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1
Amusan, Lere, and Oluwayemisi Adebola Oyekunle. "Conceptualizing innovation management development through organizational learning in the public service: any lessons for developing states?" Problems and Perspectives in Management 14, no. 3 (September 6, 2016): 266–75. http://dx.doi.org/10.21511/ppm.14(3-1).2016.13.
Повний текст джерелаАнотація:
The present economic realities, the effects of globalization, the thirst for innovation and the public’s demand for improved services have led many developing states to review their approaches to service delivery. Most public service managers and professionals spend most of their time dealing with the day-to-day pressures of delivering services, operating and reporting to senior managers, legislators and agencies. They have little or no time to think about innovation, which would ease the pressures and burdens of service delivery. The intention of this paper is to point out the fact that capacity building is the bedrock of new public management development. This paper proposes that innovation management could be used as a form of organizational learning capability in challenging the maze of diplomacy and negotiation with experienced multinational extractive industries for the benefit of developing states. This could be achieved through excellent public investments and nurturing capability, from which they execute effective innovation processes, leading to new service innovations and processes, and superior service performance results. To achieve this objective, extensive literature on innovation management and organizational learning was consulted and the need for future research. In trying to unpack the discussion in the paper, the New Public Management Theory (NPMT), which is a pro-private sectors human resources management is proposed, though other available theoretical positions are explored taking into consideration the lapses entrenched in NPMT. Keywords: innovation, NPMT, learning culture, public management, developing states, development. JEL Classification: O10
2
Chou, Wen-Chien, Shiu-Huey Chou, Ji-Shain Chiou, Bor-Sheng Ko, Shu-Wha Lin, Yueh-Chwen Hsu, and Hwei-Fang Tien. "A “canonical” Npm1 mutation Knock-in Mouse Model Revealed Subtle but Definitive Myeloid Expansion with Poor HSC Niche Interaction." Blood 118, no. 21 (November 18, 2011): 762. http://dx.doi.org/10.1182/blood.v118.21.762.762.
Повний текст джерелаАнотація:
Abstract Abstract 762 Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein, which has nucleocytoplasmic shuttling activity. Somatic mutations in NPM1 gene result in cytoplasmic dislocation of NPM1 (NPM1c) and are frequently associated with acute myeloid leukemia (AML). The pathogenetic effects of mutated NPM1 protein have been explored by animal models including transgenic or “humanized” knock-in mouse models. Here, we demonstrate the first “canonical” mouse Npm1 mutant knock-in model. Different from the previously report of humanized NPM1 mutant knock-in model, we inserted TCTG after nucleotide c.857 of murine Npm1 coding sequence (c.854–857dupTCTG), a pattern identical to human NPM1 mutation, without any “humanized” sequence. This mutation caused a shift of peptide sequence from WQWRKSL* (amino acid 286–292) to LCLAVEEISLRKGFKQFEIFCLHFCNS* (amino acid 285 to 311), a pattern mildly different from the change in human NPM1 mutation, but is still predicted to generate a nuclear export signal. NPM1c+ genotype and protein accumulation in cytoplasm were confirmed with PCR and immunocytochemistry, respectively. We found that the homozygous NPM mutant (NPMc+/c+) mice were embryonic lethal before E10.5 day, while hetrozygote (NPMwt/c+) mice survived and were fertile, and born with Mendelian ratio. Most NPMwt/c+ mice had normal hematologic parameters and remained disease-free, however, these mice developed a delayed-onset aberration on the distribution of granulocyte-monocye progenitors (GMP), monocytes, and B lymphocytes in blood, spleen, and bone marrow. Colony forming unit assay showed normal hematopoietic development of marrow hematopoietic stem cells (HSC), but poor cobblestone formation while HSCs contacted with stroma microenvironment in NPMwt/c+ mice, suggesting the NPMc mutant may affect the ability of HSCs on contact signal expression. Three (12.5%) of 24 NPMwt/c+ mice developed leukocytosis and splenomegaly mimicking myeloproliferative neoplasm of human. Microscopic examination showed panmyelosis of the bone marrow and existence of hematopoiesis in the spleen. In addition, the immune activities of NPMwt/c+ mice's splenocytes and thymocytes with mitogen stimulation were decreased. In summary, our “canonical” NPMwt/c+ mouse model demonstrated subtle but definitive phenotypes in hematopoietic cells and provided insight into the pathogenesis of NPM1 mutation in human acute myeloid leukemia. A second hit may be necessary for the development of AML in NPMwt/c+ mice since no AML was detected in these mice till 20 months of age. Disclosures: No relevant conflicts of interest to declare.
3
Manspeizer, H., E. J. Heyer, K. Lee, L. Mongero, B. Esrig, and C. R. Smith. "NPMT performance after coronary artery bypass grafting: cardiopulmonary bypass versus off-pump coronary artery bypass." Annals of Thoracic Surgery 70, no. 5 (November 2000): 1788. http://dx.doi.org/10.1016/s0003-4975(00)02079-8.
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Lukoyanov, A. V., A. O. Shorikov, and V. I. Anisimov. "Electronic structure of the NpMT 5 (M = Fe, Co, Ni; T = Ga, In) series of neptunium compounds." Physics of the Solid State 58, no. 3 (March 2016): 438–43. http://dx.doi.org/10.1134/s1063783416030215.
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Huang, Min, Xinran Li, and Beverly S. Mitchell. "Cysteine 288 Regulates NPMc+ Cytoplasmic Localization and Sensitizes Leukemic Cells to Bortezomib-Induced Apoptosis Through a Redox-Sensitive Mechanism." Blood 120, no. 21 (November 16, 2012): 532. http://dx.doi.org/10.1182/blood.v120.21.532.532.
