Succinate Dehydrogenase Loss Research Articles

Pyruvate transamination and NAD biosynthesis enable proliferation of succinate dehydrogenase-deficient cells by supporting aerobic glycolysis

Succinate dehydrogenase (SDH) is the mitochondrial enzyme converting succinate to fumarate in the tricarboxylic acid (TCA) cycle. SDH acts as a tumor suppressor with germline loss-of-function mutations in its encoding genes predisposing to aggressive familial neuroendocrine and renal cancer syndromes. Lack of SDH activity disrupts the TCA cycle, imposes Warburg-like bioenergetic features, and commits cells to rely on pyruvate carboxylation for anabolic needs. However, the spectrum of metabolic adaptations enabling SDH-deficient tumors to cope with a dysfunctional TCA cycle remains largely unresolved. By using previously characterized Sdhb-deleted kidney mouse cells, here we found that SDH deficiency commits cells to rely on mitochondrial glutamate-pyruvate transaminase (GPT2) activity for proliferation. We showed that GPT2-dependent alanine biosynthesis is crucial to sustain reductive carboxylation of glutamine, thereby circumventing the TCA cycle truncation determined by SDH loss. By driving the reductive TCA cycle anaplerosis, GPT2 activity fuels a metabolic circuit maintaining a favorable intracellular NAD+ pool to enable glycolysis, thus meeting the energetic demands of SDH-deficient cells. As a metabolic syllogism, SDH deficiency confers sensitivity to NAD+ depletion achieved by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. Beyond identifying an epistatic functional relationship between two metabolic genes in the control of SDH-deficient cell fitness, this study disclosed a metabolic strategy to increase the sensitivity of tumors to interventions limiting NAD availability.

Mitochondrial redox adaptations enable alternative aspartate synthesis in SDH-deficient cells.

The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD +to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss-of-function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC), but mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports human cell proliferation through aspartate synthesis but, unlike other ETC impairments, the effects of SDH inhibition are not ameliorated by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH-impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context depend on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production through pyruvate carboxylation and reductive carboxylation of glutamine. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. These data therefore identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal how compartmentalized redox changes can impact cellular fitness.

β-Cell Succinate Dehydrogenase Deficiency Triggers Metabolic Dysfunction and Insulinopenic Diabetes.

Mitochondrial dysfunction plays a central role in type 2 diabetes (T2D); however, the pathogenic mechanisms in pancreatic β-cells are incompletely elucidated. Succinate dehydrogenase (SDH) is a key mitochondrial enzyme with dual functions in the tricarboxylic acid cycle and electron transport chain. Using samples from human with diabetes and a mouse model of β-cell–specific SDH ablation (SDHBβKO), we define SDH deficiency as a driver of mitochondrial dysfunction in β-cell failure and insulinopenic diabetes. β-Cell SDH deficiency impairs glucose-induced respiratory oxidative phosphorylation and mitochondrial membrane potential collapse, thereby compromising glucose-stimulated ATP production, insulin secretion, and β-cell growth. Mechanistically, metabolomic and transcriptomic studies reveal that the loss of SDH causes excess succinate accumulation, which inappropriately activates mammalian target of rapamycin (mTOR) complex 1–regulated metabolic anabolism, including increased SREBP-regulated lipid synthesis. These alterations, which mirror diabetes-associated human β-cell dysfunction, are partially reversed by acute mTOR inhibition with rapamycin. We propose SDH deficiency as a contributing mechanism to the progressive β-cell failure of diabetes and identify mTOR complex 1 inhibition as a potential mitigation strategy.

A suppressor of dioxygenase inhibition in a yeast model of SDH deficiency.

