Deubiquitinase USP9X loss sensitizes renal cancer cells to mTOR inhibition.

Mammalian target of rapamycin (mTOR) is a central regulator of mammalian metabolism and physiology. Aberrant hyperactivation of the mTOR pathway promotes tumor growth and metastasis, and can also promote tumor resistance to chemotherapy and cancer drugs; this makes mTOR an attractive cancer therapeutic target. mTOR inhibitors have been approved to treat cancer; however, the mechanisms underlying drug sensitivity remain poorly understood. Here, whole exome sequencing of three chromophobe renal cell carcinoma (chRCC) patients with exceptional mTOR inhibitor sensitivity revealed that all three patients shared somatic mutations in the deubiquitinase gene USP9X. The clonal characteristics of the mutations, which were amassed by studying multiple patients' primary and metastatic samples from various years, together with the low USP9X mutation rate in unselected chRCC series, reinforced a causal link between USP9X and mTOR inhibitor sensitivity. Rapamycin treatment of USP9X-depleted HeLa and renal cancer 786-O cells, along with the pharmacological inhibition of USP9X, confirmed that this protein plays a role in patients' sensitivity to mTOR inhibitors. USP9X was not found to exert a direct effect on mTORC1, but subsequent ubiquitylome analyses identified p62 as a direct USP9X target. Increased p62 ubiquitination and the augmented rapamycin effect upon bortezomib treatment, together with the results of p62 and LC3 immunofluorescence assays, suggested that dysregulated autophagy in USP9X-depleted cells can have a synergistic effect with mTOR inhibitors. In summary, we show that USP9X constitutes a potential novel marker of sensitivity to mTOR inhibitors in chRCC patients, and represents a clinical strategy for increasing the sensitivity to these drugs.

Hsa-miR-139-5p is a prognostic thyroid cancer marker involved in HNRNPF-mediated alternative splicing.

It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow-up of 96 months. MiRNome profiles correlated with tumor-specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa-miR-139-5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease-free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa-miR-139-5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa-miR-139-5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa-miR-139-5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa-miR-139-5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence.

Transposon mutagenesis identifies chromatin modifiers cooperating with Ras in thyroid tumorigenesis and detects ATXN7 as a cancer gene.

Oncogenic RAS mutations are present in 15-30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with HrasG12V in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in Tpo-Cre/homozygous FR-HrasG12V mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway. ATXN7, a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with RAS or NF1 mutations. Expression of ATXN7 mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to ATXN7 as a previously unrecognized cancer gene.

Tumoral EPAS1 (HIF2A) mutations explain sporadic pheochromocytoma and paraganglioma in the absence of erythrocytosis.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are chromaffin-cell tumors that arise from the adrenal medulla and extra-adrenal paraganglia, respectively. The dysfunction of genes involved in the cellular response to hypoxia, such as VHL, EGL nine homolog 1, and the succinate dehydrogenase (SDH) genes, leads to a direct abrogation of hypoxia inducible factor (HIF) degradation, resulting in a pseudo-hypoxic state implicated in PCC/PGL development. Recently, somatic post-zygotic mutations in EPAS1 (HIF2A) have been found in patients with multiple PGLs and congenital erythrocytosis. We assessed 41 PCCs/PGLs for mutations in EPAS1 and herein describe the clinical, molecular and genetic characteristics of the 7 patients found to carry somatic EPAS1 mutations; 4 presented with multiple PGLs (3 of them also had congenital erythrocytosis), whereas 3 were single sporadic PCC/PGL cases. Gene expression analysis of EPAS1-mutated tumors revealed similar mRNA EPAS1 levels to those found in SDH-gene- and VHL-mutated cases and a significant up-regulation of two hypoxia-induced genes (PCSK6 and GNA14). Interestingly, single nucleotide polymorphism array analysis revealed an exclusive gain of chromosome 2p in three EPAS1-mutated tumors. Furthermore, multiplex-PCR screening for small rearrangements detected a specific EPAS1 gain in another EPAS1-mutated tumor and in three non-EPAS1-mutated cases. The finding that EPAS1 is involved in the sporadic presentation of the disease not only increases the percentage of PCCs/PGLs with known driver mutations, but also highlights the relevance of studying other hypoxia-related genes in apparently sporadic tumors. Finally, the detection of a specific copy number alteration affecting chromosome 2p in EPAS1-mutated tumors may guide the genetic diagnosis of patients with this disease.

