RNA-based high-risk HPV genotyping and identification of high-risk HPV transcriptional activity in cervical tissues.

Nearly all cervical cancers are initiated by a persistent infection with one of the high-risk human papillomaviruses (high-risk HPV). High-risk HPV DNA testing is highly sensitive but cannot distinguish between active, productive infections and dormant infections or merely deposited virus. A solution for this shortcoming may be the detection of transcriptional activity of viral oncogenes instead of mere presence of high-risk HPVs. In this study, fresh-frozen cervical tissues (n = 22) were subjected to high-risk HPV DNA detection using the line probe assay and to targeted RNA next-generation sequencing using single-molecule molecular inversion probes. Targeted RNA sequencing was applied for (1) RNA-based genotyping of high-risk HPV, giving information on specific HPV-subtype (2) discrimination of E2, E6, and E7 transcripts and (3) discovery of possible non-HPV cancer biomarkers. Data were analyzed using computational biology. Targeted RNA sequencing enabled reliable genotyping of high-risk HPV subtypes and allowed quantitative detection of E2, E6, and E7 viral gene expression, thereby discriminating cervical lesions from normal cervical tissues. Moreover, targeted RNA sequencing identified possible cervical cancer biomarkers other than high-risk HPV. Interestingly, targeted RNA sequencing also provided high-quality transcription profiles from cervical scrape samples, even after 1 week of dry storage or storage in Preservcyt fixative. This proof of concept study shows that targeted RNA sequencing can be used for high-risk HPV genotyping and simultaneous detection of high-risk HPV gene activity. Future studies are warranted to investigate the potential of targeted RNA sequencing for risk assessment for the development of cervical lesions, based on molecular analysis of cervical scrapes.

Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1mut), resulting in metabolic stress. Although IDHmut gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDHmut gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1R132H mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1mut- and IDHwt-glioma xenografts. Gene set enrichment analyses of clinical IDH1mut gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDHwt gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDHmut glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDHmut gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

Metformin Alters Human Host Responses to Mycobacterium tuberculosis in Healthy Subjects.

BACKGROUND: Metformin, the most widely administered diabetes drug, has been proposed as a candidate adjunctive host-directed therapy for tuberculosis, but little is known about its effects on human host responses to Mycobacterium tuberculosis. METHODS: We investigated in vitro and in vivo effects of metformin in humans. RESULTS: Metformin added to peripheral blood mononuclear cells from healthy volunteers enhanced in vitro cellular metabolism while inhibiting the mammalian target of rapamycin targets p70S6K and 4EBP1, with decreased cytokine production and cellular proliferation and increased phagocytosis activity. Metformin administered to healthy human volunteers led to significant downregulation of genes involved in oxidative phosphorylation, mammalian target of rapamycin signaling, and type I interferon response pathways, particularly following stimulation with M. tuberculosis, and upregulation of genes involved in phagocytosis and reactive oxygen species production was increased. These in vivo effects were accompanied by a metformin-induced shift in myeloid cells from classical to nonclassical monocytes. At a functional level, metformin lowered ex vivo production of tumor necrosis factor α, interferon γ, and interleukin 1ß but increased phagocytosis activity and reactive oxygen species production. CONCLUSION: Metformin has a range of potentially beneficial effects on cellular metabolism, immune function, and gene transcription involved in innate host responses to M. tuberculosis.

ACLY (ATP Citrate Lyase) Mediates Radioresistance in Head and Neck Squamous Cell Carcinomas and is a Novel Predictive Radiotherapy Biomarker.

Radiotherapy is an important treatment modality of head and neck squamous cell carcinomas (HNSCC). Multiple links have been described between the metabolic activity of tumors and their clinical outcome. Here we test the hypothesis that metabolic features determine radiosensitivity, explaining the relationship between metabolism and clinical outcome. Radiosensitivity of 14 human HNSCC cell lines was determined using colony forming assays and the expression profile of approximately 200 metabolic and cancer-related genes was generated using targeted RNA sequencing by single molecule molecular inversion probes. Results: Correlation between radiosensitivity data and expression profiles yielded 18 genes associated with radiosensitivity or radioresistance, of which adenosine triphosphate (ATP) citrate lyase (ACLY) was of particular interest. Pharmacological inhibition of ACLY caused an impairment of DNA damage repair, specifically homologous recombination, and lead to radiosensitization in HNSCC cell lines. Examination of a The Cancer Genome Atlas (TCGA) cohort of HNSCC patients revealed that high expression of ACLY was predictive for radiotherapy failure, as it was only associated with poor overall survival in patients who received radiotherapy (hazard ratio of 2.00, 95% CI: 1.12-3.55; p = 0.0184). These data were further validated in an independent cohort of HNSCC patients treated with chemoradiation. Furthermore, patients with poor locoregional control after radiotherapy have significantly higher nuclear ACLY protein levels. Together, we here show that ACLY affects DNA damage repair, and is a predictive factor for radiotherapy outcome in HNSCC.

