Suppression of PTEN Expression by NF-κB Prevents Apoptosis

ABSTRACT NF-κB is a heterodimeric transcription activator consisting of the DNA binding subunit p50 and the transactivation subunit p65/RelA. NF-κB prevents cell death caused by tumor necrosis factor (TNF) and other genotoxic insults by directly inducing antiapoptotic target genes. We report here that the tumor suppressor PTEN, which functions as a negative regulator of phosphatidylinositol (PI)-3 kinase/Akt-mediated cell survival pathway, is down regulated by p65 but not by p50. Moreover, a subset of human lung or thyroid cancer cells expressing high levels of endogenous p65 showed decreased expression of PTEN that could be rescued by specific inhibition of the NF-κB pathway with IκB overexpression as well as with small interfering RNA directed against p65. Importantly, TNF, a potent inducer of NF-κB activity, suppressed PTEN gene expression in IKKβ+/+ cells but not in IKKβ−/− cells, which are deficient in the NF-κB activation pathway. These findings indicated that NF-κB activation was necessary and sufficient for inhibition of PTEN expression. The promoter, RNA, and protein levels of PTEN are down-regulated by NF-κB. The mechanism underlying suppression of PTEN expression by NF-κB was independent of p65 DNA binding or transcription function and involved sequestration of limiting pools of transcriptional coactivators CBP/p300 by p65. Restoration of PTEN expression inhibited NF-κB transcriptional activity and augmented TNF-induced apoptosis, indicating a negative regulatory loop involving PTEN and NF-κB. PTEN is, thus, a novel target whose suppression is critical for antiapoptosis by NF-κB.

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A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03387-9 ◽

2019 ◽

Vol 77 (18) ◽

pp. 3627-3642 ◽

Cited By ~ 3

Author(s):

Jussi-Pekka Tolonen ◽

Minna Heikkilä ◽

Marjo Malinen ◽

Hang-Mao Lee ◽

Jorma J. Palvimo ◽

...

Keyword(s):

Target Genes ◽

Hypoxia Inducible Factor ◽

Negative Regulator ◽

Luciferase Reporter ◽

Transcription Activator ◽

Promoter Regions ◽

Α Subunit ◽

Hypoxia Response ◽

Protein Levels ◽

Oxygen Homeostasis

AbstractHypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

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The microRNA miR-8 is a conserved negative regulator of Wnt signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0807763105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15417-15422 ◽

Cited By ~ 130

Author(s):

Jennifer A. Kennell ◽

Isabelle Gerin ◽

Ormond A. MacDougald ◽

Ken M. Cadigan

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Target Genes ◽

Wnt Signaling Pathway ◽

Negative Regulator ◽

Animal Development ◽

Protein Levels ◽

Evolutionarily Conserved ◽

Multiple Levels

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

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Control of Thioredoxin Reductase Gene (trxB) Transcription by SarA in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.01202-09 ◽

2009 ◽

Vol 192 (1) ◽

pp. 336-345 ◽

Cited By ~ 31

Author(s):

Anand Ballal ◽

Adhar C. Manna

Keyword(s):

Oxidative Stress ◽

Staphylococcus Aureus ◽

Dna Binding ◽

Target Genes ◽

Thioredoxin Reductase ◽

Negative Regulator ◽

Binding Studies ◽

Oxidizing Agents

ABSTRACT Thioredoxin reductase (encoded by trxB) protects Staphylococcus aureus against oxygen or disulfide stress and is indispensable for growth. Among the different sarA family mutants analyzed, transcription of trxB was markedly elevated in the sarA mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of trxB expression. Gel shift analysis showed that purified SarA protein binds directly to the trxB promoter region DNA in vitro. DNA binding of SarA was essential for repression of trxB transcription in vivo in S. aureus. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the trxB promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the trxB gene in the sarA mutant, suggesting the presence of a SarA-independent mode of trxB induction. Analysis of two oxidative stress-responsive sarA regulatory target genes, trxB and sodM, with various mutant sarA constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes in vivo. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in S. aureus.

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NQO1 binds and supports SIRT1 function

10.1101/139907 ◽

2017 ◽

Author(s):

Peter Tsvetkov ◽

Julia Adler ◽

Yaarit Adamovich ◽

Gad Asher ◽

Nina Reuven ◽

...