A fascinating class of familial paraganglioma (PGL) neuroendocrine tumors is driven by the loss of the tricarboxylic acid (TCA) cycle enzyme succinate dehydrogenase (SDH) resulting in succinate accumulation as an oncometabolite and other metabolic derangements. Here, we exploit a Saccharomyces cerevisiae yeast model of SDH loss where accumulating succinate, and possibly reactive oxygen species, poison a dioxygenase enzyme required for sulfur scavenging. Using this model, we performed a chemical suppression screen for compounds that relieve dioxygenase inhibition. After testing 1280 pharmaceutically active compounds, we identified meclofenoxate HCl and its hydrolysis product, dimethylaminoethanol (DMAE), as suppressors of dioxygenase intoxication in SDH-loss yeast cells. We show that DMAE acts to alter metabolism so as to normalize the succinate:2-ketoglutarate ratio, improving dioxygenase function. This study raises the possibility that oncometabolite effects might be therapeutically suppressed by drugs that rewire metabolism to reduce the flux of carbon into pathological metabolic pathways.

MP39-08 GENOMIC AND METABOLIC HALLMARKS OF TCA-CYCLE-MUTANT RENAL CELL CARCINOMAS

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-08 GENOMIC AND METABOLIC HALLMARKS OF TCA-CYCLE-MUTANT RENAL CELL CARCINOMAS Angela Yoo, Cerise Tang, Mark Zucker, Kelly Fitzgerald, Phillip Rappold, Kate Weiss, Benjamin Freeman, Chung-Han Lee, Maria Carlo, Ritesh Kotecha, A Ari Hakimi, and Ed Reznik Angela YooAngela Yoo More articles by this author , Cerise TangCerise Tang More articles by this author , Mark ZuckerMark Zucker More articles by this author , Kelly FitzgeraldKelly Fitzgerald More articles by this author , Phillip RappoldPhillip Rappold More articles by this author , Kate WeissKate Weiss More articles by this author , Benjamin FreemanBenjamin Freeman More articles by this author , Chung-Han LeeChung-Han Lee More articles by this author , Maria CarloMaria Carlo More articles by this author , Ritesh KotechaRitesh Kotecha More articles by this author , A Ari HakimiA Ari Hakimi More articles by this author , and Ed ReznikEd Reznik More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Mutations in TCA cycle enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) are associated with rare kidney cancers. However, the molecular features of SDH-deficient RCC (SDHRCC) and FH-deficient RCC (FHRCC) have not been well defined. Using genomic and metabolomic profiling, we aimed to define the genetic and molecular hallmarks of TCA-cycle mutant RCC. METHODS: Patients included met either immunohistochemical (loss of FH/SDH expression and/or 2-succinocysteine positivity) or molecular (biallelic loss of FH or SDHA/B/C/D/AF2) criteria for FH- or SDH- deficiency. Both germline (if available) and somatic information were integrated to determine biallelic status. Somatic variants were identified using standard clinical pipelines, and allele-specific copy number changes were studied using the FACETS algorithm. Mass-spectrometry-based metabolomics was completed on 6 samples (3 SDHRCC and 3 FHRCC). RESULTS: 37 patients were included, 26 FHRCC and 11 SDHRCC. In the germline analysis, nearly all (16/17) SDHRCC presented with a germline mutation to SDHB, whereas only 12/20 FHRCC had pathogenic FH variants. FHRCC has a significantly higher tumor mutation burden (p=0.0165, Fig 1a) and fraction of the genome that is copy-number-altered (p = 1e-04, Fig 1b) than SDHRCC. FHRCC often arises with additional somatic mutations (Fig 1c). In contrast, only 2/11 SDHRCC patients had co-occurring mutations (Fig 1d). Notably, 100% of SDHRCC presented with deletion of chromosome 1p (location of SDHB), whereas FHRCC demonstrated high but not universal loss of 1q (FH locus). Although both FH and SDH loss truncate the TCA cycle, FHRCC and SDHRCC have metabolic differences. Urea cycle metabolites such as argininosuccinate, citrulline, homocitrulline, and arginine were elevated in FHRCC tumors but not SDHRCC tumors. FHRCC also had an excess of N6-succinyladenosine compared to SDHRCC. However, SDHRCC had an elevation of acyl-carnitine compared to FHRCC, suggesting that disruption of FADH2 oxidation through Complex II/SDH may impair normal fatty acid oxidation. CONCLUSIONS: Despite a superficial similarity, FHRCC and SDHRCC represent distinct molecular entities with unique genetic and metabolic abnormalities. Source of Funding: This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748 and the Bryan Bumsted fund © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e707-e707 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Angela Yoo More articles by this author Cerise Tang More articles by this author Mark Zucker More articles by this author Kelly Fitzgerald More articles by this author Phillip Rappold More articles by this author Kate Weiss More articles by this author Benjamin Freeman More articles by this author Chung-Han Lee More articles by this author Maria Carlo More articles by this author Ritesh Kotecha More articles by this author A Ari Hakimi More articles by this author Ed Reznik More articles by this author Expand All Advertisement Loading ...