Повний текст джерелаАнотація:
Abstract Abstract 532 Increasing evidence points to NPM1 mutations in exon 12 of the NPM1 gene (NPMc+) as a founder genetic event that defines a distinct leukemia entity accounting for approximately one-third of all AML. Unlike wild type NPM1, which resides predominantly in the nucleoli, NPMc+ mutants localize aberrantly in the leukemic-cell cytoplasm due to mutations at the C-terminus of NPM1. We have found that expression of NPMc+ in K562 and 32D cells sensitizes these cells to Bortezomib-induced apoptosis. Conversely, inducible small interfering RNA (shRNA)-induced knockdown of NPM1 in OCI-AML3 cell line, an AML cell line carrying the NPMc+ mutation, and in K562 cells over-expressing NPMc+ markedly reduced the ability of Bortezomib to induce apoptosis in both cell lines. Bortezomib-induced apoptosis in both OCI-AML3 and K562 cells was reversed with N-acetyl-1-cysteine (NAC), a ROS scavenger, suggesting that activation of ROS pathway plays an essential role in Bortezomib-induced apoptosis. Further studies showed that the cytoplasmic localization of NPMc+ in OCI-AML3 and 32D cells expressing NPMc+ increased in response to ROS activators such as diamide, hydrogen peroxide, and Bortezomib, but was reduced by NAC. These results prompted us to explore ROS-sensitive elements that might differentiate NPMc+ from wt-NPM1. We discovered that tryptophan 288 in wt-NPM1 is mutated to cysteine 288 in the majority of NPMc+ C-terminal mutants identified to date. Strikingly, mutagenesis of C288 to alanine re-localized NPMc+ completely to nucleolus, whereas mutagenesis of C21 and C104, the only two cysteines that exist in wt-NPM1, failed to alter the cytoplasmic localization of NPMc+. Unexpectedly, we also found that NPM1 oligomerization is disrupted in C21A+C104A mutants of both wt-NPM1 and NPMc+, indicating that C21 and C104 are essential for the oligomerization of NPM1. In addition, the proportion of cytoplasmic-localized endogenous wt-NPM1 also increased in response to ROS generation in K562 cells over-expressing NPMc+ or C21A+C104A-NPMc+, indicating that C288-NPMc+ partially relocates wt-NPM1 to the cytoplasm upon ROS activation through disulfide bond formation between C288-NPMc+ and C21 or C104 of wt-NPM1. Finally, we established the importance of C288-NPM1c+ as a sensitizing factor in Bortezomib-induced apoptosis by demonstrating that the sensitizing effects of NPMc+ to Bortezomib was significantly attenuated in K562 and 32D cells expressing C288A-NPMc+. In contrast, expression of C21A+C104A-NPMc+ sensitized K562 and 32D cells to Bortezomib-induced apoptosis to the same extent as NPMc+. We conclude that the mutation of tryptophan 288 to cysteine that is present in the great majority of NPM1c+ leukemic cells is the essential event that mediates the cytoplasmic localization of NPMc+ and that sensitizes leukemia cells to Bortezomib-induced apoptosis regardless of the oligomerization status of NPMc+. We also conclude that the intracellular redox level is an additional major factor that regulates the distribution of NPMc+ in the cytoplasm as opposed to the nucleolus in AML cells. Disclosures: No relevant conflicts of interest to declare.
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Bolli, Niccolò, Elspeth M. Payne, Clemens Grabher, Jeong-Soo Lee, Adam B. Johnston, Brunangelo Falini, John P. Kanki, and A. Thomas Look. "Expression of the cytoplasmic NPM1 mutant (NPMc+) causes the expansion of hematopoietic cells in zebrafish." Blood 115, no. 16 (April 22, 2010): 3329–40. http://dx.doi.org/10.1182/blood-2009-02-207225.
Повний текст джерелаАнотація:
AbstractMutations in the human nucleophosmin (NPM1) gene are the most frequent genetic alteration in adult acute myeloid leukemias (AMLs) and result in aberrant cytoplasmic translocation of this nucleolar phosphoprotein (NPMc+). However, underlying mechanisms leading to leukemogenesis remain unknown. To address this issue, we took advantage of the zebrafish model organism, which expresses 2 genes orthologous to human NPM1, referred to as npm1a and npm1b. Both genes are ubiquitously expressed, and their knockdown produces a reduction in myeloid cell numbers that is specifically rescued by NPM1 expression. In zebrafish, wild-type human NPM1 is nucleolar while NPMc+ is cytoplasmic, as in human AML, and both interact with endogenous zebrafish Npm1a and Npm1b. Forced NPMc+ expression in zebrafish causes an increase in pu.1+ primitive early myeloid cells. A more marked perturbation of myelopoiesis occurs in p53m/m embryos expressing NPMc+, where mpx+ and csf1r+ cell numbers are also expanded. Importantly, NPMc+ expression results in increased numbers of definitive hematopoietic cells, including erythromyeloid progenitors in the posterior blood island and c-myb/cd41+ cells in the ventral wall of the aorta. These results are likely to be relevant to human NPMc+ AML, where the observed NPMc+ multilineage expression pattern implies transformation of a multipotent stem or progenitor cell.
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Xu, Jie, Wu Zhang, Xiaojing Yan, Chen Zhao, Jiang Zhu, Zhu Chen, Sai-Juan Chen, and Jiong Hu. "NPM1 Mutation Contributes to Hematological Dysfunction By Disrupting H3K79 Methylation." Blood 128, no. 22 (December 2, 2016): 2702. http://dx.doi.org/10.1182/blood.v128.22.2702.2702.
Повний текст джерелаАнотація:
Abstract NPM1 is one of the most frequent acquired mutated genes in acute myeloid leukemia (AML). Previous studies have shown that NPM1 mutation (NPMc+) established the distinctive gene expression signatures, which were associated with mixed lineage leukemia (MLL)-target genes, like MEIS1 and HOXA cluster. In AML carrying MLL fusion-oncoproteins, DOT1L-mediated histone 3 lysine 79 (H3K79) methylation is implicated in the regulation of MLL-target genes. Compared with MLL abnormalities, NPM1 variants preserve the similar transcriptional characteristics. However, whether NPM1 mutation could affect the histone modification of H3K79 methylation is unknown. In this study, we showed that NPM1 mutation dysregulated the homeostasis of hematopoietic stem and progenitor cells and resulted in ageing-related myeloproliferation in NPMc+ transgenic mouse model. Interestingly, through scanning the chromatin modification related gene profiling, di- and tri- methylated H3K79 were significantly elevated in bone marrow (BM) Lin-Sca-1+c-Kit+ cells (LSKs) of NPMc+ mice comparing to wild type (WT). Meanwhile, in the leukemia cell lines and AML primary BM samples, we confirmed that NPM1 mutated cells expressed the higher level of H3K79 methylation. In vitro assays also indicated that the decrease or increase of methylated H3K79 could be regulated respectively by knockdown or overexpression of NPM1 mutant but not WT. Importantly, with DOT1L inhibitor treatment, reduced di- and tri- methylated H3K79 was observed in OCI-AML3 (NPMc+) strains but not OCI-AML2 (NPM1 WT) cells. In contrast with OCI-AML2, DOT1L inhibitor significantly promoted the cell apoptosis and restrained the cell cycle of OCI-AML3. Moreover, by the means of murine BM colony formation assay, DOT1L inhibitor obviously weakened myeloid cell proliferation in NPMc+ mice, while colony number in WT group did not change. Also, leukemia development was repressed in OCI-AML3-xenografted NOD/SCID mice with the treatment of DOT1L inhibitor. Taken together, NPM1 mutation contributes to hematological dysfunction by disrupting H3K79 methylation, which could be largely attenuated by DOT1L inhibitor. Disclosures No relevant conflicts of interest to declare.
