A novel mouse model of diffuse midline glioma initiated in neonatal oligodendrocyte progenitor cells highlights cell‐of‐origin dependent effects of H3K27M

Abstract Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells‐of‐origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv‐a avian retroviral system to generate DMG in Olig2‐expressing progenitors and Nestin‐expressing progenitors in the neonatal mouse brainstem. PDGF‐A or PDGF‐B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M‐positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF‐A and PDGF‐B‐driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA‐sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non‐overlapping between Olig2;PDGF‐B, Olig2;PDGF‐A, and Nestin;PDGF‐A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell‐of‐origin dependent with H3.3K27M promoting epithelial‐to‐mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell‐of‐origin and inhibiting EMT and angiogenesis when Nestin marks the cell‐of‐origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Via_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell‐of‐origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.

resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M-positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF-A and PDGF-B-driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA-sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non-overlapping between Olig2;PDGF-B, Olig2;PDGF-A, and Nestin;PDGF-A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell-of-origin dependent with H3.3K27M promoting epithelial-to-mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell-of-origin and inhibiting EMT and angiogenesis when Nestin marks the cell-of-origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Vi a_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell-of-origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.

K E Y W O R D S
diffuse intrinsic pontine glioma, diffuse midline glioma, H3K27M, oligodendrocyte progenitor cells

