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. 2022 Sep;70(9):1681-1698.
doi: 10.1002/glia.24189. Epub 2022 May 7.

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

Yusuke Tomita  1   2 , Yosuke Shimazu  1 , Agila Somasundaram  3 , Yoshihiro Tanaka  4   5 , Nozomu Takata  6   7 , Yukitomo Ishi  1 , Samantha Gadd  8 , Rintaro Hashizume  1   3   9 , Angelo Angione  10 , Gonzalo Pinero  10 , Dolores Hambardzumyan  10 , Daniel J Brat  11 , Christine M Hoeman  1 , Oren J Becher  1   3   9   12
Affiliations

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

Yusuke Tomita et al. Glia. 2022 Sep.

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.

Keywords: H3K27M; diffuse intrinsic pontine glioma; diffuse midline glioma; oligodendrocyte progenitor cells.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

FIGURE 1
FIGURE 1
Olig2, Sox2, and nestin expression in the neonatal mouse pons. (a–h) Representative images of P3 mouse ventral (a–c, g–i) and dorsal (d–f, j–l) sagittal and oblique sections of the pons labeled with GFP (green, to visualize Nestin‐CFP), Sox2 (red), and Olig2 (white) antibodies. Images were acquired at ×10 magnification. Scale bars represent 200 μm. Insets show magnified views of the areas marked by asterisks. (m, n) quantification of Olig2+/Sox2‐/Nestin−, Olig2+/Sox2+/Nestin−, Olig2+/Sox2−/Nestin+, and Olig2+/Sox2+/Nestin+ relative to all Olig2+ cells in sagittal (m) and oblique (n) section. Mean ± SEM, n = 3–4
FIGURE 2
FIGURE 2
H3.3K27M cooperates with PDGF‐B to increase tumor malignancy and latency in Olig2+ cells. (a) Otv‐a‐Cre;p53fl/fl, (b) Otv‐a‐Cre;p53fl/+, and (c) Otv‐a‐Cre;p53+/+ mice were injected with RCAS‐PDGF‐B and RCAS‐H3.3K27M or RCAS‐H3.3WT (PK, PDGF‐B + H3.3K27M; PH, PDGF‐B + H3.3 WT). Kaplan–Meier survival curves show a significant difference in median survival between Otv‐a‐Cre;p53fl/fl;PDGF‐B;H3.3K27M and Otv‐a‐Cre;p53fl/fl;PDGFB;H3.3WT mice. N = 21 to 38, log‐rank test. (d) Representative images of tumor tissue from the indicated mouse models, stained with H&E, or immunolabeled with antibodies against Ki67, pH3Ser10, and H3K27me3. Scale bar represents 50 μm. (e) Tumor grading according to the diagnosis of diffuse astrocytoma based on WHO 2016 classification. N = 12, Fisher's exact test. (f) Tumor dissemination rates in the indicated mouse models. N = 13 to 19, Fisher's exact test. (g, h). Quantification of percentages of Ki67+ (g) and H3K27me3+ (h) cells in H3.3K27M versus H3.3WT tumors in the three p53 backgrounds. Mean ± SEM, n = 4 to 5, Student's t test, ns, not significant
FIGURE 3
FIGURE 3
H3.3K27M does not affect survival or tumor histology when co‐expressed with PDGF‐A in Olig2+ cells. (a) Kaplan–Meier survival curves of Otv‐a‐Cre;p53fl/fl mice injected with RCAS‐PDGF‐A and RCAS‐H3.3K27M or RCAS‐H3.3 WT. N = 29 to 32, log‐rank test. (b) Representative images of tumor tissue from the indicated mouse models, stained with H&E, or immunolabeled with antibodies against Ki67, pH3Ser10, and H3K27me3. (c) Tumor grading of the indicated tumors. H3.3K27M and H3.3WT tumors were both high‐grade when induced with PDGF‐A. N = 15, Fisher's exact test. (d–f) quantification of the percentages of Ki67+ (d), pH3Ser10 (e), and H3K27me3+ (f) cells in the indicated tumors. The percentage of H3K27me3+ cells was significantly reduced in H3.3K27M tumors relative to H3.3WT. Mean ± SEM, n = 11 to 16, Student's t test, ns, not significant
FIGURE 4
FIGURE 4
