Clinical implications of the oncometabolite succinate in SDHx‐mutation carriers

Abstract Succinate dehydrogenase (SDH) mutations lead to the accumulation of succinate, which acts as an oncometabolite. Germline SDHx mutations predispose to paraganglioma (PGL) and pheochromocytoma (PCC), as well as to renal cell carcinoma and gastro‐intestinal stromal tumors. The SDHx genes were the first tumor suppressor genes discovered which encode for a mitochondrial enzyme, thereby supporting Otto Warburg's hypothesis in 1926 that a direct link existed between mitochondrial dysfunction and cancer. Accumulation of succinate is the hallmark of tumorigenesis in PGL and PCC. Succinate accumulation inhibits several α‐ketoglutarate dioxygenases, thereby inducing the pseudohypoxia pathway and causing epigenetic changes. Moreover, SDH loss as a consequence of SDHx mutations can lead to reprogramming of cell metabolism. Metabolomics can be used as a diagnostic tool, as succinate and other metabolites can be measured in tumor tissue, plasma and urine with different techniques. Furthermore, these pathophysiological characteristics provide insight into therapeutic targets for metastatic disease. This review provides an overview of the pathophysiology and clinical implications of oncometabolite succinate in SDHx mutations.

lead to severe (lethal) cardiovascular and cerebrovascular complications. Approximately, 40% of these tumors carry a germline mutation in one of more than 20 susceptibility genes, of which the SDHx genes are the most prevalent. 3 In terms of genomic features, tumors related to SDHx mutations are classified as cluster I, along with Von Hippel Lindau (VHL), fumarate hydratase (FH), malate dehydrogenase 2 (MDH2), hypoxia induced factor (HIF2α) and isocitrate dehydrogenase (IDH)-mutations and the recently identified SLC25A11. 4 Cluster I germline mutations predispose to tumors characterized by a pseudohypoxic signature, in contrast to cluster II germline mutations, which are associated with abnormal kinase signaling pathways and include mutations in the genes of rearranged during transfection (RET), neurofibromatosis (NF1), transmembrane protein 127 (TMEM127), kinesin family member 1B (KIF1B), and MYC-associated factor X (MAX). Cluster III is associated with the Wnt-signaling pathway; it includes somatic mutations of cold shock domain-containing E1 (CSDE1) and mastermind-like transcriptional coactivator 3 (MAML3) fusion genes. 5,6 SDHx genes were the first to be recognized as tumor suppressor genes encoding a mitochondrial enzyme. This resulted in an upsurge of interest in the concept of aerobic glycolysis or the "Warburg These oncometabolites modulate the activity of α-ketoglutaratedependent dioxygenases, which are involved in the induction of the pseudohypoxia pathway and inhibit histones and DNA demethylases, resulting in a hypermethylator phenotype (also known as CpG island methylator phenotype [CIMP]). The SLC25A11 gene encodes for a mitochondrial carrier protein that is part of the malate-asparate shuttle (this shuttle regenerates NADH to allow complex I to function), mediating the transport of α-ketoglutarate from the mitochondrial matrix to the cytoplasm in exchange with malate. Preliminary results show that in SLC25A11-mutated cells aspartate and glutamate concentration is increased inducing the pseudohypoxic pathway and hypermethylation. 4 Recognition of these pathophysiological characteristics provides unique opportunities for diagnostic and therapeutic strategies. Over the past years, several excellent reviews, such as those by Kucklova of SDHx-related tumors. [8][9][10] In the current review, we first present a short summary of the SDH protein and the clinical features of SDHxmutation carriers. We then focus on the oncometabolite succinate and its pivotal role in tumorigenesis in SDHx-related tumors, as well as on the implications for clinical practice, especially diagnostics and therapeutic options related to metastatic disease.