Keyword(s):

Target Genes ◽

Protective Role ◽

Class Iii ◽

Mitochondrial Stress ◽

Protein Levels ◽

Related Enzyme ◽

Metabolic Sensing ◽

Regulatory Loop ◽

Nadh Oxidoreductase

AbstractSilent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

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FoxO Transcription Factor Is Delocalized and Inactivated in Acute Myeloid Leukaemia Patients.

Blood ◽

10.1182/blood.v110.11.1251.1251 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1251-1251

Author(s):

Daniela Cilloni ◽

Cristina Panuzzo ◽

Francesca Messa ◽

Francesca Arruga ◽

Paolo Nicoli ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Mean Value ◽

Binding Activity ◽

Negative Regulator ◽

Pi3k Inhibitor Ly294002 ◽

Downstream Target ◽

Dna Binding Activity ◽

Blast Cells ◽

Nuclear Signal

Abstract The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. Although multiple mechanisms regulate FoxO activity, Akt seems to be crucial to its regulation and function. PI3K/Akt pathway has been reported to be abnormally activated in AML blast cells. The aim of this study was to investigate the function of FoxO in AML blast cells and the presence of alternative pathways responsible for FoxO3 inactivation other than PI3K-Akt. BM cells were collected from 35 AML patients at diagnosis and after chemotherapy and from 20 healthy donors. The expression levels of FoxO1, FoxO3, FoxO4 were tested by RQ-PCR, FoxO3 protein amount and localization by Western blot and immunofluorescence and the DNA binding activity by EMSA. Furthermore, downstream target genes transcribed by FoxO3 were quantified. Among these, Spred1 which codes for a negative regulator of RTK signal, including Ras mediated pathway triggered by FLT3. We have previously described the absence of Spred1 is AML patients and we have demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally, BM cells were incubated with 5 mM of the PI3K inhibitor LY294002 and 20 mM PS1145, the inhibitor of IKK kinase also responsible for FoxO phosphorylation and with the combination LY294002 plus PS1145. We found that the amount of FoxO1, FoxO3 and FoxO4 mRNA are similar in AML patients and controls. Interestingly, while FoxO3 in control cells is localized in both, nucleous and cytoplasm, is completely cytoplasmatic in AML cells and it enters the nucleous after chemotherapy. The quantification of FoxO fluorescent signal in controls shows a mean value of intensity of 21.4±2 in the nucleous and 14,6±1.7 in the cytoplasm. By contrast, in AML cells is 8,2±4 in the nucleous and 18.1±4,6 in the cytoplasm. Additionally, FoxO3 DNA binding activity in AML patients is completely absent at diagnosis and reappears after therapy. Also the mRNA of the target gene Spred1 is rather undetectable at diagnosis (mean value 2−ΔΔCt= 0,009±0,3) and is upregulated during remission (mean value 2−ΔΔ= 2±1,5) or after LY29400 incubation (mean value =0,8±0,3). LY294002 and PS1145 results in FoxO partial nuclear relocalization with a nuclear signal of 15±3 and 12±3 respectively. Interestingly, the association of PS1145 and LY294002 induces a complete nuclear shuttle with a nuclear signal of 25±4, suggesting that both pathways are implicated in FoxO inactivation. Taken together these observations suggest that FoxO inactivation may be crucial for the apoptosis arrest observed in AML. These data demonstrate that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy based on the combination of selective inhibitors such as FLT3 or Akt inhibitors or standard chemotherapy and the IKK inhibitor.

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Deubiquitinase Usp9X Regulates Cell Survival and Metabolic Signal Transduction in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.1835.1835 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1835-1835 ◽

Cited By ~ 1

Author(s):

