Nuclear Respiratory Factor 1 Binding Research Articles

HTLV-1 bZIP factor impairs DNA mismatch repair system

Adult T-cell leukemia (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Microsatellite instability (MSI) has been observed in ATL cells. Although MSI results from impaired mismatch repair (MMR) pathway, no null mutations in the genes encoding MMR factors are detectable in ATL cells. Thus, it is unclear whether or not impairment of MMR causes the MSI in ATL cells. HTLV-1 bZIP factor (HBZ) protein interacts with numerous host transcription factors and significantly contributes to disease pathogenesis and progression. Here we investigated the effect of HBZ on MMR in normal cells. The ectopic expression of HBZ in MMR-proficient cells induced MSI, and also suppressed the expression of several MMR factors. We then hypothesized that the HBZ compromises MMR by interfering with a transcription factor, nuclear respiratory factor 1 (NRF-1), and identified the consensus NRF-1 binding site at the promoter of the gene encoding MutS homologue 2 (MSH2), an essential MMR factor. The luciferase reporter assay revealed that NRF-1 overexpression enhanced MSH2 promoter activity, while co-expression of HBZ reversed this enhancement. These results supported the idea that HBZ suppresses the transcription of MSH2 by inhibiting NRF-1. Our data demonstrate that HBZ causes impaired MMR, and may imply a novel oncogenesis driven by HTLV-1.

NRF1 Alleviated Oxidative Stress of Glioblastoma Cells by Regulating NOR1.

Oxidored-nitro domain-containing protein 1 (NOR1) is a critical tumour suppressor gene, though its regulatory mechanism in oxidative stress of glioblastoma (GBM) remains unclear. Hence, further study is needed to unravel the function of NOR1 in the progression of oxidative stress in GBM. In this study, we evaluated the expression of NOR1 and nuclear respiratory factor 1 (NRF1) in GBM tissue and normal brain tissue (NBT) using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB), and investigated their relationship. We then induced oxidative stress in U251 cells through H2O2 treatment and conducted Cell Count-ing Kit-8, Transwell and wound healing assays to analyse cell proliferation, invasion and migration. Cell apoptosis was assessed by flow cytometry and TUNEL staining. We also measured the activities of superoxide dismutase and catalase, as well as the level of reactive oxygen species (ROS) using biochemical techniques. Via qRT-PCR and WB, the mRNA and protein expression levels of NOR1 and NRF1 were determined. Chromatin immunoprecipitation (ChIP) assays were applied to validate NRF1's interaction with NOR1. Our results showed that the expression of NOR1 and NRF1 was low in GBM, and their expression levels were positively correlated. H2O2-induced oxidative stress reduced NRF1 and NOR1 expression levels and increased the ROS level. The ChIP assay confirmed the binding of NRF1 to NOR1. Over-expression of NRF1 attenuated the inhibitory effect of oxidative stress on the proliferation, migration and invasion of U251 cells, which was reversed by knockdown of NOR1.

Nuclear respiratory factor 1 promotes the growth of liver hepatocellular carcinoma cells via E2F1 transcriptional activation

BackgroundRecent studies have shown that functional mitochondria are essential for cancer cells. Nuclear respiratory factor 1 (NRF1) is a transcription factor that activates mitochondrial biogenesis and the expression of the respiratory chain, but little is known about its role and underlying mechanism in liver hepatocellular carcinoma (LIHC).MethodsNRF1 expression was analyzed via public databases and 24 paired LIHC samples. Clinical-pathological information and follow-up data were collected from 165 patients with LIHC or online datasets. Furthermore, cellular proliferation and the cell cycle were analyzed by MTT, Clone-forming assay and flow cytometric analyses. NRF1 target genes were analyzed by Chromatin immunoprecipitation sequencing (ChIP-Seq). PCR and WB analysis was performed to detect the expression of related genes. ChIP and luciferase activity assays were used to identify NRF1 binding sites.ResultsOur results showed that NRF1 expression was upregulated in LIHC compared to normal tissues. NRF1 expression was associated with tumour size and poor prognosis in patients. Knockdown of NRF1 repressed cell proliferation and overexpression of NRF1 accelerated the G1/S phase transition. Additionally, data from ChIP-seq pointed out that some NRF1 target genes are involved in the cell cycle. Our findings indicated that NRF1 directly binds to the E2F1 promoter as a transcription factor and regulates its gene expression.ConclusionTherefore, this study revealed that NRF1 promotes cancer cell growth via the indirect transcriptional activation of E2F1 and is a potential biomarker in LIHC.