Assessment of Tumor Cell Death After Percutaneous Ultrasound– Guided Radiofrequency Ablation of Breast Carcinoma: A Prospective Study

Background: Current trends in breast cancer treatment include the use of less aggressive surgeries to reduce morbidity, shorten hospital stays and improve cosmetic results. The aim of the study is to assess tumor cell viability after percutaneous ultrasound (US)-guided radiofrequency ablation (RFA) for small breast cancer by a combination of staining techniques. Methods: A prospective study was conducted at a single institution from 2013 to 2017. Twenty women with invasive ductal carcinoma of the breast measuring ≤ 20 mm were treated with US-guided RFA followed immediately by surgical resection. Tumor viability pre- and post-RFA was assessed with Hematoxylin and Eosin (H&E), Nicotinamide adenine dinucleotide (NADH), Succinate dehydrogenase (SDH), Cytochrome c oxidase (COX), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Cytokeratin 18 and 19 (CK18/CK19) staining techniques. Outcomes and correlation with the different techniques were evaluated with principal component analysis Cronbach’s alpha. Results: Oxidative enzymes in frozen sections showed loss of SDH and NADH in 13 of the 16 tumors (81%) and COX in 11 of the 13 tumors (84%). In paraffin-embedded tissues, CK18 was negative or markedly reduced in 98% and CK19 in 100% of the cases. Lack of evidence of cell death was seen in 3 cases where the maximum temperature achieved at the center of the tumor was ≤ 70ºC. The reliability and internal consistency between the different staining techniques was high (Cronbach’s alpha, 0.8), with concordance between the staining results of the oxidative enzymes and of CK18/CK19. Conclusion: Loss of tumor viability in small breast tumors after US-guided percutaneous RFA could be assessed in our series with different staining methods. CK18 and CK19 could be used in paraffin-embedded tissues as surrogate markers of tumor cell viability after immediate RFA.

Gene Expression Landscape of SDH-Deficient Gastrointestinal Stromal Tumors.

Background: About 20–40% of gastrointestinal stromal tumors (GISTs) lacking KIT/PDGFRA mutations show defects in succinate dehydrogenase (SDH) complex. This study uncovers the gene expression profile (GEP) of SDH-deficient GIST in order to identify new signaling pathways or molecular events actionable for a tailored therapy. Methods: We analyzed 36 GIST tumor samples, either from formalin-fixed, paraffin-embedded by microarray or from fresh frozen tissue by RNA-seq, retrospectively collected among KIT-mutant and SDH-deficient GISTs. Pathway analysis was performed to highlight enriched and depleted transcriptional signatures. Tumor microenvironment and immune profile were also evaluated. Results: SDH-deficient GISTs showed a distinct GEP with respect to KIT-mutant GISTs. In particular, SDH-deficient GISTs were characterized by an increased expression of neural markers and by the activation of fibroblast growth factor receptor signaling and several biological pathways related to invasion and tumor progression. Among them, hypoxia and epithelial-to-mesenchymal transition emerged as features shared with SDH-deficient pheochromocytoma/paraganglioma. In addition, the study of immune landscape revealed the depletion of tumor microenvironment and inflammation gene signatures. Conclusions: This study provides an update of GEP in SDH-deficient GISTs, highlighting differences and similarities compared to KIT-mutant GISTs and to other neoplasm carrying the SDH loss of function. Our findings add a piece of knowledge in SDH-deficient GISTs, shedding light on their putative histology and on the dysregulated biological processes as targets of new therapeutic strategies.

