scholarly journals TFEB Overexpression, Not mTOR Inhibition, Ameliorates RagCS75Y Cardiomyopathy

2021 ◽  
Vol 22 (11) ◽  
pp. 5494
Author(s):  
Maengjo Kim ◽  
Linghui Lu ◽  
Alexey V. Dvornikov ◽  
Xiao Ma ◽  
Yonghe Ding ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
De Novo ◽  
Mammalian Target Of Rapamycin ◽  
Missense Variant ◽  
Neonatal Rat ◽  
Physical Interaction ◽  
Mtor Inhibition ◽  
Transcription Factor Eb ◽  
Rag Gtpase ◽  
Rat Ventricle

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR–TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.

Antileukemic activity of rapamycin in acute myeloid leukemia

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2527-2534 ◽  
Author(s):  
Christian Récher ◽  
Odile Beyne-Rauzy ◽  
Cécile Demur ◽  
Gaëtan Chicanne ◽  
Cédric Dos Santos ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Mammalian Target Of Rapamycin ◽  
Cell Types ◽  
Mtor Inhibition ◽  
Growth And Survival ◽  
Clinical Responses ◽  
De Novo Aml ◽  
Acute Myeloid

AbstractThe mammalian target of rapamycin (mTOR) is a key regulator of growth and survival in many cell types. Its constitutive activation has been involved in the pathogenesis of various cancers. In this study, we show that mTOR inhibition by rapamycin strongly inhibits the growth of the most immature acute myeloid leukemia (AML) cell lines through blockade in G0/G1 phase of the cell cycle. Accordingly, 2 downstream effectors of mTOR, 4E-BP1 and p70S6K, are phosphorylated in a rapamycin-sensitive manner in a series of 23 AML cases. Interestingly, the mTOR inhibitor markedly impairs the clonogenic properties of fresh AML cells while sparing normal hematopoietic progenitors. Moreover, rapamycin induces significant clinical responses in 4 of 9 patients with either refractory/relapsed de novo AML or secondary AML. Overall, our data strongly suggest that mTOR is aberrantly regulated in most AML cells and that rapamycin and analogs, by targeting the clonogenic compartment of the leukemic clone, may be used as new compounds in AML therapy.

scholarly journals Regulation of lysosome biogenesis by phosphoinositides and phagocytosis

2021 ◽  
Author(s):  
Christopher Choy
Keyword(s):  
Cell Function ◽  
De Novo ◽  
Mammalian Target Of Rapamycin ◽  
Protein Translation ◽  
Nuclear Accumulation ◽  
Fcγ Receptor ◽  
Lysosome Biogenesis ◽  
Transcription Factor Eb ◽  
Acidic Organelles ◽  
Pathogen Clearance

Lysosomes are acidic organelles responsible for molecular degradation, energy balance, and pathogen clearance. Consequently, lysosome dysfunction is linked to numerous diseases, including lysosome storage diseases. Notably, enhancing lysosome biogenesis ameliorates cell function and helps clear metabolites. The transcription factor EB (TFEB) is a master regulator of lysosome biogenesis, and thus a potential therapeutic target. Among known regulators of TFEB, the mammalian target of rapamycin complex 1 (mTORC1) is best understood. In nutrient-rich cells, mTORC1 is activated and represses TFEB by phosphorylation. Upon starvation, mTORC1 is inactivated and TFEB enters the nucleus, upregulating lysosomal gene expression to enhance cellular degradation for energy recovery. Numerous other TFEB-dependent pathways have been identified. We aim to understand how TFEB is regulated in two additional contexts: in lysosome enlargement during phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] depletion and in phagocytosis. First, PtdIns(3,5)P2 is required for maintaining lysosome size by an incompletely understood mechanism. We hypothesized that TFEB-mediated lysosome biogenesis contributes de novo lysosomal material. Acute depletion of PtdIns(3,5)P2-synthesizing kinase PIKfyve induced TFEB nuclear accumulation. Despite increases in transcription, little to no protein translation was observed. Furthermore, tfeb-/-cells and cells blocked with cycloheximide were similar to wild-type cells, with regard to the number and size of lysosomes during PIKfyve inhibition cells, suggesting biosynthesis is not necessary for lysosome enlargement. However, TFEB still becomes active by an known mechanism. We show that TFEB nuclear localization during PIKfyve inhibition was not due to mTORC1 inactivation but may result from GSK3 inhibition. Secondly, phagocytosis allows immune cells to sequester potential pathogens by engulfing them into phagosomes. These phagosomes are then degraded by the lysosome. We postulated that phagocytosis would enhance TFEB-mediated lysosome biogenesis to promote pathogen killing. Fcγ receptor-mediated phagocytosis activated TFEB and increased biosynthesis of select lysosomal genes, augmenting existing lysosomes and enhancing proteolysis. To understand how TFEB was activated by the Fcγ receptor, we inhibited key signaling and trafficking mediators. Particle internalization, phagosome formation, and phagosome maturation appear to be necessary for TFEB activation. Overall, our work uncovers two additional mechanisms that may govern TFEBactivation.