Luke F. Peterson ◽

Hanshi Sun ◽

Vaibhav Kapuria ◽

Malathi Kandarpa ◽

Moshe Talpaz ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Survival ◽

Board Of Directors ◽

Poor Prognosis ◽

Treatment Options ◽

Negative Regulator ◽

Drug Resistant ◽

Advisory Committees ◽

Protein Levels ◽

Induced Apoptosis

Abstract Abstract 1835 Multiple myeloma (MM) therapy has significantly improved with the addition of a number of new agents that target the proteasome and other pathways. However, most patients relapse and eventually become resistant to any drug therapy, leaving limited or no treatment options. As a result, there is tremendous interest in understanding drug resistance and regulation of MM cell survival. Toward this goal, a number of studies have assessed differential gene expression and proteome profiles between normal cells, primary myeloma and drug-resistant disease. Recent reports suggest that increased expression of the deubiquitinase (DUB) Usp9x is associated with poor prognosis of MM patients. Additional analyses indicate that Usp9x regulates a number of survival and signaling proteins including Mcl-1, a Bcl-2-family pro-survival protein previously associated with drug and apoptotic resistance in myeloma patients, and 5'-AMP kinase (AMPK), a negative regulator of the mTOR cascade. We previously described WP1130, a small molecule with Usp9x DUB inhibitory activity. This compound induced apoptosis in MM cell lines and primary cells from drug-resistant MM patients (IC50 ∼1 μM) but not in normal CD34 cells even at 5-fold higher concentrations, suggestive of a favorable therapeutic index. To determine the role of Usp9x in MM cell survival and signaling, we silenced Usp9x expression in MM cells and assessed changes in Usp9x DUB activity, Mcl-1 levels, cell survival and mTOR signaling. We also compared these results with the effects of WP1130 treatment. Constitutive knockdown of Usp9x by shRNA in H929, MM1.S and RPMI-8226 cells reduced Usp9x protein levels by >90%, but reduced Usp9x DUB activity by no more than 40%. Importantly, although Usp9x silencing reduced MM cell survival by 5–40%, major changes in Mcl-1 protein levels were not detected. In contrast, treatment of control or Usp9x-silenced cells with WP1130 resulted in a rapid reduction in both Usp9x DUB activity and Mcl-1 protein levels. These results suggest that Usp9x DUB activity was not solely dependent on Usp9x protein levels and that cells with shRNA-mediated Usp9x knockdown may activate other compensatory mechanisms to stabilize Usp9x activity. We examined the effects of Usp9x knockdown on the mTOR negative regulator AMPK, whose activation requires deubiquitination by Usp9x. Both Usp9x shRNA and WP1130 treatment stabilized ubiquitination of AMPK, resulting in inhibition of AMPK kinase activity. Such AMPK inhibition led to activation of the mTOR cascade, thereby increasing phosphorylation of Akt, S6 and GSK3β. These observations suggested that activation of mTOR through chronic Usp9x knockdown suppresses apoptosis in MM cells. Inhibition of mTOR activity (with Torin-2) in Usp9x-silenced cells rapidly induced apoptosis with a corresponding reduction in Mcl-1 protein levels. This Mcl-1 reduction was associated with decreased GSK3β activity and suppression of Mcl-1 phosphorylation at Thr159, a GSK3β phosphorylation site that negatively regulates Mcl-1/Usp9x association. Dissociation of Mcl-1/Usp9x complexes increased ubiquitination of Mcl-1 and its destruction by the proteasome. Together, these results suggest that Usp9x regulates multiple substrates with differential effects on mTOR signal transduction and apoptotic proteins. This compound impact of Usp9x on Mcl-1 and mTOR regulation may complicate the assessment Usp9x as a potential therapeutic target in MM, but may help explain the sensitivity of MM cells to WP1130. Specifically, WP1130 increases the formation of Mcl-1/Usp9x complexes by inhibiting AMPK and thereby activating mTOR/Akt/GSK3β. The Mcl-1 in complex with Usp9x becomes ubiquitinated due to rapid inhibition of Usp9x activity by WP1130. Our results predict that Usp9x inhibitors will have therapeutic activity in MM cells by modulating at least two separate pathways that regulate metabolic signaling and MM cell survival. Considering that increased Usp9x expression has been associated with short survival and poor prognosis in MM patients, Usp9x inhibitors like WP1130 could show therapeutic promise for this patient population with limited treatment options. Disclosures: Talpaz: ARIAD: Research Funding. Jakubowiak:Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria.

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Puma, noxa, p53, and p63 differentially mediate stress pathway induced apoptosis

Cell Death and Disease ◽

10.1038/s41419-021-03902-6 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Jun Wang ◽

Holly R. Thomas ◽

Zhang Li ◽

Nan Cher Yeo ◽

Hannah E. Scott ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Responses ◽

Target Genes ◽

Genotoxic Stress ◽

Negative Regulator ◽

Null Alleles ◽

Secondary Wave ◽

Apoptotic Response ◽

Induced Apoptosis

AbstractCellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.