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing.

Clear cell renal cell carcinoma (ccRCC) comprises more than 80% of all renal cancers and when metastasized leads to a 5-year survival rate of only 10%. The high rate of therapy failure and resistance development calls for reliable methods that provide information on the actionable biological pathways and predict optimal treatment protocols for individual patients. We here applied targeted RNA sequencing (t/RNA-NGS) using single molecule Molecular Inversion Probes on tumor nephrectomy samples of five ccRCC patients, comparing tumor with healthy kidney tissues. Transcriptome profiling focused on expression of genes with involvement in ccRCC biology that can be targeted with clinically available drugs. Results confirm high expression of vascular endothelial growth factor-A (VEGF-A) in tumor tissue relative to healthy-appearing kidney, in line with the angiogenic nature of ccRCC. PDGFRα and KIT, targets of the multi-kinase inhibitor sunitinib which is one of the current choices of first-line drug in metastasized ccRCC patients, were expressed at relatively low levels in tumor tissues, whereas significantly increased in normal kidney. Of all measured druggable tyrosine kinases, MET, AXL, or EGFR were expressed at higher levels in tumors than in normal kidney tissues, although intertumor differences were observed. Using cancer cell lines we show that t/RNA-NGS gene expression profiles can be used to predict in vitro sensitivity to targeted drugs. In conclusion, t/RNA-NGS analysis may provide insights into the (druggable) molecular make-up of individual renal cancers, and may guide personalized therapy of renal cell cancers.

Mapping actionable pathways and mutations in brain tumours using targeted RNA next generation sequencing.

Many biology-based precision drugs are available that neutralize aberrant molecular pathways in cancer. Molecular heterogeneity and the lack of reliable companion diagnostic biomarkers for many drugs makes targeted treatment of cancer inaccurate for many individuals. Identifying actionable hyperactive biological pathways in individual cancers may improve this situation.To achieve this we applied a novel targeted RNA next generation sequencing (t/RNA-NGS) technique to surgically obtained glioma tissues. The test combines mutation detection with analysis of biological pathway activities that are involved in tumour behavior in many cancer types (e.g. tyrosine kinase signaling, angiogenesis signaling, immune response, metabolism), via quantitative measurement of transcript levels and splice variants of hundreds of genes. We here present proof of concept that the technique, which uses molecular inversion probes, generates a histology-independent molecular diagnosis and identifies classifiers that are strongly associated with conventional histopathology diagnoses and even with patient prognosis. The test not only confirmed known glioma-associated molecular aberrations but also identified aberrant expression levels of actionable genes and mutations that have so far been considered not to be associated with glioma, opening up the possibility of drug repurposing for individual patients. Its cost-effectiveness makes t/RNA-NGS to an attractive instrument to aid oncologists in therapy decision making.

Isocitrate dehydrogenase 1-mutated cancers are sensitive to the green tea polyphenol epigallocatechin-3-gallate.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in various types of cancer and induce metabolic alterations resulting from the neomorphic activity that causes production of D-2-hydroxyglutarate (D-2-HG) at the expense of α-ketoglutarate (α-KG) and NADPH. To overcome metabolic stress induced by these alterations, IDH-mutated (IDH mut ) cancers utilize rescue mechanisms comprising pathways in which glutaminase and glutamate dehydrogenase (GLUD) are involved. We hypothesized that inhibition of glutamate processing with the pleiotropic GLUD-inhibitor epigallocatechin-3-gallate (EGCG) would not only hamper D-2-HG production, but also decrease NAD(P)H and α-KG synthesis in IDH mut cancers, resulting in increased metabolic stress and increased sensitivity to radiotherapy. METHODS: We performed 13C-tracing studies to show that HCT116 colorectal cancer cells with an IDH1 R132H knock-in allele depend more on glutaminolysis than on glycolysis for the production of D-2-HG. We treated HCT116 cells, HCT116-IDH1 R132H cells, and HT1080 cells (carrying an IDH1 R132C mutation) with EGCG and evaluated D-2-HG production, cell proliferation rates, and sensitivity to radiotherapy. RESULTS: Significant amounts of 13C from glutamate accumulate in D-2-HG in HCT116-IDH1 wt/R132H but not in HCT116-IDH1 wt/wt . Preventing glutamate processing in HCT116-IDH1 wt/R132H cells with EGCG resulted in reduction of D-2-HG production. In addition, EGCG treatment decreased proliferation rates of IDH1 mut cells and at high doses sensitized cancer cells to ionizing radiation. Effects of EGCG in IDH-mutated cell lines were diminished by treatment with the IDH1mut inhibitor AGI-5198. CONCLUSIONS: This work shows that glutamate can be directly processed into D-2-HG and that reduction of glutamatolysis may be an effective and promising new treatment option for IDH mut cancers.