scholarly journals Regulation of lysosome biogenesis by phosphoinositides and phagocytosis

2021 ◽  
Author(s):  
Christopher Choy
Keyword(s):  
Cell Function ◽  
De Novo ◽  
Mammalian Target Of Rapamycin ◽  
Protein Translation ◽  
Nuclear Accumulation ◽  
Fcγ Receptor ◽  
Lysosome Biogenesis ◽  
Transcription Factor Eb ◽  
Acidic Organelles ◽  
Pathogen Clearance

Lysosomes are acidic organelles responsible for molecular degradation, energy balance, and pathogen clearance. Consequently, lysosome dysfunction is linked to numerous diseases, including lysosome storage diseases. Notably, enhancing lysosome biogenesis ameliorates cell function and helps clear metabolites. The transcription factor EB (TFEB) is a master regulator of lysosome biogenesis, and thus a potential therapeutic target. Among known regulators of TFEB, the mammalian target of rapamycin complex 1 (mTORC1) is best understood. In nutrient-rich cells, mTORC1 is activated and represses TFEB by phosphorylation. Upon starvation, mTORC1 is inactivated and TFEB enters the nucleus, upregulating lysosomal gene expression to enhance cellular degradation for energy recovery. Numerous other TFEB-dependent pathways have been identified. We aim to understand how TFEB is regulated in two additional contexts: in lysosome enlargement during phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] depletion and in phagocytosis. First, PtdIns(3,5)P2 is required for maintaining lysosome size by an incompletely understood mechanism. We hypothesized that TFEB-mediated lysosome biogenesis contributes de novo lysosomal material. Acute depletion of PtdIns(3,5)P2-synthesizing kinase PIKfyve induced TFEB nuclear accumulation. Despite increases in transcription, little to no protein translation was observed. Furthermore, tfeb-/-cells and cells blocked with cycloheximide were similar to wild-type cells, with regard to the number and size of lysosomes during PIKfyve inhibition cells, suggesting biosynthesis is not necessary for lysosome enlargement. However, TFEB still becomes active by an known mechanism. We show that TFEB nuclear localization during PIKfyve inhibition was not due to mTORC1 inactivation but may result from GSK3 inhibition. Secondly, phagocytosis allows immune cells to sequester potential pathogens by engulfing them into phagosomes. These phagosomes are then degraded by the lysosome. We postulated that phagocytosis would enhance TFEB-mediated lysosome biogenesis to promote pathogen killing. Fcγ receptor-mediated phagocytosis activated TFEB and increased biosynthesis of select lysosomal genes, augmenting existing lysosomes and enhancing proteolysis. To understand how TFEB was activated by the Fcγ receptor, we inhibited key signaling and trafficking mediators. Particle internalization, phagosome formation, and phagosome maturation appear to be necessary for TFEB activation. Overall, our work uncovers two additional mechanisms that may govern TFEBactivation.

scholarly journals Everolimus and Malignancy after Solid Organ Transplantation: A Clinical Update