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ABA-induced cytoplasmic translocation of COP1 enhances ROS accumulation through the HY5-ABI5 pathway to modulate seed germination

10.22541/au.163257697.75025095/v1 ◽

2021 ◽

Author(s):

Qing-Bin Chen ◽

Wenjing Wang ◽

Yue Zhang ◽

Qidi Zhan ◽

Kang Liu ◽

...

Keyword(s):

Seed Germination ◽

Target Genes ◽

Seedling Survival ◽

Signal Transduction Pathway ◽

Negative Regulator ◽

Protein Levels ◽

Aba Signal Transduction ◽

Ros Accumulation ◽

Light Signals ◽

Cytoplasmic Translocation

Seed germination is a physiological process regulated by multiple factors. Abscisic acid (ABA) can inhibit seed germination to improve seedling survival under conditions of abiotic stress, and this process is often regulated by light signals. Constitutive Photomorphogenic 1 (COP1) is an upstream core repressor of light signals, and is involved in several ABA responses. Here, we demonstrate that COP1 is a negative regulator of the ABA-mediated inhibition of seed germination. Disruption of COP1 enhanced Arabidopsis seed sensitivity to ABA and increased ROS levels. In seeds, ABA induced the translocation of COP1 to the cytoplasm, resulting in enhanced ABA-induced ROS levels. Genetic evidence indicated that HY5 and ABI5 act downstream of COP1 in the ABA-mediated inhibition of seed germination. ABA-induced COP1 cytoplasmic localization increased HY5 and ABI5 protein levels in the nucleus, leading to increased expression of ABI5 target genes and ROS levels in seeds. Together, our results reveal that ABA-induced cytoplasmic translocation of COP1 activates the HY5-ABI5 pathway to promote the expression of ABA-responsive genes and the accumulation of ROS during ABA-mediated inhibition of seed germination. These findings enhance the role of COP1 in the ABA signal transduction pathway.

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Pre-Clinical Efficacy of Co-Targeting GFI1/KDM1A and BRD4 or JAK1/2 Against AML and Post-MPN Secondary AML Blast Progenitor Cells

Blood ◽

10.1182/blood-2020-140212 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-27

Author(s):

Warren Fiskus ◽

Christopher Peter Mill ◽

Christine Birdwell ◽

Bernardo H Lara ◽

Prithviraj Bose ◽

...

Keyword(s):

