Pheochromocytomas and paragangliomas are rare neuroendocrine tumours. Around 20-25% of patients develop metastases, for which there is an urgent need of prognostic markers and therapeutic stratification strategies. The presence of a MAML3-fusion is associated with increased metastatic risk, but neither the processes underlying disease progression, nor targetable vulnerabilities have been addressed. We have compiled a cohort of 850 patients, which has shown a 3.65% fusion prevalence and represents the largest MAML3-positive series reported to date. While MAML3-fusions mainly cause single pheochromocytomas, we also observed somatic post-zygotic events, resulting in multiple tumours in the same patient. MAML3-tumours show increased expression of neuroendocrine-to-mesenchymal transition markers, MYC-targets, and angiogenesis-related genes, leading to a distinct tumour microenvironment with unique vascular and immune profiles. Importantly, our findings have identified MAML3-tumours specific vulnerabilities beyond Wnt-pathway dysregulation, such as a rich vascular network, and overexpression of PD-L1 and CD40, suggesting potential therapeutic targets.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

(A) Quantitative PCR analysis of RBP1 expression of the 49 tumors included in the study compared to controls comprising five tumors carrying mutation in Krebs cycle genes and five cases carrying mutations in RET or NF1. RU: relative units. (B) SDHB immunohistochemistry of tumor_4 compared to a positive control tumor carrying a non-SDH mutation. The scale bars represent 50μm. (C) Representation of DNA methylation (M_values) of the CpG island probes located within the SDHC locus of tumor_4 compared to a blood sample from the same patient and a control DNA. (D) Immunohistochemical staining of 5-hmC in IDH3B-mutated tumor. Nuclear 5-hmC was observed only in sustentacular and some stromal cells. The scale bar represents 50μm.

Legends of the Supplementary Figures

(A) GOT2 western blot of HeLa cells stably silenced for GOT2 expression by shRNA transfection compared to non-silenced scrambled (Scr) control cells. β-actin was used as a loading control. (B) Number of GOT2 KD HeLa cells after transfection with empty vector (EV), GOT2- WT cDNA, and GOT2- c.357A>T. Cells were seeded into 12-well plates and incubated for various times, as indicated. The counts are reported as means (n=3). A t-test was applied to test for differences. n.s.: not significant.

Introduction The percentage of patients diagnosed with pheochromocytoma and paraganglioma (altogether PPGL) carrying known germline mutations in one of the over fifteen susceptibility genes identified to date has dramatically increased during the last two decades, accounting for up to 35-40% of PPGL patients. Moreover, the application of NGS to the diagnosis of PPGL detects unexpected co-occurrences of pathogenic allelic variants in different susceptibility genes. Methods Herein we uncover several cases with dual mutations in NF1 and other PPGL genes by targeted sequencing. We studied the molecular characteristics of the tumours with co-occurrent mutations, using omic tools to gain insight into the role of these events in tumour development. Results Amongst 23 patients carrying germline NF1 mutations, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic mutations in H3-3A and PRKAR1A. Three additional patients, with somatic mutations in NF1 were found carrying germline pathogenic mutations in SDHB or DLST, and a somatic truncating mutation in ATRX. Two of the cases with dual germline mutations showed multiple pheochromocytomas or extra-adrenal paragangliomas - an extremely rare clinical finding in NF1 patients. Transcriptional and methylation profiling and metabolite assessment showed an “intermediate signature” to suggest that both variants had a pathological role in tumour development. Discussion In conclusion, mutations affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signalling) co-occurring in the same patient could provide a selective advantage for the development of PPGL, and explain the variable expressivity and incomplete penetrance observed in some patients.

