A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation

Abstract

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1^R132H-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Fig. 1.. rs55705857-G is the causal glioma risk variant at 8q24.

(A) Fine-mapping of the 8q24 tag SNP allowed the discovery of rs55705857 with much lower allele frequency and much higher effect size than the originally discovered tag SNP. Of the 16 IDH mutant risk SNPs listed, rs55705857 has an OR high enough to have an effect near that of familial inheritance glioma genes. (B) Fine-mapping of the minimal-risk haplotype region surrounding the IDH-mutant glioma risk SNP rs55705857. Subjects heterozygous for the risk haplotype and with meiotic crossovers disrupting the risk haplotype fall into four groups: two including the minimal overlap region (groups B and C) and two not including the minimal overlap region (groups A and D) (red, 55 cases; blue, 22 controls). (C) Gene transcripts, conservation between human and mouse, and chromatin status surrounding rs55705857 are displayed. The red vertical line denotes the location of rs55705857. ATAC-seq data for the 8 IDH-WT and 13 IDH-mutant brain tumors and skin cutaneous melanoma (SKCM) are aligned with DiffBind log₂ fold change for H3K27ac and H3K4me1 when comparing IDH-mutant tumors against IDH-WT brain tumors. ChromHMM shows the predicted function of the genome surrounding rs55705857 on the basis of the histone marks H3K36me3, H3K4me1, H3K27ac, and H3K4me3 in IDH-WT and IDH-mutant brain tumors as well as nontumor gliosis samples sorted by rs55705857 nonrisk (A) and risk (G) alleles. (D) Comparison of GSEA results using 50 hallmark gene sets comparing IDH-mutant noncodel, IDH-mutant codel, or IDH-WT tumors versus gliosis and IDH-mutant noncodel rs55705857-G versus A allele tumors. Only gene sets with an FDR q ≤ 0.05 in at least one comparison are included and colored in the heatmap; the darker reds and blues have an FDR q < 0.001. Bottom panel shows GSEA of the indicated glioma MYC target gene signatures across the different glioma subtypes.

Fig. 2.. rs55705857 SNP resides in a brain-specific enhancer.

(A) Schematic of rs55705857 LacZ enhancer reporter construct. (B) Representative whole-mount images of LacZ-stained rs55705857 nonrisk (left) and risk (right) enhancer reporter embryos. Black arrows denote LacZ staining found in both reporter mice, while red arrows indicate LacZ staining only found in risk reporter mice. (C) Summary for enhancer activity of the nonrisk and risk allele. P-value by Fisher-Freeman-Halton exact test. n.s., not significant. (D) Representative immunofluorescent image of a sagittal section of an rs55705857-G risk allele mCherry enhancer reporter embryo at E14.5 stained for mCherry and the radial glial marker Sox2. The pontomedullary hindbrain is shown and arrows depict mCherry/Sox2 double-positive cells. Scale bars, 50 μm. Similar staining patterns were observed in the ventricular zone of the forebrain.

Fig. 3.. Idh1-mutant LGG mouse model.

(A) Schematic of conditional alleles and CRISPR virus used to generate the LGG cohorts. (B) Survival of mice with the indicated genotype transduced with an sgRNA targeting Atrx or a scrambled control sgRNA (Scr). n = 201 mice; P < 0.0001, log-rank (Mantel-Cox) test. (C) Bar graph indicating percentage of phenotypes found in mice from (B) with the indicated genotype. (D) Representative hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining of the same tumor region within a Idh1^R132H/+;Trp53^R270H/+;Atrx^−/−;Cas9-GFP brain using the indicated antibodies. Scale bars, 2.5 mm (left) and 50 μm (right).

Fig. 4.. rs55705857 cooperates with Idh1, Trp53, and Atrx mutations.

(A) Survival of mice with the indicated genotype transduced with an sgRNA targeting Atrx or a scrambled control sgRNA (Scr). n = 123 mice; P < 0.0001, log-rank (Mantel-Cox) test. (B) Bar graph indicating percentage of phenotypes found in mice from (A). (C) Representative H&E and IHC of the same tumor region within a rs557^66bp/+;Idh1^R132H/+;Trp53^fl/fl;Atrx^−/−; Cas9-GFP brain using the indicated antibodies. Scale bars, 2.5 mm (left) and 50 μm (right). (D) Survival of Nod/Scid/γ mice intracranial injected with rs557^66bp/+;Idh1^R132H/+;Trp53^{∆/ ∆};Cas9-GFP RIP cells. n = 17 mice.

Fig. 5.. rs55705857 modulates OCT2 and OCT4 binding and regulates MYC expression.

(A) The canonical OCT2/4 binding motif (top) and the rs55705857 nonrisk A allele (middle) and the rs55705857 risk G allele (bottom) are shown. (B) Sequence alignment of the human rs55705857 and its orthologous mouse locus highlighting conserved binding motifs for ASCL1/2, OCT2/4, and SOX2/4/9. The nonrisk rs55705857-A allele is marked in red. Asterisks indicate conserved amino acids. (C) Enrichment of OCT2 and SOX2 at mouse rs55705857 locus. (Top) ChIP-qPCR using rs557^66bp/+;Idh1^R132H/+;Trp53^∆/∆; Cas9-GFP RIP cells (n = 4). Immunoglobulin G (IgG) serves as a negative control and histone H3 as a positive control. P-value by two-tailed t test. (Bottom) Representative gel electrophoresis analysis of PCR amplicons from IgG, SOX2, OCT2, and H3 ChIP. (D) Enrichment of OCT4 at the mouse rs55705857 locus. (Top) ChIP-qPCR using rs557^66bp/+;Idh1^R132H/+; Trp53^∆/∆;Cas9-GFP RIP cells transfected with a V5-tagged OCT4 performed using an OCT4 and an anti-V5 antibody. IgG serves as a negative control, and histone H3 as a positive control. P-value by two-tailed t test. (Bottom) Representative gel electrophoresis analysis of PCR amplicons from IgG, H3, OCT4, and V5 ChIP. (E) The risk allele G of rs55705857 disrupts OCT binding. (Top) ChIP-qPCR showing enrichment of OCT2 at rs55705857 locus of human LGG cells heterozygous for the rs55705857 risk allele. IgG-IP serves as a negative control and histone H3 as a positive control. P-value by two-tailed t test. (Bottom) Sanger sequencing chromatograms of the SNP region from input, histone H3, and OCT2 ChIPed DNA. (F and G) Myc mRNA (F) and Myc protein (G) expression in rs55705857 AA, AG, and GG NSCs and NSC-derived OPCs. P-value by two-tailed t test. (H) Genome architecture mapping (GAM) contact matrix of the chr15:61,500,000–64,500,000 genomic window showing strong interaction between Myc and the rs55705857 locus in mouse oligodendrocytes and their precursor cells (OLGs) in the somatosensory cortex. (I) Analysis of high-frequency interacting regions at the Myc locus in rs55705857 WT versus A→G neuronal stem cells by 4C-seq. The heatmap color scale shows normalized median contact frequency. The black trendline shows the median contact frequency, and the shaded gray area indicates the 20th to 80th percentiles. The light-blue line marks the location of rs55705857, and the shaded gray box marks the location of Myc.