scholarly journals Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1

Blood ◽  
2011 ◽  
Vol 118 (11) ◽  
pp. 3096-3106 ◽  
Author(s):  
Ramesh Balusu ◽  
Warren Fiskus ◽  
Rekha Rao ◽  
Daniel G. Chong ◽  
Srilatha Nalluri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Chaperone ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
S Phase ◽  
Wild Type ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis ◽  
Export Signal

Abstract Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c+ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes NPM1c+ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1 induced p53 and p21, decreased the percentage of cells in S-phase of the cell cycle, as well as induced differentiation of the AML OCI-AML3 cells that express both NPMc+ and unmutated NPM1. Notably, knockdown of NPM1 by shRNA abolished lethal AML phenotype induced by OCI-AML3 cells in NOD/SCID mice. Knockdown of NPM1 also sensitized OCI-AML3 to all-trans retinoic acid (ATRA) and cytarabine. Inhibition of NPM1 oligomerization by NSC348884 induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA. This effect was significantly less in AML cells coexpressing FLT3-ITD, or in AML or normal CD34+ progenitor cells expressing wild-type NPM1. Thus, attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes NPM1c+ expressing AML cells to treatment with ATRA and cytarabine.

Targeting Mutant Nucleophosmin 1 (NPM1) Induces Differentiation, Loss of Survival and Sensitizes AML Cells to All-Trans Retinoic Acid, Cytarbine and FLT3 Antagonists

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 733-733
Author(s):  
Ramesh Balusu ◽  
Josiah Cox ◽  
Stacey Hembruff ◽  
Warren Fiskus ◽  
Rekha Rao ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Nuclear Export ◽  
Conflicts Of Interest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nuclear Export Signal ◽  
Monocytic Differentiation ◽  
Induced Differentiation ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis

Abstract Abstract 733 NPM1 is a nucleolar phosphoprotein that functions as an oligomeric molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML, especially those with the normal karyotype. The mutant (mt) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes mtNPM1 to be localized in the cytoplasm (NPM1c+). NPM1 has an N-terminal conserved, hydrophobic, oligomerization domain, which is common to all isoforms of NPM1 and critical for its chaperone activity. Recently, NSC348884 was identified as a small molecule inhibitor that disrupts wild-type (WT) and mtNPM1 dimer/oligomer formation, inducing apoptosis of cancer cells. Here, we determined the effects of specifically targeting mtNPM1 in cultured and primary AML cells. Treatment with shRNA specific to mtNPM1 or specific to WT and mtNPM1, as compared to the control non-targeted shRNA, induced p53, p21, p27 and CEBPα, but down regulated the levels of HOXA9 and Meis1 in the AML OCI-AML3 cells that heterozygously expressed mt NPM1. This led to an increase in the % of cell cycle G1 phase and a decline in % S phase cells, as well as marked inhibition of the colony growth of OCI-AML3 cells (p < 0.01). Knockdown of mtNPM1 induced myeloid/monocytic differentiation of OCI-AML3, as detected morphologically (∼30% over control) and induction of CD11b by flow cytometry. Knockdown of mtNPM1 also induced the proteins levels of RARα (two-fold) and sensitized OCI-AML3 cells (but not cultured AML cells with wtNPM1) in vitro to all-trans retinoic acid (ATRA) (0.25 to 2.0 μM)-induced p53, p21 and C/EBPα, as well as ATRA-induced differentiation and apoptosis (p < 0.01). Furthermore, mtNPM1 knockdown sensitized OCI-AML3 cells to cytarbine (0.5 to 2.0 μM)-induced apoptosis. Notably, mice bearing OCI-AML3 cells transduced with NPM1 shRNA and showing knockdown of total NPM1 demonstrated significantly less spleen enlargement and dramatically improved survival, as compared to the control mice (p < 0.01). Exposure to NSC348884 (1.0 to 3.0 μM) dose-dependently induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA-induced apoptosis (p < 0.01). NSC348884 treatment (3 μM) significantly disrupted the dimer (∼64 kDa) formation and increased the monomeric NPM1 levels in OCI-AML3 but not in HL-60 cells, as analyzed by native polyacrylamide gel electrophoresis (PAGE). OCI-AML3 cells transfected with FLT3-ITD lentivirus (OCI-AML3/FLT3-ITD cells) exhibited several-fold higher expression of FLT3-ITD, without causing any change in the levels of NPM1. As compared to the vector control cells, OCI-AML3/FLT3-ITD cells, as well as primary AML cells expressing both mtNPM1 and FLT3-ITD, were less sensitive to treatment with NSC348884 and/or ATRA. Notably, combination with NSC348884 (3.0 μM) and PKC412 (100 to 500 nM), a FLT3 antagonist (Novartis Pharmaceuticals), induced more apoptosis than either agent alone against OCI-AML3/FLT3-ITD and primary AML cells expressing both mtNPM1 and FLT3-ITD (p < 0.01). These findings demonstrate that knocking down of the levels and oligomerization of mtNPM1 and WT NPM1 induces differentiation and growth inhibition, as well as sensitizes AML cells with mtNPM1 to ATRA-induced differentiation and apoptosis. Furthermore our findings support the rationale of combining NPM1 antagonist with FLT3 inhibitor against AML with dual expression of mtNPM1 and FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Caspase-Dependent Cleavage of c-Abl Contributes to Apoptosis

2003 ◽  
Vol 23 (8) ◽  
pp. 2790-2799 ◽  
Author(s):  
Daniela Barilà ◽  
Alessandra Rufini ◽  
Ivano Condò ◽  
Natascia Ventura ◽  
Karel Dorey ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Death Receptor ◽  
Kinase Domain ◽  
Actin Binding ◽  
Caspase Cleavage ◽  
Nonreceptor Tyrosine Kinase ◽  
Nuclear Localization Signals ◽  
Induced Apoptosis ◽  
Export Signal