Nuclear respiratory factor 1 (NRF-1) upregulates the expression and function of reduced folate carrier (RFC) at the blood-brain barrier.

Folates are important for neurodevelopment and cognitive function. Folate transport across biological membranes is mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate transport primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems results in suboptimal folate levels in the cerebrospinal fluid (CSF) causing childhood neurological disorders. Our group has reported that upregulation of RFC at the blood-brain barrier (BBB) through interactions with specific transcription factors, that is, vitamin D receptor (VDR) could increase brain folate delivery. This study investigates the role of nuclear respiratory factor 1 (NRF-1) in the regulation of RFC at the BBB. Activation of NRF-1/PGC-1α signaling through treatment with its specific ligand, pyrroloquinoline quinone (PQQ), significantly induced RFC expression and transport activity in hCMEC/D3 cells. In contrast, transfection with NRF-1 or PGC-1α targeting siRNA downregulated RFC functional expression in the same cell system. Applying chromatin immunoprecipitation (ChIP) assay, we further demonstrated that PQQ treatment increased NRF-1 binding to putative NRF-1 binding sites within the SLC19A1 promoter, which encodes for RFC. Additionally, in vivo treatment of wild type mice with PQQ-induced RFC expression in isolated mouse brain capillaries. Together, these findings demonstrate that NRF-1/PGC-1α activation by PQQ upregulates RFC functional expression at the BBB and could potentially enhance brain folate uptake.

Integrated Chip-Seq and RNA-Seq Data Analysis Coupled with Bioinformatics Approaches to Investigate Regulatory Landscape of Transcription Modulators in Breast Cancer Cells.

The objective of this chapter is to describe step-by-step bioinformatics and functional genomics solutions for analyzing ChIP-Seq and RNA-Seq data for understanding the regulatory mechanisms of chromatin modifiers and transcription factors that can drive pathogenesis of chronic complex human diseases, such as cancer. Here we have used two transcription regulatory proteins: nuclear respiratory factor 1 (NRF1) and inhibitor of differentiation protein 3 (ID3) for ChIP-Seq and RNA-Seq data as examples for discussing the importance of selecting the appropriate computational analysis methods, software, and parameters for the processing of raw data as well as their integrative regulatory landscape analysis to obtain accurate and reliable results. Both ChIP-Seq and RNA-Seq analytic methodologies are used as instructional examples to identify NRF1 or ID3 binding to the promoters and enhancers in the genome and their effects on the activity as well as to discover target genes that can drive breast cancer.

Positive regulation of human PINK1 and Parkin gene expression by nuclear respiratory factor 1

Previous studies have demonstrated that two Parkinson'sdisease-associatedgenes PINK1 and Parkin play a key role in mitochondrial quality control. But until now, the transcriptional regulation of these two genes under normal physiological conditions are not well understood. In this study, the transcriptional regulation of PINK1 and Parkin genes by nuclear respiratory factor 1 (NRF-1) and its effect on PINK1/Parkin-mediated mitophagy were studied. The NRF-1 binding sites in the promoter regions of human PINK1 and Parkin genes were analyzed by JASPER software and were confirmed by chromatin immunoprecipitation (ChIP) assay. The transcriptional activities and the expressions of PINK1 and Parkin genes were positively regulated by NRF-1 in HEK293T cells and in SH-SY5Y cells. Furthermore, NRF-1 over-expression (OE) up-regulated the protein level of full-length PINK1 in CCCP-treated cells, indicating the enhanced PINK1/Parkin-mediated mitophagy. When NRF-1 expression was transient orstableknockdown, the CCCP-induced mitophagy was alleviated as characterized by the reduced protein level of full-length PINK1, the declined ratio of LC3 II to LC3 I, and the decreasedratioof Mt-keimafluorescenceintensityexcited at 552 nm to that excited at 488 nm. In conclusion, NRF-1 has a positive regulatory effect on the transcription of PINK1 and Parkin genes, and involves in mitochondrial quality control through regulating PINK1/Parkin-mediated mitophagy.