PDF file - 72KB, Characteristics of the human cells used for qRT-PCR

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 exhibited global hyposuccinylation, while cells expressing DLST-p.Y422C exhibited significant but more limited hyposuccinylation (Figure 1E-G, Supplementary Table S3). Some proteins with multiple down-regulated succinylation sites in DLST G374E cells were involved in the metabolic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, such as the TCA cycle and glycolysis/gluconeogenesis (Figure 1H). Overall, cells carrying PPGL-causing DLST mutations exhibited hyposuccinylation of mitochondrial proteins, which was particularly pronounced in G374E-carrying cells (Supplementary Figure S4A). Changes in mitochondrial succinylation were validated in PPGL tumors using anti-succinyllysine immunohistochemistry: DLST-mutated tumors exhibited diffuse cytoplasmic staining, whereas PPGLs with mutations in other susceptibility genes had granular and perinuclear succinylation patterns (Figure 1I). These results suggest that DLST mutations remodeled the cellular succinylome, causing mitochondrial hyposuccinylation and potentially altering the function of key proteins in PPGL development. Numerous metabolic enzymes are regulated by succinylation, either modulating their activity, degradation, or ability to interact with other proteins. For example, desuccinylation of the succinate dehydrogenase complex flavoprotein subunit A was reported to inhibit its enzymatic activity and promote clear cell renal cell carcinoma cell proliferation in vitro, and GLS desuccinylation protected it from ubiquitin-mediated degradation, promoting breast tumorigenesis [8]. Overall, histone succinylation was reduced by DLST-KO and was not fully restored to WT levels upon reintroducing DLST mutants (Supplementary Figure S4B, Supplementary Table S4). The molecular consequences of OGDHc-mediated histone succinylation have been described [4], but further studies are needed to unravel the implications of hyposuccinylation of specific histone residues. Immunofluorescence staining showed that all DLST protein variants were mainly localized in the mitochondria regardless of mutations (Figure 1J), though they were also identified in other compartments by immunoblotting (Figure 1K). Of the other OGDHc components, dihydrolipoamide dehydrogenase (DLD) was localized in the mitochondria but was also present in the nucleus, while OGDH was generally found in mitochondria but was absent in DLST-KO cells (Supplementary Figure S5). The low OGDH levels in DLST-KO cells were confirmed by immunoblotting (Figure 1K), suggesting that without DLST, OGDHc assembly is impaired and OGDH is degraded, consistent with the high OGDH degradation rates observed in the presence of OGDH mutations causing protein structural instability [9]. This result suggests that DLST plays a key role in OGDHc assembly, supporting the proposed stepwise assembly mechanism in which the stronger OGDH-DLST interaction forms before DLD is anchored [6]. Immunoblotting revealed that OGDHc assembly was unaffected by PPGL-causing DLST mutations, but a significant decrease in NADH production demonstrated the mutations’ profound effect on OGDHc activity (Supplementary Figure S6). Overall, these findings showed that DLST mutations might not affect OGDHc assembly or subcellular location but impair its enzymatic activity. Gene set enrichment analysis of RNA-sequencing data showed that when compared to DLST-WT cells, the hallmark gene sets of hypoxia and glycolysis were up-regulated, while the oxidative phosphorylation set was down-regulated in all cell lines carrying DLST alterations, including a lymphoblastoid cell line derived from a patient with the DLST-p.G374E mutation (Figure 1L, Supplementary Figure S7). This link between DLST mutations and hypoxia/glycolysis pathway up-regulation was confirmed by functional studies showing differences in glucose-dependent growth, oxygen consumption and ATP production in cells carrying DLST alterations (Supplementary Figure S8). However, DNA methylation profiling revealed no statistically significant differences between the H838 cell lines, indicating that the change in gene expression is not due to altered methylation (data not shown). This is consistent with the pseudohypoxic expression profile and absence of global hypermethylation observed in DLST-mutated tumors. It has been proposed that the shift from oxidative to glycolytic metabolism observed in esophageal squamous cell carcinoma cells could lead to global hyposuccinylation due to SucCoA depletion [10]. Our results suggest the existence of a feedback loop whereby reduced protein succinylation induces a similar metabolic shift towards a hypoxic state. In