Functional loss of a noncanonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation

Abstract

Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (Bcor^ΔE9-10 ) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCOR^ΔE9-10). While Bcor^ΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, Bcor^ΔE9-10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1^+/- ;Bcor^ΔE9-10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1^+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1^+/- ;Bcor^ΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

Keywords: BCOR; PRC1.1 complex; brain tumor; cerebellar granule cells; medulloblastoma; mouse model.

Figure 1.

Conditional Bcor mouse model is an appropriate model for human BCOR mutations. (A) BCOR mutations identified in pediatric medulloblastoma samples. (B) BCL6-binding domain; (NonAnkRep) nonankyrin repeats; (Ank) Ankyrin repeats; (PUFD) PCGF Ub-like fold discriminator domain; (maroon lollipop) nonsense mutation; (pink lollipop) frame shift mutation; (green lollipop) point mutation. (B,C) In situ hybridization (dark blue) of antisense Bcor probe (B) and Ccnd2 probe (C) at P7 in Bl6N wild-type (WT) mice. Scale bar, 50 μm. (oEGL) Outer external granule layer; (iEGL) inner external granule layer; (ML) molecular layer. Nuclei counterstained with methyl green. (D,E) In situ hybridization of antisense Bcor probe at P28 in WT (D) and in Ptch1^+/− (E) mice. Scale bar, 200 μm. (IGL) Inner granule layer; (ML) molecular layer; (PNC) preneoplastic cells. Nuclei counterstained with methyl green. (F) Increased magnification of preneoplastic region in E. Scale bar, 100 μm. (G, left) Immunohistochemistry using an anti-BCOR antibody (pink) at P7. (Right) DAPI stains nuclei in blue (merged). Maximum intensity projection of 16-μm-thick cerebellar section. Scale bar, 10 μm. (H) Immunohistochemistry using an anti-BCOR antibody (pink) and an anti-pH3 antibody (yellow) at P28 in the preneoplastic lesion of Ptch1^+/− animals. DAPI stains nuclei in blue. Single z-slice. Scale bar, 100 μm. (PNC) Preneoplastic cells; (IGL) inner granule layer. (I) Increased magnification of boxed region in H. Scale bar, 20 μm. (J) Relative mRNA expression of Bcor and Ccnd2 (quantitative PCR) of P7 granule neuron progenitors cultured in the absence (gray) or presence (blue) of Smoothened agonist (SAG). (*) P < 0.05; (***) P < 0.001, Student's t-test. Bars represent mean ± SEM. N = 6. (K) Location of LoxP sites in Bcor. Expression of Cre removes exons 9 and 10, and leads to an early translation stop. Color scheme for Bcor domains same as in A. (L) anti-BCOR Western blot of purified granule neuron progenitors in wild-type (Atoh1-Cre only) cells and in Bcor^{floxed/floxed} + Atoh1-Cre (Bcor^ΔE9–10) cells. Anti-ACTIN was used as the loading control. See also Supplemental Figures S1 and S2.

Figure 2.

Bcor^ΔE9–10 significantly reduces latency and increases penetrance in Ptch1-driven medulloblastoma. (A) Survival curve of Bcor^ΔE9–10 (black; N = 10), Ptch1^+/− (blue; N = 34), and Ptch1^+/−;Bcor^ΔE9–10 (red; N = 30) animals. (B,C) Representative H&E staining of Ptch1^+/− tumor (B) and Ptch1^+/−;Bcor^ΔE9–10 tumor (C). Notice typical round cells of classic histology (B) versus larger, pleomorphic nuclei of anaplastic histology (C). Scale bar, 10 μm. (D) Survival curve of immunodeficient animals transplanted with 8 × 10⁵ cells from Ptch1^+/− (blue; N = 13) and Ptch1^+/−;Bcor^ΔE9–10 (red; N = 13) tumors. (E,F) Representative examples of β-galactosidase staining (lacZ present in Ptch1 locus) of P28 Ptch1^+/− cerebellum (E) and Ptch1^+/−; Bcor^ΔE9–10 cerebellum (F). (Red arrows) Preneoplastic lesions. (Inset) Magnified view of preneoplastic lesion. Scale bar, 1000 μm. (G,H) Increased magnification of β-galactosidase staining similar to E and F. Preneoplastic lesions are noted by arrowheads and dotted lines. Scale bar, 500 μm. (I) Quantification of the number of preneoplastic lesions in E and F. (*) P < 0.05, Student's t-test. Bars represent mean ± standard deviation. N = 3 cerebella per genotype.

