al., 2007;Vue et al., 2014). Interestingly, in addition to ASCL1 and OLIG2, transcription factors such as NFIA, SOX2, and SOX10 are also expressed in OPCs. However, as OPCs differentiate to become mature oligodendrocytes, only OLIG2 and SOX10 are maintained while ASCL1, NFIA, and SOX2 are downregulated (Glasgow et al., 2014;Laug, Glasgow, & Deneen, 2018;Nakatani et al., 2013). This downregulation suggests that the coexpression of these transcription factors is important for maintaining OPCs in a progenitor-like state, and the loss of just one of these factors does not completely abrogate tumor formation following deletion of Nf1 and Tp53 because OPCs are still generated, and are thus susceptible to being transformed into glioma.
It is also possible that within the context of cancer, mutations to tumor Similar to the findings reported here, deletion of Nf1, Tp53, along with or without Pten at adult stages, produces glioma tumors in the brains of mice using multiple neural stem/progenitor cell type specific Cre drivers (Alcantara Llaguno et al., 2009;Alcantara Llaguno et al., 2015;Zhu et al., 2005). Tumors were induced from neural progenitors in the SVZ or OPCs, leading to the formation of two types of glioma tumors (Alcantara Llaguno et al., 2015). Type 1 tumors were found in dorsal/anterior brain regions such as striatum, hippocampus, and cortex, are highly infiltrative and aggressive, and express high levels of GFAP. Type 2 tumors, on the other hand, were found more in ventral/posterior brain regions such as the diencephalon and brainstem, exhibit well-defined boundaries, and express high levels of OLIG2 and PDGFRα. Based on difference in gene expression, Type 1 tumors are speculated to be derived from neural stem/progenitor cells in the SVZ, whereas Type 2 tumors are likely to be derived from OPCs. In the glioma model used here, in which Cre is driven in neural progenitors at embryonic stages, over 90% of the mouse glioma tumors were found in the telencephalon, predominantly in the cortex and striatum, suggesting that they may be similar to Type 1 tumors.
These tumors express high levels of GFAP, OLIG2, SOX10, NG2, and PDGFRα, although we show that the majority of GFAP expressing cells do not colocalize with the Cre-reporter, which directly marks the oncogenic cells. Whether this is also true for the Type 1 tumors described is not known since direct colocalization with GFAP was not assessed.
In conclusion, the tumors induced embryonically through deletion of Nf1;Tp53 deletion are highly heterogenous based on RNA-seq analysis, which is similar to that seen for human GBMs Sottoriva et al., 2013). This intertumor heterogeneity is likely the result of different tumors being spontaneously derived from different cell-of-origins in the various brain regions, and are thus exposed to different microenvironments such as microglia, reactive astrocytes, and immune cells, which are known to infiltrate tumor bulk and are included in the RNA-seq. Despite this heterogeneity, however, the loss of ASCL1 still significantly delays tumor progression and resulted in a significant increase in survival for mice with Ascl1 CKO tumors over those mice with Ascl1 WT tumors, illustrating an important role for ASCL1 in controlling the rate of glioma proliferation in vivo. A fundamental question remaining for future studies is whether ASCL1 and other transcription factors involved in tumor formation are similarly required for maintenance of glioma growth in the brain, and how much these transcription factors may contribute to glioma recurrence, if any, following multimodal treatments.

ACKNOWLEDGMENTS
We acknowledge Lauren K. Tyra, Erin Kibodeaux, Juan Villareal, and Trisha Savage for excellent mouse genotyping and husbandry.
We thank Dr. Francois Guillemot for Ascl1 flox mice and Dr. Luis Grant NCI P30CA118100 and made use of the Fluorescence Microscopy and Cell Imaging shared resource.

CONFLICT OF INTEREST
The authors declare no potential conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study will be made available in a repository once the manuscript is accepted for publication.