Differential gene expression of medullary thyroid carcinoma reveals specific markers associated with genetic conditions.

Medullary thyroid carcinoma accounts for 2% to 5% of thyroid malignancies, of which 75% are sporadic and the remaining 25% are hereditary and related to multiple endocrine neoplasia type 2 syndrome. Despite a genotype-phenotype correlation with specific germline RET mutations, knowledge of pathways specifically associated with each mutation and with non-RET-mutated sporadic MTC remains lacking. Gene expression patterns have provided a tool for identifying molecular events related to specific tumor types and to different clinical features that could help identify novel therapeutic targets. Using transcriptional profiling of 49 frozen MTC specimens classified as RET mutation, we identified PROM1, LOXL2, GFRA1, and DKK4 as related to RET(M918T) and GAL as related to RET(634) mutation. An independent series of 19 frozen and 23 formalin-fixed, paraffin-embedded (FFPE) MTCs was used for validation by RT-qPCR. Two tissue microarrays containing 69 MTCs were available for IHC assays. According to pathway enrichment analysis and gene ontology biological processes, genes associated with the MTC(M918T) group were involved mainly in proliferative, cell adhesion, and general malignant metastatic effects and with Wnt, Notch, NFκB, JAK/Stat, and MAPK signaling pathways. Assays based on silencing of PROM1 by siRNAs performed in the MZ-CRC-1 cell line, harboring RET(M918T), caused an increase in apoptotic nuclei, suggesting that PROM1 is necessary for survival of these cells. This is the first report of PROM1 overexpression among primary tumors.

Genome-wide association study identifies ephrin type A receptors implicated in paclitaxel induced peripheral sensory neuropathy.

BACKGROUND: Peripheral neuropathy is the dose limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat solid tumours. This toxicity exhibits great inter-individual variability of unknown origin. The present study aimed to identify genetic variants associated with paclitaxel induced neuropathy via a whole genome approach. METHODS: A genome-wide association study (GWAS) was performed in 144 white European patients uniformly treated with paclitaxel/carboplatin and for whom detailed data on neuropathy was available. Per allele single nucleotide polymorphism (SNP) associations were assessed by Cox regression, modelling the cumulative dose of paclitaxel up to the development of grade 2 sensory neuropathy. RESULTS: The strongest evidence of association was observed for the ephrin type A receptor 4 (EPHA4) locus (rs17348202, p=1.0×10(-6)), and EPHA6 and EPHA5 were among the top 25 and 50 hits (rs301927, p=3.4×10(-5) and rs1159057, p=6.8×10(-5)), respectively. A meta-analysis of EPHA5-rs7349683, the top marker for paclitaxel induced neuropathy in a previous GWAS (r(2)=0.79 with rs1159057), gave a hazard ratio (HR) estimate of 1.68 (p=1.4×10(-9)). Meta-analysis of the second hit of this GWAS, XKR4-rs4737264, gave a HR of 1.71 (p=3.1×10(-8)). Imputed SNPs at LIMK2 locus were also strongly associated with this toxicity (HR=2.78, p=2.0×10(-7)). CONCLUSIONS: This study provides independent support of EPHA5-rs7349683 and XKR4-rs4737264 as the first markers of risk of paclitaxel induced neuropathy. In addition, it suggests that other EPHA genes also involved in axonal guidance and repair following neural injury, as well as LIMK2 locus, may play an important role in the development of this toxicity. The identified SNPs could form the basis for individualised paclitaxel chemotherapy.

MAML3-fusions modulate vascular and immune tumour microenvironment and confer high metastatic risk in pheochromocytoma and paraganglioma.

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.

Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma.

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.

Analysis of Telomere Maintenance Related Genes Reveals NOP10 as a New Metastatic-Risk Marker in Pheochromocytoma/Paraganglioma.

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients.

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.