Impaired fear extinction in serotonin transporter knockout rats is associated with increased 5-hydroxymethylcytosine in the amygdala.

AIMS: One potential risk factor for posttraumatic stress disorder (PTSD) involves the low activity (short; s) allelic variant of the serotonin transporter-linked polymorphic region (5-HTTLPR), possibly due to reduced prefrontal control over the amygdala. Evidence shows that DNA methylation/demethylation is crucial for fear extinction in these brain areas and is associated with neuronal activation marker c-Fos expression. We hypothesized that impaired fear extinction in serotonin transporter knockout (5-HTT-/- ) rats is related to changes in DNA (de) methylation and c-Fos expression in the prefrontal cortex (PFC) and/or amygdala. METHODS: 5-HTT-/- and 5-HTT+/+ rats were subjected to fear extinction. 2 hours after the extinction session, the overall levels of DNA methylation (5-mC), demethylation (5-hmC), and c-Fos in fear extinction and nonfear extinction rats were measured by immunohistochemistry. RESULTS: 5-HTT-/- rats displayed decreased fear extinction. This was associated with reduced c-Fos activity in the infralimbic PFC. In the central nucleus of the amygdala, c-Fos immunoreactivity was increased in the fear extinction group compared to the no-fear extinction group, regardless of genotype. 5-hmC levels were unaltered in the PFC, but reduced in the amygdala of nonextinction 5-HTT-/- rats compared to nonextinction wild-type rats, which caught up to wild-type levels during fear extinction. 5-mC levels were stable in central amygdala in both wild-type and 5-HTT-/- extinction rats. Finally, c-Fos and 5-mC levels were correlated with the prelimbic PFC, but not amygdala. CONCLUSIONS: In the amygdala, DNA demethylation, independent from c-Fos activation, may contribute to individual differences in risk for PTSD, as conferred by the 5-HTTLPR s-allele.

Quantification and localization of oncogenic receptor tyrosine kinase variant transcripts using molecular inversion probes.

Oncogenic membrane receptor tyrosine kinases such as MET and EGFR, or auto-active variants thereof, are important targets for cancer precision therapy. Targeted inhibition of these oncogenic receptors however invariably leads to resistance, resulting from acquisition of resistance-inducing mutations or from selective outgrowth of a priori resistant tumour cells. Most applied molecular protocols cannot distinguish between intracellular and intercellular heterogeneity of oncogene (variant) expression, which may lead to misinterpretation of the molecular make-up of a cancer and suboptimal application of targeted therapies. We here combined two related techniques to allow semiquantitative and localized in situ detection of specific transcript splice variants using single molecule molecular inversion probe (smMIP)-based next generation sequencing and padlock probe-based rolling circle amplification, respectively. We show highly specific padlock probe-based multiplex detection of MET, METΔ7-8 and METΔ14 transcripts, lacking exons 7-8 and exon 14 respectively, and of EGFR and the auto-active EGFRvIII, lacking exons 2-7. The combination of quantitative transcript variant detection with smMIPs and transcript localization using padlock probes can be used for detection of oncogenic transcripts on the single-cell level, allowing study of tumour heterogeneity. Visualization of tumour heterogeneity can shed light on the biology underlying drug resistance and potentially improve targeted therapeutics.

Profiling of the metabolic transcriptome via single molecule molecular inversion probes.

Cancer-specific metabolic alterations are of high interest as therapeutic targets. These alterations vary between tumor types, and to employ metabolic targeting to its fullest potential there is a need for robust methods that identify candidate targetable metabolic pathways in individual cancers. Currently, such methods include 13C-tracing studies and mass spectrometry/ magnetic resonance spectroscopic imaging. Due to high cost and complexity, such studies are restricted to a research setting. We here present the validation of a novel technique of metabolic profiling, based on multiplex targeted next generation sequencing of RNA with single molecule molecular inversion probes (smMIPs), designed to measure activity of and mutations in genes that encode metabolic enzymes. We here profiled an isogenic pair of cell lines, differing in expression of the Von Hippel Lindau protein, an important regulator of hypoxia-inducible genes. We show that smMIP-profiling provides relevant information on active metabolic pathways. Because smMIP-based targeted RNAseq is cost-effective and can be applied in a medium high-throughput setting (200 samples can be profiled simultaneously in one next generation sequencing run) it is a highly interesting approach for profiling of the activity of genes of interest, including those regulating metabolism, in a routine patient care setting.

