Gut virome-colonising Orthohepadnavirus genus is associated with ulcerative colitis pathogenesis and induces intestinal inflammation in vivo.

OBJECTIVES: Ulcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is far from fully understood. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the hepatitis B virus X protein (HBx), in UC aetiopathogenesis. DESIGN: HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing and correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors.C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control, with or without antibiotic treatment. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferation assay, chromatin immunoprecipitation assay with sequencing and RNA-sequencing were performed on in vitro models of the gut barrier. HBx-silencing experiments were performed in vitro and in vivo. RESULTS: HBx was detected in about 45% of patients with UC and found to induce colonic inflammation in mice, while its silencing reverted the colitis phenotype in vivo. HBx acted as a transcriptional regulator in epithelial cells, provoking barrier leakage and altering both innate and adaptive mucosal immunity ex vivo and in vivo. CONCLUSION: This study described HBx as a contributor to the UC pathogenesis and provides a new perspective on the virome as a target for tailored treatments.

De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways.

Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia patients in chronic phase fail to respond to imatinib or to second-generation inhibitors and progress to blast crisis. Until now, improvements in the understanding of the molecular mechanisms responsible for chronic myeloid leukemia transformation from chronic phase to the aggressive blast crisis remain limited. Here we present a large parallel sequencing analysis of 10 blast crisis samples and of the corresponding autologous chronic phase controls that reveals, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A). Additional analyses on a cohort of 24 blast crisis, 41 chronic phase as well as 40 acute myeloid leukemia and 38 atypical chronic myeloid leukemia patients at onset confirmed that UBE2A mutations are specifically acquired during chronic myeloid leukemia progression, with a frequency of 16.7% in advanced phases. In vitro studies show that the mutations here described cause a decrease in UBE2A activity, leading to an impairment of myeloid differentiation in chronic myeloid leukemia cells.

Recurrent ETNK1 mutations in atypical chronic myeloid leukemia.

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples (P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders.

BCR/ABL1 and BCR are under the transcriptional control of the MYC oncogene.

BACKGROUND: Chronic Myeloid Leukaemia (CML) is caused by the BCR/ABL1 fusion gene. Both the presence and the levels of BCR/ABL1 expression seem to be critical for CML progression from chronic phase (CP) to blast crisis (BC). After the oncogenic translocation, the BCR/ABL1 gene is under the transcriptional control of BCR promoter but the molecular mechanisms involved in the regulation of oncogene expression are mostly unknown. METHODS: A region of 1443bp of the functional BCR promoter was studied for transcription factor binding sites through in-silico analysis and Chromatin Immunoprecipitation experiments. BCR and BCR/ABL1 expression levels were analysed in CML cell lines after over-expression or silencing of MYC transcription factor. A luciferase reporter assay was used to confirm its activity on BCR promoter. RESULTS: In the present study we demonstrate that MYC and its partner MAX bind to the BCR promoter, leading to up-regulation of BCR and BCR/ABL1 at both transcriptional and protein levels. Accordingly, silencing of MYC expression in various BCR/ABL1 positive cell lines causes significant downregulation of BCR and BCR/ABL1, which consequently leads to decreased proliferation and induction of cell death. CONCLUSIONS: Here we describe a regulatory pathway modulating BCR and BCR/ABL1 expression, showing that the BCR promoter is under the transcriptional control of the MYC/MAX heterodimer. Since MYC is frequently over-expressed in BC, this phenomenon could play a critical role in BCR/ABL1 up-regulation and blast aggressiveness acquired during CML evolution.

Age and dPCR can predict relapse in CML patients who discontinued imatinib: the ISAV study.