Poly(ADP-ribose) polymerase-1 inhibits mitochondrial respiration by suppressing PGC-1α activity in neurons

Poly(ADP-ribose) polymerase-1 (PARP1) is a ubiquitous nuclear enzyme that regulates DNA repair and genomic stability. In oxidative genotoxic conditions, PARP1 activity is enhanced significantly, leading to excessive depletion of nicotinamide adenine dinucleotide (NAD+) and mitochondrial dysfunction. We hypothesized that PARP1-induced NAD+ depletion inhibits NAD+-dependent sirtuin deacetylase activity, thereby interfering with the mitochondrial regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). The DNA alkylator, N′-Nitro-N-nitroso-N-methylguanidine (MNNG), induced NAD+ depletion, inhibited sirtuin deacetylase activity and enhanced acetylation of PGC-1α. This was associated with reduced interaction between PGC-1α and nuclear respiratory factor 1 (NRF-1), which is a nuclear transcription factor that drives mitochondrial replication by regulating mitochondrial transcription factor A (TFAM). MNNG also reduced binding of NRF-1 to the tfam upstream promoter region and reduced TFAM mRNA, mitochondrial DNA copy number and respiratory function. MNNG effects were mitigated by PARP1 inhibition and genetic loss of function, by enhancing intracellular NAD+ levels, and with sirtuin (SIRT1) gain of function, supporting a mechanism dependent on PARP1 activity, NAD+-depletion and SIRT1 inhibition. This and other work from our group supports a destructive sequelae of events related to PARP1-induced sirtuin inhibition and sirtuin-mediated regulation of transcription.

Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding.

Chromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats – dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA.

Resveratrol Promotes Mitochondrial Biogenesis and Protects against Seizure-Induced Neuronal Cell Damage in the Hippocampus Following Status Epilepticus by Activation of the PGC-1α Signaling Pathway.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is known to regulate mitochondrial biogenesis. Resveratrol is present in a variety of plants, including the skin of grapes, blueberries, raspberries, mulberries, and peanuts. It has been shown to offer protective effects against a number of cardiovascular and neurodegenerative diseases, stroke, and epilepsy. This study examined the neuroprotective effect of resveratrol on mitochondrial biogenesis in the hippocampus following experimental status epilepticus. Kainic acid was microinjected into left hippocampal CA3 in Sprague Dawley rats to induce bilateral prolonged seizure activity. PGC-1α expression and related mitochondrial biogenesis were investigated. Amounts of nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (Tfam), cytochrome c oxidase 1 (COX1), and mitochondrial DNA (mtDNA) were measured to evaluate the extent of mitochondrial biogenesis. Increased PGC-1α and mitochondrial biogenesis machinery after prolonged seizure were found in CA3. Resveratrol increased expression of PGC-1α, NRF1, and Tfam, NRF1 binding activity, COX1 level, and mtDNA amount. In addition, resveratrol reduced activated caspase-3 activity and attenuated neuronal cell damage in the hippocampus following status epilepticus. These results suggest that resveratrol plays a pivotal role in the mitochondrial biogenesis machinery that may provide a protective mechanism counteracting seizure-induced neuronal damage by activation of the PGC-1α signaling pathway.

Nuclear Respiratory Factor 1 Negatively Regulates the P1 Promoter of the Peroxisome Proliferator-Activated Receptor-γ Gene and Inhibits Chicken Adipogenesis.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipogenesis, and alterations in its function are associated with various pathological processes related to metabolic syndrome. Recently, we found that the chicken PPARγ gene is regulated by three alternative promoters (P1, P2 and P3), producing five different transcript isoforms and two protein isoforms. In this study, the P1 promoter structure was characterized. Bioinformatics identified six putative nuclear respiratory factor 1 (NRF1) binding sites in the P1 promoter, and a reporter assay showed that NRF1 inhibited the activity of the P1 promoter. Of the six putative NRF1 binding sites, individual mutations of three of them abolished the inhibitory effect of NRF1 on P1 promoter activity. Furthermore, a ChIP assay indicated that NRF1 directly bound to the P1 promoter, and real-time quantitative RT-PCR analysis showed that NRF1 mRNA expression was negatively correlated with PPARγ1 expression (Pearson’s r = -0.148, p = 0.033). Further study showed that NRF1 overexpression inhibited the differentiation of the immortalized chicken preadipocyte cell line (ICP1), which was accompanied by reduced PPARγ1 mRNA expression. Taken together, our findings indicated that NRF1 directly negatively regulates the P1 promoter of the chicken PPARγ gene and inhibits adipogenesis.

Nuclear Respiratory Factor 1 (NRF-1) Controls the Activity Dependent Transcription of the GABA-A Receptor Beta 1 Subunit Gene in Neurons.