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Sportoletti, Paolo, Emanuela Varasano, Roberta Rossi, Oxana Bereshchenko, Debora Cecchini, Ilaria Gionfriddo, Niccolò Bolli, et al. "The human NPM1 mutation A perturbs megakaryopoiesis in a conditional mouse model." Blood 121, no. 17 (April 25, 2013): 3447–58. http://dx.doi.org/10.1182/blood-2012-08-449553.
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Okuwaki, M., A. Sumi, M. Hisaoka, A. Saotome-Nakamura, S. Akashi, Y. Nishimura, and K. Nagata. "Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling." Nucleic Acids Research 40, no. 11 (February 22, 2012): 4861–78. http://dx.doi.org/10.1093/nar/gks162.
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Martelli, M. P., V. Pettirossi, N. Manes, A. Liso, F. Mezzasoma, F. Cecchetti, M. F. De Marco, et al. "Selective Silencing of the NPM1 Mutant Protein and Apoptosis Induction upon ATRA In Vitro Treatment of AML Cells Carrying NPM1 Mutations." Blood 110, no. 11 (November 16, 2007): 868. http://dx.doi.org/10.1182/blood.v110.11.868.868.
Повний текст джерелаАнотація:
Abstract We previously identified a new AML category carrying NPM1 mutations which lead to aberrant cytoplasmic expression of the nucleolar protein NPM1, hence the term NPMc+ AML[Falini et al, NEJM 2005]. This leukemia accounts for about one-third of adult AML and shows distinctive biological and clinical features[Falini et al, Blood 2007]. Notably, AML carrying NPM1 mutations in the absence of FLT3-ITD are characterized by a favourable prognosis. However, still a proportion of NPMc+ AML cannot be cured by conventional treatments and new therapeutic strategies need to be explored. We previously identified OCI/AML3 as the only human AML cell line carrying cytoplasmic mutated NPM (type A) in the absence of FLT3-ITD[Quentmeier et al, Leukemia 2005]. Because of these features and the ability to engraft in NOD/SCID mice, the OCI-AML3 represents a remarkable tool for the study of NPMc+ AML. Previous findings that ATRA exerts growth inhibitory effects on the OCI/AML3 prompt us to investigate the molecular mechanisms underlying the response to ATRA, with focus on the NPM mutant protein. As cellular model for our studies, we also used primary leukemia cells originated from a patient with NPMc+ AML (mutation A) bearing FLT3-ITD (Mont1) that have been propagated in NOD/SCID mice for 5 years without loss of initial characteristics. Early cell cycle arrest and proapoptotic effects of pharmacological doses of ATRA were confirmed in both cellular models in vitro. Morphological signs of differentiation were not evident. Western blot analysis using specific antibodies showed marked downregulation of the leukemic NPM1 mutant protein upon ATRA treatment, preceding apoptosis activation. On the other hand, wild-type NPM1 protein levels remained unchanged, leading to a condition of NPM1 haploinsufficiency. Semi-quantitative RT-PCR for NPM mutant A showed no change in mRNA expression following treatment, suggesting a regulation of the NPM mutant protein expression at post-transcriptional level. Indeed, concomitant treatment with proteasome-inhibitors partly reverted this effect. Downregulation of NPM mutant protein preceded activation of caspase-8 and caspase-3, PARP-cleavage and Bax activation. No NF-kB activation was observed upon ATRA treatment. Activation of the p53-dependent pathway was a later event, as expected in conditions of NPM1 haploinsufficiency. Importantly, these results were confirmed in the primary NPMc+ AML cells from patient Mont1. Activation of caspase-8 suggests that the response to ATRA in NPMc+ AML cells may be mediated through the death receptor pathway. Although protein levels of TRAIL, TRAIL receptors and TNF-alpha receptors seem to be unaffected, it might be possible that the NPM1 mutant protein modulates the signalling through death cell receptors. Analysis of ATRA-induced transcriptome and proteome modifications in NPMc+ AML is ongoing and will be also presented, as well as further pre-clinical studies on patients’ primary AML cells and in NOD/SCID mice. In conclusion, our data suggest that NPM mutant protein might be involved in the in vitro response to ATRA in AML cells carrying NPM1 mutations. Figure Figure
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Martelli, Maria Paola, Valentina Pettirossi, Elisabetta Bonifacio, Federica Mezzasoma, Nicla Manes, Debora Cecchini, Maruska Capanni, et al. "Evidence for CD34+ Hematopoietic Progenitor Cell Involvement in Acute Myeloid Leukemia with NPM1 Gene Mutation: Implications for the Cell of Origin." Blood 112, no. 11 (November 16, 2008): 307. http://dx.doi.org/10.1182/blood.v112.11.307.307.
Повний текст джерелаАнотація:
Abstract Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity >90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure
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Falini, Brunangelo, Ildo Nicoletti, Massimo F. Martelli, and Cristina Mecucci. "Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features." Blood 109, no. 3 (October 2, 2006): 874–85. http://dx.doi.org/10.1182/blood-2006-07-012252.
Повний текст джерелаАнотація:
Abstract The nucleophosmin (NPM1) gene encodes for a multifunctional nucleocytoplasmic shuttling protein that is localized mainly in the nucleolus. NPM1 mutations occur in 50% to 60% of adult acute myeloid leukemia with normal karyotype (AML-NK) and generate NPM mutants that localize aberrantly in the leukemic-cell cytoplasm, hence the term NPM-cytoplasmic positive (NPMc+ AML). Cytoplasmic NPM accumulation is caused by the concerted action of 2 alterations at mutant C-terminus, that is, changes of tryptophan(s) 288 and 290 (or only 290) and creation of an additional nuclear export signal (NES) motif. NPMc+ AML shows increased frequency in adults and females, wide morphologic spectrum, multilineage involvement, high frequency of FLT3-ITD, CD34 negativity, and a distinct gene-expression profile. Analysis of mutated NPM has important clinical and pathologic applications. Immunohistochemical detection of cytoplasmic NPM predicts NPM1 mutations and helps rationalize cytogenetic/molecular studies in AML. NPM1 mutations in absence of FLT3-ITD identify a prognostically favorable subgroup in the heterogeneous AML-NK category. Due to their frequency and stability, NPM1 mutations may become a new tool for monitoring minimal residual disease in AML-NK. Future studies should focus on clarifying how NPM mutants promote leukemia, integrating NPMc+ AML in the upcoming World Health Organization leukemia classification, and eventually developing specific antileukemic drugs.
