DLST mutations in pheochromocytoma and paraganglioma cause proteome hyposuccinylation and metabolic remodeling

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Dear Editor, Of all human tumors, pheochromocytomas and paragangliomas (PPGLs) have the highest heritability rate. Over 15% of PPGLs harbor mutations in genes encoding tricarboxylic acid (TCA) cycle-related enzymes that cause oncometabolite accumulation and drive tumorigenesis via metabolic adaptation to hypoxia and global hypermethylation [1]. The dihydrolipoamide S-succinyltransferase (DLST) gene was recently described as a new PPGL susceptibility gene [2]. DLST is a component of the 2-oxoglutarate dehydrogenase (OGDH) complex (OGDHc) that catalyzes the conversion of alpha-ketoglutarate to succinyl-coenzyme A (SucCoA) in the TCA cycle. It also plays an understudied role in protein succinylation, a highly conserved post-translational modification (PTM) involving succinyl group transfer from SucCoA to protein lysine residues [3]. Succinylation causes major chemical and structural changes to proteins and has been linked to the development of diseases, including cancer [4, 5]. Here, we further characterized DLST-mutated PPGLs, explored the molecular mechanisms underlying their tumorigenesis, and examined the impact of DLST mutations on the succinylome. Transcriptome analysis was performed for PPGLs carrying the recurrent DLST-p.G374E mutation and a PPGL carrying the DLST-p.Y422C variant, whose deleterious effect was supported by a comprehensive study (Supplementary Figure S1). Hierarchical clustering revealed that DLST-mutated tumors showed a pseudohypoxic transcriptional profile (Figure 1A), supported by the expression of canonical hypoxia-related genes (Supplementary Figure S2). Conversely, unsupervised clustering of DNA methylation data separated DLST-mutated tumors from PPGLs with mutations of other TCA cycle-related genes (Figure 1B), suggesting that DLST-mutated tumors lacked a hypermethylated phenotype. Thus, we investigated alternative tumorigenic mechanisms. PPGL-causing DLST mutations remodel the cellular succinylome and up-regulate the hypoxic pathway. (A) Unsupervised clustering of transcriptomic data (z-scores) for genes expressed differentially in PPGLs (Burnichon et al. 2011) grouped DLST-mutated tumors (shown in green) with cases featuring SDH/VHL/EPAS1 (in blue, red, and purple, respectively) mutations (Cluster 1) and separately from Cluster 2 PPGLs (tumors carrying mutations in kinase signaling-related genes; in black), evidencing their pseudohypoxic nature. (B) Hierarchical clustering using DNA methylation data from a list of probes found differentially methylated in SDHB- or FH-mutated tumors showing a CpG island methylator phenotype (Ricketts CJ et al. PLoS One. 2022;17(12):e0278108), separated DLST-mutated tumors (green) from hypermethylated tumors carrying mutations in SDH genes (blue). (C) Structure of DLST's homooligomeric 24-mer. Three DLST monomers are highlighted in dark blue, yellow and purple. The upper panels show the structure of DLST WT, while the bottom left and bottom right panels show the p.G374E and p.Y422C mutants, respectively. SucCoa denotes Succinyl-CoA (only the succinyl group is shown). (D-G) Volcano plots showing the number of differentially succinylated sites (Log2FC < -1 or > 1, P < 0.05) in DLST KO (D), DLST H424A (E), DLST G374E (F), and DLST Y422C (G) cells when compared to DLST WT cells. Blue, red and grey dots represent differentially hyposuccinylated sites, differentially hypersuccinylated sites, and non-significantly succinylated sites and/or with a Log2FC between -1 and 1, respectively. (H) Representation of sites exhibiting significant differential succinylation (colored circles) in TCAc enzymes (left panel) and the Glycolysis/Gluconeogenesis pathway (right panel) when comparing DLST G374E and WT DLST cells. Both pathways exhibit global hyposuccinylation (blue color). (I) Anti-succinyllysine immunohistochemistry images of PPGLs harboring the DLST mutations p.Y422C (left top) and p.G374E (left bottom) and known mutations