Diagnostic algorithm for gastrointestinal stromal tumor (GIST) using patient registry data impacts pathology guidelines.

e23519 Background: The Life Raft Group (LRG) identified that succinate dehydrogenase (SDH) deficient GISTs are under-recognized. In the LRG Registry only 30% of KIT/PDGFRA wildtype patients had received the advanced mutational testing (or SDHB staining) required to identify this unique subtype of GIST with a different natural history and response to treatment, and very strong familial associations. We proposed that two changes would significantly increase the diagnosis of SDH-deficient GIST patients: 1. SDHB staining for all GIST patients with a stomach primary tumor at initial biopsy, 2. Initial pathology report should contain strong recommendations for specific mutational testing. Methods: The LRG collaborated with key medical opinion leaders on a diagnostic algorithm that illustrated the need for wider use of targeted diagnostic procedures and submitted the algorithm to the College of American Pathologists (CAP) GIST editorial board. Results: Because SDH-deficient GISTs require germline genetic analysis and, if mutation related, subsequent surveillance for paragangliomas/pheochromocytomas, and cascade genetic testing for family members, it is necessary to screen all gastric GISTs for loss of SDH by immunohistochemistry. This is best accomplished by immunohistochemical staining for SDHB, which is lost in all genetic subtypes of SDH-deficient GISTs. If SDHB is absent, additional staining for SDHA may be performed, as this protein is selectively lost in SDHA-mutant GISTs and this may help focus genetic analysis. All patients with SDH-deficient GIST should be referred to a genetic counselor. The CAP incorporated both SDHB and SDHA testing paradigms into the updated GIST protocols which were published in August 2019. Conclusions: We believe this will result in significantly better diagnosis and treatment for SDH-deficient GIST patients. We encourage other groups that advise on guidelines to develop similar recommendations. This is a great example of a patient advocacy group utilizing real world evidence derived from a patient registry to influence protocols for treatments that affect GIST patients on a large scale.

Mitochondrial Dysfunction Promotes Diabetes via A Previously Unrecognized Mechanism: Protein Succinylation

Mitochondrial dysfunction is a central contributor to the development of β‐cell failure and Type 2 Diabetes (T2D). Succinate dehydrogenase subunit B (SDHB) is a key component of the Succinate Dehydrogenase (SDH) protein complex that strongly impacts mitochondrial function via 1) oxidation of succinate to fumarate in the TCA cycle and 2) electron transfer in the electron transport chain as Complex II. Loss of SDH activity has been observed in diabetic β‐cells (humans and rodents); however, the relevance of this observation to diabetes pathogenesis remains poorly understood. Consistent with prior work, we found reduced SDHB expression in the pancreatic islets of human diabetics. To investigate the links between mitochondrial dysfunction, β‐cell failure and diabetes, we generated mice with a β‐cell‐specific knockout of SDHB (SDHBβKO). We found that SDHBβKO mice develop early onset diabetes. At 5 weeks, prediabetic SDHBβKO mice demonstrate mildly elevated glucose levels with low insulin levels and normal insulin sensitivity. These in vivo findings were confirmed by a reduction in glucose‐stimulated insulin secretion in SDHBβKO islets. Furthermore, glucose‐induced β‐cell replication was impaired in SDHBβKO mice; consistent with impaired adaptive β‐cell expansion. Given the prominent role of glucose metabolism in controlling β‐cell insulin secretion and replication, we assessed the mitochondrial bioenergetics by Seahorse Assay and found that basal and glucose‐stimulated respiration were significantly reduced in SDHBβKO islets. Notably, loss of SDH is expected to increase succinate accumulation and promote protein succinylation, a recently described bulky post‐translational modification. Indeed, SDHBβKO islets demonstrated a dramatic increase in succinate levels (16‐fold) and protein succinylation (6‐fold), providing a potential mechanism for global mitochondrial dysfunction. Accordingly, expression of the desuccinylation enzyme, Sirt5, was increased in SDHBβKO islets. Metabolomics by mass spectrometry and transcriptomics by high‐throughput RNA sequencing revealed significantly altered mitochondrial metabolism. Finally, protein succinylation levels were increased in islets of diabetic db/db mice indicating lysine‐succinylation may be characteristic of diabetes. Taken together, these results demonstrate a surprisingly necessary role for the SDH complex in β‐cell function and introduce a pathogenic mechanism of diabetes based upon β‐cell metabolic dysfunction.Support or Funding InformationStanford Diabetes Research Center (SRDC) Pilot & Feasibility Grants Program (P30DK116074), Endocrinology Training grant (T32DK007217) and Child Health Research Institute at Stanford (UL1TR001085)