While the exact role of β1 subunit-containing GABA-A receptors (GABARs) in brain function is not well understood, altered expression of the β1 subunit gene (GABRB1) is associated with neurological and neuropsychiatric disorders. In particular, down-regulation of β1 subunit levels is observed in brains of patients with epilepsy, autism, bipolar disorder and schizophrenia. A pathophysiological feature of these disease states is imbalance in energy metabolism and mitochondrial dysfunction. The transcription factor, nuclear respiratory factor 1 (NRF-1), has been shown to be a key mediator of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Using a variety of molecular approaches (including mobility shift, promoter/reporter assays, and overexpression of dominant negative NRF-1), we now report that NRF-1 regulates transcription of GABRB1 and that its core promoter contains a conserved canonical NRF-1 element responsible for sequence specific binding and transcriptional activation. Our identification of GABRB1 as a new target for NRF-1 in neurons suggests that genes coding for inhibitory neurotransmission may be coupled to cellular metabolism. This is especially meaningful as binding of NRF-1 to its element is sensitive to the kind of epigenetic changes that occur in multiple disorders associated with altered brain inhibition.

Interplay between NRF1, E2F4 and MYC transcription factors regulating common target genes contributes to cancer development and progression

Nuclear respiratory factor 1 (NRF1), historically perceived as a protein regulating genes controlling mitochondrial biogenesis, is now widely recognized as a multifunctional protein and as a key player in the transcriptional modulation of genes implicated in various cellular functions. Here, we present emerging data supporting novel roles of NRF1 in cancer development and progression through its interplay with the transcription factors E2F4 and MYC. To identify common human NRF1, E2F4 and MYC target genes, we analyzed the Encyclopedia of DNA Elements (ENCODE) NRF1 ChIP-Seq data. By doing so, we identified 9253 common target genes with NRF1, E2F4 and MYC binding motifs. NRF1 binding motifs were found to be present in genes operating in signaling pathways governing all hallmarks of malignant transformation and progression, including proliferation, invasion, self-renewal and apoptosis. In addition to controlling mitochondrial biogenesis NRF1, in conjunction with E2F4 and MYC, may play a critical role in the acquisition of human cancer characteristics. Additionally, NRF1 may orchestrate both MYC and E2F4 to regulate common target genes linked to multiple networks in the development and progression of cancer. A comprehensive understanding of this dynamic interplay will set the stage, not only for the design of novel treatment strategies, but also for the discovery of pan-cellular transcription factor regulatory strategies to predict cancer risk, therapy response and patient prognosis.

Abstract 1552: Integrative bioinformatics analyses of ChIP seq and RNA seq data reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer

Abstract The expression of redox sensitive transcription factor -nuclear respiratory factor 1 (NRF1) expression significantly correlates with histological grades and prognosis of breast cancer. However, the molecular mechanism by which NRF1 may contribute in the development of breast cancer is not clear. In this study we examined whether NRF1 is a molecular risk factor of breast cancer. We also identified regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Steps followed for the investigation included the selection of mRNA microarray dataset from TCGA and Metabric, selection and analysis of ChIP-Seq datasets from different breast cancer cell lines to identify NRF1 target genes possibly involved in breast cancer through Molecular Pathway, Gene Ontology and statistical analysis. Analysis of mRNA expression in breast cancer patients showed that NRF1 was significantly overexpressed in breast cancer tissues compared to normal tissues. ChIP-Seq analysis of two different breast cancer cell line - MCF7 and T7D showed that there are more than 8,000 genes, which contain NRF1 binding DNA motif(s). The NRF1 binding to ChIP DNA sequences of target gene is cell context dependent. High percentage of known breast cancer (~50%) susceptibility genes are transcriptional targets of NRF1. Network analysis revealed that NRF1 regulates genes, which are key regulators of epithelial-mesenchymal transition (EMT), stemness, cell apoptosis, cell cycle regulation, chromosomal integrity, and DNA damage and repair. Furthermore, we observed that NRF1 regulates target genes of both breast cancer and cancer KEGG pathways. Many of the MicroRNAs involved in cancer and genes of signaling pathways involved in cancer initiation and progression such as MAPK, PI3K/AKT and Notch signaling are also transcriptional targets of NRF1. In summary, this study reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Dysregulation of NRF1 signaling pathways may contribute in the development of breast cancer. Clinical confirmation of our study will have significant impact on our understanding of the role of NRF1 as a valuable biomarker for breast cancer diagnosis and prognosis and will provide strong rationale for the future studies to further develop NRF1 for breast cancer therapeutic target. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jairo Ramos, Deodutta Roy. Integrative bioinformatics analyses of ChIP seq and RNA seq data reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1552. doi:10.1158/1538-7445.AM2017-1552

Mitochondrial quality-control dysregulation in conditional HO-1-/- mice.

