Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/ mTOR axis in metastatic pheochromocytoma/ paraganglioma

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar y los autores pertenecientes a la UAMPheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients’ liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients’ management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitorsThis work was supported by the Instituto de Salud Carlos III (ISCIII), Acción Estratégica en Salud, cofounded by FEDER, [grant number PI14/00240, PI17/01796 to M.R., PI15/00783 to A.C], the Paradifference Foundation [no grant number applicable to M.R.], the ANR [ANR-2011-JCJC-00701 MODEOMAPP to AP.G-R], the European Union [FP7/2007-2013 n° 259735, Horizon 2020 n° 633983 to AP.G-R], Epigénétique et Cancer [EPIG201303 METABEPIC to AP.G-R], the the Ligue Nationale contre le Cancer ["Cartes d'Identité des Tumeurs (CIT) program" to AP.G-R], the Institut National du Cancer, the Direction Générale de l’Offre de Soins [PRT-K 2014, COMETE-TACTIC, INCa-DGOS_8663 to AP.G-R], the Deutsche Forschungsgemeinschaft (DFG) [CRC/Transregio 205/1 “The Adrenal: Central Relay in Health and Disease“ to F.B, M.F and G.E], the Rafael del Pino Foundation [Becas de Excelencia Rafael del Pino 2017 to B.C], the Severo Ochoa Excellence Programme [project SEV-2011-0191 to M.C-F], La Caixa Foundation [B004235 to JM.R-R], the Spanish Ministry of Education, Culture and Sport [grant number FPU16/05527 to M.S.], the Site de Recherche Intégré sur le Cancer-SIRIC [CARPEM Project to N.B.] and the AECC Foundation [grant number AIO15152858 to C.M-C

Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma

Adrenal gland diseases; Cancer genomics; Prognostic markersMalalties de les glàndules suprarenals; Genòmica del càncer; Marcadors pronòsticsEnfermedades de las glándulas suprarrenales; Genómica del cáncer; Marcadores pronósticosThe 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.This work was supported by Project PI17/01796 and PI20/01169 to M.R. [Instituto de Salud Carlos III (ISCIII), Acción Estratégica en Salud, cofinanciado a través del Fondo Europeo de Desarrollo Regional (FEDER)], Paradifference Foundation [no grant number applicable to M.R.], Pheipas Association [no grant number applicable to M.R.], the Clinical Research Priority Program of the University of Zurich for the CRPP HYRENE to F.B., the Deutsche Forschungsgemeinschaft (DFG) within the CRC/Transregio 205/1 (Project No. 314061271-TRR205 to to F.B., M.F., N.B., and G.E.) and the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation (Project No. PID2019-111356RA-I00 to G.M.). B.C. was supported by the Rafael del Pino Foundation (Becas de Excelencia Rafael del Pino 2017). A.M.M.-M. was supported by CAM (S2017/BMD-3724; TIRONET2-CM). A.F.-S. and J.L. received the support of a fellowship from La Caixa Foundation (ID 100010434; LCF/BQ/DR21/11880009 and LCF/BQ/DR19/11740015, respectively). M.M., S.M., and M.S. were supported by the Spanish Ministry of Science, Innovation and Universities “Formación del Profesorado Universitario— FPU” fellowship with ID number FPU18/00064, FPU19/04940 and FPU16/05527. A.D.-T. is supported by the Centro de Investigacion Biomédica en Red de Enfermedades Raras (CIBERER). L.J.L.-G. was supported both by the Banco Santander Foundation and La Caixa Postdoctoral Junior Leader Fellowship (LCF/BQ/PI20/11760011). C.M.-C. was supported by a grant from the AECC Foundation (AIO15152858 MONT). We thank the Spanish National Tumor Bank Network (RD09/0076/00047) for the support in obtaining tumorsamples and all patients, physicians and tumor biobanks involved in the study

Novel DNMT3A Germline Variant in a Patient with Multiple Paragangliomas and Papillary Thyroid Carcinoma

Over the past few years, next generation technologies have been applied to unravel the genetics of rare inherited diseases, facilitating the discovery of new susceptibility genes. We recently found germline DNMT3A gain-of-function variants in two patients with head and neck paragangliomas causing a characteristic hypermethylated DNA profile. Here, whole-exome sequencing identifies a novel germline DNMT3A variant (p.Gly332Arg) in a patient with bilateral carotid paragangliomas, papillary thyroid carcinoma and idiopathic intellectual disability. The variant, located in the Pro-Trp-Trp-Pro (PWWP) domain of the protein involved in chromatin targeting, affects a residue mutated in papillary thyroid tumors and located between the two residues found mutated in microcephalic dwarfism patients. Structural modelling of the variant in the DNMT3A PWWP domain predicts that the interaction with H3K36me3 will be altered. An increased methylation of DNMT3A target genes, compatible with a gain-of-function effect of the alteration, was observed in saliva DNA from the proband and in one independent acute myeloid leukemia sample carrying the same p.Gly332Arg variant. Although further studies are needed to support a causal role of DNMT3A variants in paraganglioma, the description of a new DNMT3A alteration in a patient with multiple clinical features suggests a heterogeneous phenotypic spectrum related to DNMT3A germline variants