| INTRODUCTION
Diffuse midline glioma (DMG) accounts for 15%-20% of pediatric brain tumors and represents the leading cause of death in children with brain tumors (Buczkowicz & Hawkins, 2015). The critical location and diffuse nature of the tumor precludes surgical resection, and radiotherapy offers only transient symptomatic relief. Despite numerous clinical trials, no chemotherapy approach has prolonged the survival of children with DMG. The failure of these trials is partly due to the adaptation of established treatments for adult glioma, which have been evaluated in adult patients, for children (Hargrave et al., 2006).
Approximately 85% of DMG contain the somatic gain-of-function K27M mutation in histones H3.1 and H3.3 but these mutations are extremely rare in adult gliomas (Bender et al., 2013;Chan et al., 2013;Schwartzentruber et al., 2012;Wu et al., 2012). Nearly 60% of the K27M mutations in DMG involve a single allele in H3F3A encoding H3.3 . H3K27M mutations are thought to drive DMG formation (Chan et al., 2013;Schwartzentruber et al., 2012;Wu et al., 2012). The H3K27M mutation reduces global histone H3K27 methylation (H3K27me3) by inhibiting polycomb repressive complex 2 (PRC2) activity (Lewis et al., 2013). As H3K27me3 primarily correlates with gene repression, H3K27M mutations result in a net gain in cellular transcriptional activity (Chan et al., 2013;Funato et al., 2014;Lewis et al., 2013). However, for certain genes such as the tumor suppressor p16 or Ink4a, associated H3K27me3 is retained or even increased in some experimental models resulting in increased gene repression (Bender et al., 2013;Brien et al., 2021;Cordero et al., 2017;Mohammad et al., 2017). Thus, the mutation's effects on H3K27 methylation and gene expression are aimed at a combination of gene activation and suppression that collectively promote tumor growth.
Others and we have previously shown that the oncogenic properties of the H3.3K27M mutation are dependent on the glioma cell-oforigin. For example, in Nestin+ neural progenitor cells (NPCs), H3.3K27M combined with platelet-derived growth factor B (PDGF-B) expression and p53 loss induces DMG, alters gene-expression, and reduces the survival of tumor-bearing mice (Cordero et al., 2017;Lewis et al., 2013). But an effect on tumor latency is not seen with Pax3+ cells, which include neurons and undifferentiated progenitors (Misuraca et al., 2016). Further, H3.3K27M increases the proliferation of neural stem cells derived from human embryonic stem cells, but not so for early astrocytes (Funato et al., 2014). Recent studies have implicated oligodendrocyte progenitor cells (OPCs) of the neonatal brainstem as the likely cell-of-origin for DMG (Filbin et al., 2018;Lindquist et al., 2016;Nagaraja et al., 2017;Tate et al., 2015), consistent with prior studies showing a spatiotemporal correlation between Olig2+ precursor proliferation in human and mouse brainstem and brainstem glioma occurrence in children (Monje et al., 2011).
Single cell RNA-sequencing (RNA-seq) analysis has revealed that the majority of the cells in H3K27M gliomas express OPC-like transcriptomes (Filbin et al., 2018). The most active promoter enhancers in H3.3K27M tumor cells are associated with genes of the oligodendroglial lineage (Nagaraja et al., 2017), and OPCs are the most proliferative precursor cell-type in the neonatal ventral pons, coinciding spatiotemporally with the development of midline gliomas (Lindquist et al., 2016). Despite these findings, the characteristics of H3.3K27Mbearing diffuse gliomas initiated in OPCs are not known.
Here, we describe a novel genetically engineered mouse model (GEMM) of diffuse intrinsic pontine glioma (DIPG, a subset of DMG that occurs in the pons) initiated in neonatal Olig2+ progenitors. We used the RCAS/Tv-a avian retroviral system to introduce gene alterations that are commonly found in human DIPG (H3.3K27M, PDGF-B, or PDGF-A to activate PDGFRA signaling, and p53 inactivation) (Khuong-Quang et al., 2012;Mackay et al., 2017;Wu et al., 2012) in Olig2+ progenitors. The resulting diffuse midline high grade gliomas exhibited histological and immunophenotypical characteristics such as infiltration and leptomeningeal dissemination that closely resemble the human disease. Surprisingly, H3.3K27M expression had only a minor effect on tumor latency and no significant effect on proliferation relative to H3.3WT tumors, in contrast with H3K27M tumors originating in Nestin+ cells. GSEA analysis revealed that H3.3K27M has cell-of-origin dependent effects on key gene-sets with known roles in tumorigenesis. For example, H3.3K27M mutant tumors were significantly positively enriched for EMT and angiogenesis when Olig2 marks the cell-of-origin but H3.3K27M tumors were negatively enriched for angiogenesis and EMT when nestin marked the cell-of-origin. Thus, our study provides a novel DIPG mouse model initiated in Olig2+ OPCs that can be used for mechanistic studies and preclinical evaluation of novel therapies for DIPG. Our results highlight cell-of-origin dependent effects of H3.3K27M with a more oncogenic role for H3K27M in Nestin+ cells relative to Olig2+ cells.
The genomic structure and characteristics of these mice have been previously reported (Schüller et al., 2008). Otv-a-Cre mice were intercrossed with conditional null p53 mice (Jackson Labs, C57BL/6J background) to generate Otv-a-Cre;p53 fl/fl and Otv-a-Cre;p53 fl/+ mice, as previously described (Mehta et al., 2011). Nestin-Tv-a;p53 fl/fl (Ntv-a;p53 fl/fl ) mice were created by crossing Ntv-a and p53 fl/fl mice (Barton et al., 2013). Nestin-CFPnuc mice express cyan fluorescent protein (CFP) fused to a nuclear localization signal under the control of the Nestin enhancer (Encinas et al., 2006;Misuraca et al., 2016). All animals were maintained in accordance with the Northwestern University Animal Care and Use Committee guidelines (animal protocol IS00012699).

| Immunofluorescence
Nestin-CFPnuc mice were euthanized at P3, brains were extracted and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h and cryopreserved in 30% sucrose in PBS for 24-48 h. After embedding in OCT compound, blocks were sectioned using a Cryostat (TN50, Tanner Scientific, Inc.) into 10 μm thick sagittal or oblique coronal sections to visualize the whole pons (Lindquist et al., 2016). Sections were rehydrated in PBS with 0.1% Triton X-100 (PBS-T).