Modeling succinate dehydrogenase loss disorders in C. elegans through effects on hypoxia-inducible factor.

Mitochondrial disorders arise from defects in nuclear genes encoding enzymes of oxidative metabolism. Mutations of metabolic enzymes in somatic tissues can cause cancers due to oncometabolite accumulation. Paraganglioma and pheochromocytoma are examples, whose etiology and therapy are complicated by the absence of representative cell lines or animal models. These tumors can be driven by loss of the tricarboxylic acid cycle enzyme succinate dehydrogenase. We exploit the relationship between succinate accumulation, hypoxic signaling, egg-laying behavior, and morphology in C. elegans to create genetic and pharmacological models of succinate dehydrogenase loss disorders. With optimization, these models may enable future high-throughput screening efforts.

Succinate detection using in vivo 1H-MR spectroscopy identifies germline and somatic SDHx mutations in paragangliomas.

Germline mutations in genes encoding succinate dehydrogenase (SDH) are frequent in patients with pheochromocytoma and paraganglioma (PPGL). They lead to SDH inactivation, mediating a massive accumulation of succinate, which constitutes a highly specific biomarker of SDHx-mutated tumors when measured in vitro. In a recent pilot study, we showed that magnetic resonance spectroscopy (1H-MRS) optimized for succinate detection (SUCCES) could detect succinate in vivo in both allografted mouse models and PPGL patients. The objective of this study was to prospectively assess the diagnostic performances of 1H-MRS SUCCES sequence for the identification of SDH deficiency in PPGL patients. Forty-nine patients presenting with 50 PPGLs were prospectively enrolled in our referral center for 1H-MRS SUCCES. Two observers blinded to the clinical characteristics and genetic status analyzed the presence of a succinate peak and confronted the results to a composite gold standard combining PPGL genetic testing and/or in vitro protein analyses in the tumor. A succinate peak was observed in 20 tumors, all of which had proven SDH deficiency using the gold standard (17 patients with germline SDHx mutations, 2 with a somatic SDHD mutation, and 1 with negative SDHB IHC and SDH loss of function). A false negative result was observed in 3 tumors. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 1H-MRS SUCCES were respectively 87%, 100%, 100%, 90%, and 94%. Detection of succinate using 1H-MRS is a highly specific and sensitive hallmark of SDH-deficiency in PPGLs.

Loss of succinate dehydrogenase B immunohistochemical expression distinguishes pulmonary chondromas from hamartomas.