Imatinib is effective for the treatment of chronic myeloid leukemia (CML). However even undetectable BCR-ABL1 by Q-RT-PCR does not equate to eradication of the disease. Digital-PCR (dPCR), able to detect 1 BCR-ABL1 positive cell out of 10(7) , has been recently developed. The ISAV study is a multicentre trial aimed at validating dPCR to predict relapses after imatinib discontinuation in CML patients with undetectable Q-RT-PCR. CML patients under imatinib therapy since more than 2 years and with undetectable PCR for at least 18 months were eligible. Patients were monitored by standard Q-RT-PCR for 36 months. Patients losing molecular remission (two consecutive positive Q-RT-PCR with at least 1 BCR-ABL1/ABL1 value above 0.1%) resumed imatinib. The study enrolled 112 patients, with a median follow-up of 21.6 months. Fifty-two of the 108 evaluable patients (48.1%), relapsed; 73.1% relapsed in the first 9 months but 14 late relapses were observed between 10 and 22 months. Among the 56 not-relapsed patients, 40 (37.0% of total) regained Q-RT-PCR positivity but never lost MMR. dPCR results showed a significant negative predictive value ratio of 1.115 [95% CI: 1.013-1.227]. An inverse relationship between patients age and risk of relapse was evident: 95% of patients <45 years relapsed versus 42% in the class ≥45 to <65 years and 33% of patients ≥65 years [P(χ(2) ) < 0.0001]. Relapse rates ranged between 100% (<45 years, dPCR+) and 36% (>45 years, dPCR-). Imatinib can be safely discontinued in the setting of continued PCR negativity; age and dPCR results can predict relapse.

FusionAnalyser: a new graphical, event-driven tool for fusion rearrangements discovery.

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2-ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data.

Gene expression signature of non-involved lung tissue associated with survival in lung adenocarcinoma patients.

Lung adenocarcinoma patients of similar clinical stage and undergoing the same treatments often have marked interindividual variations in prognosis. These clinical discrepancies may be due to the genetic background modulating an individual's predisposition to fighting cancer. Herein, we hypothesized that the lung microenvironment, as reflected by its expression profile, may affect lung adenocarcinoma patients' survival. The transcriptome of non-involved lung tissue, excised from a discovery series of 204 lung adenocarcinoma patients, was evaluated using whole-genome expression microarrays (with probes corresponding to 28 688 well-annotated coding sequences). Genes associated with survival status at 60 months were identified by Cox regression analysis (adjusted for gender, age and clinical stage) and retested in a validation series of 78 additional cases. RNA-Seq analysis from non-involved lung tissue of 12 patients was performed to characterize the different isoforms of candidate genes. Ten genes for which the loge-transformed hazard ratios expressed the same direction of effect in the discovery (P < 1.0 × 10(-3)) and validation series comprised the gene expression signature associated with survival: CNTNAP1, PKNOX1, FAM156A, FRMD8, GALNTL1, TXNDC12, SNTB1, PPP3R1, SNX10 and SERPINH1. RNA sequencing highlighted the complex expression pattern of these genes in non-involved lung tissue from different patients and permitted the detection of a read-through gene fusion between PPP3R1 and the flanking gene (CNRIP1) as well as a novel isoform of CNTNAP1. Our findings support the hypothesis that individual genetic characteristics, evidenced by the expression pattern of non-involved tissue, influence the outcome of lung adenocarcinoma patients.

Analysis of Intestinal Microbiota and Metabolic Pathways before and after a 2-Month-Long Hydrolyzed Fish and Rice Starch Hypoallergenic Diet Trial in Pruritic Dogs.

