Homoharringtonine deregulates MYC transcriptional expression by directly binding NF-κB repressing factor
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ABSTRACT:
PROVIDER: PRJNA495949 | ENA |
REPOSITORIES: ENA
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Project description:Homoharringtonine (HHT), a previously known protein synthesis inhibitor, has anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myeloid leukemia (AML) with favorable and intermediate prognosis, especially in t(8;21) subtype. Here we provide evidence to show that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+, LICs) in t(8;21) murine leukemia model and exerts down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). By using biochemistry approach, NF-κB-repressing factor (NKRF) was discovered to be bound directly by HHT via the second double-strand RNA binding motif (DSRM2) domain, which is the nuclear localization signal (NLS) of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding site to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from nucleus (including nucleoli) to cytoplasm through occupying the DSRM2 domain, strengthens p65-NKRF interaction, and interferes with the p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in consistence with MYC downregulation. Our work has thus identified a mechanism different to, but in concert with, the classical mode of action of HHT and justifies its further clinical exploration in the treatment of AML.
2018-12-31 | GSE121175 | GEO
Project description:Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.
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Project description:Genes directly regulated by NF-κB in human hepatocellular carcinoma HepG2
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Project description:Over activation of NF-κB has close relationship with hepatitis and hepatocellular carcinoma (HCC). In this study, by manipulating NF-κB activity with its recognized activator TNFα and using ChIP-seq and RNA-seq techniques, we identified 699 NF-κB direct target genes (DTGs) in a widely used HCC cell line, HepG2, including 399 activated and 300 repressed genes. In these NF-κB DTGs, 216 genes (126 activated and 90 repressed genes) are among the current HCC gene signature. Functional annotation revealed that NF-κB DTGs in HepG2 cell are mainly related with many typical NF-κB-related biological processes, such as immune system process, response to stress, response to stimulus, defense response and signaling pathways of NF-kappa B. Some NF-κB DTGs are also involved in Hepatitis C and B pathways. The NF-κB DTGs were further confirmed by detecting the NF-κB binding and expression of 14 genes with ChIP-PCR and RT-PCR.
2017-06-05 | GSE98984 | GEO
Project description:Over activation of NF-κB has close relationship with hepatitis and hepatocellular carcinoma (HCC). In this study, by manipulating NF-κB activity with its recognized activator TNFα and using ChIP-seq and RNA-seq techniques, we identified 699 NF-κB direct target genes (DTGs) in a widely used HCC cell line, HepG2, including 399 activated and 300 repressed genes. In these NF-κB DTGs, 216 genes (126 activated and 90 repressed genes) are among the current HCC gene signature. Functional annotation revealed that NF-κB DTGs in HepG2 cell are mainly related with many typical NF-κB-related biological processes, such as immune system process, response to stress, response to stimulus, defense response and signaling pathways of NF-kappa B. Some NF-κB DTGs are also involved in Hepatitis C and B pathways. The NF-κB DTGs were further confirmed by detecting the NF-κB binding and expression of 14 genes with ChIP-PCR and RT-PCR.
2017-06-05 | GSE98983 | GEO
Project description:NF-κB has an essential role in innate immune response and inflammation and is involved in cancer development and progression. We apply the SEC-PCP-SILAC method incorporating metabolic labeling, size exclusion chromatography and protein correlation profiling to construct a complex network of interactome rearrangement in response to NF-κB modulation in breast cancer cells. Our interaction network represents a complex insight into the dynamics of MCF-7 protein interactome associated with NF-κB pathway. Our dataset could serve as a basis for future studies characterizing role of NF-κB in breast cancer cellular pathways. This PRIDE project includes results from SILAC labeled and label-free replicates from the SEC-PCP-SILAC analysis of protein complexes in MCF-7 cells with inhibited and uninhibited NF-κB pathway, results from the immunoprecipitation experiment aimed at interaction partners of NF-κB factor RELA, analysis of total proteome after NF-κB inhibition, and results from SEC fractionation of untreated and unlabeled MCF-7 cells.
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Project description:Transcriptional profiling of human control and Néstor-Guillermo Progeria Syndrome (NGPS) fibroblasts and induced pluripotent stem cells (iPSCs). Somatic cell reprogramming involves rejuvenation of adult cells and relies on the ability to erase age-associated molecular marks. Accordingly, reprogramming efficiency declines with ageing, and age-associated features such as genetic instability, cell senescence or telomere shortening negatively affect this process. However, the regulatory mechanisms that constitute age-associated barriers for cell reprogramming remain largely unknown. Here, by using cells from patients with premature ageing, we demonstrate that NF-κB activation is a critical barrier for the generation of induced pluripotent stem cells (iPSCs) in ageing. We show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs generation of iPSCs by eliciting reprogramming repressors DOT1L and YY1, reinforcing cell senescence signals and down-regulating pluripotency genes. We also show that genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo Progeria Syndrome (NGPS) and Hutchinson-Gilford Progeria Syndrome (HGPS) patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo ameliorates the accelerated ageing phenotype and extends lifespan in a progeroid animal model. Collectively, our results provide evidence for a novel role of NF-κB in the control of cell fate transitions and reinforce the interest of studying age-associated molecular impairments to implement cell reprogramming methodologies, and to identify new targets of rejuvenation strategies. Control and NGPS fibroblasts were reprogrammed. RNA was extracted and transcriptional profiling was obtained with GeneChip Human Exon 1.0 ST Arrays.
2015-07-01 | E-GEOD-65170 | biostudies-arrayexpress
Project description:Transcriptional profiling of human control and Néstor-Guillermo Progeria Syndrome (NGPS) mesenchymal stem cells (MSCs). Somatic cell reprogramming involves rejuvenation of adult cells and relies on the ability to erase age-associated molecular marks. Accordingly, reprogramming efficiency declines with ageing, and age-associated features such as genetic instability, cell senescence or telomere shortening negatively affect this process. However, the regulatory mechanisms that constitute age-associated barriers for cell reprogramming remain largely unknown. Here, by using cells from patients with premature ageing, we demonstrate that NF-κB activation is a critical barrier for the generation of induced pluripotent stem cells (iPSCs) in ageing. We show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs generation of iPSCs by eliciting reprogramming repressors DOT1L and YY1, reinforcing cell senescence signals and down-regulating pluripotency genes. We also show that genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo Progeria Syndrome (NGPS) and Hutchinson-Gilford Progeria Syndrome (HGPS) patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo ameliorates the accelerated ageing phenotype and extends lifespan in a progeroid animal model. Collectively, our results provide evidence for a novel role of NF-κB in the control of cell fate transitions and reinforce the interest of studying age-associated molecular impairments to implement cell reprogramming methodologies, and to identify new targets of rejuvenation strategies. Control and NGPS MSCs were differentiated into bone in the presence or absence of sodium salicylate. Total RNA was extracted and global gene expression was analyzed.
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