Selective MET Kinase Inhibition in MET-Dependent Glioma Models Alters Gene Expression and Induces Tumor Plasticity.

The receptor tyrosine kinase (RTK) MET represents a promising tumor target in a subset of glioblastomas. Most RTK inhibitors available in the clinic today, including those inhibiting MET, affect multiple targets simultaneously. Previously, it was demonstrated that treatment with cabozantinib (MET/VEGFR2/RET inhibitor) prolonged survival of mice carrying orthotopic patient-derived xenografts (PDX) of the MET-addicted glioblastoma model E98, yet did not prevent development of recurrent and cabozantinib-resistant tumors. To exclude VEGFR2 inhibition-inflicted blood-brain barrier normalization and diminished tumor distribution of the drug, we have now investigated the effects of the novel MET-selective inhibitor Compound A in the orthotopic E98 xenograft model. In vitro, Compound A proved a highly potent inhibitor of proliferation of MET-addicted cell lines. In line with its target selectivity, Compound A did not restore the leaky blood-brain barrier and was more effective than cabozantinib in inhibiting MET phosphorylation in vivo Compound A treatment significantly prolonged survival of mice carrying E98 tumor xenografts, but did not prevent eventual progression. Contrasting in vitro results, the Compound A-treated xenografts displayed high levels of AKT phosphorylation despite the absence of phosphorylated MET. Profiling by RNA sequencing showed that in vivo transcriptomes differed significantly from those in control xenografts.Implications: Collectively, these findings demonstrate the plasticity of paracrine growth factor receptor signaling in vivo and urge for prudency with in vitro drug-testing strategies to validate monotherapies. Mol Cancer Res; 15(11); 1587-97. ©2017 AACR.

Diabetes Mellitus and Increased Tuberculosis Susceptibility: The Role of Short-Chain Fatty Acids.

Type 2 diabetes mellitus confers a threefold increased risk for tuberculosis, but the underlying immunological mechanisms are still largely unknown. Possible mediators of this increased susceptibility are short-chain fatty acids, levels of which have been shown to be altered in individuals with diabetes. We examined the influence of physiological concentrations of butyrate on cytokine responses to Mycobacterium tuberculosis (Mtb) in human peripheral blood mononuclear cells (PBMCs). Butyrate decreased Mtb-induced proinflammatory cytokine responses, while it increased production of IL-10. This anti-inflammatory effect was independent of butyrate's well-characterised inhibition of HDAC activity and was not accompanied by changes in Toll-like receptor signalling pathways, the eicosanoid pathway, or cellular metabolism. In contrast blocking IL-10 activity reversed the effects of butyrate on Mtb-induced inflammation. Alteration of the gut microbiota, thereby increasing butyrate concentrations, can reduce insulin resistance and obesity, but further studies are needed to determine how this affects susceptibility to tuberculosis.

The Symptomatic Treatment of Acquired Dystonia: A Systematic Review.

BACKGROUND: Acquired dystonia is caused by an acquired or exogenous event. Although the therapeutic armamentarium used in clinical practice is more or less similar to that used for inherited or idiopathic dystonia, formal proof of the efficacy of these interventions in acquired dystonia is lacking. METHODS: The authors attempt to provide a comprehensive and systematic review of the current evidence for medical and allied health care treatment strategies in acquired dystonias. The PubMed, Cochrane Library, MEDLINE, Web of Science, PiCarta, and PsycINFO databases were searched up to December 2015, including randomized controlled trials, patient-control studies, and case series or single case reports containing a report on clinical outcome. RESULTS: There are level 3 practice recommendations for botulinum toxin injections and globus pallidus pars interna deep brain stimulation for tardive dystonia and dystonic cerebral palsy as well as intrathecal baclofen for dystonic cerebral palsy. There are insufficient and conflicting data on the effect (vs. the hazard) of other pharmacological interventions, and limited work has been done on other forms of neurostimulation and allied health care. Because no class A1 or A2 studies were identified, level 1 or 2 practice recommendations could not be deducted for a specific treatment intervention. CONCLUSIONS: To improve the current medical and allied health care treatment options for patients with acquired dystonia, high-quality trials that examine the efficacy of therapies need to be performed.