SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas

•Generation and phenotypic characterization of chromaffin cell SDHB knockout mice•SDHB loss and succinate accumulation are insufficient for pheochromocytoma initiation•Dual SDHB/NF1 chromaffin cell knockout mice develop SDHx-like pheochromocytomas•Robust survival of SDHB-deficient chromaffin cells requires an ample redox environment Inherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation. Inherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation. Succinate belongs to a family of so-called “oncometabolites,” a collection of TCA cycle-derived cellular metabolites that can pathologically accumulate to promote cellular metabolic reprogramming and drive neoplasia (Dang et al., 2009Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. et al.Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Google Scholar; Yang et al., 2013Yang M. Soga T. Pollard P.J. Oncometabolites: linking altered metabolism with cancer.J. Clin. Invest. 2013; 123: 3652-3658Google Scholar). Normally, succinate is oxidized to fumarate by the mitochondrial enzyme succinate dehydrogenase (SDH), complex II of the electron transport chain and a component of the TCA cycle. Germline loss-of-function mutations in succinate dehydrogenase (SDH) genes (SDHA, SDHB, SDHC, SDHD, or SDHAF2) cause the autosomal dominant hereditary pheochromocytoma and paraganglioma tumor syndrome (hPPGLs) (Baysal et al., 2000Baysal B.E. Ferrell R.E. Willett-Brozick J.E. Lawrence E.C. Myssiorek D. Bosch A. Van Der Mey A. Taschner P.E. Rubinstein W.S. Myers E.N. et al.Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma.Science. 2000; 287: 848-851Google Scholar). The hPPGL syndrome exhibits incomplete penetrance, as stochastic loss of the wild-type allele, generally in the context of a partial chromosomal deletion, is required for tumor formation (Fishbein et al., 2017Fishbein L. Leshchiner I. Walter V. Danilova L. Robertson A.G. Johnson A.R. Lichtenberg T.M. Murray B.A. Ghayee H.K. Else T. et al.Comprehensive molecular characterization of pheochromocytoma and paraganglioma.Cancer Cell. 2017; 31: 181-193Google Scholar). Tumors with SDHx mutations exhibit impaired succinate-to-fumarate oxidase activity (Rapizzi et al., 2012Rapizzi E. Ercolino T. Canu L. Giache V. Francalanci M. Pratesi C. Valeri A. Mannelli M. Mitochondrial function and content in pheochromocytoma/paraganglioma of succinate dehydrogenase mutation carriers.Endocr. Relat. Cancer. 2012; 19: 261-269Google Scholar; Favier et al., 2009Favier J. Briere J.J. Burnichon N. Riviere J. Vescovo L. Benit P. Giscos-Douriez I. De Reynies A. Bertherat J. Badoual C. et al.The Warburg effect is genetically determined in inherited pheochromocytomas.PLoS One. 2009; 4: e7094Google Scholar) and accumulate succinate to levels as high as 100-fold normal (Lendvai et al., 2014Lendvai N. Pawlosky R. Bullova P. Eisenhofer G. Patocs A. Veech R.L. Pacak K. Succinate-to-fumarate ratio as a new metabolic marker to detect the presence of SDHB/D-related paraganglioma: initial experimental and ex vivo findings.Endocrinology. 2014; 155: 27-32Google Scholar). The current model of SDHx tumorigenesis proposes that high levels of succinate inhibit 2-oxoglutarate (2-OG)-dependent enzymes, leading to epigenetic changes and pseudohypoxia that promote tumorigenesis. Whether succinate accumulation is sufficient to promote tumor formation and how the loss of SDH enzymatic activity leads to tumorigenesis remains uncertain. It is well demonstrated that succinate levels in SDHx-PPGL tumor tissue are substantially elevated relative to non-SDHx tumors and normal organ levels (Lussey-Lepoutre et al., 2016Lussey-Lepoutre C. Bellucci A. Morin A. Buffet A. Amar L. Janin M. Ottolenghi C. Zinzindohoue F. Autret G. Burnichon N. et al.In vivo detection of succinate by magnetic Resonance spectroscopy as a hallmark of SDHx mutations in paraganglioma.Clin. Cancer Res. 2016; 22: 1120-1129Google Scholar) (Richter et al., 2014Richter S. Peitzsch M. Rapizzi E. Lenders J.W. Qin N. De Cubas A.A. Schiavi F. Rao J.U. Beuschlein F. Quinkler M. et al.Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/paragangliomas due to succinate dehydrogenase deficiency.J. Clin. Endocrinol. Metab. 2014; 99: 3903-3911Google Scholar). In vitro data show that knockdown of SDHx subunits or chemical inhibition of SDH leads to succinate accumulation in diverse cell lines, although the extent of succinate elevation varies substantially (Xiao et al., 2012Xiao M. Yang H. Xu W. Ma S. Lin H. Zhu H. Liu L. Liu Y. Yang C. Xu Y. et al.Inhibition of alpha-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors.Genes Dev. 2012; 26: 1326-1338Google Scholar; Letouze et al., 2013Letouze E. Martinelli C. Loriot C. Burnichon N. Abermil N. Ottolenghi C. Janin M. Menara M. Nguyen A.T. Benit P. et al.SDH mutations establish a hypermethylator phenotype in paraganglioma.Cancer Cell. 2013; 23: 739-752Google Scholar). Consequently, the prevailing hypothesis, based on analysis of SDHx and other mitochondrial protein mutation-caused tumors (isocitrate dehydrogenase [IDH]1/2- and fumarate hydratase [FH]-related glioblastoma and renal cell carcinoma, respectively), is that succinate accumulation acts as an “oncometabolite” by competitively inhibiting 2-OG-dependent enzymes, which leads to DNA and histone hypermethylation, and hypoxia-inducible factor (HIF) stabilization. The importance of these pathways in PPGL tumorigenesis is also supported by the overlapping phenotype caused by mutation of other enzymes in these pathways: VHL, DNA methyltransferase-3α (DNMT3A), and HIF2α, which similarly augment HIF and DNA methylation (Remacha et al., 2018Remacha L. Curras-Freixes M. Torres-Ruiz R. Schiavi F. Torres-Perez R. Calsina B. Leton R. Comino-Mendez I. Roldan-Romero J.M. Montero-Conde C. et al.Gain-of-function mutations in DNMT3A in patients with paraganglioma.Genet. Med. 2018; 20: 1644-1651Google Scholar; Fishbein et al., 2017Fishbein L. Leshchiner I. Walter V. Danilova L. Robertson A.G. Johnson A.R. Lichtenberg T.M. Murray B.A. Ghayee H.K. Else T. et al.Comprehensive molecular characterization of pheochromocytoma and paraganglioma.Cancer Cell. 2017; 31: 181-193Google Scholar). The 2-OG-dependent dioxygenase enzymes include the PHD, TET, and KDM/JMJ families (Losman et al., 2020Losman J.A. Koivunen P. Kaelin Jr., W.G. 2-Oxoglutarate-dependent dioxygenases in cancer.Nat. Rev. Cancer. 2020; 20: 710-726Google Scholar). These enzymes, which modify the epigenetic landscape (TETs and KDM/JmJs) and regulate the adaptive cellular response to hypoxia via HIF1/2α stability (PHDs), utilize Fe2+, O2, and ascorbate as co-factors and hydroxylate or demethylate their substrates via concomitant oxidation of 2-OG to succinate. The TET enzyme family (TET1, TET2, TET3) are DNA demethylases that mediate the conversion of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmC). TET inhibition by succinate manifests histologically in SDHx tumors as a loss of 5hmC immunostaining (Hoekstra et al., 2015aHoekstra A.S. De Graaff M.A. Briaire-De Bruijn I.H. Ras C. Seifar R.M. Van Minderhout I. Cornelisse C.J. Hogendoorn P.C. Breuning M.H. Suijker J. et al.Inactivation of SDH and FH cause loss of 5hmC and increased H3K9me3 in paraganglioma/pheochromocytoma and smooth muscle tumors.Oncotarget. 