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Garcia, Jacqueline S., Min Huang, Bruno C. Medeiros, and Beverly S. Mitchell. "Preclinical Activity of a New Proteasome Inhibitor, MLN9708, in Acute Myelogenous Leukemias Expressing NPM1 Mutated Protein." Blood 124, no. 21 (December 6, 2014): 993. http://dx.doi.org/10.1182/blood.v124.21.993.993.
Повний текст джерелаАнотація:
Abstract ABSTRACT: The nucleophosmin-1 (NPM1) mutation represents the most common genetic lesion (30-35%) in adult acute myeloid leukemia (AML) with the mutation resulting in the cytoplasmic delocalization of the NPM protein (NPMc+, most commonly mutation type A, c.860_863dupTCTG). Outside of conventional chemotherapy, treatment options for those with relapsed disease are extremely limited. We have previously discovered that leukemic cells expressing NPMc+ are preferentially sensitive to the cytotoxic effects of the first generation proteasome inhibitor, bortezomib, as result of the induction of superoxide in these cells (Huang M, et al., Leukemia, 2013). However, bortezomib has demonstrated only modest anti-leukemic activity as a single agent in early phase clinical trials that have included primarily heavily pretreated patients not stratified according to mutational genotype. In addition, the high incidence of clinically significant peripheral neuropathy in these studies led to early discontinuation of treatment. MLN9708 (Ixazomib) is a novel second generation proteasome inhibitor that is orally bioavailable and has shown promising activity and improved tolerability in early phase clinical trials in lymphoma and in multiple myeloma, where it is currently in a Phase III clinical trial. The efficacy of MLN9708 in relapsed patients with AML has not yet been examined. We have investigated the in vitro effects of MLN9708 on the cytotoxicity, generation of reactive oxygen species (ROS), and cellular glutathione in both cultured NPMc+ AML cell lines and primary leukemic samples. Cultured OCI-AML3 cells (human AML cell line featuring the NPMc+ mutation) were approximately 50-fold more sensitive to MLN9708 with a mean half inhibitory concentrations (IC50 ± SD) of 67 nM ± 1.83 nM at 24 hours than were an AML cell line (THP-1) expressing wild type NPM1. Knockdown of both NPM1/NPMc+ by inducible NPM1 shRNA expression significantly attenuated the MLN9708-mediated cytotoxicity, as determined by MTS colorimetric and flow cytometric apoptosis assays. As with bortezomib, MLN9708 treatment at 75 nM induced a 2.1-fold (p < 0.002) increase in superoxide-specific DHE (PE)-derived fluorescence as compared to controls. Pre-incubation of cells with 25 mM N-acetylcysteine, a ROS scavenger, dramatically reduced cytotoxicity, strongly implicating the generation of ROS in the mechanism of MLN9708-induced cytotoxicity. Knockdown of NPM1/ NPMc+ expression resulted in an increase of glutathione levels of 2.6-fold (p < 0.04) in untreated cells, and 2.3-fold (p < 0.005) and 2.9-fold (p < 0.004) after a 22 hour incubation with MLN9708 at pharmacologically achievable doses of 75 nM and 150 nM, respectively (Figure 1). In addition, a concomitant 4-fold reduction (p < 0.04) in the NADP+/NADPH ratio was observed in the NPM1/NPMc+ depleted OCI-AML3 cells. In vitro apoptosis assays of primary leukemic blasts isolated from five patients with relapsed NPMc+ AML showed an IC50 for MLN9708 ranging from 49.963-467.487nM (183.8 nM ± 176.8 nM). Based on these promising in vitro studies, a Phase 2 clinical trial of single-agent oral MLN9708 (clinicaltrials.gov identifier: NCT02030405) has been initiated for patients with relapsed or refractory NPMc+ AML. We conclude that the expression of NPMc+ renders AML cells more susceptible to MLN9708 toxicity and that this effect results at least in part from the generation of superoxide. Lower glutathione levels in NPMc+ expressing cells suggests an underlying defect in the antioxidant defense pathway. Demonstration of the sensitivity of NPMc+ AML cells to MLN9708 as a single agent may serve as a platform for the development of novel combination strategies with this drug. Acknowledgments: Research was facilitated by the ASH Clinical Research Training Institute and AACR/ASCO Clinical Cancer Research training program. J.S.G is funded by the Leukemia & Lymphoma Society Career Development Award and ASCO YIA award through a grant supported by Mr. Aaron Sasson. J.S.G. is further supported by the Stanford Hematology Fellowship Program. Figure 1 Effect of NPM1/NPMc+ depletion in OCI-AML3 cells on intracellular glutathione levels Figure 1. Effect of NPM1/NPMc+ depletion in OCI-AML3 cells on intracellular glutathione levels Disclosures Off Label Use: MLN9708 is an investigational compound by Millennium Pharmaceuticals, Inc. (MPI). Through an MTA we have secured the compound for in vitro analysis. This data has led to a clinical trial and drug for that study is supplied by MPI.. Medeiros:Agios: Consulting - Ad board Other.
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Liso, Arcangelo, Filippo Castiglione, Antonio Cappuccio, Fabrizio Stracci, Christian Thiede, Gerhard Ehninger, Susanne Schnittger, et al. "One-Mutation Model Can Explain Age Incidence in AML Carrying Nucleophosmin (NPM1) Mutations." Blood 110, no. 11 (November 16, 2007): 4312. http://dx.doi.org/10.1182/blood.v110.11.4312.4312.
Повний текст джерелаАнотація:
Abstract Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) mutations and cytoplasmic NPM (NPMc+ AML) accounts for about one-third of all AML patients, and exhibits distinctive biological and clinical features. The role of NPM1 mutations in leukemogenesis remains elusive. Mathematical models have been developed that, starting from cancer incidence data, allow to infer the somatic mutation rate, or the number of genetic events required to cause cancer. We collected data on age at diagnosis of AML patients from four centers in three different countries, and calculated age-specific rates of NPMc+ AML. A total of 4,155 AML patients were investigated. NPM1 mutations these were detected in 1288. Patients carrying NPM1 mutations with age below 20 years and above 59 years were excluded from the study because of the low number of younger cases and because older patients are not always referred to major institutions for diagnosis and treatment. To investigate NPMc+ AML we adapted one-mutation model published by Michor et al (PNAS, 2006; 103: 14931). The mathematical model consider a population of N (hemopoietic stem) cells that at beginning are wild-type. These cells proliferate according to the Moran process. The growth follows a logistic law with a saturation term. Our process follows the “classical” Moran process up to the appearance of a successful mutant. After that, the clone expands to a limiting population size. This is done to account for the dramatic expansion of the initial compartment peculiar of AML. Finally the rate of AML detection is proportional to the number of mutated cells. Experimental incidence curves of AML in Germany (Ge), Netherlands (Nl), and Italy (It) plotted simultaneously with predicted one-mutation model estimates are shown in Fig. 1. Linear regression of curves representing age-specific rate of diagnoses per year showed similar slopes (about 4 on a double-log scale) in different countries. The one-event model reproduces well the “exponential phenotype” of NPMc+ AML. In conclusion the model is in accordance with the hypothesis that NPM1 mutations by themselves are sufficient to cause NPMc+ AML. Alternatively, it is still possible that NPM1 mutations might cooperate with other molecular alterations to cause AML. In particular, since NPM1 mutations cause haploinsufficiency of wild-type NPM in leukemic cells and in knock-out mice NPM haploinsufficiency results in a MDS-like syndrome and given that the NPM1 mutant has oncogenic properties, these alterations could act in concert to cause AML. Indeed, the effect of these two alterations occurring simultaneously could be seen as a single genetic event. Figure Figure
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Kuželová, Kateřina, Christoph Röllig, Zdenek Racil, Juliane S. Stickel, Barbora Brodská, Johannes Schetelig, Christian Thiede, et al. "HLA Class I-Specific Effects in AML with Mutated Nucleophosmin." Blood 128, no. 22 (December 2, 2016): 5230. http://dx.doi.org/10.1182/blood.v128.22.5230.5230.