The heme oxygenase-1 (Hmox1; HO-1) pathway was tested for defense of mitochondrial quality control in cardiomyocyte-specific Hmox1 KO mice (HO-1[CM]-/-) exposed to oxidative stress (100% O2). After 48 hours of exposure, these mice showed persistent cardiac inflammation and oxidative tissue damage that caused sarcomeric disruption, cardiomyocyte death, left ventricular dysfunction, and cardiomyopathy, while control hearts showed minimal damage. After hyperoxia, HO-1(CM)-/- hearts showed suppression of the Pgc-1α/nuclear respiratory factor-1 (NRF-1) axis, swelling, low electron density mitochondria by electron microscopy (EM), increased cell death, and extensive collagen deposition. The damage mechanism involves structurally deficient autophagy/mitophagy, impaired LC3II processing, and failure to upregulate Pink1- and Park2-mediated mitophagy. The mitophagy pathway was suppressed through loss of NRF-1 binding to proximal promoter sites on both genes. These results indicate that cardiac Hmox1 induction not only prevents heme toxicity, but also regulates the timing and registration of genetic programs for mitochondrial quality control that limit cell death, pathological remodeling, and cardiac fibrosis.

Nuclear respiratory factor-1 (NRF-1) regulated hypoxia-inducible factor-1α (HIF-1α) under hypoxia in HEK293T.

Hypoxia-inducible factor 1α (HIF-1α) is a master regulator of oxygen homeostasis. Under hypoxia, the active HIF1-α subunits are mainly regulated through increased protein stabilization. Little is known concerning HIF-1α transcriptional regulation. Nuclear respiratory factor 1 (NRF-1) is a DNA-binding transcription factor that regulates mitochondrial biogenesis. In this study, we showed that NRF-1was a repressor of HIF-1α. The cellular depletion of NRF-1 by siRNA targeting leads to increased HIF-1αtranscriptional activity. EMSA, ChIP and luciferase activity allowed the identification of two functional NRF-1 binding sites within HIF-1α promoter. This study therefore identifies NRF-1 as a novel regulator of HIF-1α. © 2016 IUBMB Life, 68(9):748-755, 2016.

Evaluating the role of NRF-1 in the regulation of the goldfish COX4-1 gene in response to temperature.

Cold acclimation in fish typically increases muscle mitochondrial enzymes. In mammals, stressors that increase mitochondrial content are mediated though transcriptional regulators, including nuclear respiratory factor-1 (NRF-1). Focusing on the goldfish gene for cytochrome c oxidase (COX) subunit 4-1, we analysed the regulatory regions in various contexts to identify a mechanistic link between NRF-1 and cold-induced mitochondrial proliferation. Promoter analysis implicated two putative NRF-1 sites: one in the proximal promoter and a second in exon 1, which encodes the 5' untranslated region (5'-UTR). Transfection into mouse myoblasts showed that deletion of a region that included the proximal NRF-1 site reduced promoter activity by 30%; however, mutagenesis of the specific sequence had no effect. Thermal sensitivity analyses performed in rainbow trout gonadal fibroblasts (RTG-2) showed no effect of temperature (4 vs 19°C) on reporter gene expression. Likewise, reporters injected into muscle of thermally acclimated goldfish (4 vs 26°C) showed no elevation in expression. There was no difference in thermal responses of COX4-1 promoter reporters constructed from homologous regions of eurythermal goldfish and stenothermal zebrafish genes. NRF-1 chromatin immunoprecipitation of thermally acclimated goldfish muscle showed no temperature effect on NRF-1 binding to either the proximal promoter or 5'-UTR. It remains possible that the cold-induced upregulation of COX4-1 expression is a result of NRF-1 binding to distal regulatory regions or through indirect effects on other transcription factors. However, the proximal promoter does not appear to play a role in mediating the thermal response of the COX4-1 gene in fish.