Progenitor Cells ◽

Research Funding ◽

Target Genes ◽

Amine Oxidase ◽

Colony Growth ◽

Protein Domain ◽

Rna Seq ◽

Protein Levels ◽

Gene Sets ◽

Induced Apoptosis

Transcriptional regulators (TFs) involved in cell-growth, differentiation and survival of AML stem/progenitor cells (LSCs) include RUNX1, PU.1, CEBPα, c-Myb and c-Myc. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2). LSD1 is part of the repressor complexes involving GFI1, CoREST and HDAC1/2, that regulate active super-enhancers/enhancers (SEs/Es) and their target genes, mediating transcriptional repression and differentiation block in LSCs. GFI1 is a zinc-finger transcriptional repressor involved in AML development and differentiation. GFI1 contains an N-terminal domain through which it binds to the CoREST/LSD1/HDAC1/2 complex to regulate differentiation in LSCs. CRISPR-suppressor scanning revealed that enzymatic activity of LSD1 was not required for LSC differentiation, instead disruption of binding of LSD1 to GFI1 and CoREST induced differentiation in LSCs. LSD1 and GFI1 expression correlates with worse prognosis in MDS/AML. In present studies, we demonstrate first-time ever that knockout (KO) or degradation of LSD1 utilizing CRISPR-Cas9 or LSD1-FKBP12(F36V) and dTAG-13, respectively, disrupted LSD1-binding to GFI1/1B and CoREST, inhibiting colony growth and inducing differentiation markers (CD86 and CD11b) and morphologic differentiation of AML and post-MPN sAML blast progenitor cells (BPCs). CRISPR-mediated knockout of LSD1 in the AML OCI-AML5 and sAML SET2 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at SEs/Es (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed DNMT1, CoREST, c-Myc, CDK6, and c-KIT, while inducing GFI1, PU.1, CEBPα, p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq and by Western analyses). This correlated with growth inhibition, % differentiation and apoptosis of AML and sAML cells. CRISPR-mediated GFI1-KO ± the irreversible LSD1 inhibitor (LSD1i) (INCB059872, INCB), repressed GFI1 levels, yet enhanced expressions of PU.1, p21 and CD11b and significantly increased % morphologic differentiation. Treatment with INCB (0.25 to 1.0 µM) also disrupted binding of LSD1 to GFI1 and to CoREST, increased GFI1/1B and PU.1 and repressed c-Myc protein levels, while significantly inhibiting colony growth, inducing differentiation and loss of viability of AML and post-MPN sAML (SET2 and HEL92.1.7) cells, as well as patient-derived AML and post-MPN sAML blasts (p < 0.01). Following INCB treatment, ATAC-Seq analysis demonstrated gained peaks in GFI1 and PU.1-target genes. Following H3K27Ac ChIP-seq analysis rank-ordering of SEs (ROSE) plot highlighted active SEs of RUNX1, GFI1, BCL2, PU.1, IRF8 and SMYD3, accompanied by increased H3K27Ac occupancy at the chromatin of GFI1 and PU.1 targets. Notably, INCB treatment also increased BRD4 occupancy, especially at the GFI1 and PU.1-target genes. RNA-Seq analysis showed that INCB treatment perturbed mRNA expressions, with positive normalized enrichment scores (NES) for interferon α, inflammatory-response, GFI1-targets and E2F-target gene-sets, and negative NES for c-Myc-targets and oxidative-phosphorylation gene-sets. RNA-Seq analyses of INCB-treated compared to untreated OCI-AML5 and SET-2 cells also demonstrated log2 fold-increase in the mRNA expressions of GFI1, PU.1 and CEBPα target-genes. Utilizing a protein domain-scanning CRISPR-Cas9 sgRNA screen followed by LSD1i treatment, present studies also demonstrate co-dependencies, including BRD4, in AML cells. BET inhibitor (BETi) treatment also depleted LSD1 protein levels, and co-treatment with the BETi OTX015 and INCB induced synergistic lethality in AML and post-MPN sAML blasts (Combination Indices < 1.0). Pre-treatment with INCB re-sensitized JAKi-resistant sAML cells to ruxolitinib-induced apoptosis and BETi-resistant post-MPN sAML cells to BETi-induced apoptosis. Notably, co-treatment with INCB (1.5 mg/kg) and ruxolitinib (20 mg/kg) or OTX015 (50 mg/kg), administered orally for 21 days, compared to ruxolitinib alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with luciferized sAML HEL92.1.7 xenografts (p < 0.01). Collectively, these findings support further pre-clinical development of LSD1i-based combinations with ruxolitinib and BETi against post-MPN sAML. Disclosures Bose: CTI BioPharma: Honoraria, Research Funding; NS Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding; Blueprint Medicines Corporation: Honoraria, Research Funding; Promedior, Inc.: Research Funding; Incyte Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kartos Therapeutics: Honoraria, Research Funding. Kadia:Incyte: Research Funding; Pulmotec: Research Funding; Cellenkos: Research Funding; Celgene: Research Funding; Amgen: Research Funding; Genentech: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Cyclacel: Research Funding; Novartis: Honoraria; Ascentage: Research Funding; Astellas: Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Astra Zeneca: Research Funding; BMS: Honoraria, Research Funding. Verstovsek:CTI Biopharma Corp: Research Funding; AstraZeneca: Research Funding; Sierra Oncology: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding; PharmaEssentia: Research Funding; Blueprint Medicines Corp: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Gilead: Research Funding; Protagonist Therapeutics: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Celgene: Consultancy, Research Funding; ItalPharma: Research Funding.

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NQO1 Binds and Supports SIRT1 Function

Frontiers in Pharmacology ◽

10.3389/fphar.2021.671929 ◽

2021 ◽

Vol 12 ◽

Author(s):

Peter Tsvetkov ◽

Julia Adler ◽

Romano Strobelt ◽

Yaarit Adamovich ◽

Gad Asher ◽

...

Keyword(s):

Target Genes ◽

Protective Role ◽

Class Iii ◽

Mitochondrial Stress ◽

Protein Levels ◽

Related Enzyme ◽

Metabolic Sensing ◽

Regulatory Loop ◽

Nadh Oxidoreductase

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

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