Abstract
Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts ...diagnostic detection, even for known and recurrent configurations. Here, we develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. FUDGE couples target-selected and strand-specific CRISPR-Cas9 activity for fusion gene driver enrichment — without prior knowledge of fusion partner or breakpoint-location — to long read nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints within 2 days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay for diagnostic pan-cancer fusion detection.
Genomic rearrangements that give rise to oncogenic gene fusions can offer actionable targets for cancer therapy. Here we present a systematic analysis of oncogenic gene fusions among a clinically ...well-characterized, prospectively collected set of 278 primary colon cancers spanning diverse tumor stages and clinical outcomes. Gene fusions and somatic genetic variations were identified in fresh frozen clinical specimens by Illumina RNA-sequencing, the STAR fusion gene detection pipeline, and GATK RNA-seq variant calling. We considered gene fusions to be pathogenically relevant when recurrent, producing divergent gene expression (outlier analysis), or as functionally important (e.g., kinase fusions). Overall, 2.5% of all specimens were defined as harboring a relevant gene fusion (kinase fusions 1.8%). Novel configurations of
, and
gene fusions resulting from chromosomal translocations were identified. An R-spondin fusion was found in only one tumor (0.35%), much less than an earlier reported frequency of 10% in colorectal cancers. We also found a novel fusion involving USP9X-ERAS formed by chromothripsis and leading to high expression of ERAS, a constitutively active RAS protein normally expressed only in embryonic stem cells. This USP9X-ERAS fusion appeared highly oncogenic on the basis of its ability to activate AKT signaling. Oncogenic fusions were identified only in lymph node-negative tumors that lacked BRAF or KRAS mutations. In summary, we identified several novel oncogenic gene fusions in colorectal cancer that may drive malignant development and offer new targets for personalized therapy.
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Here, we describe a novel approach for rapid discovery of a set of tumor-specific genomic structural variants (SVs), based on a combination of low coverage cancer genome sequencing using Oxford ...Nanopore with an SV calling and filtering pipeline. We applied the method to tumor samples of high-grade ovarian and prostate cancer patients and validated on average ten somatic SVs per patient with breakpoint-spanning PCR mini-amplicons. These SVs could be quantified in ctDNA samples of patients with metastatic prostate cancer using a digital PCR assay. The results suggest that SV dynamics correlate with and may improve existing treatment-response biomarkers such as PSA. https://github.com/UMCUGenetics/SHARC .
Adult granulosa cell tumors (AGCTs) arise from the estrogen-producing granulosa cells. Treatment of recurrence remains a clinical challenge, as systemic anti-hormonal treatment or chemotherapy is ...only effective in selected patients. We established a method to rapidly screen for drug responses in vitro using direct patient-derived cell lines in order to optimize treatment selection. The response to 11 monotherapies and 12 combination therapies, including chemotherapeutic, anti-hormonal, and targeted agents, were tested in 12 AGCT-patient-derived cell lines and an AGCT cell line (KGN). Drug screens were performed within 3 weeks after tissue collection by measurement of cell viability 72 h after drug application. The potential synergy of drug combinations was assessed. The human maximum drug plasma concentration (Cmax) and steady state (Css) thresholds obtained from available phase I/II clinical trials were used to predict potential toxicity in patients. Patient-derived AGCT cell lines demonstrated resistance to all monotherapies. All cell lines showed synergistic growth inhibition by combination treatment with carboplatin, paclitaxel, and alpelisib at a concentration needed to obtain 50% cell death (IC50) that are below the maximum achievable concentration in patients (IC50 < Cmax). We show that AGCT cell lines can be rapidly established and used for patient-specific in vitro drug testing, which may guide treatment decisions. Combination treatment with carboplatin, paclitaxel, and alpelisib was consistently effective in AGCT cell lines and should be further studied as a potential effective combination for AGCT treatment in patients.
High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to ...identify patients that will profit from a specific therapy do not exist.
In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a) to enrich specimens for brain tumor initiating cells and (b) to confront cells with a therapeutic agent before expression profiling.
As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.