in other susceptibility genes as controls (right). The granular pattern and perinuclear staining observed in the images on the right suggest mitochondrial staining, whereas cells from DLST-mutated PPGLs exhibit more homogeneous cytoplasmic staining. The tumor harboring the p.Y422C DLST mutation (top left) has an intermediate pattern with fewer granules and some perinuclear staining, in accordance with proteomic succinylation data obtained using our cell model. (J) Immunofluorescence analysis of the different cell lines showing that DLST (green) colocalizes with the Mitotracker dye (red, MITO in the figure) and is thus predominantly localized in the mitochondria regardless of its mutations in all cells other than DLST KO cells lacking the DLST protein. Nuclei are stained in blue with DAPI. (K) Immunoblotting assay showing that DLST is present in the cytosol and nucleus compartments as well as the mitochondria-enriched fraction (named mitochondria in the figure) regardless of PPGL-causing mutations. OGDH levels are significantly reduced in DLST KO cells lacking DLST. GAPDH and TBP (nucleus) were used as loading controls, and COXIV as a mitochondrial marker. (L) Representation of normalized enrichment scores (NES) relative to DLST WT cells for the GSEA Hallmark gene sets of Hypoxia, Glycolysis and Oxidative phosphorylation in cells harboring DLST alterations (from left to right: DLST H424A, DLST G374E, DLST Y422C, DLST KO and LCL-G374E). Abbreviations: PPGL, pheochromocytoma and paraganglioma; SucCoA, succinyl coenzyme A; TCAc, tricarboxylic acid cycle; DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; NES, normalized enrichment score; GSEA, gene set enrichment analysis; LCL, lymphoblastoid cell line; Succ-K IHC, succinylated lysine immunohistochemistry; Log2FC, Log2 fold change. Structural modeling of the recently described homooligomeric DLST 24-mer [6] showed that glycine 374 is physically close to aspartic acid 428 of a nearby DLST monomer (Figure 1C, left panel). The DLST-p.G374E mutation substitutes this glycine with a glutamic acid, potentially introducing a repulsive interaction between the two anionic side chains that could alter the oligomer's structure. Conversely, the DLST-p.Y422C mutation could disrupt a likely interaction between tyrosine 422 and threonine 383 from an adjacent DLST monomer (Figure 1C, right panel), altering the substrate pocket's three-dimensional conformation. DLST mutations identified in PPGL patients thus likely alter the homooligomeric DLST assembly and hence the native structure of the OGDHc. To determine whether DLST alterations may affect the cellular succinylome, mass spectrometry was applied to previously generated DLST knockout (KO) H838 cell lines into which DLST constructs, including the wild-type (WT), the two aforementioned PPGL-causing mutants, and the catalytically dead p.H424A mutant [2] were stably introduced (Supplementary Figure S3A-B). DLST-KO cells exhibited dramatically reduced succinylation levels (Figure 1D, Supplementary Table S1) that were not caused by reduced protein abundance (Supplementary Figure S3C). The number of hyposuccinylated lysines differed between proteins, ranging from 1 to 20 (Supplementary Figure S3D). Moreover, hyposuccinylated proteins were involved in multiple pathways and mapped to all cellular compartments (Supplementary Figure S3E, Supplementary Table S2), indicating a widespread down-regulation of succinylation. These findings enable us to experimentally link the absence of DLST to a profound decrease in this PTM. Although succinylation was once believed to be a pH-dependent non-enzymatic reaction regulated by donor (SucCoA) concentration, Gibson et al. [7] found that succinylation efficiency increased in the presence of α-ketoglutarate and the OGDHc, suggesting that OGDHc could catalyze the succinylation of proteins. Our results support the role of DLST (and hence the OGDHc) as a major regulator of protein succinylation. Compared to DLST-WT cells, those expressing DLST-p.G374E and DLST-p.H424A exhi

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