ABSTRACT The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program.

scholarly journals Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins

2003 ◽  
Vol 373 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Jose A. RODRIGUEZ ◽  
Simone W. SPAN ◽  
Frank A. E. KRUYT ◽  
Giuseppe GIACCONE
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Cowpox Virus ◽  
Amino Acid Region ◽  
Localization Signal ◽  
Induced Apoptosis ◽  
Export Signal

The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments.

scholarly journals The microtubule plus-end tracking protein Bik1 is required for chromosome congression

2021 ◽  
Author(s):  
Alexander Julner ◽  
Marjan Abbasi ◽  
Victoria Menendez Benito
Keyword(s):  
Cell Cycle ◽  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Nuclear Export Signal ◽  
Microtubule Dynamics ◽  
Dependent Manner ◽  
Chromosome Congression ◽  
A Cell ◽  
Cycle Dependent ◽  
Export Signal

During mitosis, sister chromatids congress on either side of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have pivotal roles in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are also involved. Here, we show that the microtubule plus-end tracking protein Bik1 (the budding yeast ortholog of CLIP-170) is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell-cycle-dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to a slower cell cycle progression characterized by a delayed metaphase-anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in Bik1-NES and does not localize to the unclustered kinetochores characteristic of Bik1-NES cells. Thus, we propose that Bik1 functions together with Cin8 to regulate kinetochore-microtubule dynamics for correct kinetochore positioning and chromosome congression.

Targeting Levels, Aberrant Localization or Oligomerization of Mutant Nucleophosmin Induces Differentiation and Loss of Survival of Human AML Cells with Mutant NPM1.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2656-2656 ◽  
Author(s):  
Ramesh Balusu ◽  
Warren C. Fiskus ◽  
Rekha Rao ◽  
Kathleen M. Buckley ◽  
Yongchao Wang ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Nuclear Export ◽  
Conflicts Of Interest ◽  
Nuclear Export Signal ◽  
Immunoblot Analysis ◽  
Clonogenic Survival ◽  
Npm1 Mutation ◽  
Highly Active ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis

Abstract Abstract 2656 Poster Board II-632 NPM1 is a nucleolar phosphoprotein that forms oligomers and functions as a molecular chaperone for both proteins and nucleic acids. NPM1 normally shuttles between the nucleus and cytoplasm but is mutated and aberrantly localized to the cytoplasm in 35% of patients with AML, Mutant (m) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES), which causes mNPM1 to remain in the cytoplasm. In AML, the presence of mNPM1 is often co-detected with internal tandem duplications of FLT-3, which confers a poor prognosis. The effects of the cytosolic mNPM1 on the biology and drug-sensitivity of AML are unclear. In the present studies we determined the expression and biologic effects of mNPM1 in cultured and primary AML cells. Utilizing RT-PCR, immunoblot analysis and immunoflourescence microscopy (with a polyclonal anti-mNPM1 antibody, we confirmed that in the cultured AML OCI-AML3 cells (hetrozygous for NPM1 mutation) mNPM1 protein was localized in the cytosol, while the un-mutated NPM1 was nuclear. In AML HL-60 cells NPM1 was un-mutated and entirely nuclear. As compared to control siRNA, treatment with siRNA to NPM1 for 72 hours significantly induced p21 expression, decreased % of S phase cells in the cell cycle, as well as induced morphologic differentiation of OCI-AML3 but not of HL-60 cells. This was associated with marked induction of CEBPα in OCI-AML3 cells. NPM1 siRNA also attenuated the expression of HOXA9 and MEIS1 (which are known to be leukemogenic), associated with a marked loss of clonogenic survival of OCI-AML3 cells. NPM1 siRNA mediated depletion of Meis1, which is known to transactivate FLT3 tyrosine kinase in AML progenitor cells, was associated with down regulation of FLT-3 expression in OCI-AML3 cells. Importantly, treatment with NPM1 siRNA significantly sensitized OCI-AML3 more than HL-60 cells to all-trans retinoic acid (ATRA, 0.25 to 2.0 uM) and Ara-C (0.5 to 5.0 uM). Treatment with leptomycin B (5 nM for 24 hours), which is an inhibitor of the NES receptor CRM1/exportin-1, shifted the localization of mNPM1 from the cytosol to the nucleus, abrogated the levels of Meis1 and HoxA9 and induced apoptosis of OCI-AML3 but not HL-60 cells. Recently, treatment with NSC348884, a small molecule inhibitor of NPM1 oligomerzation, was shown to induce apoptosis of colon and prostate cancer cells. Our studies determined that NSC348884 (3.0 uM) was highly active in disrupting the oligomerization of mNPM1 and induce apoptosis of 90 % of OCI-AML but only 20% of HL-60 cells. Additionally, treatment with 5.0 uM of NSC34884 induced apoptosis of approximately 75% of primary AML cells with mNPM1, without inducing apoptosis of normal CD34+ progenitor cells. Collectively, these findings suggest that strategies targeting mNPM1 levels or oligomerization, or reversing the aberrant cytosolic localization of mNPM1, would induce relatively selective differentiation and apoptosis, as well as sensitize AML cells with mNPM1 to Ara-C and ATRA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport

2002 ◽  
Vol 156 (5) ◽  
pp. 817-828 ◽  
Author(s):  
Anne Brunet ◽  
Fumihiko Kanai ◽  
Justine Stehn ◽  
Jian Xu ◽  
Dilara Sarbassova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Ligand Binding ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Rich Region ◽  
Present Evidence ◽  
Α Helix ◽  
Export Signal

14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal α-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

Sign in / Sign up

Export Citation Format

Share Document