Figure 3.

Igf2 is overexpressed in Ptch1^+/−; Bcor^ΔE9–10 tumors. (A) Volcano plot of differentially expressed genes between Ptch1^+/− and Ptch1^+/−;Bcor^ΔE9–10 tumors. (B) Relative mRNA expression of Igf2 (qPCR) of P7 GNPs (gray) in the indicated genotypes, Ptch1^+/− tumors (light blue), and Ptch1^+/−;Bcor^ΔE9–10 tumors (dark blue). (n.s.) Not significant, Student's t-test. Bars of P7 GNPs represent mean ± SEM. N = 6 (WT, Bcor^ΔE9–10 GNPs) or N = 8 (Ptch1^+/−, Ptch1^+/−;Bcor^ΔE9–10 GNPs). (C) Anti-IGF2 Western blot of total protein extracts from P7 GNPs or tumor samples from the indicated genotypes. Anti-ACTIN was used as the loading control. (D–I) ISH of antisense Igf2 probe (dark blue) in cerebella of animals from the indicated genotypes. (IGL) Inner granule layer; (EGL) external granule layer; (ML) molecular layer; (m) meninges; (PNC) preneoplastic cells. (D,E) P7. Scale bar, 100 μm. (F,G) P28. Scale bar, 200 μm. (H,I) Adult tumor. Scale bar, 100 μm. (J) Reads per kilobase of transcript per million mapped reads (RPKM) of IGF2 from the ICGC cohort of SHH medulloblastoma samples (Northcott et al. 2017). Black samples are wild type at the BCOR locus. (K) Survival curve of immunodeficient animals transplanted with 1 × 10⁶ Ptch1^+/− GNPs transduced with retrovirus overexpressing eGFP alone (black; N = 8), Igf2-eGFP (red; N = 7), or Mycn-eGFP (blue; N = 5). (L) Representative example of resulting Igf2-eGFP tumor from K. Scale bar, 2000 μm. (M) DAPI (top left; blue), eGFP (bottom left; green), and Ki67 (top right; magenta) immunohistochemistry of Igf2-eGFP-driven tumor. (IGL) Inner granule layer; (Cb) cerebellum. Scale bar, 100 μm. (N) Increased magnification of boxed region in M. Scale bar, 50 μm. (O) Representative histology sample of Igf2-eGFP-driven tumor. Scale bar, 10 μm. (P) Relative mRNA expression of Igf2 in tumor cells from the indicated genotypes, Bars represent mean + SEM. N = 7 for Ptch1^+/−, N = 4 for Igf2-eGFP, or N = 5 for Ptch1^+/−; Bcor^ΔE9–10. See also Supplemental Figures S3 and S4 and Supplemental Tables S1 and S2.

Figure 4.

The PUFD of BCOR is required for Igf2 repression through H2AK119 Ubiquitination. (A) Coimmunoprecipitation of BCOR or BCOR^ΔE9–10 with RING1B in Ptch1^+/− and Ptch1^+/−;Bcor^ΔE9–10 tumor cells, respectively. (IgG) Immunoglobulin G control. Five percent input. (IP) Anti-RING1B. (Top blot) Anti-BCOR. (Bottom blot) Anti-RING1B. (B) Relative mRNA expression of Igf2 in two Ptch1 CRISPR tumors (gray), and two Ptch1;Bcor^ΔPUFD CRISPR-engineered tumors (blue). (C) Heat map of significant differentially bound peaks from BCOR chromatin immunoprecipitation (ChIP; yellow) between tumors from the indicated genotypes and the status of H2AK119Ub ChIP signals (red) within these genomic loci. (D) Combined BCOR and H2AK119Ub ChIP peaks in the indicated genotypes. Significantly different peak heights represented with yellow (BCOR) and red (H2AK119Ub) bars (BCOR peaks called first). Igf2 locus is represented in blue. (E) Quantification of BCOR ChIP signal at Igf2 locus. Differential peak calling with DiffBind. (*) P-adj < 0.05. (F) Quantification of H2AK119Ub ChIP signal at Igf2 locus. Differential peak calling with DiffBind. (*) P-adj < 0.05. (G) Model of PRC1.1 complex disruption. (Open lollipops) Unmethylated CpG; (TSS) transcriptional start site; (yellow dots) H2AK119Ub histone marks; (red diamonds) H3K27me3 histone marks. See also Supplemental Tables S3 and S4 and Supplemental Figure S5.