Intestinal microbiota alterations were described in allergic individuals and may improve with diets. Farmina Ultra Hypo (FUH), a hydrolyzed fish/rice starch hypoallergenic diet, is able to improve clinical signs in allergic dogs. Study objectives were to determine microbiota differences in allergic dogs before and after feeding with FUH for eight weeks. Forty skin allergic dogs were evaluated clinically before and after the diet. Unresponsive dogs were classified as canine atopic dermatitis (CAD); responsive dogs relapsing after challenge with previous foods were classified as being food reactive (AFR), and those not relapsing as doubtful (D). Sequencing of feces collected pre- and post-diet was performed, with comparisons between and within groups, pre- and post-diet, and correlations to possible altered metabolic pathways were sought. Microbiota in all dogs was dominated by Bacteroidota, Fusobacteriota, Firmicutes and Proteobacteria, albeit with large interindividual variations and with some prevalence changes after the diet. In general, bacteria producing short-chain fatty acids were increased in all samples. CAD dogs showed pre-and post-diet microbiota patterns different from the other two groups. Bacteria taxa were enriched post-diet only in the AFR group. Changes in metabolic pathways were observed mainly in the CAD group. FUH may be able to improve intestinal microbiota and thus clinical signs of skin allergy.

Identification of genetic polymorphisms modulating nausea and vomiting in two series of opioid-treated cancer patients.

Nausea and vomiting are often associated with opioid analgesia in cancer patients; however, only a subset of patients develop such side effects. Here, we tested the hypothesis that the occurrence of nausea and vomiting is modulated by the genetic background of the patients. Whole exome sequencing of DNA pools from patients with either low (n = 937) or high (n = 557) nausea and vomiting intensity, recruited in the European Pharmacogenetic Opioid Study, revealed a preliminary association of 53 polymorphisms. PCR-based genotyping of 45 of these polymorphisms in the individual patients of the same series confirmed the association for six SNPs in AIM1L, CLCC1, MUC16, PDE3A, POM121L2, and ZNF165 genes. Genotyping of the same 45 polymorphisms in 264 patients of the Italian CERP study, also treated with opioids for cancer pain, instead confirmed the association for two SNPs in ZNF568 and PDE3A genes. Only one SNP, rs12305038 in PDE3A, was confirmed in both series, although with opposite effects of the minor allele on the investigated phenotype. Overall, our findings suggest that genetic factors are indeed associated with nausea and vomiting in opioid-treated cancer patients, but the role of individual polymorphisms may be weak.

Integrated Genomic, Functional, and Prognostic Characterization of Atypical Chronic Myeloid Leukemia.

Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.

Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells.

BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants.

SETBP1 induces transcription of a network of development genes by acting as an epigenetic hub.

SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel-Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel-Giedion syndrome caused by SETBP1 mutations.

Erratum: OncoScore: a novel, Internet-based tool to assess the oncogenic potential of genes.

This corrects the article DOI: 10.1038/srep46290.

OncoScore: a novel, Internet-based tool to assess the oncogenic potential of genes.

The complicated, evolving landscape of cancer mutations poses a formidable challenge to identify cancer genes among the large lists of mutations typically generated in NGS experiments. The ability to prioritize these variants is therefore of paramount importance. To address this issue we developed OncoScore, a text-mining tool that ranks genes according to their association with cancer, based on available biomedical literature. Receiver operating characteristic curve and the area under the curve (AUC) metrics on manually curated datasets confirmed the excellent discriminating capability of OncoScore (OncoScore cut-off threshold = 21.09; AUC = 90.3%, 95% CI: 88.1-92.5%), indicating that OncoScore provides useful results in cases where an efficient prioritization of cancer-associated genes is needed.

Synergistic activity of ALK and mTOR inhibitors for the treatment of NPM-ALK positive lymphoma.