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/paraganglioma

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through models, and define specific therapeutic options according to tumor genomic features. : We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized . : A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, =4.67·10), and was found associated with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated a repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. : Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/ mTOR axis in metastatic pheochromocytoma/ paraganglioma

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients’ liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients’ management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors

Genomic dissection to identify pheochromocytoma’s Achiles’ heel: novel markers of metastatic disease and therapeutic strategies

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 26-05-2021Esta Tesis tiene embargado el acceso al texto completo hasta el 26-11-2022Pheochromocytomas and paragangliomas (PPGLs) are highly heterogeneous rare neuroendocrine tumors, associated with mutations in one of the at least 20 susceptibility genes described so far related to the disease. Approximately 15% of the PPGLs are metastatic with a 5-year survival rate of 40%. Although some genotype-phenotype correlations have been recognized for patients with PPGLs, disease management continues to be difficult due to their highly variable clinical behavior, and the lack of reliable predictive markers of metastatic PPGL (mPPGL). Moreover, the poor understanding of the mechanisms driving the metastatic behavior hinders the establishment of an efficient therapeutic strategy for mPPGLs. Thus, the principal aim of this Thesis was the discovery of novel molecular markers able to predict the risk of metastasis, helping to improve patients’ clinical management and treatment options. Firstly, we confirmed MDH2 as a novel PPGL susceptibility gene responsible of less than 1% of PPGL cases. For this purpose, we screened a large series of PPGL patients and established a workflow to classify variants of unknown significance. We also described the connection between germline mutations in MDH2 and a noradrenergic and metastatic phenotype. Secondly, by analyzing a large series of miRNome tumor data, we established a miRNA signature associated with metastatic risk and time to progression, which could also be detected in PPGL patients’ serum. MiR21-3p and miR-183-5p were identified as the best markers to predict metastasis, and were found linked in vitro to pro-metastatic characteristics, such as a neuroendocrine-to-mesenchymal transition phenotype, and an enhanced cell migration rate. Through the integration of the miRNome, transcriptome and proteome, and the use of the in vitro models, we deciphered a miR-21-3p/TSC2/mTOR axis with therapeutic relevance for mPPGL patients. In the third study, we aimed to widen the list of mPPGL informative miRNAs. We carried out an extensive analysis of miR-483-5p, uncovering that it is potentially released by the metastases, and consequently higher levels are detected in the circulation of mPPGL patients. Integrative analyses with transcriptomic data suggested its role in metastasis-related processes, and discovered ALCAM, a gene related to tumor immune evasion, as the best target of miR-483-5p. Finally, by performing whole-exome-and transcriptome-sequencing in a large series of mPPGLs with extensive clinical annotated data, a higher tumor mutational burden and microsatellite instability was observed in metastatic tumors, which were both correlated with ATRX/TERT alterations and shorter progression-free survival. Although we encountered a large inter-tumoral heterogeneity at mutational level among mPPGLs, we could evince a functionally relevant enrichment of mutated genes involved in extracellular matrix organization, cell adhesion, and neuron projection morphogenesis. Moreover, this study revealed several biological processes deregulated in metastatic primary tumors at the transcriptional level that are of great significance in terms of further understanding the mechanisms leading the metastatic behavior. One of them is an immunosuppressive phenotype, further evidenced by deconvolution analysis of RNAseq data. In summary, in this Thesis we report novel molecular markers able to assess metastatic potential at diagnosis and to guide the surveillance of those patients with high metastatic risk. Furthermore, we bring to light new data that could help to understand the mechanisms contributing to metastasis, and aid in the discovery of pheochromocytoma’s Achilles heel.The research presented in this Doctoral thesis has been granted with the following awards: 2019 ENS@T PPGL scientific award (November 2019 in Uppsala, Sweden) for the work Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/ paraganglioma to Bruna Calsina and Luis J Castro-Vega. 2018 ENS@T PPGL scientific award (November 2018 in Florence, Italy) for the work Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients to Bruna Calsina and María Currás-Freixes. 2nd oral presentation award at VI CNIO Lab Day (December 2016 in Madrid, Spain) for the work miR213p and miR-183-5p: novel risk markers for metastatic PPGL to Bruna Calsina

Pheochromocytomas and Paragangliomas: Bypassing Cellular Respiration

Abstract: Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors that show the highest heritability of all human neoplasms and represent a paradoxical example of genetic heterogeneity. Amongst the elevated number of genes involved in the hereditary predisposition to the disease (at least nineteen) there are eleven tricarboxylic acid (TCA) cycle-related genes, some of which are also involved in the development of congenital recessive neurological disorders and other cancers such as cutaneous and uterine leiomyomas, gastrointestinal tumors and renal cancer. Somatic or germline mutation of genes encoding enzymes catalyzing pivotal steps of the TCA cycle not only disrupts cellular respiration, but also causes severe alterations in mitochondrial metabolite pools. These latter alterations lead to aberrant accumulation of “oncometabolites„ that, in the end, may lead to deregulation of the metabolic adaptation of cells to hypoxia, inhibition of the DNA repair processes and overall pathological changes in gene expression. In this review, we will address the TCA cycle mutations leading to the development of PPGL, and we will discuss the relevance of these mutations for the transformation of neural crest-derived cells and potential therapeutic approaches based on the emerging knowledge of underlying molecular alterations

Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients

PURPOSE MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes

Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients

Item does not contain fulltex