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Hallvard Holdaas ◽  
Paolo De Simone ◽  
Andreas Zuckermann
Keyword(s):  
Organ Transplantation ◽  
Mtor Inhibitor ◽  
De Novo ◽  
Case Reports ◽  
Meta Analysis ◽  
Mammalian Target Of Rapamycin ◽  
Solid Organ Transplantation ◽  
Population Data ◽  
Solid Organ ◽  
Mtor Inhibition

Malignancy after solid organ transplantation remains a major cause of posttransplant mortality. The mammalian target of rapamycin (mTOR) inhibitor class of immunosuppressants exerts various antioncogenic effects, and the mTOR inhibitor everolimus is licensed for the treatment of several solid cancers. In kidney transplantation, evidence from registry studies indicates a lower rate ofde novomalignancy under mTOR inhibition, with some potentially supportive data from randomized trials of everolimus. Case reports and small single-center series have suggested that switch to everolimus may be beneficial following diagnosis of posttransplant malignancy, particularly for Kaposi’s sarcoma and nonmelanoma skin cancer, but prospective studies are lacking. A systematic review has shown mTOR inhibition to be associated with a significantly lower rate of hepatocellular carcinoma (HCC) recurrence versus standard calcineurin inhibitor therapy. One meta-analysis has concluded that patients with nontransplant HCC experience a low but significant survival benefit under everolimus monotherapy, so far unconfirmed in a transplant population. Data are limited in heart transplantation, although observational data and case reports have indicated that introduction of everolimus is helpful in reducing the recurrence of skin cancers. Overall, it can be concluded that, in certain settings, everolimus appears a promising option to lessen the toll of posttransplant malignancy.

mTOR inhibition with temsirolimus causes acute increases in glomerular permeability, but inhibits the dynamic permeability actions of puromycin aminonucleoside

2015 ◽  
Vol 308 (10) ◽  
pp. F1056-F1064 ◽  
Author(s):  
Josefin Axelsson ◽  
Anna Rippe ◽  
Bengt Rippe
Keyword(s):  
High Performance ◽  
De Novo ◽  
Mammalian Target Of Rapamycin ◽  
Size Exclusion ◽  
Ang Ii ◽  
Puromycin Aminonucleoside ◽  
Glomerular Permeability ◽  
Mtor Inhibition ◽  
Transplant Patients ◽  
Exclusion Chromatography

Inhibitors of the mammalian target of rapamycin (mTORi) can produce de novo proteinuria in kidney transplant patients. On the other hand, mTORi has been shown to suppress disease progression in several animal models of kidney disease. In the present study, we investigated whether glomerular permeability can be acutely altered by the mTORi temsirolimus and whether mTORi can affect acute puromycin aminonucleoside (PAN) or angiotensin II (ANG II)-induced glomerular hyperpermeability. In anesthetized Wistar rats, the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Temsirolimus was administered as a single intravenous dose 30 min before the start of the experiments in animals infused with PAN or ANG II or in nonexposed animals. Polydispersed FITC-Ficoll-70/400 (molecular radius 10–80 Å) and 51Cr-EDTA infusion was given during the whole experiment. Measurements of Ficoll in plasma and urine were performed sequentially before the temsirolimus injection (baseline) and at 5, 15, 30, 60, and 120 min after the start of the experiments. Urine and plasma samples were analyzed by high-performance size-exclusion chromatography (HPSEC) to assess glomerular sieving coefficients (θ) for Ficoll10-80Å. Temsirolimus per se increased baseline glomerular permeability to Ficoll50-80Å 45 min after its administration, a reactive oxygen species (ROS)-dependent phenomenon. PAN caused a rapid and reversible increase in glomerular permeability, peaking at 5 min, and again at 60–120 min, which could be blocked by the ROS scavenger tempol. mTORi abrogated the second permeability peak induced by PAN. However, it had no effect on the immediate ANG II- or PAN-induced increases in glomerular permeability.