conclusion, we show in this study that global protein succinylation levels depend strongly on DLST, advancing our understanding of this PTM's mechanistic role and highlighting DLST as a promising therapeutic target for treating diseases linked to dysregulated succinylation. Furthermore, we show that DLST mutations found in PPGL patients can remodel the cellular succinylome and cause a transcriptional shift from oxidative phosphorylation to a hypoxic cellular state. Sara Mellid contributed to experimental design, data acquisition, analysis, interpretation, and manuscript writing. Fernando García and Javier Muñoz contributed to data acquisition, analysis and interpretation. Alberto Díaz-Talavera and Ángel Mario Martínez-Montes contributed to bioinformatics and statistical analysis. Juan María Roldán-Romero, Eduardo Gil, Carlos Valdivia, Scherezade Jiménez, Manuel Pérez-Martínez, Ana Cerezo, Clara María Santiveri, Manel Esteller, Ramón Campos-Olivas and Javier Coloma helped conducting the experiments. Bruna Calsina, María Monteagudo, Rocío Letón, María Santos, Javier Lanillos, Natalia Martínez-Puente, Javier de Nicolás-Hernández, Emiliano Honrado and Eduardo Caleiras contributed to data analysis and interpretation. Cristina Montero-Conde, Luis Javier Leandro-García, Cristina Rodríguez-Antona and Mercedes Robledo provided suggestions and contributed to critical revision of the manuscript. Alberto Cascón contributed to the conception, experimental design, data interpretation, and critical revision of the manuscript. All authors reviewed and edited the manuscript. All authors read and approved the final manuscript. The authors have nothing to report. The authors declare no conflict of interest. This work was supported by the Instituto de Salud Carlos III (ISCIII) through the “Acción Estratégica en Salud” (AES) (projects PI18/00454 and PI22/01490 to A.C. and PI20/01169 to M.R.), cofounded by the European Regional Development Fund (ERDF). Sara Mellid was supported by the Spanish Ministry of Science, Innovation and Universities “Formación del Profesorado Universitario- FPU” fellowship with ID number FPU19/04940. Luis J. Leandro-García is supported by ‘la Caixa’ Foundation (ID 100010434) under agreement LCF/BQ/PI20/11760011. Not applicable. The study was approved by the ethics committee of Instituto de Salud Carlos III (CEI PI54_2016-v2) and obtained patients’ consent to participate. Data and materials supporting these findings will be available upon reasonable request. The DNA methylation data are available in the National Center for Biotechnology Information GEO database under the accession numbers GSE210809, GSE111336 and GSE123185. The raw data of the RNA-seq used in this study are available in GEO under the accession number GSE210808 and in the European Genome-phenome archive under the accession number EGAS00001006044. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD036782. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:Approximately 15% of adult GIST patients harbor tumors that are wild-type for KIT and PDGFRα genes (KP-wtGIST). These tumors usually have SDH deficiencies, exhibit a more indolent behavior and are resistant to imatinib. Underlying oncogenic mechanisms in KP-wtGIST include overexpression of HIF1α high IGFR signaling through the MAPK pathway or BRAF activating mutation, among others. As regorafenib inhibits these signaling pathways, it was hypothesized that it could be more active as upfront therapy in advanced KP-wtGIST. METHODS:Adult patients with advanced KP-wtGIST after central confirmation by NGS, naïve of systemic treatment for advanced disease, were included in this international phase II trial. Eligible patients received regorafenib 160 mg per day for 21 days every 28 days. The primary endpoint was disease control rate (DCR), according to RECIST 1.1 at 12 weeks by central radiological assessment. RESULTS:From May 2016 to October 2020, 30 patients were identified as KP-wtGIST by Sanger sequencing and 16 were confirmed by central molecular screening with NGS. Finally, 15 were enrolled and received regorafenib. The study was prematurely closed due to the low accrual worsened by COVID outbreak. The DCR at 12 weeks was 86.7% by central assessment. A subset of 60% experienced some tumor shrinkage, with partial responses and stabilization observed in 13% and 87% respectively, by central assessment. SDH-deficient GIST showed better clinical outcome than other KP-wtGIST. CONCLUSIONS:Regorafenib activity in KP-wtGIST compares favorably with other tyrosine kinase inhibitors, especially in the SDH-deficient GIST subset and it should be taken into consideration as upfront therapy of advanced KP-wtGIST. TRIAL REGISTRATION:ClinicalTrials.gov Identifier: NCT02638766.