| SUCCINATE DEHYDROGENASE
SDH is a hetero-tetrameric mitochondrial enzyme that plays a role in the TCA cycle and in the mitochondrial electron transport chain as complex II (Figure 1). SDH catalyzes the oxidation of succinate to fumarate in the TCA cycle and transfers electrons to the ubiquinone (coenzyme Q) pool in the respiratory chain. SDH subunit A (SDHA) and subunit B (SDHB) comprise the catalytic subunits in the hydrophilic head that protrudes into the mitochondrial matrix. SDH subunit C (SDHC) and subunit D (SDHD) are the ubiquinone-binding and membrane-anchoring subunits. SDH assembly factor (SDHAF) is required for the flavination of SDHA, which is essential for the formation of the SDH complex. The SDHA gene is located on chromosome 5p15.33 and contains 16 exons. 11 SDHA is the major catalytic subunit, converting succinate to fumarate. It contains the binding site for succinate. The gene encoding for SDHB is located on chromosome 1p35-36.1 and has eight exons 12 ; the SDHB protein contains three Fe-S centers and mediates electron transfer to the ubiquinone pool.
The gene encoding SDHC is located at 1q21 and has six exons, 13 and the SDHD gene is located on chromosome 11q23 and has four exons. 14 SDHC and SDHD bind ubiquinone, generating protons eventually leading to the production of ATP.

| PHENOTYPE OF SDHX MUTATION CARRIERS
Although different SDHx mutations occur in genes encoding for a single enzyme, the clinical picture for each subunit differs with regard to penetrance, manifestations and rate of malignancy. International guidelines advice to screen all germline mutation carriers, however with different screenings strategies for different SDHx mutation carriers. 15 Screenings advices do not only differ between the different mutations, but also over time, because studies on penetrance differ over time regarding the population studied (index included or not), the imaging methods used and the duration of follow-up. Adherence to screening, leads to the detection of smaller PCC/PGL and might even lead to an improved survival for patients who develop metastases, although this is based on only few patients. 16 Until now, a clear explanation for the difference of the clinical picture between different SDHx mutations is lacking, except for the hypothesis that the extent of SDH deficiency or loss depends on the subunit. Apart from the differences, all SDHx mutations are characterized by the (potential) presence of PGL/PCC. SDHx-associated tumors harbor germline and somatic mutations, consistent with Knudson's second-hit hypothesis. 12 This hypothesis states that the combination of an inactivating germline mutation as a first hit, and somatic loss of function of the wild type allele as a second hit, is essential for tumor development. 17 This second hit usually is an inactivation of the normal allele, that is, loss of 1p as was shown in a large genomic study. 18 Germline SDHx mutations have been associated with neoplasms other than PGL/ PCC, such as RCC, [19][20][21] GISTs and possibly pituitary adenoma. [22][23][24] In addition, somatic SDHx mutations have been described in T-cell leukemia. 25 Because the discovery of SDHx genes is relatively recent, the full clinical phenotype of these carriers remains to be sufficiently clarified. The following paragraphs describe the currently known phenotype of each SDHx subunit ( Table 1). The question of why SDHx mutations predispose to tumors in a select subset of tissues remains elusive.