ALK-positive Anaplastic Large Cell Lymphoma (ALCL) represents a subset of Non-Hodgkin Lymphoma whose treatment benefited from crizotinib development, a dual ALK/MET inhibitor. Crizotinib blocks ALK-triggered pathways such as PI3K/AKT/mTOR, indispensable for survival of ALK-driven tumors.Despite the positive impact of targeted treatment in ALCL, resistant clones are often selected during therapy. Strategies to overcome resistance include the design of second generation drugs and the use of combined therapies that simultaneously target multiple nodes essential for cells survival. We investigated the effects of combined ALK/mTOR inhibition. We observed a specific synergistic effect of combining ALK inhibitors with an mTOR inhibitor (temsirolimus), in ALK+ lymphoma cells. The positive cooperation resulted in an increased inhibition of mTOR effectors, compared to single treatments, a block in G0/G1 phase and induction of apoptosis. The combination was able to prevent the selection of resistant clones, while long-term exposure to single agents led to the establishment of resistant cell lines, with either ALK inhibitor or temsirolimus. In vivo, mice injected with Karpas 299 cells and treated with low dose combination showed complete regression of tumors, while only partial inhibition was obtained in single agents-treated mice. Upon treatment stop the combination was able to significantly delay tumor relapses. Re-challenge of relapsed tumors at a higher dose led to full regression of xenografts in the combination group, but not in mice treated with lorlatinib alone. In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for ALK+ ALCL patients.

Read-through transcripts in normal human lung parenchyma are down-regulated in lung adenocarcinoma.

Read-through transcripts result from the continuous transcription of adjacent, similarly oriented genes, with the splicing out of the intergenic region. They have been found in several neoplastic and normal tissues, but their pathophysiological significance is unclear. We used high-throughput sequencing of cDNA fragments (RNA-Seq) to identify read-through transcripts in the non-involved lung tissue of 64 surgically treated lung adenocarcinoma patients. A total of 52 distinct read-through species was identified, with 24 patients having at least one read-through event, up to a maximum of 17 such transcripts in one patient. Sanger sequencing validated 28 of these transcripts and identified an additional 15, for a total of 43 distinct read-through events involving 35 gene pairs. Expression levels of 10 validated read-through transcripts were measured by quantitative PCR in pairs of matched non-involved lung tissue and lung adenocarcinoma tissue from 45 patients. Higher expression levels were observed in normal lung tissue than in the tumor counterpart, with median relative quantification ratios between normal and tumor varying from 1.90 to 7.78; the difference was statistically significant (P < 0.001, Wilcoxon's signed-rank test for paired samples) for eight transcripts: ELAVL1-TIMM44, FAM162B-ZUFSP, IFNAR2-IL10RB, INMT-FAM188B, KIAA1841-C2orf74, NFATC3-PLA2G15, SIRPB1-SIRPD, and SHANK3-ACR. This report documents the presence of read-through transcripts in apparently normal lung tissue, with inter-individual differences in patterns and abundance. It also shows their down-regulation in tumors, suggesting that these chimeric transcripts may function as tumor suppressors in lung tissue.

NPM/ALK mutants resistant to ASP3026 display variable sensitivity to alternative ALK inhibitors but succumb to the novel compound PF-06463922.

ALK is involved in the onset of several tumors. Crizotinib (XalkoriTM), a potent ALK inhibitor, represents the current front-line treatment for ALK+ NSCLC and shows great clinical efficacy. However, resistant disease often develops after initial response. ASP3026 is a novel second-generation ALK inhibitor with activity on crizotinib-resistant ALK-L1196M gatekeeper mutant. As resistance is likely to be a relevant hurdle for any drug, we sought to determine the resistance profile of ASP3026 in the context of NPM/ALK+ ALCL. We selected six ASP3026-resistant cell lines by culturing human ALCL cells in the presence of increasing concentrations of drug. The established resistant cell lines carry several point mutations in the ALK kinase domain (G1128S, C1156F, I1171N/T, F1174I, N1178H, E1210K and C1156F/D1203N were the most frequent) that are shown to confer resistance to ASP3026 in the Ba/F3 cell model. All mutants were profiled for cross-resistance against a panel of clinically relevant inhibitors including ceritinib, alectinib, crizotinib, AP26113 and PF-06463922. Finally, a genetically heterogeneous ASP3026-resistant cell line was exposed to second-line treatment simulations with all inhibitors. The population evolved according to relative sensitivity of its mutant subclones to the various drugs. Compound PF-06463922 did not allow the outgrowth of any resistant clone, at non-toxic doses.