Pheochromocytomas, catecholamine-secreting tumors of neural crest origin, are frequently hereditary1. However, the molecular basis for the majority of these tumors is unknown2. We identified the transmembrane-encoding TMEM127 gene, on chromosome 2q11, as a novel pheochromocytoma susceptibility gene. In a cohort of 103 samples, truncating germline TMEM127 mutations were detected in one-third of familial and about 3% of sporadic-appearing tumors without a known genetic cause. The wild-type allele was consistently deleted in tumor DNA, suggesting a two-hit mechanism of inactivation. Pheochromocytomas with TMEM127 mutations are transcriptionally related to NF1-mutant tumors and, similarly, show hyperphosphorylation of mTOR targets. Accordingly, in vitro gain- and loss-of-function analyses indicate that TMEM127 is a negative regulator of mTOR. TMEM127 dynamically associates with the endomembrane system and colocalizes with perinuclear (activated) mTOR, suggesting a subcompartmental-specific effect. Our studies unveil TMEM127 as a novel tumor suppressor gene and validate the power of hereditary tumors for elucidating cancer pathogenesis.

HEOCHROMOCYTOMAS AND paragangliomas are chromaffin cell tumors of neural crest origin that arise from the adrenal medulla or extra-adrenal sympathetic paraganglia, respectively, and are frequently catecholamine secreting. 1 These tumors are usually benign and can occur as a single entity or as part of various hereditary tumor syndromes. Genetically, pheochromocytomas and paragangliomas are heterogeneous , with at least one-third of cases resulting from germline but not somatic mutations in 1 of several independent genes: RET, VHL, NF1, and succinate dehydrogenase (SDH) subunit B, C, and D genes. 2-5 More recently, other candidate susceptibil-Author Affiliations are listed at the end of this article.

Pheochromocytomas and paragangliomas are highly vascular tumors of the autonomic nervous system. Germline mutations, including those in hypoxia-related genes, occur in one-third of the cases, but somatic mutations are infrequent in these tumors. Using exome sequencing of 6 paired constitutive and tumor DNA from sporadic pheochromocytomas and paragangliomas, we identified a somatic mutation in the HIF2A (EPAS1) gene. Screening of an additional 239 pheochromocytomas/paragangliomas uncovered three other HIF2A variants in sporadic (4/167, 2.3%), but not in hereditary tumors or controls. Three of the mutations involved proline 531, one of the two residues that controls HIF2α stability by hydroxylation. The fourth mutation, on Ser71, was adjacent to the DNA binding domain. No mutations were detected in the homologous regions of the HIF1A gene in 132 tumors. Mutant HIF2A tumors had increased expression of HIF2α target genes, suggesting an activating effect of the mutations. Ectopically expressed HIF2α mutants in HEK293, renal cell carcinoma 786-0 or rat pheochromocytoma PC12 cell lines showed increased stability, resistance to VHL-mediated degradation, target induction and reduced chromaffin cell differentiation. Furthermore, mice injected with cells expressing mutant HIF2A developed tumors, and those with Pro531Thr- and Pro531Ser mutations had shorter latency than tumors from mice with wild-type HIF2A. Our results support a direct oncogenic role for HIF2A in human neoplasia and strengthen the link between hypoxic pathways and pheochromocytomas and paragangliomas.

Recently, the KIF1B beta gene on 1p36, a region commonly deleted in neural crest cancers, was found to be a proapoptotic factor for sympathetic precursors. KIF1B beta mutations were detected in pheochromocytomas and neuroblastomas, two sympathetic lineage tumors, suggesting a role for this gene in cancer. Here, we studied five individuals from a three-generation cancer-prone family with a KIF1B beta germline variant and seven of their tumors, both of neural crest and nonneural origin. Genetic studies including sequencing, copy number analysis and fluorescence in situ-hybridization (FISH) showed retention of both KIF1B beta alleles in all neural crest-derived tumors in this family, consistent with haploinsufficiency or methylation of the wild-type allele. In contrast, the lung adenocarcinoma from one mutation carrier had somatic loss of the wild-type allele in agreement with a classical two-hit inactivation. Global transcription analysis of KIF1B beta mutant pheochromocytomas revealed that these tumors are transcriptionally related to pheochromocytomas with RET and NF1 mutations but independent from SDH- and VHL-associated tumors. Furthermore, KIF1B beta-mutant tumors are uniquely enriched for pathways related to glutamate metabolism and the oxidative stress response. Our data start to delineate the signals that are disrupted by KIF1B beta dysfunction in pheochromocytomas and suggest that loss of this gene may also be permissive to the development of nonneural crest malignancies. This may imply the existence of a tissue-specific gene dosage requirement for its tumorigenesis.

Purpose Pheochromocytomas and paragangliomas (PPGLs) are genetically heterogeneous tumors of neural crest origin, but the molecular basis of most PPGLs is unknown. Experimental Design We performed exome or transcriptome sequencing of 43 samples from 41 patients. A validation set of 136 PPGLs was used for amplicon-specific resequencing. In addition, a subset of these tumors was used for microarray-based transcription, protein expression and histone methylation analysis by western blot or immunohistochemistry. In vitro analysis of mutants was performed in cell lines. Results We detected mutations in chromatin remodeling genes, including histone-methyltransferases, histone-demethylases and histones in 11 samples from 8 patients (20%). In particular, we characterized a new cancer syndrome involving PPGLs and giant cell tumors of bone (GCT) caused by a postzygotic G34W mutation of the histone 3.3 gene, H3F3A. Furthermore, mutations in kinase genes were detected in samples from 15 patients (37%). Among those, a novel germline kinase domain mutation of MERTK detected in a patient with PPGL and medullary thyroid carcinoma was found to activate signaling downstream of this receptor. Recurrent germline and somatic mutations were also detected in MET, including a familial case and sporadic PPGLs. Importantly, in each of these three genes mutations were also detected in the validation group. Additionally, a somatic oncogenic hotspot FGFR1 mutation was found in a sporadic tumor. Conclusions This study implicates chromatin-remodeling and kinase variants as frequent genetic events in PPGLs, many of which have no other known germline driver mutation. MERTK, MET, and H3F3A emerge as novel PPGL susceptibility genes.