Pulmonary chondromas, which are rare cartilaginous neoplasms that often arise in the setting of Carney triad, are morphologically similar to pulmonary hamartomas, which are much more common. There is evidence that succinate dehydrogenase (SDH) deficiency drives neoplasia in patients with Carney triad, and SDHB immunohistochemistry can be used as a surrogate markerto detect SDH deficiency. The aim of this study was to investigate the utility of SDHB immunohistochemistry in distinguishing pulmonary chondromas from hamartomas. Immunohistochemistry for SDHB (clone 21A11AE7) was performed on histological sections from six cases of pulmonary chondroma and 33 cases of pulmonary hamartoma. SDHB expression was retained in all 33 pulmonary hamartomas, and lost in the majority of evaluable chondromas (five of six). Of the five patients with chondromas showing SDHB loss, four had definitiveCarney triad. Most patients with pulmonary hamartomas were older males with small solitary masses, whereas chondromas often presented as multiple masses in young females. Loss of SDHB immunohistochemical expression can be useful for differentiating pulmonary chondromas from hamartomas, and potentially identifying patients with Carney triad.

A conditional mouse model of complex II deficiency manifesting as Leigh-like syndrome.

Leigh syndrome embodies degenerative disorders with a collection of symptoms secondary to inborn errors of metabolism. Combinations of hypomorphic and loss-of-function alleles in many genes have been shown to result in Leigh syndrome. Interestingly, deficiency for the tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH) can lead to Leigh-like syndrome in some circumstances and to cancer (paraganglioma, renal cell carcinoma, gastrointestinal stromal tumor) in others. In our experiments originally intended to create an inducible whole-body SDH-loss mouse model of tumorigenesis, we generated a condition reminiscent of Leigh-like syndrome that is lethal to mice within 4 wk. Remarkably, as has been shown for other mitochondrial diseases, chronic hypoxia offers substantial protection to mice from this condition after systemic SDH loss, allowing survival in the context of profoundly impaired oxidative metabolism.-Al Khazal, F., Holte, M. N., Bolon, B., White, T. A., LeBrasseur, N., Maher, L. J. III. A conditional mouse model of complex II deficiency manifesting as Leigh-like syndrome.

Huntington’s Disease: Astrocytes Shift to Fatty Acid Metabolism

A recent study by Polyzos et al. (Cell Metab., 2019) shows that astrocytes in a Huntington disease (HD) mouse model switch from glycolysis to fatty acid oxidation (FAO), causing increased superoxide radical anion production and loss of succinate dehydrogenase (SD) activity. Blocking mitochondria reactive oxygen species (ROS) with an antioxidant compound called XJB-5-151 reversed lipofuscin formation and protected the mice.

Clinical implications of the oncometabolite succinate in SDHx-mutation carriers.

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.

The loss of succinate dehydrogenase B expression is frequently identified in hemangioblastoma of the central nervous system

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that plays an important role in both the Krebs cycle and the electron transport chain. SDH inactivation is associated with tumorigenesis in certain types of tumor. SDH consists of subunits A, B, C and D (SDHA, SDHB, SDHC, and SDHD, respectively). Immunohistochemistry for SDHB is a reliable method for detecting the inactivation of SDH by mutations in SDHA, SDHB, SDHC, SDHD and SDH complex assembly factor 2 (SDHAF2) genes with high sensitivity and specificity. SDHB immunohistochemistry has been used to examine the inactivation of SDH in various types of tumors. However, data on central nervous system (CNS) tumors are very limited. In the present study, we investigated the loss of SDHB immunoexpression in 90 cases of CNS tumors. Among the 90 cases of CNS tumors, only three cases of hemangioblastoma showed loss of SDHB immunoexpression. We further investigated SDHB immunoexpression in 35 cases of hemangioblastoma and found that 28 (80%) showed either negative or weak-diffuse pattern of SDHB immunoexpression, which suggests the inactivation of SDH. Our results suggest that SDH inactivation may represent an alternative pathway in the tumorigenesis of hemangioblastoma.

