scholarly journals Deubiquitinase USP9X loss Sensitizes Cancer Cells to mTOR Inhibitors through p62 Dysregulation

2021 ◽  
Author(s):  
Juan M. Roldán-Romero ◽  
Maria Santos ◽  
Javier Lanillos ◽  
Pablo Maroto ◽  
Georgia Anguera ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Mtor Inhibitor ◽  
Mtor Inhibitors ◽  
High Sensitivity ◽  
Causal Link ◽  
Synthetic Lethal ◽  
Bortezomib Treatment ◽  
Whole Exome ◽  
Mechanistic Investigation

Abstract BackgroundMutations in MTOR and TSC1/2 can only explain part of variability in mTOR inhibitor response. Here, we performed a comprehensive molecular characterization of tumors with high sensitivity to these drugs to uncover novel mechanisms of response.MethodsChromophobe renal cell carcinoma (chRCC) tumors were analyzed by whole-exome sequencing. Rapamycin sensitivity, expression of mTOR pathway effectors, ubiquitylome analyses to identify USP9X substrates, p62 immunoprecipitation and autophagy assessment through immunofluorescence assays for p62 and LC3 were performed in USP9X depleted HeLa and 786-O cells for mechanistic investigation. ResultsWhole-exome sequencing of chRCC patients with high mTOR inhibitor sensitivity, uncovered USP9X deubiquitinase as the only mutated gene shared by these tumors. The clonal characteristics of the mutations, revealed by studying multiple primary and metastatic samples, together with the low USP9X mutation rate in unselected chRCC series, implied a causal link between USP9X and mTOR inhibitor sensitivity. The high sensitivity was recapitulated in vitro, and while no direct effect on mTORC1 was detected, an unbiased ubiquitylome analysis revealed p62 as a direct USP9X target. Bortezomib treatment, which also led to p62 ubiquitination, increased rapamycin effect. Finally, immunofluorescence assays for p62 and LC3 confirmed dysregulated autophagy in USP9X depleted cells, supporting a synthetic lethal interaction between rapamycin-induced autophagy via mTOR-axis and USP9X loss.ConclusionsOur study uncovers USP9X as a potential novel marker of sensitivity to mTOR inhibitors and suggests the inhibition of this gene as a clinically exploitable strategy able to increase sensitivity to these drugs through a novel p62 regulatory mechanism.

scholarly journals Multiple Variant Calling Pipelines in Wheat Whole Exome Sequencing

2021 ◽  
Vol 22 (19) ◽  
pp. 10400
Author(s):  
H. Busra Cagirici ◽  
Bala Ani Akpinar ◽  
Taner Z. Sen ◽  
Hikmet Budak
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Variant Calling ◽  
High Sensitivity ◽  
Whole Exome ◽  
The Impact ◽  
Multiple Reference ◽  
Reference Genomes ◽  
Selection Of ◽  
Variant Identification

The highly challenging hexaploid wheat (Triticum aestivum) genome is becoming ever more accessible due to the continued development of multiple reference genomes, a factor which aids in the plight to better understand variation in important traits. Although the process of variant calling is relatively straightforward, selection of the best combination of the computational tools for read alignment and variant calling stages of the analysis and efficient filtering of the false variant calls are not always easy tasks. Previous studies have analyzed the impact of methods on the quality metrics in diploid organisms. Given that variant identification in wheat largely relies on accurate mining of exome data, there is a critical need to better understand how different methods affect the analysis of whole exome sequencing (WES) data in polyploid species. This study aims to address this by performing whole exome sequencing of 48 wheat cultivars and assessing the performance of various variant calling pipelines at their suggested settings. The results show that all the pipelines require filtering to eliminate false-positive calls. The high consensus among the reference SNPs called by the best-performing pipelines suggests that filtering provides accurate and reproducible results. This study also provides detailed comparisons for high sensitivity and precision at individual and population levels for the raw and filtered SNP calls.

scholarly journals Establishment and drug screening of patient-derived extrahepatic biliary tract carcinoma organoids

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhiwei Wang ◽  
Yinghao Guo ◽  
Yun Jin ◽  
Xiaoxiao Zhang ◽  
Hao Geng ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Biliary Tract ◽  
Drug Screening ◽  
Periodic Acid ◽  
Biliary Tract Carcinoma ◽  
Clinical Effects ◽  
Whole Exome ◽  
Genetic Profiles