For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Environmental integrons are ubiquitous in natural microbial communities, but they are mostly uncharacterized and their role remains elusive. Thus far, research has been hindered by methodological ...limitations. Here, we successfully used an innovative approach combining CRISPR‐Cas9 enrichment with long‐read nanopore sequencing to target, in a complex microbial community, a putative adaptive environmental integron, InOPS, and to unravel its complete structure and genetic context. A contig of 20 kb was recovered containing the complete integron from the microbial metagenome of oil‐contaminated coastal sediments. InOPS exhibited typical integron features. The integrase, closely related to integrases of marine Desulfobacterota, possessed all the elements of a functional integron integrase. The gene cassettes harboured mostly unknown functions hampering inferences about their ecological importance. Moreover, the putative InOPS host, likely a hydrocarbonoclastic marine bacteria, raises questions as to the adaptive potential of InOPS in response to oil contamination. Finally, several mobile genetic elements were intertwined with InOPS highlighting likely genomic plasticity, and providing a source of genetic novelty. This case study showed the power of CRISPR‐Cas9 enrichment to elucidate the structure and context of specific DNA regions for which only a short sequence is known. This method is a new tool for environmental microbiologists working with complex microbial communities to target low abundant, large or repetitive genetic structures that are difficult to obtain by classical metagenomics. More precisely, here, it offers new perspectives to comprehensively assess the eco‐evolutionary significance of environmental integrons.
Résumé
Les intégrons environnementaux sont omniprésents dans les communautés microbiennes naturelles, mais la plupart ne sont pas caractérisés et leur rôle reste obscur. A ce jour, les limitations méthodologiques ont restreint leur étude. Ici, nous avons utilisé avec succès une approche innovante, combinant l’enrichissement par CRISPR‐Cas9 et le séquençage nanopore longs‐fragments, pour cibler, dans une communauté microbienne complexe, un intégron environnemental potentiellement adaptatif, InOPS, et pour révéler sa structure complète et son contexte génétique. Un contig de 20 kb contenant l’intégron complet a été obtenu à partir du métagénome microbien de sédiments côtiers contaminés par du pétrole. InOPS présente les caractéristiques typiques d’un intégron. Son intégrase, proche des intégrases des Desulfobacterota marines, possède tous les éléments d’une intégrase d’intégron fonctionnelle. Les cassettes de gène ont des fonctions pour la plupart inconnues, ce qui empêche d’inférer leur importance écologique. De plus, l’hôte présumé d’InOPS, probablement une bactérie marine hydrocarbonoclaste, interroge sur le potentiel adaptatif d’InOPS en réponse à la contamination par le pétrole. En outre, la présence de plusieurs éléments génétiques mobiles dans le contig met en évidence une probable plasticité génomique qui pourrait être source de remaniements génétiques. Cette étude de cas a montré la puissance de l’enrichissement par CRISPR‐Cas9 pour élucider la structure et le contexte de régions d’ADN spécifiques pour lesquelles seule une courte séquence est connue. Cette méthode fournit un nouvel outil aux microbiologistes environnementaux travaillant avec des communautés microbiennes complexes pour cibler des structures génétiques peu abondantes, larges ou répétées, qui sont difficiles à obtenir par métagénomique classique. Plus précisément, elle offre ici de nouvelles perspectives pour évaluer de manière exhaustive l’importance éco‐évolutive des intégrons environnementaux.
Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The
gene is frequently involved in oncogenic gene fusions, with fusion ...frequencies of 0.2%-3% throughout different cancers. However,
fusions rarely occur in the same gene configuration, potentially challenging personalized therapy design. In particular, the impact of the wide variety of fusion partners on the oncogenic role of
during tumor growth and drug response is unknown. Here, we used patient-derived colorectal cancer organoids to functionally characterize and cross-compare
fusions containing various partner genes (
, and
) with respect to cellular behavior, downstream signaling activation, and response to targeted therapies. We demonstrate that 5' fusion partners mainly promote canonical oncogenic
activity by replacing the auto-inhibitory N-terminal region. In addition, the 5' partner of BRAF fusions influences their subcellular localization and intracellular signaling capacity, revealing distinct subsets of affected signaling pathways and altered gene expression. Presence of the different BRAF fusions resulted in varying sensitivities to combinatorial inhibition of MEK and the EGF receptor family. However, all
fusions conveyed resistance to targeted monotherapy against the EGF receptor family, suggesting that
fusions should be screened alongside other MAPK pathway alterations to identify patients with metastatic colorectal cancer to exclude from anti-EGFR-targeted treatment. IMPLICATIONS: Although intracellular signaling and sensitivity to targeted therapies of
fusion genes are influenced by their 5' fusion partner, we show that all investigated
fusions confer resistance to clinically relevant EGFR inhibition.
