Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.

Department of Proteomics Institute of Hematology and Blood Transfusion U Nemocnice 1 128 20 Prague Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 121 16 Prague Czech Republic

Zobrazit více v PubMed

Yang K., Yang J., Yi J. Nucleolar Stress: Hallmarks, sensing mechanism and diseases. Cell Stress. 2018;2:125–140. doi: 10.15698/cst2018.06.139. PubMed DOI PMC

Colombo E., Alcalay M., Pelicci P.G. Nucleophosmin and its complex network: A possible therapeutic target in hematological diseases. Oncogene. 2011;30:2595–2609. doi: 10.1038/onc.2010.646. PubMed DOI

Colombo E., Marine J.C., Danovi D., Falini B., Pelicci P.G. Nucleophosmin regulates the stability and transcriptional activity of p53. Nat. Cell Biol. 2002;4:529–533. doi: 10.1038/ncb814. PubMed DOI

Lambert B., Buckle M. Characterisation of the interface between nucleophosmin (NPM) and p53: Potential role in p53 stabilisation. FEBS Lett. 2006;580:345–350. doi: 10.1016/j.febslet.2005.12.025. PubMed DOI

Daniely Y., Dimitrova D.D., Borowiec J.A. Stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation. Mol. Cell. Biol. 2002;22:6014–6022. doi: 10.1128/MCB.22.16.6014-6022.2002. PubMed DOI PMC

Dhar S.K., St Clair D.K. Nucleophosmin blocks mitochondrial localization of p53 and apoptosis. J. Biol. Chem. 2009;284:16409–16418. doi: 10.1074/jbc.M109.005736. PubMed DOI PMC

Saxena A., Rorie C.J., Dimitrova D., Daniely Y., Borowiec J.A. Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization. Oncogene. 2006;25:7274–7288. doi: 10.1038/sj.onc.1209714. PubMed DOI

Matt S., Hofmann T.G. The DNA damage-induced cell death response: A roadmap to kill cancer cells. Cell Mol. Life Sci. 2016;73:2829–2850. doi: 10.1007/s00018-016-2130-4. PubMed DOI PMC

Lindstrom M.S. NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling. Biochem. Res. Int. 2011;2011:195209. doi: 10.1155/2011/195209. PubMed DOI PMC

Brodska B., Sasinkova M., Kuzelova K. Nucleophosmin in leukemia: Consequences of anchor loss. Int. J. Biochem. Cell Biol. 2019;111:52–62. doi: 10.1016/j.biocel.2019.04.007. PubMed DOI

Meani N., Alcalay M. Role of nucleophosmin in acute myeloid leukemia. Expert Rev. Anticancer Ther. 2009;9:1283–1294. doi: 10.1586/era.09.84. PubMed DOI

Federici L., Falini B. Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization. Protein Sci. 2013;22:545–556. doi: 10.1002/pro.2240. PubMed DOI PMC

Michael D., Oren M. The p53-Mdm2 module and the ubiquitin system. Semin. Cancer Biol. 2003;13:49–58. doi: 10.1016/S1044-579X(02)00099-8. PubMed DOI

Brooks C.L., Gu W. P53 Regulation by Ubiquitin. FEBS Lett. 2011;585:2803–2809. doi: 10.1016/j.febslet.2011.05.022. PubMed DOI PMC

Marine J.C., Lozano G. Mdm2-mediated ubiquitylation: p53 and beyond. Cell Death Differ. 2010;17:93–102. doi: 10.1038/cdd.2009.68. PubMed DOI

Gjerset R.A. DNA damage, p14ARF, nucleophosmin (NPM/B23), and cancer. J. Mol. Histol. 2006;37:239–251. doi: 10.1007/s10735-006-9040-y. PubMed DOI

Vogelstein B., Lane D., Levine A.J. Surfing the p53 network. Nature. 2000;408:307–310. doi: 10.1038/35042675. PubMed DOI

Gallagher S.J., Kefford R.F., Rizos H. The ARF tumour suppressor. Int. J. Biochem. Cell Biol. 2006;38:1637–1641. doi: 10.1016/j.biocel.2006.02.008. PubMed DOI

Korgaonkar C., Hagen J., Tompkins V., Frazier A.A., Allamargot C., Quelle F.W., Quelle D.E. Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function. Mol. Cell. Biol. 2005;25:1258–1271. doi: 10.1128/MCB.25.4.1258-1271.2005. PubMed DOI PMC

Nalabothula N., Indig F.E., Carrier F. The Nucleolus Takes Control of Protein Trafficking Under Cellular Stress. Mol. Cell. Pharmacol. 2010;2:203–212. PubMed PMC