Inhibition of the Histone Demethylase LSD1 Combined with Caloric Restriction or IGF1/Insulin Inhibition Leads to Durable Responses in a Preclinical Model of Acute Myeloid Leukemia

Introduction. There is increasing interest in therapeutic modulation of metabolic pathways in cancer. Tumor cells preferentially use aerobic glycolysis to meet their energetic demands. However, glycolysis inhibition alone is unable to bring durable responses because of limited therapeutic index and because of previously underappreciated metabolic adaptability in tumor cells, which can switch to alternative substrate usage when specific nutrients are limiting. The molecular basis of metabolic adaptation is poorly understood. Recently, the histone demethylase LSD1 (Lysine-Specific Demethylase 1) has been implicated in the control of oxidative phosphorylation (OXPHOS) in adipocytes through its interaction with NRF1 (Nuclear Respiratory Factor 1), a master regulator of metabolic gene transcription (1). We hypothesized that LSD1 could regulate metabolic adaptability and be a therapeutic target upon metabolic modulation through Caloric Restriction (CR) in Acute Myeloid Leukaemia (AML) and specifically in APL (Acute Promyelocytic Leukaemia), which we showed to be sensitive to body fatness in the clinic (2).Methods. APLs were generated in mice expressing the PML-RARa fusion under the control of the Cathepsin G promoter (3). Primary leukemias were transplanted into recipients subjected to 30% CR or Standard Diet (SD). We scored the effect of CR alone or in combination with the LSD1 inhibitor IEO368 (4) on mouse survival, Leukemia Initiating Cell (LIC) frequency and epigenomic, transcriptomic and metabolic parameters.Results. Compared to SD controls, CR-fed recipients experienced an initial dramatic decrease in the total leukemic burden accompanied by cell cycle slowdown (“adaptation phase”); this was followed by a delayed disease progression that brought animals to death (“terminal phase”) (median survival 91 vs 51 days, p=0.038). Limiting-dilution transplantation of CR-conditioned leukemias revealed increased frequency of LICs (estimated frequency 1/3064 cells in SD vs 1/947 in CR, p=0.003) and increased aggressiveness (median survival reduced to 49 vs 70.5 days with 5000 cells injected, p<0.0001). Thus, CR limits the expansion of leukemic cells but enriches for cells with increased ability to regrow.RNAseq of leukemic cells purified during the terminal phase (but not earlier) showed that a dramatic transcriptional reprogramming in CR, characterized by upregulation of genes controlling OXPHOS, Krebs cycle and nucleotide and protein biosynthesis, and downregulation of insulin signaling and glucose transporters. Flow cytometry with Mitotracker Red confirmed increased mitochondrial activity. Thus, leukemic cells exposed to CR put in place adaptive transcriptional changes to allow survival in a nutrient/growth factor deprived environment.To investigate the basis of these transcriptional changes, we revised ChIPseq analysis of LSD1 binding in human APL cell lines and found a significant enrichment for i) NRF1 consensus binding motif and ii) promoters of genes encoding for OXPHOS and Krebs cycle enzymes. NRF1 binding to OXPHOS/Krebs enzymes was confirmed on mouse leukemias by ChIPseq. These data suggested that the CR-induced adaptive changes could be mediated by LSD1/NRF1.Strikingly, co-treatment of leukemic mice with CR and our LSD1 inhibitor IEO368 (4) resulted in macroscopic and microscopic eradication of disease (see figure, p=0.0018 compared to SD). In these conditions, leukemic cells completely disappeared in 4/6 mice after 4 weeks. LSD1 inhibition alone was also effective but did not produce bona fide disease eradication. Importantly, some of the features of the CR-LSD1 interaction could be modeled by combining LSD1 and an IGF1/Insulin inhibitor. In vivo, this combination was synergistic and led to durable responses (median survival 121 vs 50 days in untreated controls, p=0.0143, vs 65.5 and 78.5 days with Insulin/IGF1 Inhibitor and IEO368 respectively).Conclusion: the combination of LSD1 inhibition and insulin/IGF1 signaling reduction by pharmacological or dietary intervention appears as a highly effective therapeutic strategy and deserves further investigation. Ongoing preclinical studies will verify its applicability to other models of AML.