Chronic stress-related psychiatric conditions and comorbid somatic pathologies are an enormous public health concern in modern society. The etiology of these disorders is complex, with stressors ...holding a chronic and psychosocial component representing the most acknowledged risk factor. During the last decades, research on the metabotropic glutamate receptor (mGlu) system advanced dramatically and much attention was given to the role of the metabotropic glutamate receptor subtype 7 (mGlu7) in acute stress-related behavior and physiology. However, virtually nothing is known about the potential involvement of mGlu7 in chronic psychosocial stress-related conditions. Using the chronic subordinate colony housing (CSC, 19 days) in male mice, we addressed whether central mGlu7 is altered upon chronic psychosocial stressor exposure and whether genetic ablation of mGlu7 interferes with the multitude of chronic stress-induced alterations. CSC exposure resulted in a downregulation of mGlu7 mRNA transcript levels in the prefrontal cortex, a brain region relevant for stress-related behaviors and physiology. Interestingly, mGlu7 deficiency relieved multiple chronic stress-induced alterations including the CSC-induced anxiety-prone phenotype; mGlu7 ablation also ameliorated CSC-induced physiological and immunological consequences such as hypothalamo-pituitary-adrenal (HPA) axis dysfunctions and colonic inflammation, respectively. Together, our findings provide first evidence for the involvement of mGlu7 in a wide range of behavioral and physiological alterations in response to chronic psychosocial stressor exposure. Moreover, the stress-protective phenotype of genetic mGlu7 ablation suggests mGlu7 pharmacological blockade to be a relevant option for the treatment of chronic stress-related emotional and somatic dysfunctions.
This article is part of the Special Issue entitled ‘Metabotropic Glutamate Receptors, 5 years on’.
•CSC exposure dysregulated mGlu7 transcript levels in the prefrontal cortex in wildtype mice.•Genetic ablation of mGlu7 relieved the CSC-induced anxiety-prone phenotype.•mGlu7 deficiency ameliorated CSC-induced physiological and immunological consequences.•mGlu7 seems to be involved in the development of CSC-induced alterations.
Highlights • The role of mGlu5 in modulating consequences of chronic psychosocial stress using the CSC paradigm was investigated. • Genetic ablation and pharmacological blockade of mGlu5 decreased ...the vulnerability to CSC-induced alterations. • CSC exposure dysregulated hippocampal mGlu5 binding sites in wildtype mice. • mGlu5 seems to be involved in chronic psychosocial stress-induced affective and somatic consequences.
We studied the interaction between mGlu7 and α1-adrenergic receptors in heterologous expression systems, brain slices, and living animals. L-2-Amino-4-phosphonobutanoate (L-AP4), and ...l-serine-O-phosphate (L-SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the α1-adrenergic receptor agonist, phenylephrine, in HEK 293 cells co-expressing α1-adrenergic and mGlu7 receptors. The inibitory action of L-AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C-terminal portion of type-2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, UO126 and PD98059. This suggests that the functional interaction between mGlu7 and α1-adrenergic receptors was mediated by the βγ-subunits of the Gi protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced α1-adrenergic receptor-mediated PI hydrolysis. In mouse cortical slices, both L-AP4 and L-SOP were able to attenuate norepinephrine- and phenylephrine-stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L-AP4 failed to affect norepinephrine-stimulated PI hydrolysis in cortical slices from mGlu7−/− mice, but retained its inhibitory activity in slices from mGlu4−/− mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant-like effects in the forced swim test. The action of phenylephrine was attenuated by L-SOP, which was inactive per se. Finally, both phenylephrine and L-SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that α1-adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress-related disorders.
•mGlu7 receptors and α1-adrenergic receptors functionally interact.•The interaction is shown in heterologous expression systems, brain tissue and living mice.•This interaction is mediated by the βγ-subunits of the Gi proteins.•The interaction requires the activation of the MAP kinase pathway.•This receptor cross-talk might be targeted in the treatment of stress related disorders.
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