MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells
2020; Elsevier BV; Volume: 88; Linguagem: Inglês
10.1016/j.exphem.2020.07.004
ISSN1873-2399
AutoresStefania Bottardi, Romain Guièze, Vincent Bourgoin, Nasser Fotouhi‐Ardakani, Aurore Dougé, Anaïs Darracq, Yahia A. Lakehal, Marc G. Berger, Luigina Mollica, Jacques‐Olivier Bay, James G. Omichinski, Éric Milot,
Tópico(s)Acute Myeloid Leukemia Research
Resumo•MNDA downregulates MCL-1 and BCL-2 in lymphoid cells.•MNDA is associated with (pre)mRNA.•MNDA is recruited to gene regulatory regions. The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course. The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course. Myeloid nuclear differentiation antigen (MNDA) is a member of the hematopoietic interferon (IFN)-inducible nuclear proteins with a 200–amino-acid repeat (IFI200/HIN-200) family. 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Likewise, the MNDA-mediated apoptosis is affected when proteasome activity is blocked with MG132 [18Fotouhi-Ardakani N Kebir DE Pierre-Charles N et al.Role for myeloid nuclear differentiation antigen in the regulation of neutrophil apoptosis during sepsis.Am J Respir Crit Care Med. 2010; 182: 341-350Crossref PubMed Scopus (30) Google Scholar,19Milot E Fotouhi-Ardakani N Filep JG Myeloid nuclear differentiation antigen, neutrophil apoptosis and sepsis.Front Immunol. 2012; 3: 397Crossref PubMed Scopus (23) Google Scholar]. In support of these observations, it has been found that in neutrophils from patients with severe sepsis, cytoplasmic relocalization of MNDA is impaired, MCL-1 accumulates, and apoptosis is delayed. MNDA is also expressed in B lymphocytes, lymphoid cell lines, specific types of lymphoma, as well as chronic lymphocytic leukemia (CLL) cells [20Geng Y Choubey D. 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Production and stability of mRNAs depend on a number of intertwined processes such as transcriptional and elongation rates, pre-mRNA modifications, alternative splicing, epi-transcriptomic decoration, nuclear cytoplasm export, and activation of decay pathways, which can result in degradation or translational blocking. Throughout these different steps, the RNA molecules physically interact with a plethora of RNA-binding proteins. Collectively, these processes, which are responsible for RNA biology, stability, and degradation, are accountable for the dynamic concentration of RNA molecules in the cell [42Harries LW. RNA biology provides new therapeutic targets for human disease.Front Genet. 2019; 10: 205Crossref PubMed Scopus (15) Google Scholar]. We investigated whether MNDA can influence apoptosis by controlling the expression of the anti-apoptotic factors MCL-1 and BCL-2 in CLL cells. The prognostic value of MNDA expression was addressed by the analysis of human CLL samples. We found an inverse correlation between the expression levels of MNDA and those of MCL-1 and BCL-2 in the majority of the CLL samples tested. Higher MNDA levels characterized Binet A samples, whereas lower MNDA levels were observed in CLL samples with the high-risk cytogenetic abnormality del (11q) or del (17p). In accordance with its nuclear localization in CLL cells, our results revealed that MNDA is recruited at these genes, associates with Mcl1 and Bcl2 (pre-)mRNAs, and favors their rapid decay following genotoxic stress. Altogether, these results provide a molecular mechanism to explain the inverse correlation between the expression levels of MNDA and MCL-1 or BCL-2 and, thereby, the prognostic significance of MNDA expression in CLL. Peripheral blood lymphocytes from 62 CLL patients were processed with written informed consent, collected, and then cryopreserved by the CRB-Auvergne (certified according to NF 96–900 standard) as biobank of the Clermont-Ferrand University Hospital. The study was approved by the Ethical Commission of the University Hospital of Clermont-Ferrand and performed in accordance with the Declaration of Helsinki. Cells were stained with CD19 and CD5 antibodies to identify clonally amplified CLL clones. When required, CLL cells were isolated by flow cytometry using CD19 antibodies. Guidelines for clinical criteria were as reported in Halleck et al. [43Hallek M Cheson BD Catovsky D et al.iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL.Blood. 