PDF file, 794KB, Supplementary figure S1. Gross MAX deletion analysis Supplementary figure S2. Immunohistochemical assessment. Supplementary figure S3. Pedigree of 3 families with affected relatives in more than one generation.

PDF file - 99K, Clinical characteristics of the patients

Genes included in the targeted next-generation sequencing panel

DNA methylation (M-values) of the CpG island probes located within six PCC/PGL susceptibility genes encoding Krebs cycle enzymes (SDHA, SDHAF2, SDHB, SDHD, FH, and MDH2) in the 11 analyzed tumors, compared to in vitro methylated DNA (IVD).

Supplementary Information and Tables 1-5 from Association Study of 69 Genes in the Ret Pathway Identifies Low-penetrance Loci in Sporadic Medullary Thyroid Carcinoma

(A) Aspartate/glutamate ratios assessed by LC-MS in GOT2 KD Hela cells transfected with empty vector (EV), GOT2 wild-type (WT) cDNA, GOT2- c.223T>G cDNA, and GOT2- c.357A>T cDNA. The ratios were reported as means (n=3). Error bars represent standard deviations. A t-test was applied to test for differences between GOT WT and GOT2- c.223T>G and - c.357A>T transfected cells. n.s.: not significant. (B) Succinate/fumarate ratios assessed by LC-MS in OGDHL KD Hela cells transfected with EV, OGDHL WT cDNA, and OGDHL- c.750G>T cDNA. The ratios were reported as means (n=3). Error bars represent standard deviations. A t-test was applied to test for differences. n.s.: not significant

PDF file - 79KB, Oligonucleotides used for TUBB1 sequencing, cloning and mutagenesis

Supplementary Table 1: Clinicopathological features, TERT-related molecular data and telomere lengths of analyzed thyroid tumor series expressed in a.u.f. (arbitrary units of fluorescence). Na: not available data.Supplementary methods table 1: Log2CPMs (log2 counts per million) normalized gene expression matrix of 26 genes involved in telomere maintenance (rows) for the 106 tumor series (columns). Tumor/gene expression cells of TERT probes with more than 3 normalized reads are colored in pink. The TERT_AVG row corresponds to the mean expression values of the 4 TERT probes for each tumor. Tumor cells with at least 1 probe with more than 3 reads are colored in pink and these were considered positive for TERT expression in subsequent analyses. Outlier gene expression thresholds used to generate the gene expression print in Figure 1B (TERC, TCAB1, DKC1, NAT10, TINF2 and POT1) are indicated. Two-tailed Mann-Whitney U-test p-value and FDR-adjusted p-value for the differential gene expression analysis between tumors classes (DF and PP) are included (colums DF and DG, respectively). DF: tumors from disease-free patients; PP: clinically aggressive tumors (PP, n=48).

BACKGROUND:The limited knowledge about the PTEN hamartoma tumor syndrome (PHTS) makes its diagnosis a challenging task. We aimed to define the clinical and genetic characteristics of this syndrome in the Spanish population and to identify new genes potentially associated with the disease.RESULTS:We reviewed the clinical data collected through a specific questionnaire in a series of 145 Spanish patients with a phenotypic features compatible with PHTS and performed molecular characterization through several approaches including next generation sequencing and whole exome sequencing (WES). Macrocephaly, mucocutaneous lesions, gastrointestinal polyposis and obesity are prevalent phenotypic features in PHTS and help predict the presence of a PTEN germline variant in our population. We also find that PHTS patients are at risk to develop cancer in childhood or adolescence. Furthermore, we observe a high frequency of variants in exon 1 of PTEN, which are associated with renal cancer and overexpression of KLLN and PTEN. Moreover, WES revealed variants in genes like NEDD4 that merit further research.CONCLUSIONS:This study expands previously reported findings in other PHTS population studies and makes new contributions regarding clinical and molecular aspects of PHTS, which are useful for translation to the clinic and for new research lines.