Sensing and responding to energetic stress: The role of the AMPK-PGC1α-NRF1 axis in control of mitochondrial biogenesis in fish

Remodeling the muscle metabolic machinery in mammals in response to energetic challenges depends on the energy sensor AMP-activated protein kinase (AMPK) and its ability to phosphorylate PPAR γ coactivator 1 α (PGC1α), which in turn coactivates metabolic genes through direct and indirect association with DNA-binding proteins such as the nuclear respiratory factor 1 (NRF1) (Wu et al., 1999). The integrity of this axis in fish is uncertain because PGC1α i) lacks the critical Thr177 targeted by AMPK and ii) has mutations that may preclude binding NRF1. In this study we found no evidence that AMPK regulates mitochondrial gene expression through PGC1α in zebrafish and goldfish. AICAR treatment of zebrafish blastula cells increased phosphorylation of AMPK and led to changes in transcript levels of the AMPK targets mTOR and hexokinase 2. However, we saw no increases in mRNA levels for genes associated with mitochondrial biogenesis, including PGC1α, NRF1, and COX7C, a cytochrome c oxidase subunit. Further, AMPK phosphorylated mammalian peptides of PGC1α but not the corresponding region of zebrafish or goldfish in vitro. In vivo cold acclimation of goldfish caused an increase in mitochondrial enzymes, AMP and ADP levels, however AMPK phosphorylation decreased. In fish, the NRF1-PGC1α axis may be disrupted due to insertions in fish PGC1α orthologs within the region that serves as NRF1 binding domain in mammals. Immunocopurification showed that recombinant NRF1 protein binds mammalian but not fish PGC1α. Collectively, our studies suggest that fish have a disruption in the AMPK-PGC1α-NRF1 pathway due to structural differences between fish and mammalian PGC1α.

Abstract 4559: Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor

Abstract Although several nuclear regulatory proteins may be targeted by estrogen, our efforts have focused on the redox nuclear respiratory factor-1 (NRF-1), because our recent study showed that NRF-1 gene expression significantly increases with the progression of breast tumor grades. NRF-1 overexpression supported in vitro tumor formation. To determine if NRF-1 is required for 17 beta estradiol (E2)-induced neoplastic phenotype, we have generated NRF-1 mutants by site directed mutagenesis. We have generated stable clones that express NRF-1 mutant. The in vitro tumor formation was detected by anchorage independent growth and 3D spheroid assays. NRF-1 overexpression enhanced in vitro tumor spheroid formation, cell migration and cell invasion. Our flow cytometry analysis showed that overexpression of wild-type NRF-1 increased the percent of transformed MCF-10A cells in S phase compared to vector alone. Dominant negative NRF-1 protein showed reduction in the tumor formation. In addition, shRNA targeting NRF-1 resulted in the inhibition of anchorage independent growth of MCF-10A cells in both vehicle control and E2-treated cells. Since NRF-1 is a substrate of the kinase AKT, we determined whether NRF-1 phosphorylation was increased in MCF-10A cells after treatment with a carcinogenic regimen of E2. We observed more than a 2-fold increase in phospho-NRF-1 in E2 treated (100pg/ml for 30 min) MCF-10A cells and phosphorylation of NRF-1 was inhibited by co-treatment with either biological (CAT or MnSOD) or chemical (20μM ebselen) ROS modifiers. These results suggest that E2-induced phosphorylation of NRF-1 is influenced by ROS signaling messengers. We found that E2 treated MCF-10A cells showed increased NRF-1 binding to the promoters of Cdc2, PRC1, PCNA, Cyclin B1, and CDC25C genes. NRF-1 binding induced by E2 treatment was inhibited by the overexpression of CAT and MnSOD. NRF-1 phosphorylation site specific to kinase AKT, Thr-109, was mutated to Asp (D) or Ala (A), and the NRF-1 acetylation site specific to acetyl-transferase PCAF, Lys- 89, was mutated to Gln (glutamine)-Q or Ala (A). We examine the effect of one of the NRF-1 mutants on E2-induced in vitro tumor formation. We observed that the expression of NRF-1 (T109A) phosphodeficient mutant (NRF-1PMT) significantly inhibited E2-induced cell transformation, whereas empty vector did not influence E2-induced colony formation. Together these findings support the idea that NRF-1 may play an important role in E2-induced malignant transformation of breast epithelial cells. Thus, our data is consistent with the hypothesis that in addition to the estrogen receptor activity, NRF-1 activation contributes to the susceptibility to develop malignant phenotype in response to exposure to estrogen. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR Citation Format: Lazaro Mesa, Jayanta Das, Alok Deoraj, Victor Okoh, Deodutta Roy. Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2015-4559

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.