Li Y.P., Busch R.K., Valdez B.C., Busch H. C23 interacts with B23, a putative nucleolar-localization-signal-binding protein. Eur. J. Biochem. 1996;237:153–158. doi: 10.1111/j.1432-1033.1996.0153n.x. PubMed DOI

Mitrea D.M., Cika J.A., Guy C.S., Ban D., Banerjee P.R., Stanley C.B., Nourse A., Deniz A.A., Kriwacki R.W. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. Elife. 2016:5. doi: 10.7554/eLife.13571. PubMed DOI PMC

Chen D., Huang S. Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells. J. Cell Biol. 2001;153:169–176. doi: 10.1083/jcb.153.1.169. PubMed DOI PMC

Olson M.O., Dundr M. The moving parts of the nucleolus. Histochem. Cell Biol. 2005;123:203–216. doi: 10.1007/s00418-005-0754-9. PubMed DOI

Phair R.D., Misteli T. High mobility of proteins in the mammalian cell nucleus. Nature. 2000;404:604–609. doi: 10.1038/35007077. PubMed DOI

Holoubek A., Heřman P., Sýkora J., Brodská B., Humpolickova J., Kráčmarová M., Gášková D., Hof M., Kuzelová K. Monitoring of nucleophosmin oligomerization in live cells. Methods Appl. Fluoresc. 2018;6:035016. doi: 10.1088/2050-6120/aaccb9. PubMed DOI

Sasinkova M., Herman P., Holoubek A., Strachotova D., Otevrelova P., Grebenova D., Kuzelova K., Brodska B. NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization. Sci. Rep. 2021;11:1084. doi: 10.1038/s41598-020-80224-1. PubMed DOI PMC

Sasinkova M., Holoubek A., Otevrelova P., Kuzelova K., Brodska B. AML-associated mutation of nucleophosmin compromises its interaction with nucleolin. Int. J. Biochem. Cell Biol. 2018;103:65–73. doi: 10.1016/j.biocel.2018.08.008. PubMed DOI

Chen Y., Hu J. Nucleophosmin1 (NPM1) abnormality in hematologic malignancies, and therapeutic targeting of mutant NPM1 in acute myeloid leukemia. Ther. Adv. Hematol. 2020;11 doi: 10.1177/2040620719899818. PubMed DOI PMC

Falini B., Mecucci C., Tiacci E., Alcalay M., Rosati R., Pasqualucci L., La Starza R., Diverio D., Colombo E., Santucci A., et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N. Engl. J. Med. 2005;352:254–266. doi: 10.1056/NEJMoa041974. PubMed DOI

Bullinger L., Dohner K., Dohner H. Genomics of Acute Myeloid Leukemia Diagnosis and Pathways. J. Clin. Oncol. 2017;35:934–946. doi: 10.1200/JCO.2016.71.2208. PubMed DOI

Forghieri F., Comoli P., Marasca R., Potenza L., Luppi M. Minimal/Measurable Residual Disease Monitoring in NPM1-Mutated Acute Myeloid Leukemia: A Clinical Viewpoint and Perspectives. Int. J. Mol. Sci. 2018;19:3492. doi: 10.3390/ijms19113492. PubMed DOI PMC

Falini B., Brunetti L., Martelli M.P. How I diagnose and treat NPM1-mutated AML. Blood. 2021;137:589–599. doi: 10.1182/blood.2020008211. PubMed DOI

Bolli N., De Marco M.F., Martelli M.P., Bigerna B., Pucciarini A., Rossi R., Mannucci R., Manes N., Pettirossi V., Pileri S.A., et al. A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF. Leukemia. 2009;23:501–509. doi: 10.1038/leu.2008.326. PubMed DOI

Brodska B., Kracmarova M., Holoubek A., Kuzelova K. Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM. PLoS ONE. 2017;12:e0175175. doi: 10.1371/journal.pone.0175175. PubMed DOI PMC

Falini B., Bolli N., Shan J., Martelli M.P., Liso A., Pucciarini A., Bigerna B., Pasqualucci L., Mannucci R., Rosati R., et al. Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML. Blood. 2006;107:4514–4523. doi: 10.1182/blood-2005-11-4745. PubMed DOI

Falini B., Albiero E., Bolli N., De Marco M.F., Madeo D., Martelli M., Nicoletti I., Rodeghiero F. Aberrant cytoplasmic expression of C-terminal-truncated NPM leukaemic mutant is dictated by tryptophans loss and a new NES motif. Leukemia. 2007;21:2052–2054. doi: 10.1038/sj.leu.2404839. PubMed DOI

Den Besten W., Kuo M.L., Williams R.T., Sherr C.J. Myeloid leukemia-associated nucleophosmin mutants perturb p53-dependent and independent activities of the Arf tumor suppressor protein. Cell Cycle. 2005;4:1593–1598. doi: 10.4161/cc.4.11.2174. PubMed DOI