Повний текст джерелаАнотація:
Abstract Background We have recently identified a skewed distribution of class I human leukocyte antigens (HLA) among patients with AML and mutated nucleophosmin gene (NPMc+ AML). A lower frequency of several HLA allelic groups in the NPMc+ AML patient cohort was in good correlation with the results of theoretical predictions for high-affinity immunopeptides derived from NPM1, suggesting that an anti-NPM1 immune response could prevent leukemia development in patients with a suitable HLA class I type. Aim We here present more detailed analysis of HLA-specific features, in a much larger cohort (N = 357) of AML patients with NPM1 mutation (type A/D) from several centers in the Czech Republic, Germany and Poland. Results Most importantly, we confirmed the lower incidence of B´40 and C´07 allelic groups in NPMc+ AML compared to the normal values. The moderate decrease of A´02 allelic group frequency became statistically significant in this larger patient cohort (see Table). On the other hand, no difference in HLA class II frequencies was found compared to the normal distribution. Furthemore, patients with B´07 allelic group had a significantly better prognosis, but only in the absence of Flt-3-ITD mutations (p = 0.049, see Figure). HLA typing was performed by molecular methods in a subgroup of patients (N = 73), allowing for discrimination between C´07 alleles. In this subgroup, C*07:01, but not C*07:02/04 expression was associated with better overall survival (p = 0.036), in agreement with theoretical predictions. Conclusion Our results support the hypothesis that anti-NPM1 immune response reduces AML development and contributes to a better outcome of NPMc+ AML patients with suitable HLA class I type (including at least A´02, B´07, B´40 and C*07:01). Furthermore, NPMc+ immunogenicity is caused rather by its aberrant localization than by the generation of a unique aminoacid sequence. Figure Figure. Disclosures Schetelig: Sanofi: Honoraria. Thiede:AgenDix: Employment, Other: Ownership.
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Cheng, Ke, Paolo Sportoletti, Keisuke Ito, John G. Clohessy, Julie Teruya-Feldstein, Jeffery L. Kutok, and Pier Paolo Pandolfi. "The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model." Blood 115, no. 16 (April 22, 2010): 3341–45. http://dx.doi.org/10.1182/blood-2009-03-208587.
Повний текст джерелаАнотація:
Abstract Although NPM1 gene mutations leading to aberrant cytoplasmic expression of nucleophosmin (NPMc+) are the most frequent genetic lesions in acute myeloid leukemia, there is yet no experimental model demonstrating their oncogenicity in vivo. We report the generation and characterization of a transgenic mouse model expressing the most frequent human NPMc+ mutation driven by the myeloid-specific human MRP8 promoter (hMRP8-NPMc+). In parallel, we generated a similar wild-type NPM trans-genic model (hMRP8-NPM). Interestingly, hMRP8-NPMc+ transgenic mice developed myeloproliferation in bone marrow and spleen, whereas nontransgenic littermates and hMRP8-NPM transgenic mice remained disease free. These findings provide the first in vivo evidence indicating that NPMc+ confers a proliferative advantage in the myeloid lineage. No spontaneous acute myeloid leukemia was found in hMPR8-NPMc+ or hMRP8-NPM mice. This model will also aid in the development of therapeutic regimens that specifically target NPMc+.
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Czepiel, Kathryn, Kelly Nichol, Mason Tippy, Nirmalee Abayasekara, Jaclyn Lee, Ethan Wajswol, Anjali Khanna, Nancy Berliner та Arati Khanna-Gupta. "Haploinsufficiency of NPM1 in AML Derived NPM1 Mutant (NPMc+) Expressing Cells Contributes to Aberrant C/EBPα Activity". Blood 124, № 21 (6 грудня 2014): 2188. http://dx.doi.org/10.1182/blood.v124.21.2188.2188.