2018; 131: 2745-2760Crossref PubMed Scopus (551) Google Scholar]. Karyotype complexity refers to the presence of three or more chromosomal abnormalities on tumour cell karyotype. Cytogenetic subgroups were identified according to a hierarchical classification, whereby 1 = 17p deletion, 2 = 11q deletion, 3 = trisomy 12, 4 = normal, and 5 = 13q deletion [26Dohner H Stilgenbauer S Benner A et al.Genomic aberrations and survival in chronic lymphocytic leukemia.N Engl J Med. 2000; 343: 1910-1916Crossref PubMed Scopus (2629) Google Scholar]. For the Binet system of classification, patients were grouped into three categories: A, B, and C. Stages A and B both have a hemoglobin count ≥10 g/dL and platelet count ≥100/L, but differ in characterization of lymphadenopathy; stage A has fewer than three enlarged areas, whereas stage B has three or more enlarged areas. Stage C is characterized by a hemoglobin count <10 g/dL and platelet count of <100,000/mm3 irrespective of lymphadenopathy. The median survival for stage A is 8.5–9 years, for stage B 5–6 years, and for stage C 1.5 years [44Binet JL Auquier A Dighiero G et al.A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis.Cancer. 1981; 48: 198-206Crossref PubMed Scopus (1446) Google Scholar]. The human Burkitt's lymphoma-derived B-cell line Namalwa and the promyeloblast cell line HL-60 were purchased from the American Type Culture Collection (Rockville, MD; ATCC CRL-1432, ATCC CCL-240) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C. Flag-HA epitope-tagged MNDA (FH-MNDA)–expressing Namalwa cells were obtained using the MMLV-based pOZ-FH-N retroviral expression plasmid, which allows expression of double epitope (Flag-HA)–tagged MNDA protein, according to the protocol described in Nakatani et al. [45Nakatani Y Ogryzko V. Immunoaffinity purification of mammalian protein complexes.Methods Enzymol. 2003; 370: 430-444Crossref PubMed Scopus (225) Google Scholar]. Chromatin immunoprecipitation (ChIP) assays were carried out as reported in Bottardi et al. [46Bottardi S Ross J Bourgoin V et al.Ikaros and GATA-1 combinatorial effect is required for silencing of human gamma-globin genes.Mol Cell Biol. 2009; 29: 1526-1537Crossref PubMed Scopus (49) Google Scholar] and according to the manufacturer's instructions (MilliporeSigma, Burlington, MA), with minor modifications. Cells (106 cells per antibody condition) were fixed with 1% formaldehyde (HCHO) for 10 min at 37°C. Reactions were quenched by the addition of ice-cold glycine (20 mmol/L final). Chromatin was reduced in size by sonication to obtain fragments of 400 to 600 bp. About 1/30th of immunoprecipitated and unbound (input) material was used as a template for quantitative polymerase chain reaction (qPCR) with SYBR green (Bimake) on an ABI 7300 Fast real-time PCR system (Applied Biosystems, Foster City, CA). Quantification was carried out according to the "percentage input method" (https://www.thermofisher.com/ca/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html) or the "2–ΔΔCt method" for ChIP with anti-HA antibodies. The HA tag antibody is a highly specific ChIP-grade antibody previously used in ChIP-seq experiments [47Liu N Song J Xie Y et al.Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis.Proc Natl Acad Sci USA. 2019; 116: 890-899Crossref PubMed Scopus (7) Google Scholar]. All data shown are the results of at least four independent ChIP experiments with qPCR from each ChIP performed in triplicate and averaged (standard deviation). To correctly interpret amplification values obtained by qPCR, the efficiency of all primer sets was carefully checked, and primer pairs displaying an amplification efficiency ranging from 95% to 102% were chosen. Antibodies and primer sets are listed in the Supplementary Data (online only, available at www.exphem.org). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays were carried out as reported in Bottardi et al. [46Bottardi S Ross J Bourgoin V et al.Ikaros and GATA-1 combinatorial effect is required for silencing of human gamma-globin genes.Mol Cell Biol. 2009; 29: 1526-1537Crossref PubMed Scopus (49) Google Scholar] with minor modifications. Total RNA was extracted with RNazol (MilliporeSigma) and treated with DNase I (Thermo Fisher, Waltham, MA). RNA was retrotranscribed with the All-in-One cDNA Synthesis SuperMix (Bimake.com), and cDNA was used as a template for qPCR with SYBR green (Bimake) on an ABI 7300 fast real-time PCR system (Applied Biosystem). Quantification was carried out with the Pfaffl equation [48Pfaffl MW. A new mathematical model for relative quantification in real-time QRT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (23987) Google Scholar]R=(Etarget)ΔCPtarget(control−sample)(Eref)ΔCPref(control−sample)where Etarget = the efficiency of the gene of interest; Eref = the efficiency of the control gene; and CP = the crossing point. For most of the experiments, "control" refers to mock or nontreated cells, and "sample" to MNDA or treated cells. All data shown are the results of at least four independent experiments with qPCR from each experiment performed in triplicate and averaged (standard deviation). Primer sets are listed in the Supplementary Data. RNA stability was assessed by studying the mRNA degradation rate in actinomycin D-treated cells or by metabolic labeling and purification of nascent RNA. For actinomycin D treatment, Mcl1, Bcl2, and p16 mRNA decay was measured following inhibition of de novo transcription by actinomycin D. Namalwa cells were treated with 10 μg/mL cisplatin together with 10 μg/mL actinomycin D for 1, 4, or 8 hours. RNA isolation, and qPCR reactions were carried out as described in the previous paragraph. Metabolic labeling and purification of nascent RNA were carried out with the Click-iT RNA capture kit (Thermo Fisher), according to the manufacturer's protocol. Briefly, cells were cultured with 2 mmol/L 5-ethynyl uridine (EU) for 18 hours. Then, EU was washed out with RPMI complete medium, and cells were incubated with 10 μg/mL cisplatin or 0.9% NaCl (diluent control) for 4 hours. After RNazol (MilliporeSigma) extraction, 1 μg of RNA was biotinylated and precipitated. Biotinylated RNA (500 ng per condition) was collected by streptavidin T1 magnetic bead purification, retrotranscribed, and subject to real-time quantitative PCR as described in the previous paragraphs. In general, 5 million cells were suspended in 150 μL of Laemmli buffer and sonicated. Then, 10 to 15 μL of protein extract was loaded onto 10% or 15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein quantification was performed with the MultiGauge software. Cells were stained as reported in Fotouhi-Ardakani et al. [18Fotouhi-Ardakani N Kebir DE Pierre-Charles N et al.Role for myeloid nuclear differentiation antigen in the regulation of neutrophil apoptosis during sepsis.Am J Respir Crit Care Med. 2010; 182: 341-350Crossref PubMed Scopus (30) Google Scholar]. Cells were layered on polylysine-treated glass slides and fixed in 4% paraformaldehyde for 30 min at room temperature. Slides were then washed twice in phosphate-buffered solution (PBS) and blocked with 5% non-fat dry milk after a permeabilization step with 0.5% Triton/PBS. Primary (goat anti-MNDA) as well as secondary (rabbit anti-goat fluorescein isothiocyanate [FITC]-conjugated) antibodies were diluted in PBS containing 0.1% bovine serum albumin (BSA) and 0.01% Triton. Cells were counterstained with 4′,6-diaminido-2-phenylindole (DAPI). Images were acquired using a DeltaVision Elite imaging system (GE Healthcare, Chicago, IL) equipped with 100× objective lens (Olympus IX71, Tokyo, Japan) and a 15-bit EDGE/sCMOS camera (PCO). Images were deconvoluted at 200 nm using the SoftWoRx (version 6.2.0) software. Apoptosis was assessed with flow cytometry (fluorescence-activated cell sorting [FACS]) using FITC-conjugated Annexin V in combination with propidium iodide (PI). One million cells were washed once in PBS and resuspended in HEPES buffer (10 mmol/L HEPES, 135 mmol/L NaCl, 5 mmol/L CaCl2). FITC-conjugated Annexin V antibodies were then added, and cells were incubated for 15 min at 37°C. PI solution (1 μg/mL final) was added just before FACS analysis. Mitochondrial transmembrane potential was monitored by FACS with CMXRos at a final concentration of 200 nmol/L, freshly prepared from 1 mmol/L stock solution in DMSO. Staining was performed per the manufacturer's instructions (https://www.thermofisher.com/order/catalogue/product/M7512#/M7512). For cell cycle analysis, 2 × 106 cells were washed with cold PBS, fixed with 75% cold ethanol, and incubated overnight at −20˚C. Cells were then washed with PBS and treated with 100 μg/mL RNase A (MilliporeSigma) for 20 min at 37˚C, and DNA was stained with 50 μg/mL PI. FACS assays were carried out on a FACSCalibur Cell Analyser (BD Biosciences, La Jolla, CA) with CellQuestPro software (BD Biosciences). Namalwa or HL-60 cells (30 × 106) were fixed with 0.3% formaldehyde (ultrapure, MilliporeSigma) for 10 min and then quenched with 125 mmol/L glycine for 5 min at room temperature. Cell pellets were washed twice in PBS, resuspended in 150 μL of ice-cold 0.1× complete radio-immunoprecipitation assay (RIPA) lysis buffer (0.1× RIPA buffer [MilliporeSigma], 1 mmol/L dithiothreitol [DTT], 0.2 U/μL RNAseOUT [Thermo Fisher], 25 μl/mL Protease Inhibitor Cocktail [MilliporeSigma], 1 mM phenylmethylsulfonyl fluoride [PMSF], 140 mM NaCl), and placed on ice for 10 min. Pellets were sonicated and cellular disruption was assessed by microscopic observation. Cell lysates were centrifuged at 13,000 rpm for 30 min at 4°C; 10% of the sample was kept as input and 90% was used in RNA IP. Magnetic beads (Dynabeads, Thermo Fisher) and HA or MNDA antibodies were added to the lysates, and tubes were incubated overnight on an end-to-end rotator. RIP washes were performed as follows: three washes in 0.1× complete RIPA lysis buffer plus two additional washes in PBS containing protease inhibitors, 1 mmol/L DTT, and 0.2 U/μL RNAseOUT. RNA elution and protein digestion were carried out by adding 200 μL of elution buffer (200 mmol/L NaCl, 50 mmol/L Tris pH 8.0, 0.05% v/v Nonidet P-40, 1 mmol/L DTT, 0.1% SDS, 10 mmol/L EDTA, 0.2 U/μL RNaseOUT, and 30 μg proteinase K), with agitation, at 55°C for 30 min. To reverse the crosslink, tubes were heated at 65°C for 2 hours, and RNA was purified and DNaseI-digested using quick RNA mini-preps from Zymo Research (Irvine, CA). Purified RNA was retrotranscribed with an all-in-one cDNA synthesis kit (Bimake) and used as template for qRT-PCR analysis. Spearman's rank correlation coefficient (or Spearman's ρ), Pearson's correlation, and Student t test and distribution were used to determine correlations, dependence, and statistical significance. Survival curves were analyzed according to the Kaplan–Meier method using SPSS software. Protein structure was investigated via the PSI–blast-based secondary structure prediction (PSIPRED; Bioinformatics Group, Department of Computer Science, University College London, London, UK) method. Antibodies, reagents, and oligonucleotide sequences are detailed in the Supplementary Data. MNDA promotes apoptosis and participates in degradation of the anti-apoptotic factor MCL-1 in myeloid lineages [18Fotouhi-Ardakani N Kebir DE Pierre-Charles N et al.Role for myeloid nuclear different
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