2015; 6: 38777-38788Google Scholar), while epigenetic DNA analysis reveals a substantial increase in CpG Island methylation (Letouze et al., 2013Letouze E. Martinelli C. Loriot C. Burnichon N. Abermil N. Ottolenghi C. Janin M. Menara M. Nguyen A.T. Benit P. et al.SDH mutations establish a hypermethylator phenotype in paraganglioma.Cancer Cell. 2013; 23: 739-752Google Scholar). The JmjC-domain enzyme family are histone demethylases that mediate the removal of histone lysine methyl groups (Laukka et al., 2018Laukka T. Myllykoski M. Looper R.E. Koivunen P. Cancer-associated 2-oxoglutarate analogues modify histone methylation by inhibiting histone lysine demethylases.J. Mol. Biol. 2018; 430: 3081-3092Google Scholar). These post-translational modifications influence chromatin structure, gene expression, and DNA replication/repair. Succinate inhibition of JmjC enzymes leads to an increase in multiple histone 3 lysine methyl marks, such as H3K9me3, H3K27me3, and H3K4me3 (Letouze et al., 2013Letouze E. Martinelli C. Loriot C. Burnichon N. Abermil N. Ottolenghi C. Janin M. Menara M. Nguyen A.T. Benit P. et al.SDH mutations establish a hypermethylator phenotype in paraganglioma.Cancer Cell. 2013; 23: 739-752Google Scholar; Hoekstra et al., 2015aHoekstra A.S. De Graaff M.A. Briaire-De Bruijn I.H. Ras C. Seifar R.M. Van Minderhout I. Cornelisse C.J. Hogendoorn P.C. Breuning M.H. Suijker J. et al.Inactivation of SDH and FH cause loss of 5hmC and increased H3K9me3 in paraganglioma/pheochromocytoma and smooth muscle tumors.Oncotarget. 2015; 6: 38777-38788Google Scholar). While there are conflicting data with respect to HIF activation in PPGLs, it is clear that SDHx tumors exhibit a pseudohypoxic transcriptional signature, which is suggestive of HIF1/2 activation but does not exclude alternative mechanisms. Indeed, transcriptional analysis of PPGLs may be used to group tumors into two predominant clusters, reflecting the underlying driver mutation. Cluster 1 tumors, which includes several tricarboxylic acid cycle enzyme genes (SDHx, IDH1/2, FH, and MDH2), VHL, EPAS1, and EGLN1/2 mutations, all share a pseudohypoxic transcriptional profile and are hypermethylated. By contrast, Cluster 2 tumors, including NF1, RET, and HRAS mutations, are enriched in kinase-signaling transcripts and have a lower degree of DNA methylation (Fishbein et al., 2017Fishbein L. Leshchiner I. Walter V. Danilova L. Robertson A.G. Johnson A.R. Lichtenberg T.M. Murray B.A. Ghayee H.K. Else T. et al.Comprehensive molecular characterization of pheochromocytoma and paraganglioma.Cancer Cell. 2017; 31: 181-193Google Scholar; Dahia et al., 2005Dahia P.L. Ross K.N. Wright M.E. Hayashida C.Y. Santagata S. Barontini M. Kung A.L. Sanso G. Powers J.F. Tischler A.S. et al.A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas.PLoS Genet. 2005; 1: 72-80Google Scholar). Although the transcriptomics suggest succinate-mediated HIF stabilization, HIF1α immunostaining of human pheochromocytomas does not distinguish between SDHx-mutated and wild-type (wt)-SDHx tumors (Favier et al., 2009Favier J. Briere J.J. Burnichon N. Riviere J. Vescovo L. Benit P. Giscos-Douriez I. De Reynies A. Bertherat J. Badoual C. et al.The Warburg effect is genetically determined in inherited pheochromocytomas.PLoS One. 2009; 4: e7094Google Scholar; Bernardo-Castineira et al., 2019Bernardo-Castineira C. Saenz-De-Santa-Maria I. Valdes N. Astudillo A. Balbin M. Pitiot A.S. Jimenez-Fonseca P. Scola B. Tena I. Molina-Garrido M.J. et al.Clinical significance and peculiarities of succinate dehydrogenase B and hypoxia inducible factor 1alpha expression in parasympathetic versus sympathetic paragangliomas.Head Neck. 2019; 41: 79-91Google Scholar) and HIF2α immunostaining is not universally positive in SDHx pheochromocytomas (Pollard et al., 2006Pollard P.J. El-Bahrawy M. Poulsom R. Elia G. Killick P. Kelly G. Hunt T. Jeffery R. Seedhar P. Barwell J. et al.Expression of HIF-1alpha, HIF-2alpha (EPAS1), and their target genes in paraganglioma and pheochromocytoma with VHL and SDH mutations.J. Clin. Endocrinol. Metab. 