Abstract Background Patient-derived organoids (PDO) have been proposed as a novel in vitro method of drug screening for different types of cancer. However, to date, extrahepatic biliary tract carcinoma (eBTC) PDOs have not yet been fully established. Methods We collected six samples of gallbladder carcinoma (GBC) and one sample of extrahepatic cholangiocarcinoma (eCCA) from seven patients to attempt to establish eBTC PDOs for drug screening. We successfully established five GBC and one eCCA PDOs. Histological staining was used to compare structural features between the original tissues and cancer PDOs. Whole exome sequencing (WES) was performed to analyze the genetic profiles of original tissues and cancer PDOs. Drug screening, including gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, infigratinib, and ivosidenib, was measured and verified by clinical effects in certain cases. Results Different PDOs exhibited diverse growth rates during in vitro culture. Hematoxylin and eosin staining demonstrated that the structures of most cancer PDOs retained the original structures of adenocarcinoma. Immunohistological and periodic acid-schiff staining revealed that marker expression in cancer PDOs was similar to that of the original specimens. Genetic profiles from the four original specimens, as well as paired cancer PDOs, were analyzed using whole exome sequencing. Three of the four PDOs exhibited a high degree of similarity when compared to the original specimens, except for GBC2 PDO, which only had a concordance of 74% in the proportion of single nucleotide polymorphisms in the coding sequence. In general, gemcitabine was found to be the most efficient drug for eBTC treatment, as it showed moderate or significant inhibitory impact on cancer growth. Results from drug screening were confirmed to a certain extent by three clinical cases. Conclusions Our study successfully established a series of eBTC PDOs, which contributed to the field of eBTC PDOs. Additional enhancements should be explored to improve the growth rate of PDOs and to preserve their immune microenvironment.

scholarly journals Metabolomics and Whole-Exome Sequencing in Patients with Differential Sensitivity to Sevoflurane: A Protocol for a Prospective Observational Trial

2021 ◽  
Vol 12 ◽  
Author(s):  
Yiyong Wei ◽  
Donghang Zhang ◽  
Yunxia Zuo
Keyword(s):  
Steady State ◽  
Observational Study ◽  
Exome Sequencing ◽  
Prospective Observational Study ◽  
High Sensitivity ◽  
Volatile Anesthetics ◽  
Differential Sensitivity ◽  
Whole Exome ◽  
Low Sensitivity ◽  
End Tidal

Introduction: Different sensitivity to volatile anesthetics in Drosophila, nematodes and mice is related to mutation of energy metabolism genes. In clinical practice, we find that the end-tidal sevoflurane concentration (ETsevo) differs among patients at the same depth of anesthesia, indicating that the sensitivity to sevoflurane varies among patients. However, the underlying mechanism remains unclear. The sensitivity of an anesthetic is associated with the postoperative outcomes of patients and the mechanism of action of volatile anesthetics. We therefore propose this protocol to determine whether differences in metabolite profile and genetic variations contribute to patients’ sensitivity to volatile anesthetics.Methods and Analysis: This is a single-centre, prospective observational study. 720 patients undergoing abdominal surgery were included. General anesthesia was induced with inhaled sevoflurane, a bolus of sufentanil (0.2–0.4 μg/kg) and cis-atracurium (0.2–0.3 mg/kg). The end-tidal sevoflurane concentration (ETsevo) was adjusted to maintain a BIS (bispectral index) value between 40–60. The mean ETsevo from 20 min after endotracheal intubation to 2 h after the beginning of surgery (steady state) was calculated for each patient. Patients were further divided into a high-sensitivity group (mean ETsevo – SD) and a low-sensitivity group (mean ETsevo + SD) to investigate the sensitivity to sevoflurane. Cases were paired from the high-sensitivity group (group H) and low-sensitivity group (group L) according to age, sex, body mass index (BMI), ASA physical status classification, vital signs, BIS, ephedrine use, sufentanildose, and cis-atracurium dose at anesthesia induction and steady state. Differences in metabolite levels, single nucleotide polymorphisms (SNPs) and protein-coding gene sequence variations between group H and group L will be determined through plasma metabolomics, whole-exome sequencing (WES), genome-wide association study (GWAS), and bioinformatics analyses. These results will be analysed to determine the reasons for the differential sensitivity to sevoflurane in humans.Ethics and Dissemination: This prospective observational study protocol has received ethical approval from the Ethical Committee of West China Hospital of Sichuan University on May 19, 2017 (Approval No. 78). Informed consent will be obtained before patient enrolment. The results will be submitted to international peer-review journals.Trial Registration Number: ChiCTR1800014327.

scholarly journals Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy

2019 ◽  
Vol 116 (45) ◽  
pp. 22730-22736 ◽  
Author(s):  
Luca Zammataro ◽  
Salvatore Lopez ◽  
Stefania Bellone ◽  
Francesca Pettinella ◽  
Elena Bonazzoli ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Cell Lines ◽  
Copy Number ◽  
Mtor Pathway ◽  
In Vivo Models ◽  
Whole Exome

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.