O’Brate A., Giannakakou P. The importance of p53 location: Nuclear or cytoplasmic zip code? Drug Resist. Updat. 2003;6:313–322. doi: 10.1016/j.drup.2003.10.004. PubMed DOI

Comel A., Sorrentino G., Capaci V., Del Sal G. The cytoplasmic side of p53′s oncosuppressive activities. FEBS Lett. 2014;588:2600–2609. doi: 10.1016/j.febslet.2014.04.015. PubMed DOI

Senapedis W.T., Baloglu E., Landesman Y. Clinical translation of nuclear export inhibitors in cancer. Semin. Cancer Biol. 2014;27:74–86. doi: 10.1016/j.semcancer.2014.04.005. PubMed DOI

Lane D.P., Cheok C.F., Lain S. P53-Based Cancer Therapy. Cold Spring Harb. Perspect. Biol. 2010;2:a001222. doi: 10.1101/cshperspect.a001222. PubMed DOI PMC

Marcus J.M., Burke R.T., Doak A.E., Park S., Orth J.D. Loss of p53 expression in cancer cells alters cell cycle response after inhibition of exportin-1 but does not prevent cell death. Cell Cycle. 2018;17:1329–1344. doi: 10.1080/15384101.2018.1480224. PubMed DOI PMC

Mao L., Yang Y. Targeting the nuclear transport machinery by rational drug design. Curr. Pharm. Des. 2013;19:2318–2325. doi: 10.2174/1381612811319120018. PubMed DOI

Nguyen K.T., Holloway M.P., Altura R.A. The CRM1 nuclear export protein in normal development and disease. Int. J. Biochem. Mol. Biol. 2012;3:137–151. PubMed PMC

Das A., Wei G., Parikh K., Liu D. Selective inhibitors of nuclear export (SINE) in hematological malignancies. Exp. Hematol. Oncol. 2015;4 doi: 10.1186/s40164-015-0002-5. PubMed DOI PMC

Zhang Y., Xiong Y. A p53 amino-terminal nuclear export signal inhibited by DNA damage-induced phosphorylation. Science. 2001;292:1910–1915. doi: 10.1126/science.1058637. PubMed DOI

Stommel J.M., Marchenko N.D., Jimenez G.S., Moll U.M., Hope T.J., Wahl G.M. A leucine-rich nuclear export signal in the p53 tetramerization domain: Regulation of subcellular localization and p53 activity by NES masking. EMBO J. 1999;18:1660–1672. doi: 10.1093/emboj/18.6.1660. PubMed DOI PMC

Turner J.G., Dawson J., Sullivan D.M. Nuclear export of proteins and drug resistance in cancer. Biochem. Pharmacol. 2012;83:1021–1032. doi: 10.1016/j.bcp.2011.12.016. PubMed DOI PMC

Gravina G.L., Senapedis W., McCauley D., Baloglu E., Shacham S., Festuccia C. Nucleo-cytoplasmic transport as a therapeutic target of cancer. J. Hematol. Oncol. 2014;7:1–9. doi: 10.1186/s13045-014-0085-1. PubMed DOI PMC

Gu X., Ebrahem Q., Mahfouz R.Z., Hasipek M., Enane F., Radivoyevitch T., Rapin N., Przychodzen B., Hu Z., Balusu R., et al. Leukemogenic nucleophosmin mutation disrupts the transcription factor hub that regulates granulomonocytic fates. J. Clin. Investig. 2018;128:4260–4279. doi: 10.1172/JCI97117. PubMed DOI PMC

Kunchala P., Kuravi S., Jensen R., McGuirk J., Balusu R. When the good go bad: Mutant NPM1 in acute myeloid leukemia. Blood Rev. 2018;32:167–183. doi: 10.1016/j.blre.2017.11.001. PubMed DOI

Brodska B., Holoubek A., Otevrelova P., Kuzelova K. Low-Dose Actinomycin-D Induces Redistribution of Wild-Type and Mutated Nucleophosmin Followed by Cell Death in Leukemic Cells. J. Cell. Biochem. 2016;117:1319–1329. doi: 10.1002/jcb.25420. PubMed DOI

Grebenova D., Holoubek A., Roselova P., Obr A., Brodska B., Kuzelova K. PAK1, PAK1 Delta 15, and PAK2: Similarities, differences and mutual interactions. Sci. Rep. 2019;9:17171. doi: 10.1038/s41598-019-53665-6. PubMed DOI PMC

Herman P., Holoubek A., Brodska B. Lifetime-based photoconversion of EGFP as a tool for FLIM. Biochim. Biophys. Acta Gen. Subj. 2019;1863:266–277. doi: 10.1016/j.bbagen.2018.10.016. PubMed DOI

Patting M. Evaluation of Time-Resolved Fluorescence Data: Typical Methods and Problems, Standardization and Quality Assurance in Fluorescence Measurements I. Springer Ser. Fluoresc. 2008;5:233–258.