| SDHA mutations
Mutations in the SDHA gene were originally described as a cause of autosomal recessive juvenile encephalopathy, also known as Leigh syndrome. 26 Later on, in 2010, a 32 year old woman with an abdominal PGL was reported to have a heterozygous SDHA germline mutation. 27 Mutations in the SDHA gene remain a rare cause of PGL and F I G U R E 1 Succinate dehydrogenase (SDH) complex (simplified). The catalytic subunits SDH subunit A contains the flavin cofactor (FAD) which accepts electrons from succinate and passes them to Fe-S center in the SDH subunit B subunit. The electrons are then passed the ubiquinone pool embedded in SDHC and SDHD subunits. Reduced Q (QH2 = ubiquinol) transfers electrons within the mitochondrial inner membrane space to complex III but also for a combination of index and non-index patients. 40 Their Kaplan-Meier analysis showed an estimated risk for the combined manifestation of PCC, sympathetic PGL and HNPGL in non-index patients to be 22% at age 60. 40 In their retrospective cohort analysis (index and non-index patients) the penetrance was 24% at age 60 years. 40 Males seem to be slightly more at risk than females of developing a PGL. 40,41 SDHB-related PGL/PCC are associated with a high risk of metastasis and poor prognosis. Earlier studies report a higher metastatic rate (31%-97%) 42-45 than more recent studies. 40 In a meta-analysis including 12 studies comprising both asymptomatic SDHB carriers and carriers with manifest non-metastatic disease, van Hulsteijn et al reported a metastatic rate of 17%. 46 The risk of metastasis in HNPGL in SDHB mutation carriers appears to be lower compared to PGL developing at other sites. 15 In a recent meta-analysis of the outcomes of metastatic PGL and PCC, Hamidi et al found that the overall mortality in SDHB mutation carriers ranged from 35% to 55% (n = 96) compared to an overall mortality of 53% (95% confidence interval 43%-63%) in the whole group of PGL/PCC. 47 In the past, several studies have shown an association between SDHB-related metastatic PGL/PCC and a shorter survival in patients compared to sporadic metastatic PGL/PCC. 48,49 In a recent analysis of Hescot et al, not the SDHB mutation status but hypersecretion of metanephrines and chromogranin A was found to be a significant prognostic factor for worst overall survival. 50 Other SDHB-associated tumors include RCC, although the risk for this manifestation seems low, varying between 4.7% and 8%. 21,40 GISTs are reported to occur in approximately 2% of SDHB carriers. 51 Pituitary adenoma have been reported in nine cases, but only three had proven LoH (loss of heterozygosity) and abnormal SDHB expression, thus confirming involvement of SDHB mutation. 24 Tufton et al reported a case of a SDHB mutation carrier with pituitary carcinoma. 52

| SDHC mutations
Mutations in the SDHC gene account for 1% to 2% of PGL/PCC cases. 28 SDHC typically manifest as benign, non-functional HNPGL, although sympathetic PGL and PCC are also reported. 53

| SDHD mutations
A mutation in the SDHD gene accounts for approximately 5% to 9% of all cases of PGL/PCC. 28,29 This gene follows an autosomal dominant inheritance, modified by maternal imprinting. The predominant clinical feature of SDHD carriers is the development of (multiple) HNPGLs, as 85% of carriers develop tumors at this site. 51 PCC and sympathetic PGL occur less frequently in 10% to 25% and 20% to 25% of carriers, respectively. Penetrance for 160 non-index SDHD mutation carriers was 43% at age 60. 40 Metastatic risk in SDHD carriers is low and occurs in 7% to 8% of cases. 15,60 Other associated tumors include RCC and GIST, although the lifetime risk for this manifestation is very low (<1%). 40

| SDHAF2 mutations
The SDHAF2 gene, like SDHD, is affected by maternal imprinting; therefore, only those carriers who inherit the mutation via the paternal line will develop the disease. Only a few cases of PGL/PCC associated with SDHAF2 mutations have been described, and these account for <1% of all cases of PGL. 29 Germline pathogenic variants in   complex II can also produce a significant number. 91,92  as changes in anaplerotic pathways and in oxidative phosphorylation ( Figure 3).

| Warburg effect
As stated above, SDHx-related tumors display the Warburg effect.  104

| SDH and immunohistochemistry
In the vast majority of SDHx-associated tumors, loss of SDHB protein expression can be detected by immunohistochemical staining with a high sensitivity and specificity (100% and 84%, respectively). 33 109  The cornerstone treatment for patients with benign SDHx related PGL/PCC is surgery. 117 As described above, SDHB-mutation carriers are those especially at risk of metastatic disease. Even for patients with metastatic PGL/PCC, resection of the primary tumor seems to be associated with a better overall survival. 126 Metastases frequently occur in lymph nodes (distant and regional), bones, liver and lungs.