Succinate dehydrogenase mutation underlies global epigenomic divergence in gastrointestinal stromal tumor

Abstract

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance.

Figure 1

A, Histomorphology of GISTs and comparison reference tissues. B, unsupervised PCA (left) and hierarchical clustering (right) sharply segregate SDH-mutant (SDHA, B, C, or D) and kinase-mutant (KIT, PDGFRA, BRAF, or NF1) GIST samples and reveal marked methylation in the SDH mutants. SDH mutation is also correlated with a nearly normal genomic copy number (G-CNN), whereas kinase mutation is correlated with a copy number abnormal genome (G-CAN). PCA and heatmaps were derived from the 476 autosomal targets remaining after a methylation β variance filter across the 63 GIST samples was set to 0.5. C, unsupervised PCA (left) and hierarchical clustering (right) of GISTs and normal reference tissues reveal distant divergence of SDH-mutant GIST. PCA and heatmaps were derived from the 552 autosomal targets remaining after a methylation β variance filter across the 109 samples was set to 0.5. mut, mutant.

Figure 2

Visual display of the proportions of hyper- and hypomethylated differentially methylated targets (DMT) relative to reference muscularis tissue in SDH- versus kinase-mutant GIST. A, 2-D hierarchical clustering of 24 SDH-mutant GIST (dark blue) and 10 reference muscularis samples (cyan); CpG targets (y-axis) are filtered for significant DMT between the 2 sample groups (GoldenGate methylation assay, group delta β > 0.1 and P < 0.05, n = 666 targets). Of the DMT, 457 are hypermethylated and 209 are hypomethylated in SDH-mutant GIST. B, 2-D hierarchical clustering of 39 kinase-mutant GIST (green) and 10 reference baseline muscularis samples (cyan); CpG targets are filtered for significant DMT between the 2 sample groups (GoldenGate methylation assay, group delta β > 0.1 and P < 0.05, n = 222 targets). Of the DMT, 19 are hypermethylated, and 203 hypomethylated, in kinase-mutant GIST. C, 2-D hierarchical clustering of the union sets of all tissues [SDH-mutant GIST (n = 24), kinase-mutant GIST (n = 39), and reference muscularis (n = 10)] and DMT (n = 748 targets) from A and B. D, hyper- and hypomethylated DMT composition for SDH-deficient versus kinase-mutant GIST (Infinium 450 K methylation data). E, 2-D hierarchical clustering of SDH-deficient GIST (n = 26, dark blue) and reference muscularis tissues (n = 7, cyan) with Infinium 450 K methylation data; targets included on the y-axis are the top hyper- and hypomethylated DMT selected in proportion to their fraction of total DMT (DMT defined as group delta β > 0.1 and P < 0.05. Displayed on the heatmap is the union set of 1.7 K hyper- and 1.3 K hypomethylated DMT, which reflects their proportions of total DMT). F, 2-D hierarchical clustering of kinase-mutant GIST (n = 44, green) and reference muscularis tissues (n = 7, cyan) with Infinium 450 K methylation data; targets included on the y-axis are the top hyper- and hypomethylated DMT in proportion to their fraction of total DMT (DMT defined as group delta β > 0.1 and P < 0.05. Displayed on the heatmap is the union set of 0.3 K hyper- and 2.7 K hypomethylated DMT, which reflects their proportions of total DMT). G, fold enrichment of genomic annotations of hypermethylated targets in SDH-deficient GIST based on their proportion of all array targets. CpG target anatomic and functional annotations are according to the Illumina Infinium 450 K methylation assay manifest. * DNase hypersensitive site (DHS) is the only significantly enriched annotation (P = 0.0022) after Bonferroni correction. TSS200: within a distance of 200 bp of transcription start site. UTR, untranslated region. H, representative IHC results for 5-hmC⁺ (left) and 5-hmC loss in GIST tumors (right). Sixteen of 24 methyl-divergent GIST had 5-hmC loss, versus 1 of 12 methyl-centrist.

Figure 3

Human SDH-deficient tumor genotype-epigenotype validation model. A, histomorphology of pheochromocytoma (pheo), paraganglioma (pgl), and comparison reference adrenal gland. B, left, PCA segregates pgl/pheo according to oncogenotype and reveals greater divergence from baseline adrenal for SDH mutants. The plot includes 20 SDH-mutant pgl/pheo, 9 SDH-wt pgl/pheo, and 15 reference adrenal specimens. The PCA plot data are 340 autosomal targets filtered for methylation β variance > 0.5 among the 44 samples. C, number of significant array target hyper- and hypomethylations as a function of tumor SDH mutation status (group delta β > 0.1 and P < 0.05). There are substantially more target hypermethylations in both SDH-mutant GIST and pgl/pheo compared with wild-type counterparts. D, 2-D hierarchical clustering of the 306 significant DMT between SDH-mutant pgl/pheo and adrenal reference (group delta β > 0.1 and p < 0.05). E, 2-D hierarchical clustering of the 303 significant DMT between SDH-wild-type pgl/pheo and adrenal reference (group delta β > 0.1 and P < 0.05). F, hierarchical clustering of 20 SDH-mutant pgl/pheo, 9 wild-type pgl/pheo, and 15 reference baseline adrenal samples based on the 340 union targets from D and E. mut, mutant; wt, wild-type.

Figure 4

Comparison of isocitrate dehydrogenase (IDH)-mutant glioma with SDH-mutant GIST. A, histomorphology of high-grade glioma, low-grade glioma, and comparison reference glial tissue. (Reference neuronal tissue is previously shown in Fig. 1A.) B, left, PCA segregates glial neoplasms according to oncogenotype, and reveals greater divergence from baseline glia for IDH mutants. The plot includes 7 IDH1-mutant glial tumors, 20 IDH-wt glial tumors, and 12 glia and 13 neuronal reference tissues. The PCA plot data are 386 autosomal targets filtered for methylation β variance > 0.5 among the 46 samples (GoldenGate methylation data). Right: Unsupervised 2-D hierarchical clustering of the same data. C, left, hypomethylated DMT (group delta β > 0.1 and P < 0.05, n = 140 targets) identified in IDH-mutant glioma relative to reference glial tissue. Right, hypermethylated DMT (group delta β > 0.1 and P < 0.05, n = 388 targets) identified in IDH-mutant glioma relative to reference glial tissue (GoldenGate methylation data). D, unsupervised hierarchical clustering of all tumors in the study. Top colorbar: tumor type; bottom colorbar: oncogenotype. The y-axis data are 575 autosomal targets filtered for methylation β variance > 0.5 among the 113 samples. Oncogenotype drives higher level segregation. Also evident is the marked hypermethylation of SDH/IDH-mutant tumors of different lineage and anatomic sites. E, unsupervised PCA plot of 186 study samples annotated as normal tissue, SDH/IDH-mutant tumor, or SDH/IDH-wt/kinase-mutant tumor (var 0.5, 649 targets). F, quantities of significant hyper- and hypomethylated DMT in different tumor lineages as a function of mutant versus wt SDH/IDH status. mut, mutant; wt, wild-type.