Strachotova D., Holoubek A., Kucerova H., Benda A., Humpolickova J., Vachova L., Palkova Z. Ato protein interactions in yeast plasma membrane revealed by fluorescence lifetime imaging (FLIM) Biochim. Biophys. Acta. 2012;1818:2126–2134. doi: 10.1016/j.bbamem.2012.05.005. PubMed DOI

Heikal A., Hess S., Webb W. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent proteins (EGFP): Acid-base specifity. Chem. Phys. 2001;274:37–55. doi: 10.1016/S0301-0104(01)00486-4. DOI

Hingorani K., Szebeni A., Olson M.O. Mapping the functional domains of nucleolar protein B23. J. Biol. Chem. 2000;275:24451–24457. doi: 10.1074/jbc.M003278200. PubMed DOI

Enomoto T., Lindstrom M.S., Jin A., Ke H., Zhang Y. Essential role of the B23/NPM core domain in regulating ARF binding and B23 stability. J. Biol. Chem. 2006;281:18463–18472. doi: 10.1074/jbc.M602788200. PubMed DOI

Wallrabe H., Periasamy A. Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 2005;16:19–27. doi: 10.1016/j.copbio.2004.12.002. PubMed DOI

Bastiaens P.I., Squire A. Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell. Trends Cell Biol. 1999;9:48–52. doi: 10.1016/S0962-8924(98)01410-X. PubMed DOI

Kenworthy A.K. Molecular Imaging: FRET Microscopy and Spectroscopy. In: Periasamy A., Day R., editors. Photobleaching FRET Microscopy. Oxford University Press; New York, NY, USA: 2005. p. 146.

Lakowicz J.R. Principles of Fluorescence Spectroscopy. Springer; New York, NY, USA: 2006.

Suhling K., Siegel J., Phillips D., French P.M., Leveque-Fort S., Webb S.E., Davis D.M. Imaging the environment of green fluorescent protein. Biophys. J. 2002;83:3589–3595. doi: 10.1016/S0006-3495(02)75359-9. PubMed DOI PMC

Kojima K., Kornblau S.M., Ruvolo V., Dilip A., Duvvuri S., Davis R.E., Zhang M., Wang Z., Coombes K.R., Zhang N., et al. Prognostic impact and targeting of CRM1 in acute myeloid leukemia. Blood. 2013;121:4166–4174. doi: 10.1182/blood-2012-08-447581. PubMed DOI PMC

Garzon R., Savona M., Baz R., Andreeff M., Gabrail N., Gutierrez M., Savoie L., Mau-Sorensen P.M., Wagner-Johnston N., Yee K., et al. A phase 1 clinical trial of single-agent selinexor in acute myeloid leukemia. Blood. 2017;129:3165–3174. doi: 10.1182/blood-2016-11-750158. PubMed DOI PMC

Vousden K.H., Vande Woude G.F. The ins and outs of p53. Nat. Cell Biol. 2000;2:E178–E180. doi: 10.1038/35036427. PubMed DOI

Nakayama R., Zhang Y.X., Czaplinski J.T., Anatone A.J., Sicinska E.T., Fletcher J.A., Demetri G.D., Wagner A.J. Preclinical activity of selinexor, an inhibitor of XPO1, in sarcoma. Oncotarget. 2016;7:16581–16592. doi: 10.18632/oncotarget.7667. PubMed DOI PMC

Ferreira B.I., Cautain B., Grenho I., Link W. Small Molecule Inhibitors of CRM1. Front. Pharmacol. 2020;11:625. doi: 10.3389/fphar.2020.00625. PubMed DOI PMC

Mahipal A., Malafa M. Importins and exportins as therapeutic targets in cancer. Pharmacol. Ther. 2016;164:135–143. doi: 10.1016/j.pharmthera.2016.03.020. PubMed DOI

Otevrelova P., Brodska B. Chemotherapy-induced survivin regulation in acute myeloid leukemia. Appl. Sci. 2021;11:460. doi: 10.3390/app11010460. DOI

Russo L.C., Ferruzo P.Y.M., Forti F.L. Nucleophosmin Protein Dephosphorylation by DUSP3 Is a Fine-Tuning Regulator of p53 Signaling to Maintain Genomic Stability. Front. Cell Dev. Biol. 2021;9:624933. doi: 10.3389/fcell.2021.624933. PubMed DOI PMC

Interferometric excitation fluorescence lifetime imaging microscopy