Повний текст джерелаАнотація:
Abstract Nucleophosmin 1 (NPM-1) is a highly conserved, ubiquitously expressed nucleolar protein that functions as a molecular chaperone shuttling protein-binding partners between the nucleolus, nucleus and cytoplasm. NPM-1 and has been assigned more than a dozen functions in the cell, including ribosome biogenesis and centrosome duplication. The NPM-1 gene maps to chromosome 5q35, a region that is the target of deletions in both de novo and therapy-associated MDS in humans. Additionally, heterozygous mutations in the NPM-1 gene have been identified in 60% of cytogenetically normal adult AMLs. Mutant NPM-1, (NPMc+), commonly results in the generation of a novel nuclear export signal (NES) leading to cellular mislocalization of NPMc+ from the nucleolus and nucleus to the cytoplasm. The role of NPMc+ in contributing to AML however remains unresolved to date. Two hypotheses to explain the role of NPMc+ in leukemogenesis have been advanced. The first purports that aberrant cytoplasmic mislocalization of NPMc+ also mislocalizes a number of NPM1-cargo proteins into the cytoplasm including for example, the tumor suppressor Arf, leading to the activation of the c-MYC oncogene, thereby contributing to leukeomogenesis. A second hypothesis states that reduction in wild type levels of NPM-1 in the nucleolus as a result of both heterozygosity as well as mislocalization into the cytoplasm following association with NPMc+, contributes to tumorigenesis. To address these hypotheses we generated a series of IL-3-dependent cell lines from the bone marrow of NPM1+/+ and NPM+/-mice. In addition, we stably transduced an NPMc+ expression vector into the NPM+/- cells resulting in the NPM+/-c+ line, thereby providing cellular models for both NPM1 haploinsufficieny as well as mutant NPM-1 associated AML. We then sought to examine the role of the master myeloid transcription factor C/EBPα in contributing to NPM-1-associated AML. C/EBPα is a single exon, bzip transcription factor that generates four isoforms derived from separate in-frame AUGs resulting in the translation of a nucleolar p50, a full length p42, a p40 and a dominant negative p30 isoform. We found that in NPM+/-c+ and OCI-AML3 cells (derived from a CN-AML patient harboring the NPMc+ mutation), only the p40 isoform of C/EBPα migrated to the cytoplasm while the full length p42 isofom remained in the nucleus. The p40 isoform of C/EBPα lacks the first 14 N-terminal amino acids when compared to the full length p42 isoform, and its function has not been well described in the literature. However, we have demonstrated that like the full length p42 isoform, the p40 isoform is also capable of transcriptional activation as measured by a reporter gene assay. Hence mislocalization of p40 could affect the overall activity of C/EBPα in NPMc+ expressing cells. We next sought to determine the contribution of NPM-1 haploinsufficieny to C/EBPα activity using our NPM+/- cells. We showed that a) expression levels of the dominant negative p30 isoform of C/EBPα are elevated in NPM+/- cells thereby blocking the activity of wildtype p42 b) C/EBPα p42 protein in the nucleus is rendered inactive due to phosphorylation by an unknown kinase at the S21position and c) expression of the downstream targets of C/EBPα (lactoferrin, MMP8, MMP9, gp91phox) are blocked in NPM+/- cells. These observations together suggest that in CN-AML where one copy of NPM-1 is wildtype and one copy is mutant (NPMc+), both haploinsufficient levels of wild type NPM-1 as well as mutant NPMc+ contribute to aberrant myeloid differentiation by inactivating C/EBPα activity. Our data thus provide new insights into the molecular mechanisms of mutant NPM-1 associated AML. Disclosures No relevant conflicts of interest to declare.
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Lit, Benny Man Wai, Yok Lam Kwong, and Kit Fai Wong. "Immunohistochemical detection of cytoplasmic nucleophosmin in formalin-fixed paraffin-embedded marrow trephine biopsies in acute myeloid leukaemia." Journal of Clinical Pathology 69, no. 5 (October 23, 2015): 409–14. http://dx.doi.org/10.1136/jclinpath-2015-203175.
Повний текст джерелаАнотація:
AimsNucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalisation of nucleophosmin (NPMc+) are the most common genetic abnormality in acute myeloid leukaemia (AML). In this study, we tested whether immunohistochemical (IHC) detection of cytoplasmic NPM1 (cNPM1) in formalin-fixed bone marrow trephine biopsies correlated with NPM1 mutations and the prognostic impact of NPM1 and fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) gene mutations was also assessed.MethodsA total of 71 Chinese adult de novo AML cases were evaluated for cNPM1 by IHC where the bone marrow trephines were fixed in 10% buffered formalin and decalcified by 5% EDTA. NPM1 and FLT3-ITD gene mutations were also investigated using PCR, fragment analysis and direct DNA sequencing.ResultsIHC analysis of cNPM1 had a very good sensitivity (86.7%) and excellent specificity (96.4%) for NPM1 mutation. The positive predictive value was 86.7% and the negative predictive value was 96.4%. NPM1 mutations and FLT3-ITD were closely associated (p=0.003). Patients with mutated NPM1 and without FLT3-ITD mutation have a longer overall survival (p=0.042) than patients with both NPM1 and FLT3-ITD mutations.ConclusionsOur results showed that IHC detection of cNPM1 in formalin-fixed trephine biopsies correlated well but not entirely with NPM1 mutation. Furthermore, NPM1 mutations were significantly more frequent in FLT3-ITD than FLT3-wild-type cases.
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Balusu, Ramesh, Warren Fiskus, Rekha Rao, Daniel G. Chong, Srilatha Nalluri, Uma Mudunuru, Hongwei Ma, et al. "Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1." Blood 118, no. 11 (September 15, 2011): 3096–106. http://dx.doi.org/10.1182/blood-2010-09-309674.
Повний текст джерелаАнотація:
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.
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Wang, Youping, Zichu Zhao, Jingyu Hu, Fei Zhao, Jing He, Jun Fang, Chun Zhang, Guohui Cui та Yan Chen. "Low-Dose Deguelin, Selective Targeting of Mt NPM1, Induces Differentiation in NPMc+ AML Cells By Regulation the Axis of SIRT1/CEBPβ/G-CSFR, Instead of HDAC1/3". Blood 124, № 21 (6 грудня 2014): 2231. http://dx.doi.org/10.1182/blood.v124.21.2231.2231.
Повний текст джерелаАнотація:
Abstract AML with NPM1 mutation (Mt NPM1) showing aberrant cytoplasmic dislocation of NPM1 exhibit relative good response to induction therapy, however, still a proportion of NPMc+ AML can not be cured by conventional treatments. Therefore, the aim of the present study was to determine the therapeutic efficacy of deguelin, a natural rotenoid isolated from several plant species, which has been demonstrated extensive biological activity in various tumors with low lethal effect. In the 54th ASH meeting in 2012, we reported deguelin selectively reduced Mt NPM1, induced differentiation and potentiated apoptosis in acute myeloid leukemia cells carrying NPM1 mutation. In this paper, we will focus on the investigation of the effect and molecular of mechanisms of deguelin treatment in nontoxic concentration with long time duration on AML cells carrying Mt NPM1. Deguelin treatment at the differentiation-induced concentration of 2µM for 5 days significantly induced granulocytic/monocytic differentiation such as a lower nucleocytoplasmic ratio, peripherally located nucleus, and chromatin condensation in 66.7% of OCI-AML3 cells carrying Mt NPM1. However, these phenomena were not presented in the OCIM2 cells harboring Wt NPM1. The NBT reduction assays that used as another marker of differentiation of myeloid leukemia exhibited increased NBT reduction activity, and Fluorescence-activated cell sorting (FACS) analysis demonstrated significantly increased expression levels of CD11b/CD14/CSF-3R in deguelin-treated OCI-AML3 cells (CD11b: from 2.8% to 62%; CD14: from 2.6% to 32.3%, CSF-3R: from 0.7% to 21.6%, P£¼0.01). Furthermore, immunoblot analysis revealed deguelin significantly reduced the levels of Mt NPM1 and HDACs (HDAC1/HDAC3/SIRT1), but not Wt NPM1, accompanied by increasing the expression of proteins and mRNAs of CEBP¦Â and G-CSFR. In addition, immunoprecipitation and immunoblot analysis confirmed deguelin significantly down-regulated Mt NPM1 via the ubiquitin-proteasome pathway. In consistent with above results, sh-Mt NPM1 and sh-SIRT1 treatment induced differentiation of OCI-AML3 (sh-Mt NPM1: CD11b/CD14/CSF-3R respectively: from 15.5% to 62.3%; from 13.9% to 47.1%; from 0.6% to 18.4%; sh-SIRT1: CD11b/CD14/CSF-3R respectively: from 18.2% to 47.1%; from 19.2% to 43.7%; from 0.4% to 13.4%, P£¼0.01), which confirmed by morphological examination and NBT reduction assays, and also upregulated the levels of proteins and mRNAs of CEBP¦Â and G-CSFR. sh-Mt NPM1 treatment reduced the expression of SIRT1, which did not change the levels of HDAC1/3. In other sides, sh-SIRT1 treatment did not alter the levels of Mt NPM1, suggesting that the decline of SIRT1 was partially accountable for granulocytic/monocytic differentiation of AML cells with Mt NPM1. Finally, overexpression of Mt NPM1 was performed to determine whether the depletion of Mt NPM1 directly induces granulocytic/monocytic cell differentiation. Compared to GFP-expressing cells, the proportion of the differentiation antigens (CD11b/CD14) and differentiation gene CSF-3R declined in Mt NPM1-expressing cells after deguelin treatment (Mt NPM1-expressing-treated deguelin-induced CD11b/CD14/CSF-3R-positive cells respectively: from 47.5% to 23.1%; from 29% to 18.8%; from 23.2% to 6.5%, P£¼0.01). Importantly, the upregulation of CEBP¦Â and G-CSFR by deguelin was appreciably hampered in Mt NPM1 expressing cells, compared with GFP-expressing cells. Taken together, our studies suggest deguelin, selective silencing of Mt NPM1, which induces differentiation via the axis of SIRT1/CEBP¦Â/G-CSFR (Fig1) may represent a novel active differentiating agent like ATRA for NPMc+ AML. Footnotes * Asterisk with author names denotes non-ASH members Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Florio, Daniele, Sara La Manna, Concetta Di Natale, Marilisa Leone, Flavia Anna Mercurio, Fabiana Napolitano, Anna Maria Malfitano, and Daniela Marasco. "Insights into Network of Hot Spots of Aggregation in Nucleophosmin 1." International Journal of Molecular Sciences 23, no. 23 (November 25, 2022): 14704. http://dx.doi.org/10.3390/ijms232314704.
Повний текст джерелаАнотація:
In a protein, point mutations associated with diseases can alter the native structure and provide loss or alteration of functional levels, and an internal structural network defines the connectivity among domains, as well as aggregate/soluble states’ equilibria. Nucleophosmin (NPM)1 is an abundant nucleolar protein, which becomes mutated in acute myeloid leukemia (AML) patients. NPM1-dependent leukemogenesis, which leads to its aggregation in the cytoplasm (NPMc+), is still obscure, but the investigations have outlined a direct link between AML mutations and amyloid aggregation. Protein aggregation can be due to the cooperation among several hot spots located within the aggregation-prone regions (APR), often predictable with bioinformatic tools. In the present study, we investigated potential APRs in the entire NPM1 not yet investigated. On the basis of bioinformatic predictions and experimental structures, we designed several protein fragments and analyzed them through typical aggrsegation experiments, such as Thioflavin T (ThT), fluorescence and scanning electron microscopy (SEM) experiments, carried out at different times; in addition, their biocompatibility in SHSY5 cells was also evaluated. The presented data clearly demonstrate the existence of hot spots of aggregation located in different regions, mostly in the N-terminal domain (NTD) of the entire NPM1 protein, and provide a more comprehensive view of the molecular details potentially at the basis of NPMc+-dependent AML.
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Brown, Patrick, Emily McIntyre, Rachel Rau, Soheil Meshinchi, Norman Lacayo, Gary Dahl, Todd A. Alonzo, Myron Chang, Robert J. Arceci, and Donald Small. "The incidence and clinical significance of nucleophosmin mutations in childhood AML." Blood 110, no. 3 (August 1, 2007): 979–85. http://dx.doi.org/10.1182/blood-2007-02-076604.
Повний текст джерелаАнотація:
Abstract Frameshift mutations in exon 12 of the nucleophosmin gene (NPM1) result in aberrant cytoplasmic localization of the NPM protein (NPMc+) and occur in 25% to 35% of adult acute myeloid leukemia (AML). In adults with AML, NPMc+ has been associated with normal karyotype, FLT3/ITD mutations, high remission induction rates, and improved survival (particularly in patients lacking FLT3/ITD). NPMc+ has not been well characterized in childhood AML. This study examines the incidence and clinical significance of NPMc+ in 295 children with newly diagnosed AML treated on a large cooperative group clinical trial (POG-9421). We find that NPMc+ is relatively uncommon in childhood AML (23 of 295 patients, 8%); and is significantly associated with FLT3/ITD mutations (P = .046), female sex (P = .029), older age (P = .047), and normal cytogenetics (P < .001). There is a favorable impact of NPMc+ on survival in children lacking FLT3/ITD (5-year EFS, 69% vs 35%; hazard ratio, 0.39; P = .051), which is similar in magnitude to the favorable impact of t(8;21) and inv(16). We conclude that NPMc+ is relatively rare in childhood AML, particularly in younger children. NPMc+ does not abrogate the negative prognostic influence of FLT3/ITD mutations, but may contribute to risk stratification in children who lack FLT3/ITD mutations by identifying a group with superior prognosis.
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McDuff, Fiona K. E., C. Elizabeth Hook, Reuben M. Tooze, Brian J. Huntly, Pier Paolo Pandolfi, and Suzanne D. Turner. "Determining the contribution of NPM1 heterozygosity to NPM-ALK-induced lymphomagenesis." Laboratory Investigation 91, no. 9 (June 27, 2011): 1298–303. http://dx.doi.org/10.1038/labinvest.2011.96.
Повний текст джерела
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Khanna-Gupta, Arati, Jian Chen, Matthew Silver, Hong Sun, Nirmalee Abayasekara, Stephanie Halene, Paolo Sportoletti, Pier Paolo Pandolfi та Nancy Berliner. "Nucleophosmin-1 Interacts with CCAAT Enhancer Binding Protein Alpha (C/EBPα) to Facilitate Granulocyte Maturation: Implications in MDS and AML." Blood 114, № 22 (20 листопада 2009): 2768. http://dx.doi.org/10.1182/blood.v114.22.2768.2768.