Postmortem Whole Exome Sequencing Identifies Novel EIF2B3 Mutation With Prenatal Phenotype in 2 Siblings

2017 ◽  
Vol 32 (10) ◽  
pp. 867-870 ◽  
Author(s):  
Hannah Song ◽  
Sina Haeri ◽  
Hannes Vogel ◽  
Marjo van der Knaap ◽  
Keith Van Haren
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Clinical Care ◽  
Mutation Screening ◽  
Medical Decision ◽  
Homozygous Mutation ◽  
Vitro Fertilization ◽  
Whole Exome ◽  
The Impact

Objective: We describe 2 male siblings with a severe, prenatal phenotype of vanishing white matter disease and the impact of whole exome sequencing on their diagnosis and clinical care. Methods: The 2 children underwent detailed clinical characterization, through clinical and laboratory testing, as well as prenatal and postnatal imaging. Biobanked blood from the 2 siblings was submitted for whole exome sequencing at Baylor Laboratories. Results: Both male children had abnormal prenatal neuroimaging and suffered precipitous, fatal neurologic decline. Neuropathologic findings included subependymal pseudocysts, microcalcifications, and profound lack of brain myelin and sparing of peripheral nerve myelin. A novel homozygous mutation in the EIF2B3 gene (c.97A>G [p.Lys33Glu]) was found in both children; both parents were heterozygous carriers. The family subsequently conceived a healthy child via in vitro fertilization with preimplantation mutation screening. Conclusion: These histories expand the prenatal phenotype of eIF2b-related disorders and poignantly illustrate the impact that unbiased genomic sequencing can have on the diagnosis and medical decision making for families affected by childhood neurodegenerative disorders.

Combination of the mTOR inhibitor rapamycin and CC-5013 has synergistic activity in multiple myeloma

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4188-4193 ◽  
Author(s):  
Noopur Raje ◽  
Shaji Kumar ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mtor Inhibitor ◽  
Mtor Inhibitors ◽  
Mitogen Activated Protein Kinase ◽  
Synergistic Activity ◽  
Growth And Survival ◽  
Mitogen Activated Protein ◽  
Induced Apoptosis

Abstract Previous studies have demonstrated the in vitro and in vivo activity of CC-5013 (Revlimid), an immunomodulatory analog (IMiD) of thalidomide, in multiple myeloma (MM). In the present study, we have examined the anti-MM activity of rapamycin (Rapamune), a specific mTOR inhibitor, combined with CC-5013. Based on the Chou-Talalay method, combination indices of less than 1 were obtained for all dose ranges of CC-5013 when combined with rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or adherence to bone marrow stromal cells (BMSCs). Combining rapamycin and CC-5013 induced apoptosis of MM cells. Differential signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3′-kinase/Akt kinase (PI3K/Akt) pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies, therefore, provide the framework for clinical evaluation of mTOR inhibitors combined with IMiDs to improve patient outcome in MM.

scholarly journals A novel intron mutation in FBN-1 gene identified in a pregnant woman with Marfan syndrome

Hereditas ◽  
2021 ◽  
Vol 158 (1) ◽  
Author(s):  
Yuduo Wu ◽  
Hairui Sun ◽  
Yihua He ◽  
Hongjia Zhang
Keyword(s):  
Pregnant Woman ◽  
Exome Sequencing ◽  
Marfan Syndrome ◽  
Connective Tissue Diseases ◽  
Genetic Mutation ◽  
Gene Splicing ◽  
Whole Exome ◽  
Alternative Donor ◽  
Intron Mutation

AbstractMarfan syndrome (MFS) is one of the most common hereditary connective tissue diseases, with great individual heterogeneity. We reported a Chinese pregnancy with Clinical diagnosis of MFS, performed whole-exome sequencing, and screened for the genetic abnormality. We also conducted an in vitro mini-gene splicing assay to demonstrate the predicted harmful effects of an intronic variant of FBN-1. Exome sequencing identified a novel intronic variant (c.6497-13 T>A) in intron 53 of the FBN-1 gene (NM_000138.4). It’s predicted to insert 11 bp of intron 53 into the mature mRNA. The mini-gene splicing experiment demonstrated that c.6497-13 T>A could result in 11 bp retention in intron 53 to exon 54 (c.6496_6497ins gtttcttgcag) and the use of an alternative donor causing the frameshift p.Asp2166Glyfs*23. According to the results, the pregnant woman chose to continue the pregnancy and gave birth to a healthy baby. This study expands the genetic mutation spectrum of MFS patients and indicates the importance of intron sequencing.

scholarly journals Digenic Variants in the FGF21 Signaling Pathway Associated with Severe Insulin Resistance and Pseudoacromegaly

2020 ◽  
Vol 4 (12) ◽  
Author(s):  
Stephen I Stone ◽  
Daniel J Wegner ◽  
Jennifer A Wambach ◽  
F Sessions Cole ◽  
Fumihiko Urano ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Hormone Secretion ◽  
Growth Hormone Secretion ◽  
Unknown Etiology ◽  
Fibroblast Growth Factor 21 ◽  
Severe Insulin Resistance ◽  
Whole Exome