H3.3K27M affects survival and tumor histology when co‐expressed with PDGF‐A in Nestin + cells. (a) Kaplan–Meier survival curves of Nestin‐Tv‐a;p53fl/fl mice injected with RCAS‐PDGF‐A, RCAS‐Cre, and RCAS‐H3.3K27M or RCAS‐H3.3 WT. N = 24, log‐rank test. (b) Representative images of tumor tissue from the indicated mouse models, stained with H&E, or immunolabeled with antibodies against Ki67, pH3Ser10, and H3K27me3. (c) Tumor grading of the indicated tumors. H3.3K27M and H3.3WT tumors were both high‐grade when induced with PDGF‐A. N = 8 to 11, Fisher's exact test. (d–f) Quantification of the percentages of Ki67+ (d), pH3Ser10+ (e), and H3K27me3+ (f) cells in the indicated tumors. The percentages of Ki‐67+, pH3Ser10+ were significantly increased and the percentage of H3K27me3+ cells were significantly reduced in H3.3K27M tumors relative to H3.3WT. Mean ± SEM, n = 8 to 11, Student's t test
FIGURE 5
FIGURE 5
Nestin and PDGF‐A derived tumors express higher H3K27M levels than Olig2 and PDGF‐B derived tumors. (a) Representative IHC images of tumor tissue from the indicated mouse models, stained with H3K27M and GFP antibodies. Otv‐a‐Cre;p53fl/fl mice were injected with RCAS‐H3.3K27M‐GFP and RCAS‐PDGF‐B or RCAS–PDGF‐A. Ntv‐a;p53fl/fl mice were injected with RCAS‐H3.3K27M‐GFP, Cre and RCAS‐PDGF‐B or RCAS–PDGF‐A. (b, c). Quantification of the above IHC data showing percentages of H3K27M+ (b) and GFP+ (c) cells. PDGF‐B‐derived tumors in Ntv‐a;p53fl/fl mice had higher levels of H3K27M+ and GFP+ cells than those in Otv‐a‐Cre;p53fl/fl mice. Similar differences can be seen between PDGF‐A‐derived tumors in Ntv‐a;p53fl/fl mice relative to Otv‐a‐Cre;p53fl/fl mice. Mean ± SEM, n = 4 to 12, ANOVA with Tukey's post hoc test, ns, not significant
FIGURE 6
FIGURE 6
Transcriptomic analysis of Olig2‐initiated tumors with PDGF‐A overexpression. (a) Otv‐a‐Cre;p53fl/fl mice were injected with RCAS‐PDGF‐A and RCAS‐H3.3K27M or RCAS‐H3.3WT. Volcano plot comparing gene expression in H3.3K27M and H3.3WT tumors. There were a total of 907 significantly differentially expressed genes including 320 upregulated and 587 downregulated genes in the H3.3K27M group compared with the controls. N = 5. (b) Principal component analysis comparing Otv‐a‐Cre;p53fl/fl;PDGF‐A;H3.3K27M and Otv‐a‐Cre;p53fl/fl;PDGF‐A;H3.3WT tumors showing partial overlap. (c) Unsupervised hierarchical clustering of differentially regulated genes (p adj  <.05) between H3.3K27M and H3.3WT tumors (n = 5 each). (d) Gene ontology pathway analysis of upregulated and downregulated genes. (e–g) GSEA plots for interferon alpha response (e), interferon gamma response (f), and H3K27me3 promoters (g) (Mikkelsen et al., 2007) in H3.3K27M tumors relative to H3.3WT. NES, normalized enrichment score
FIGURE 7
FIGURE 7
Transcriptomic analysis of nestin‐initiated tumors with PDGF‐A overexpression. (a) Ntv‐a;p53fl/fl mice were injected with RCAS‐PDGF‐A, RCAS‐Cre, and RCAS‐H3.3K27M or RCAS‐H3.3WT. Volcano plot comparing gene expression of H3.3K27M and H3.3WT tumors. There were a total of 213 significantly differentially expressed genes including 123 upregulated and 90 downregulated genes in the H3.3K27M group compared with the controls. N = 7 to 9. (b) Principal component analysis comparing Ntv‐a;p53fl/fl;PDGF‐A;H3.3K27M and Ntv‐a;p53fl/fl;PDGF‐A;H3.3WT tumors showing partial overlap. (c) Unsupervised hierarchical clustering of differentially regulated genes (p adj  <.05) between H3.3K27M and H3.3WT tumors (n = 7 to 9). (d–f) GSEA plots for inflammatory response (d), mitotic spindle (e), and H3K27me3 promoters (f) (Mikkelsen et al., 2007) in H3.3K27M tumors relative to H3.3WT. NES, normalized enrichment score
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