| Tumor grading
Tumor tissue was harvested, fixed in 20% formalin for 24-48 h, and embedded in paraffin by the Northwestern University Mouse Histology and Phenotyping Laboratory. Embedded tissue was cut into 5 μm sections using a Leica RM2235 microtome. Hematoxylin and eosin (H&E) staining was performed using standard protocols. Tumors were graded by a blinded neuropathologist using the following criteria according to the WHO classification 2016 (Louis et al., 2016): Grade II glioma by moderately increased cellular density without mitosis; Grade III glioma by increased cellular density with mitosis; and Grade IV glioma by the presence of microvascular proliferation and/or pseudopalisading necrosis. WHO Grade III and IV tumors were defined as high-grade. Tumor dissemination was defined as tumor aggregation, existing separately from the main tumor mass (Wagner et al., 2006), in the subependymal spaces of the lateral ventricle or the subcortical space.

| Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was isolated using RNeasy kit (Qiagen) and cDNA synthesized using Superscript IV and OligodT primers (Invitrogen) per manufacturers' instructions. Real-time detection and quantification of cDNA were performed with the QuantiStudio™ 6 Flex kit (Applied Biosystems, #4485691). qRT-PCR was done using Power SYBR Green qPCR Master mix (Applied Biosystems, #4367659). Gene expression levels were normalized to that of endogenous beta-actin. All experiments were done in triplicates, and relative gene expression levels calculated using the ΔΔCt method. Primer sequences are listed in Table S2.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism (Version 9.1.0) for all data. Survival rate was estimated using Kaplan Meier Curve analysis and a statistical significance was evaluated by log-rank test. Continuous variables are expressed as mean ± standard deviation (SD) or median (interquartile range: IQR). Categorical variables are expressed as n (%). Continuous variables were compared using Mann-Whitney U test, Student's t test, or one way ANOVA with Tukey's post hoc test. Categorical variables were compared using Fisher's exact tests.

| The majority of Olig2+ in the neonatal brainstem co-express Sox2
We have previously described H3.3K27M glioma initiated in Nestin+ cells in the murine ventral pons (Cordero et al., 2017). Several studies, however, suggest that DIPG likely originates from Olig2+ cells with OPC-like features (Anderson et al., 2017;Filbin et al., 2018). As cell clusters with OPC-like features express both Nestin and Olig2 , we first evaluated the expression patterns of Olig2 and Nestin, and the extent of overlap of these markers, in the developing mouse brainstem. We immunolabeled sagittal and oblique brain sections (Lindquist et al., 2016)
H&E-stained brain sections revealed increased necrosis, microvascular proliferation, mitosis, and higher overall cell density both in 3.3 | H3.3K27M does not impact tumor latency or proliferation of PDGF-A-driven DIPG initiated in neonatal Olig2+ cells PDGF-B overexpression has been widely used in mouse models of DIPG (Barton et al., 2013;Becher et al., 2010;Misuraca et al., 2016). However, recently published molecular meta-analysis shows that PDGF-B is rarely upregulated and/or amplified in human DIPG (Paugh et al., 2011;Puget et al., 2012), although PDGFRA alterations are frequently observed (Mackay et al., 2017). Therefore, we generated a GEMM of DIPG by overexpressing PDGF-A to selectively activate the PDGFRA signaling pathway (Westermark et al., 1995). Otv-a-Cre;p53 fl/fl ;PDGF-A;H3.3K27M mice had signi- There was no correlation between survival and sex based on cox hazard regression analysis (Table S3). All the infected mice developed intracranial tumors, similar to the PDGF-B-based model, and the majority of the PDGF-A-driven tumors were observed in the brainstem (Table S5) There was a significant correlation between H3.3K27M mutation and survival time based on cox hazard regression analysis (  Therefore, we stained H3.3K27M-tumor tissues initiated in Otv-a-Cre;p53 fl/fl mice with the microglial marker Iba1 to determine whether the difference in H3K27M positivity between PDGF-A and PDGF-B tumors is due to a difference in the microglial population (Hoeman et al., 2019;Ross et al., 2020), but found no significant difference between these two groups (PDGF-B, 10.5 ± 4.3%, PDGF-A, 7.9 ± 2.7%, p = .0835, Student's t test) ( Figure S4A,B). Because exogenous H3K27M is tagged to GFP in our mouse models, we also stained tumor tissues with an antibody against GFP. The fraction of GFP+ cells was higher than that of H3K27M+ cells in all the tumor models except for Ntv-a;p53 fl/fl mice with PDGF-A induction (Otv-a-Cre;  Table S7). We performed qRT-PCR analysis of all of these genes, but only Gm2694 and Egr4 were significantly downregulated ( Figure S5D). Interestingly, GSEA analysis showed significant positive enrichment in oligodendrocyte markers and myelin sheath ( Figure S5E.F, Table S8). Thus, H3.3K27M had a modest effect on the transcriptome of PDGF-B-induced DIPG relative to H3.3WT when initiated in Olig2+ cells, although the heterogeneity in tumor locations might be a contributing factor (Table S4).
Next, we performed RNA-seq analysis of PDGF-A-induced tumors from Otv-a-Cre;p53 fl/fl mice expressing H3.3WT or H3.3K27M  (Mikkelsen et al., 2007) in H3.3K27M tumors relative to H3.3WT. NES, normalized enrichment score and principal component analysis showed that H3.3K27M tumors overlapped partially with H3.3WT tumors (Figure 6b,c). Gene ontology using DAVID software indicated that the upregulated genes were enriched in several processes including "cell adhesion" and "cell proliferation" (Figure S6). Using normalized expression data, we conducted GSEA analysis to further examine the effects of H3.3K27M on the transcriptome focusing on features of DMG cells including cell proliferation, epithelial-to-mesenchymal transition (EMT), and angiogenesis (Table S10). We identified a significant correlation between H3.3K27M and upregulation of genes characterized by H3K27 trimethylation and high-CpG-density promoters (Mikkelsen et al., 2007) (Figure 6d).  (Table S12)