2006; 91: 4593-4598Google Scholar). The role of succinate inhibition of PHDs and its relationship to the pseudohypoxic transcriptional profile remains incompletely understood. Given the mechanistic uncertainty of SDHx tumorigenesis, numerous efforts to generate mouse models of Cluster 1 PPGLs have been made; however, these attempts have been unsuccessful, leading some to suggest its impossibility (reviewed in Lepoutre-Lussey et al., 2016Lepoutre-Lussey C. Thibault C. Buffet A. Morin A. Badoual C. Benit P. Rustin P. Ottolenghi C. Janin M. Castro-Vega L.J. et al.From Nf1 to Sdhb knockout: successes and failures in the quest for animal models of pheochromocytoma.Mol. Cell Endocrinol. 2016; 421: 40-48Google Scholar; Lussey-Lepoutre et al., 2018Lussey-Lepoutre C. Buffet A. Morin A. Goncalves J. Favier J. Rodent models of pheochromocytoma, parallels in rodent and human tumorigenesis.Cell Tissue Res. 2018; 372: 379-392Google Scholar). Approaches taken include global and conditional Sdhd disruption (Piruat et al., 2004Piruat J.I. Pintado C.O. Ortega-Saenz P. Roche M. Lopez-Barneo J. The mitochondrial SDHD gene is required for early embryogenesis, and its partial deficiency results in persistent carotid body glomus cell activation with full responsiveness to hypoxia.Mol. Cell Biol. 2004; 24: 10933-10940Google Scholar; Diaz-Castro et al., 2012Diaz-Castro B. Pintado C.O. Garcia-Flores P. Lopez-Barneo J. Piruat J.I. Differential impairment of catecholaminergic cell maturation and survival by genetic mitochondrial complex II dysfunction.Mol. Cell Biol. 2012; 32: 3347-3357Google Scholar), conditional Sdhb deletion, and combined dual Sdhb/Pten loss with PSA-Cre (Lepoutre-Lussey et al., 2016Lepoutre-Lussey C. Thibault C. Buffet A. Morin A. Badoual C. Benit P. Rustin P. Ottolenghi C. Janin M. Castro-Vega L.J. et al.From Nf1 to Sdhb knockout: successes and failures in the quest for animal models of pheochromocytoma.Mol. Cell Endocrinol. 2016; 421: 40-48Google Scholar) as well as Sdhc/p53 loss with Th-Cre (Al Khazal et al., 2020Al Khazal F. Kang S. Nelson Holte M. Choi D.S. Singh R. Ortega-Saenz P. Lopez-Barneo J. Maher 3rd, L.J. Unexpected obesity, rather than tumorigenesis, in a conditional mouse model of mitochondrial complex II deficiency.FASEB J. 2020; 35: e21227Google Scholar). All of these attempts failed to demonstrate elevated succinate in viable SDHx-deficient mouse chromaffin cells. Despite this history, we endeavored to address the question of whether the oncometabolite succinate is sufficient to initiate tumorigenesis via 2-OG enzyme inhibition by conditionally disrupting SDHB expression in the mouse chromaffin cells and studying the metabolic, ultrastructural, and transcriptomic consequences. To conditionally disrupt Sdhb in the mouse adrenal medulla, we used mice with LoxP sites flanking exon 3 and bred them to animals expressing Cre recombinase driven by the tyrosine hydroxylase promoter (Th-Cre) (Savitt et al., 2005Savitt J.M. Jang S.S. Mu W. Dawson V.L. Dawson T.M. Bcl-x is required for proper development of the mouse substantia nigra.J. Neurosci. 2005; 25: 6721-6728Google Scholar). We bred Th-Cre SDHBf/f animals to the mT/mG Cre-reporter mice (Muzumdar et al., 2007Muzumdar M.D. Tasic B. Miyamichi K. Li L. Luo L. A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Google Scholar) to rigorously follow the recombined cell population. As previously reported (Savitt et al., 2005Savitt J.M. Jang S.S. Mu W. Dawson V.L. Dawson T.M. Bcl-x is required for proper development of the mouse substantia nigra.J. Neurosci. 2005; 25: 6721-6728Google Scholar), incomplete Th-Cre-driven recombination occurred in adrenal gland chromaffin cells. To determine the impact of SDHB loss on chromaffin cell survival, we compared the percentage of GFP+ medulla areas between SDHBf/+Rosamtmg/+Th-Cre (HET) and SDHBf/fRosamtmg/+Th-Cre (SDHB) in adult (6-month-old) animals (Figure 1A ). Males from both genotypes had 60% GFP+ medulla area (Figure 1C). Loss of SDHB expression was verified by performing western blots on surgically isolated adrenal medullas (Figure 1B). As anticipated from incomplete recombination, modest residual SDHB expression was present in SDHB medulla. Notably, Ki-67 