Abstract Insulin-mediated pseudoacromegaly (IMPA) is a rare disease of unknown etiology. Here we report a 12-year-old female with acanthosis nigricans, hirsutism, and acromegalic features characteristic of IMPA. The subject was noted to have normal growth hormone secretion, with extremely elevated insulin levels. Studies were undertaken to determine a potential genetic etiology for IMPA. The proband and her family members underwent whole exome sequencing. Functional studies were undertaken to validate the pathogenicity of candidate variant alleles. Whole exome sequencing identified monoallelic, predicted deleterious variants in genes that mediate fibroblast growth factor 21 (FGF21) signaling, FGFR1 and KLB, which were inherited in trans from each parent. FGF21 has multiple metabolic functions but no known role in human insulin resistance syndromes. Analysis of the function of the FGFR1 and KLB variants in vitro showed greatly attenuated ERK phosphorylation in response to FGF21, but not FGF2, suggesting that these variants act synergistically to inhibit endocrine FGF21 signaling but not canonical FGF2 signaling. Therefore, digenic variants in FGFR1 and KLB provide a potential explanation for the subject’s severe insulin resistance and may represent a novel category of insulin resistance syndromes related to FGF21.

Cellular and molecular effects of the mTOR inhibitor everolimus

2015 ◽  
Vol 129 (10) ◽  
pp. 895-914 ◽  
Author(s):  
Uttara Saran ◽  
Michelangelo Foti ◽  
Jean-François Dufour
Keyword(s):  
Mtor Inhibitor ◽  
Mtor Inhibitors ◽  
Cancer Therapies ◽  
Breast Cancers ◽  
Mtor Inhibition ◽  
Growth And Survival ◽  
Molecular Effects ◽  
Mtor Complex 1

mTOR (mechanistic target of rapamycin) functions as the central regulator for cell proliferation, growth and survival. Up-regulation of proteins regulating mTOR, as well as its downstream targets, has been reported in various cancers. This has promoted the development of anti-cancer therapies targeting mTOR, namely fungal macrolide rapamycin, a naturally occurring mTOR inhibitor, and its analogues (rapalogues). One such rapalogue, everolimus, has been approved in the clinical treatment of renal and breast cancers. Although results have demonstrated that these mTOR inhibitors are effective in attenuating cell growth of cancer cells under in vitro and in vivo conditions, subsequent sporadic response to rapalogues therapy in clinical trials has promoted researchers to look further into the complex understanding of the dynamics of mTOR regulation in the tumour environment. Limitations of these rapalogues include the sensitivity of tumour subsets to mTOR inhibition. Additionally, it is well known that rapamycin and its rapalogues mediate their effects by inhibiting mTORC (mTOR complex) 1, with limited or no effect on mTORC2 activity. The present review summarizes the pre-clinical, clinical and recent discoveries, with emphasis on the cellular and molecular effects of everolimus in cancer therapy.

Combination of the mTOR Inhibitor Rapamycin and Revlimid™ (CC-5013) Has Synergistic Activity in Multiple Myeloma (MM).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1492-1492 ◽  
Author(s):  
Noopur Raje ◽  
Shaji Kumar ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Mtor Inhibitor ◽  
Mtor Inhibitors ◽  
Mitogen Activated Protein Kinase ◽  
Signaling Cascades ◽  
Synergistic Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Growth And Survival ◽  
Mitogen Activated Protein

Abstract Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) mediate growth of MM cells via activation of the mitogen-activated protein kinase (MAPK), JAK2/STAT3, and phosphatidylinositol 3′-kinase/Akt kinase (PI3-K/Akt) signaling cascades. We have previously demonstrated the in vitro and in vivo activity of Revlimid™ (CC-5013), an immunomodulatory analog (IMiD) of thalidomide, in MM. In the present study, we have examined the anti-MM activity of rapamycin, a specific mTOR inhibitor, combined with Revlimid™. This combination was highly synergistic at 0.1nmol/L of rapamycin with 0.1mmol/L of Revlimid™, and remained synergistic at higher concentrations. Based on the Chou-Talalay method, combination indices of < 1 were noted for all dose ranges of Revlimid™ and rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional (doxorubicin, melphalan, dexamethasone) chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by IL-6, IGF-1, or adherence to bone marrow stromal cells (BMSCs). Cytotoxicity triggered by a combination of rapamycin with Revlimid™ resulted in apoptosis of MM cells. Furthermore, differential signaling cascades, including the MAPK and PI3-K/Akt pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies therefore provide the framework for the clinical evaluation of targeted agents like mTOR inhibitors combined with immunomodulatory agents like Revlimid™ to improve patient outcome in MM.

Sign in / Sign up

Export Citation Format

Share Document