| DISCUSSION
Several studies have suggested that neonatal OPCs are a likely cell-oforigin for DIPG (Filbin et al., 2018;Monje et al., 2011). Single cell RNA-seq of human DIPG samples has shown that H3K27M DIPG tumors primarily contain OPC-like cells (Filbin et al., 2018). The basic helix-loop-helix transcription factor Olig2 is widely expressed in proliferating cells in the pons (Tate et al., 2015) and is highly expressed in 70%-80% of DIPG samples (Puget et al., 2012). Anderson et al. have shown that only DIPG cell lines retaining Olig2 could form robust Olig2-positive brainstem glioma (Anderson et al., 2017). We and others have previously published DIPG models arising from Nestin+ progenitors (Cordero et al., 2017;Larson et al., 2019), which also include OPCs and other progenitor cell populations . But there are no GEMMs characterizing midline gliomas initiated in OPCs. Here, we established GEMMs of DIPG initiated in Olig2+ cells.
We generated a murine DIPG model by overexpressing H3.3K27M and PDGF-B or PDGF-A, and by incorporating p53 loss into Olig2+ cells. Surprisingly, our data show that H3.3K27M has almost no effect on mice survival or cell proliferation relative to H3.3WT. In addition, we and p53 inactivation in an orthotopic xenograft model (Haag et al., 2021). This is also in agreement with our previous work showing an oncogenic effect of H3.3K27M when combined with PDGF-B and p53 loss in Nestin+ progenitors (Cordero et al., 2017). Mackay et al.
previously described the genomic profiles of 157 cases of pediatric high-grade glioma and showed that H3.3K27M coexisted with PDGFRA amplifications in many of the ventral pons tumors (Mackay et al., 2017).
Also, Haag et al showed that many OPC-like genes were upregulated in human NSCs with H3.3K27M induction (Haag et al., 2021). If the pri-  (Buczkowicz et al., 2014). Also, H3.3K27M significantly altered the transcriptome and upregulated genes characterized by H3K27me3, relative to H3.3WT ( Figure 6). This suggests that H3.3K27M impacts gene-expression in DIPG initiated in Olig2+ cells, but its effects on tumorigenesis are difficult to discern with relatively crude measurements such as histological and survival analyses.
The number of significant differentially expressed genes between H3.3K27M mutant tumors and H3.3WT control tumors was relatively small in our analysis relative to analysis in the human disease. Our data showed that H3.3K27M significantly changed the expression of 907 genes in Otv-a-Cre;p53 fl/fl mice model, which was still much smaller than the number of significantly differentially expressed genes altered by H3.3K27M in human DIPG specimens.