immunostaining was not significantly different between HET and SDHB medullas (Figure 1D). Therefore, in contrast to expectations and prior mouse modeling attempts, SDHB loss neither provoked nor inhibited chromaffin cell proliferation. To determine if loss of SDHB resulted in succinate accumulation, we measured abundance of succinate in the adrenal medulla by desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) (Figure 1E). DESI-MSI is a nondestructive, ambient ionization method that preserves tissue metabolite distribution, enables detection of minute metabolite alterations, and discriminates between the cortex and medulla of the small murine adrenal gland (Gouw et al., 2019Gouw A.M. Margulis K. Liu N.S. Raman S.J. Mancuso A. Toal G.G. Tong L. Mosley A. Hsieh A.L. Sullivan D.K. et al.The MYC oncogene cooperates with sterol-regulated element-binding protein to regulate lipogenesis essential for neoplastic growth.Cell Metab. 2019; 30: 556-572.e555Google Scholar; Wiseman et al., 2006Wiseman J.M. Ifa D.R. Song Q. Cooks R.G. Tissue imaging at atmospheric pressure using desorption electrospray ionization (DESI) mass spectrometry.Angew. Chem. Int. Ed. Engl. 2006; 45: 7188-7192Google Scholar; Vijayalakshmi et al., 2020Vijayalakshmi K. Shankar V. Bain R.M. Nolley R. Sonn G.A. Kao C.S. Zhao H. Tibshirani R. Zare R.N. Brooks J.D. Identification of diagnostic metabolic signatures in clear cell renal cell carcinoma using mass spectrometry imaging.Int. J. Cancer. 2020; 147: 256-265Google Scholar). Because whole-gland metabolomics gives an averaged chemical footprint across the cortex and medulla, DESI-MSI was crucial to spatially resolve the distributions of succinate and other metabolite levels between these two adrenal compartments. Indeed, succinate was selectively increased (7.6-fold by averaged abundance) in the medulla of SDHB adrenals compared with controls (Figure 1F). Given that each DESI-MSI pixel (∼120 μm2) included 60–100 cells, and approximately 60% of those cells were Cre-recombined, the actual succinate accumulation per SDHB−/− cell was likely higher. The DESI-MSI spectra also contained peaks for epinephrine and norepinephrine; however, these metabolites were unchanged (Figure 1F). These data provided definitive evidence that mouse SDHB-deficient chromaffin cells were viable and exhibited substantial succinate accumulation. Having verified that SDHB loss in mouse chromaffin cells was viable and led to succinate accumulation, we analyzed the effects of SDHB loss at an ultrastructural and an immunohistochemical level. Transmission electron micrographs (TEM) of SDHB medulla revealed large clusters of swollen mitochondria in a subset of chromaffin cells (Figures 2A and S1A). Importantly, these clusters were never observed in HET adrenals. This mitochondrial phenotype occurred in SDHB-null cells but not adjacent SDHB-intact cells, reflecting incomplete Th-Cre-mediated recombination (Figure S1A). Notably, similar mitochondrial ultrastructural changes are characteristically observed in SDHx tumors (Douwes Dekker et al., 2003Douwes Dekker P.B. Hogendoorn P.C. Kuipers-Dijkshoorn N. Prins F.A. Van Duinen S.G. Taschner P.E. Van Der Mey A.G. Cornelisse C.J. SDHD mutations in head and neck paragangliomas result in destabilization of complex II in the mitochondrial respiratory chain with loss of enzymatic activity and abnormal mitochondrial morphology.J. Pathol. 2003; 201: 480-486Google Scholar; Szarek et al., 2015Szarek E. Ball E.R. Imperiale A. Tsokos M. Faucz F.R. Giubellino A. Moussallieh F.M. Namer I.J. Abu-Asab M.S. Pacak K. et al.Carney triad, SDH-deficient tumors, and Sdhb+/- mice share abnormal mitochondria.Endocr. Relat. Cancer. 