scholarly journals Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ba Van Vu ◽  
Quyet Nguyen ◽  
Yuki Kondo-Takeoka ◽  
Toshiki Murata ◽  
Naoki Kadotani ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Copy Number ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Pyricularia Oryzae ◽  
Transcriptional Gene Silencing ◽  
Physical Interaction ◽  
Uncharacterized Protein ◽  
Form Complex

AbstractTransposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungusPyricularia oryzae. Genetic and physical interaction studies revealed thatRecAdomain-containing proteins, includingP. oryzaehomologs ofRad51, Rad55, andRad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly,P. oryzaemutants of specific RNA silencing components (MoDCL1andMoAGO2)were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

scholarly journals FEN1 Blockade for Platinum Chemo-Sensitization and Synthetic Lethality in Epithelial Ovarian Cancers

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1866
Author(s):  
Katia A. Mesquita ◽  
Reem Ali ◽  
Rachel Doherty ◽  
Michael S. Toss ◽  
Islam Miligy ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
High Throughput Screening ◽  
Nuclear Translocation ◽  
Synthetic Lethality ◽  
Excision Repair ◽  
Progression Free Survival ◽  
Ovarian Cancer Cells ◽  
Physical Interaction ◽  
Base Excision

FEN1 plays critical roles in long patch base excision repair (LP-BER), Okazaki fragment maturation, and rescue of stalled replication forks. In a clinical cohort, FEN1 overexpression is associated with aggressive phenotype and poor progression-free survival after platinum chemotherapy. Pre-clinically, FEN1 is induced upon cisplatin treatment, and nuclear translocation of FEN1 is dependent on physical interaction with importin β. FEN1 depletion, gene inactivation, or inhibition re-sensitizes platinum-resistant ovarian cancer cells to cisplatin. BRCA2 deficient cells exhibited synthetic lethality upon treatment with a FEN1 inhibitor. FEN1 inhibitor-resistant PEO1R cells were generated, and these reactivated BRCA2 and overexpressed the key repair proteins, POLβ and XRCC1. FEN1i treatment was selectively toxic to POLβ deficient but not XRCC1 deficient ovarian cancer cells. High throughput screening of 391,275 compounds identified several FEN1 inhibitor hits that are suitable for further drug development. We conclude that FEN1 is a valid target for ovarian cancer therapy.

scholarly journals A novel de novo DDX3X missense variant in a female with brachycephaly and intellectual disability: a case report

2021 ◽  
Vol 47 (1) ◽  
Author(s):  
Giada Moresco ◽  
Jole Costanza ◽  
Carlo Santaniello ◽  
Ornella Rondinone ◽  
Federico Grilli ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Clinical Presentation ◽  
De Novo ◽  
Clinical Signs ◽  
Missense Variant ◽  
Psychomotor Development ◽  
Helicase Domain ◽  
Protein Activity ◽  
Mrna Metabolism ◽  
Pathogenic Variants

Abstract Background De novo pathogenic variants in the DDX3X gene are reported to account for 1–3% of unexplained intellectual disability (ID) in females, leading to the rare disease known as DDX3X syndrome (MRXSSB, OMIM #300958). Besides ID, these patients manifest a variable clinical presentation, which includes neurological and behavioral defects, and abnormal brain MRIs. Case presentation We report a 10-year-old girl affected by delayed psychomotor development, delayed myelination, and polymicrogyria (PMG). We identified a novel de novo missense mutation in the DDX3X gene (c.625C > G) by whole exome sequencing (WES). The DDX3X gene encodes a DEAD-box ATP-dependent RNA-helicase broadly implicated in gene expression through regulation of mRNA metabolism. The identified mutation is located just upstream the helicase domain and is suggested to impair the protein activity, thus resulting in the altered translation of DDX3X-dependent mRNAs. The proband, presenting with the typical PMG phenotype related to the syndrome, does not show other clinical signs frequently reported in presence of missense DDX3X mutations that are associated with a most severe clinical presentation. In addition, she has brachycephaly, never described in female DDX3X patients, and macroglossia, that has never been associated with the syndrome. Conclusions This case expands the knowledge of DDX3X pathogenic variants and the associated DDX3X syndrome phenotypic spectrum.

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