Alterations in Nuclesome Core Particle Dynamics in the Context of Paraganglioma

Paraganglioma (PGL) is a rare form of neuroendocrine cancer for which the underlying molecular basis is poorly understood. A mysterious hallmark of many PGL tumors is the inactivation of succinate dehydrogenase (SDH), a mitochondrial enzyme of the TCA cycle. Why does SDH loss induce cancer in certain cells? As a result of SDH loss, PGL tumor cells accumulate succinate, which may have epigenetic consequences. For example, certain lysine residues on histone proteins in nucleosomes are subject to succinylation by succinyl‐CoA as a post translational modification (PTM) that converts the lysine cation to an anion. We hypothesize that the structure and stability of the nucleosome core particle (NCP) is affected by succinylation PTMs through weakening of electrostatic histone‐DNA and histone‐histone interactions. Proteomic analysis of an SDH‐loss cell line model of PGL previously demonstrated that the greatest increase in nucleosomal lysine succinylation maps to H4K79 when compared to a control cell line. The mammalian H4K79 residue lies within the nucleosome core near wrapped DNA. We specifically hypothesize that the charge inversion conferred by H4K79 succinylation, destabilizes the NCP. Point mutations at H4K79 have been generated as lysine succinylation mimics at this site. Biophysical and in vitro transcription assays are being utilized to validate the assembly of NCPs incorporating lysine succinylation mimics at this site and to interrogate their resulting stabilities. Analyses of alterations in NCP dynamics in variant NCP will help to elucidate the role of lysine succinylation of histones in the altered chromatin landscape observed during PGL oncogenesis.Support or Funding InformationGM75148‐09‐10 PREPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 1291: Master regulator analysis of paragangliomas carrying SDH, VHL, or MAML3 genetic alterations

Abstract Background: Succinate dehydrogenase (SDH) loss and mastermind-like 3 (MAML3) translocation are two clinically important genetic alterations that occur in subtypes of human paraganglioma and pheochromocytoma (PPGL) neuroendocrine tumors that correlate with increased rates of metastasis. Although hypotheses exist for how global activation of pseudohypoxia and epigenomic hypermethylation are driven via dioxygenase poisoning in the context of SDH loss, it remains unclear how these defects drive tumorigenesis. Additionally, MAML3 translocation has recently been identified as a genetic alteration in PGL and is poorly understood. We hypothesize that a key to understanding the tumorigenic basis for these genetic alterations is the deconvolution of the gene expression profiles in these tumors and identification of the transcription factors responsible for the observed transcriptional changes. Methods: In the present work, we leverage publicly-available human tumor gene expression profiling experiments (N=179) to reconstruct a PPGL tumor-specific transcriptional network. Inferred network structure was subsequently validated by examining the frequency of known transcription factor DNA-binding motifs in the inferred regulons. We subsequently use the inferred transcriptional networks and known gene expression signatures for specific PPGL subtypes to perform a master regulator analysis nominating specific transcription factors that may control observed oncogenic patterns of gene expression. The findings of the master regulator analysis were confirmed by duplication of the analysis on a second PPGL-specific inferred transcriptional network (N=188 specimens). Results: A small number of master regulator transcription factors are likely drivers of the observed subtype-specific gene expression patterns in SDH loss- and MAML3 translocation-positive PPGL. Unexpectedly, hypoxia-inducible transcription factors were not identified as master regulators in SDH-loss tumors. Additionally, analysis of the MAML3 translocation subtype master regulators revealed that a single transcription factor, IRX4, may control 37% of the deferentially expressed genes in this highly malignant tumor subtype. Conclusions: These observations from unbiased analyses suggest a basis for future development of targeted therapies for specific PPGL molecular subtypes. Citation Format: John A. Smestad, Louis J. Maher. Master regulator analysis of paragangliomas carrying SDH, VHL, or MAML3 genetic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1291.