2015; 22: 345-352Google Scholar). The TEM findings led us to predict that the mitochondrial clusters represented a general increase in mitochondrial protein that could be used as an immunohistochemical marker for SDHB deficiency. Direct staining for SDHB protein was inconsistent, given a low basal expression level in normal mouse chromaffin cells, where cortical SDHB expression is easily visualized (Figure S1B). Immunostaining of HET and SDHB adrenal glands for several mitochondrial proteins, including cytochrome c and TFAM, demonstrated robust induction in GFP+ cells of SDHB medulla, but not the HET medulla (Figures 2B and S1C). Hence, SDHB disruption caused mitochondrial swelling, clustering, and an apparently expanded mass that was associated with increased mitochondrial protein expression, including the master mitochondrial mass regulator TFAM. To address the question of whether succinate accumulation in SDHB animals resulted in inhibition of TET, PHD, and KDM enzymes yielding the predicted changes in histone and DNA methylation as well as HIF protein stabilization, we performed a series of co-staining experiments. We co-stained GFP with H3K9me3, 5hmC, and Hif1α and compared the staining intensity of each marker between the GFP+ and GFP– populations within a section (Figures 2C, S1D, and S1E). We also looked for HIF2α but failed to detect nuclear HIF2α in any sample. As shown in Figure 2C, H3K9me3 immunostaining was specifically increased in GFP+ cells from SDHB animals but not HET animals, consistent with succinate-mediated inhibition of a KDM4 enzyme. 5hmC staining of HET controls revealed substantial variability in staining intensity throughout the medulla, with some cell clusters staining intensely and others weakly (Figure S1D). Calculation of the ratio of the 5hmC intensity in GFP+/GFP– medulla populations did not indicate a loss of 5hmC in GFP+ SDHB cells. This result was consistent with the observations that robust changes in 5hmC required cellular division (Lu et al., 2012Lu C. Ward P.S. Kapoor G.S. Rohle D. Turcan S. Abdel-Wahab O. Edwards C.R. Khanin R. Figueroa M.E. Melnick A. et al.IDH mutation impairs histone demethylation and results in a block to cell differentiation.Nature. 2012; 483: 474-478Google Scholar), a phenotype not substantiated by Ki-67 staining (Figure 1D). Surprisingly, we found substantial HIF1α expression in a subset of normal chromaffin cells and a paradoxical loss of HIF1α expression in the GFP+ SDHB population (Figure S1E). Aged (>12 months) SDHB animals failed to develop tumors despite succinate accumulation. Therefore, we sought to promote SDHB cell proliferation by providing an additional driving force. While several Cluster 2 genes, such as neurofibromin 1 (NF1) and RET, have been modeled in mice, modeling of Cluster 1 gene (SDHx, VHL, EPAS1)-related PPGLs has been unsuccessful. Accordingly, we asked whether combining Cluster 1 and Cluster 2 gene disruption would enable development of SDHx-like pheochromocytomas. Toward this end, we evaluated publicly available TCGA transcriptomic data from five SDHx and six NF1 (germline mutation) human pheochromocytomas (Fishbein et al., 2017Fishbein L. Leshchiner I. Walter V. Danilova L. Robertson A.G. Johnson A.R. Lichtenberg T.M. Murray B.A. Ghayee H.K. Else T. et al.Comprehensive molecular characterization of pheochromocytoma and paraganglioma.Cancer Cell. 2017; 31: 181-193Google Scholar). Interestingly, two of the six germline NF1 pheochromocytomas were transcriptionally classified as Cluster 1 (pseudohypoxic), illustrating phenotypic overlap between some NF1 and SDHx pheochromocytomas. Additionally, loss of the NF1 locus (17q11) has been observed in some SDHB PPGLs (Fishbein et al., 2017Fishbein L. Leshchiner I. Walter V. Danilova L. Robertson A.G. Johnson A.R. Lichtenberg T.M. Murray B.A. Ghayee H.K. Else T. et al.Comprehensive molecular characterization of pheochromocytoma and paraganglioma.Cancer Cell. 2017; 31: 181-193Google Scholar). These data, combined with the knowledge that SDHB and NF1 tumors both exhibit loss of the 1p chromosome, suggested possible overlapping tumorigenic mechanisms. On the basis of apparently overlapping mechanisms of NF1 and SDHx pheochromocytoma formation, we tested whether combined NF1 and SDHB disruption could drive SDHx-like pheochromocytoma formation in mice. NF1 is a GTPase-activating protein that negatively regulates the RAS/MAPK and PI3K/mTOR pathways (Kiuru and Busam, 2017Kiuru M. Busam K.J. The NF1 gene in tumor syndromes and melanoma.Lab Invest. 