Abstract 2996: Insulator dysfunction and epigenetic oncogene activation in SDH-deficient gastrointestinal stromal tumor

Abstract Metabolic lesions with profound effects on epigenetic regulation are widely implicated in cancer, yet the mechanistic links between this epigenetic dysregulation and tumorigenesis remain unclear. Succinate dehydrogenase (SDH) deficiency, responsible for a subset of gastrointestinal stromal tumors (GISTs), causes accumulation of the metabolite succinate and DNA hypermethylation. We identified convergent mechanisms involving altered chromosomal conformation and pseudo-hypoxia that mediate the tumorigenic effects of SDH deficiency in GIST. To investigate epigenetic alterations in this disease, we created epigenetic maps of 14 clinical GIST specimens; including KIT and PDGFRA mutant, and SDH-deficient tumors. We characterized the landscapes of enhancers, genetic regulatory elements which can drive gene expression, through histone H3 lysine 27 acetylation chromatin immunoprecipitation sequencing (ChIP-seq). We characterized both the DNA methylation and CTCF occupancy of insulators, elements which help control chromatin conformation and restrict enhancer-gene interactions, through hybrid selection bisulfite sequencing and CTCF ChIP-seq, respectively. Analyzing these data, we uncovered thousands of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. One of the strongest disrupted insulators protected the receptor tyrosine kinase and known driver of GIST, c-KIT, from a nearby superenhancer. Chromatin conformation studies confirmed an SDH-deficient-specific interaction of this superenhancer with the KIT gene. CRISPR-mediated excision of the insulator in an SDH-intact GIST model resulted in enhancer interaction and KIT upregulation. Immunohistochemical studies confirm strong expression of c-KIT in SDH-deficient GIST clinical samples. SDH deficiency has also been reported to cause pseudohypoxia in tumors. We confirmed that the enhancer landscape of SDH-deficient tumors had a signature of pseudohypoxia. Additionally, following pseudohypoxia induction in a SDH-intact GIST model, the c-KIT ligand Stem Cell Factor (SCF/KITLG) was upregulated 12-fold. While activating KIT mutations drive the majority (~75%) of GIST tumors and are mutually exclusive with SDH deficiency, we show that a primary consequence of SDH loss is in fact induction of KIT signaling. Our findings demonstrate how metabolic lesions can provide alternate epigenetic mechanisms to activate classic tumorigenic pathways in the absence of canonical genetic mutations. Citation Format: William A. Flavahan, Yotam Drier, Sarah E. Johnstone, Daniel R. Tarjan, Esmat Hegazi, Ewa T. Sicinska, Matthew L. Hemming, Chandrajit P. Raut, Jason L. Hornick, George D. Demetri, Bradley E. Bernstein. Insulator dysfunction and epigenetic oncogene activation in SDH-deficient gastrointestinal stromal tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2996.

Characterization and metabolic synthetic lethal testing in a new model of SDH-loss familial pheochromocytoma and paraganglioma.

Succinate dehydrogenase (SDH)-loss pheochromocytoma and paraganglioma (PPGL) are tumors driven by metabolic derangement. SDH loss leads to accumulation of intracellular succinate, which competitively inhibits dioxygenase enzymes, causing activation of pseudohypoxic signaling and hypermethylation of histones and DNA. The mechanisms by which these alterations lead to tumorigenesis are unclear, however. In an effort to fundamentally understand how SDH loss reprograms cell biology, we developed an immortalized mouse embryonic fibroblast cell line with conditional disruption of Sdhc and characterize the kinetics of Sdhc gene rearrangement, SDHC protein loss, succinate accumulation, and the resultant hypoproliferative phenotype. We further perform global transcriptomic, epigenomic, and proteomic characterization of changes resulting from SDHC loss, identifying specific perturbations at each biological level. We compare the observed patterns of epigenomic derangement to another previously-described immortalized mouse chromaffin cell model of SDHB loss, and compare both models to human SDH-loss tumors. Finally, we perform analysis of SDHC synthetic lethality with lactate dehydrogenase A (LDHA) and pyruvate carboxylase (PCX), which are important for regeneration of NAD+ and aspartate biosynthesis, respectively. Our data show that SDH-loss cells are selectively vulnerable to LDH genetic knock-down or chemical inhibition, suggesting that LDH inhibition may be an effective therapeutic strategy for SDH-loss PPGL.