2017; 97: 146-157Google Scholar). In prior work, approximately 18% of mice heterozygous for a NF1 loss-of-function mutation developed pheochromocytomas (Jacks et al., 1994Jacks T. Shih T.S. Schmitt E.M. Bronson R.T. Bernards A. Weinberg R.A. Tumour predisposition in mice heterozygous for a targeted mutation in Nf1.Nat. Genet. 1994; 7: 353-361Google Scholar). We reasoned that if the SDHB mice failed to develop pheochromocytomas as a consequence of a missing replication trigger, simultaneous loss of NF1 would be sufficient to induce tumor formation. Toward this end, we bred SDHBf/f and NF1f/f animals (Zhu et al., 2001Zhu Y. Romero M.I. Ghosh P. Ye Z. Charnay P. Rushing E.J. Marth J.D. Parada L.F. Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain.Genes Dev. 2001; 15: 859-876Google Scholar) to generate the following experimental genotypes: SDHBf/+NF1f/+Rosamtmg/+Th-Cre (HET/HET), SDHBf/fNF1f/+Rosamtmg/+Th-Cre (SDHB/HET), SDHBf/+NF1f/fRosamtmg/+Th-Cre (HET/NF1), and SDHBf/fNF1f/fRosamtmg/+Th-Cre (SDHB/NF1). When adrenal medullas from all four genotypes were examined at 8 weeks of age, both SDHB/NF1 (35.5%) and HET/NF1 (34.1%) glands had significantly more GFP+ area compared with SDHB/HET glands (22%) (Figures 3A–3C ). The difference between HET/HET and SDHB/HET %GFP+ area at 8 weeks was not statistically different. When animals were aged to 22 weeks, differences were more prominent. At 22 weeks, 71% of the SDHB/NF1 medulla area was GFP+ compared with 53% in HET/HET and 60% in HET/NF1 medullas. Notably, the patterns of SDHB/NF1 and HET/NF1 GFP+ cell expansion were distinct. In the SDHB/NF1 medullas, GFP+ cell clusters were uniformly distributed; however, GFP+ cell clusters in HET/NF1 medullas were mostly similar to HET/HET medullas with discrete areas of densely packed GFP+ cells, suggestive of nodular hyperplasia (Figure 3A). Importantly, SDHB/NF1 medulla exhibited the highest replication activity (Ki-67+ nuclei; Figure 3D), demonstrating a cooperative proliferative effect of concurrent SDHB and NF1 gene disruption. The increased SDHB/NF1 medulla replication (Ki-67) and expanded GFP+ area led to increased adrenal gland mass. Both SDHB/NF1 and HET/NF1 glands were approximately 24% heavier than control glands (Figure S2A) at 22 weeks. Paradoxically, SDHB/HET glands were 47% larger than those of controls; however, this was related to increased cortical area and fat accumulation (see below). Next, we investigated whether SDHB/NF1 medullary cells, like their SDHB counterpart, exhibited succinate accumulation and the striking mitochondrial phenotype. Indeed, TEM ultrastructure analysis revealed that HET/NF1 mitochondria were indistinguishable from Cre negative controls, whereas SDHB/NF1 cells contained large clusters of swollen mitochondria with remarkably intact cristea, as observed in SDHB/HET medullas (Figure 4A ). DESI-MSI analysis of HET/NF1 and SDHB/NF1 adrenal sections confirmed elevated succinate abundance in the SDHB/NF1 but not HET/NF1 medullas, which were indistinguishable from those of controls (Figures 4B and 4C). Catecholamine levels in these 5-month-old animals were not significantly changed from those of controls (Figure S2C). These data indicated a dominant effect of the SDHB-associated phenotype in the context of concurrent NF1 deficiency. Next, we explored whether the hypermethylation phenotype of replicating SDHB/NF1 cells was distinct from SDHB chromaffin cells. In contrast to SDHB/HET adrenal glands, HET/NF1 and SDHB/NF1 samples demonstrated s