Повний текст джерелаАнотація:
Abstract Abstract 2768 Poster Board II-744 NPM-1, a gene that resides on chromosome 5q, is a highly conserved, ubiquitously expressed nucleolar phosphoprotein that belongs to the nucloeplasmin family of nuclear chaperones. NPM-1 is a multifunctional protein and has been implicated in ribosome biogenesis and the transport of pre-ribosomal particles, maintenance of genomic stability by the control of cellular ploidy, in DNA repair and in the regulation of DNA transcription by controlling chromatin condensation/decondensation. NPM1−/− mice die at mid-gestation (E11.5) due to severe anemia, underscoring the role of this gene in embryonic development. NPM-1 has been shown to be deleted or involved with chromosomal translocations in several hematologic malignancies. Mutant NPM-1 (NPMc+) has been found to be aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Additionally, mutations in the NPM1 gene have been described in 50% of normal karyotype AML patients. This makes NPM-1 one of the most frequently mutated genes in AML. The NPM-1 gene maps to a region on chromosome 5q that is the target of deletions in both denovo and therapy-associated MDS in humans, and is deleted in 50% of MDS associated with 5q deletions. NPM-1+/− mice develop a hematological syndrome similar to that observed in MDS patients. NPM-1 thus appears to act as a haploinsufficient tumor suppressor in the hematological compartment, the molecular basis of which remains unexplored. The NPM-1 deficient model provides an excellent platform to interrogate not only the molecular basis of MDS but also to study AML progression. To further explore the role of NPM1 in myeloid malignancy, we have generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. We demonstrate compromised neutrophil function and neutrophil-specific gene expression in the NPM1+/− cells similar to that observed in Npm1+/− mouse bone marrow, and attribute these observations to decreased expression of the master myeloid regulator C/EBPα in the Npm1+/− cells. We have demonstrated that overexpression of wildtype NPM1 but not mutant NPM1 (NPMc+) upregulates C/EBPα expression, and that overexpression of C/EBPα in Npm1 deficient cells rescues the myeloid phenotype. We further demonstrate by co-immunoprecipitation and oligonucleotide pulldown assays that NPM1 and C/EBPα physically interact. Our observations suggest that as a result of protein-protein interactions at specific cis elements in the promoters of C/EBPα target genes, C/EBPα and NPM1 act in concert to facilitate normal neutrophil gene expression and function. Interestingly, mutations in the C/EBPα gene have also been implicated in normal karyotype AML. We demonstrate for the first time a connection between these two components of the myeloid compartment, dysregulation of which can lead to disruption of the myeloid maturation program. Taken together, our observations may explain the defective myeloid phenotype observed when NPM1 expression is compromised in MDS and AML. We are now poised to examine the effects of knocking down genes such as RPS14 (the pathogenic gene in the 5q- syndrome) or overexpressing ones such as FLT3-ITD (associated with MDS/AML harboring NPM1 mutations) in our factor-dependent NPM1+/− cell line, to determine if these genes cooperate with NPM1 deficiency to render the cells factor independent. This cell system should be a useful model that will allow us to further dissect the role of NPM-1 in MDS and AML at the molecular level. Disclosures: No relevant conflicts of interest to declare.
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Boissel, Nicolas, Aline Renneville, Valeria Biggio, Nathalie Philippe, Xavier Thomas, Jean-Michel Cayuela, Christine Terre, et al. "NPM Mutations in Adult AML with Normal Karyotype: A Retrospective Study of the Acute Leukemia French Association (ALFA)." Blood 106, no. 11 (November 16, 2005): 2359. http://dx.doi.org/10.1182/blood.v106.11.2359.2359.
Повний текст джерелаАнотація:
Abstract NPM mutation (NPMm) has been recently reported as the most frequent mutation in AML, especially in the presence of a normal karyotype. Association of NPMm with an increased complete remission (CR) rate has been suggested, but the long-term prognosis is not known. The abnormal mutated NPM protein shows an aberrant cytoplasmic localization that allows an immunohistochemical detection of NPM status. However, all mutations reported are insertion/deletion of exon 12 resulting in a global insertion of 4 nucleotides. We therefore developed a NPMm detection test based on DNA PCR amplification and fragment length analysis. As previously reported, we observed a higher frequency of NPMm in AML with normal karyotype (47%, 50/106) than in CBF AML (0%, 0/7) or in AML with poor risk cytogenetic (13%, 4/32). We further evaluated the clinical profile and the prognosis of NPM mutations in a retrospective cohort of 106 patients with normal karyotype treated according to the ALFA-9000 or ALFA-9802 protocols between 1990 and 2004. In these patients aged 17–65 years, NPMm was significantly associated with a high white blood cell count (69 vs 18 G/L, p<.001) and an involvement of the monocytic lineage (FAB AML-M4/M5, p=.009). NPMm was also associated with a low rate of CEBPA mutation (CEBPAm, 10% vs 27%, p=.05) and a higher rate of FLT3 internal tandem duplication (FLT3/ITD, 38% vs 25%, p=NS). We did not find any correlation between NMPm and CR rate (86% vs 88%, p=NS). Long-term outcome did not differ between NPMm and NPM wild-type (NPMwt) patients neither in univariate analysis (6y-OS, 43% vs 37%; 6y-RFS, 47% vs 34%, p=NS) nor in multivariate analysis after adjustment on covariates significantly associated with prognosis in univariate analysis (age, CEBPAm, FLT3/ITD). NPM mutational status was available for 15 patients at relapse time. Ten patients out of 15 had NPM mutations at diagnosis and still displayed NPM mutations at relapse time. None of the 5 NPMwt patients acquired NPM mutation at relapse. Recently, we developed a RQ PCR technique to study minimal residual disease. Fifteen patients were evaluated at diagnosis and follow-up. In the majority of the cases a sensitivity of 10−4 was obtained. The high frequency, the stability, and the homogeneity of NPMm provide a promising minimal residual disease marker that we are prospectively evaluating. Prospective studies are also needed to confirm the definitive role of NPM mutation in the prognosis of AML patients.
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Oelschlaegel, Uta, Sina Koch, Markus Schaich, Frank Kroschinsky, Stefani Parmentier, Gerhard Ehninger, and Christian Thiede. "A Rapid Flow Cytometric Method for the Detection of NPM1 Mutated Patients with Acute Myeloid Leukemia (AML)." Blood 112, no. 11 (November 16, 2008): 1490. http://dx.doi.org/10.1182/blood.v112.11.1490.1490.
Повний текст джерелаАнотація:
Abstract In about 30% of patients with AML the predominantly nucleolar protein nucleophosmin (NPM1) is dislocated into the cytoplasm (NPM1c), caused by mutations in exon 12 of the NPM1 gene. NPM1-mutations are associated with a specific subgroup of AML with particular clinical, cytogenetic, and prognostic characteristics, and NPM1-mutations are part of the new WHO classification, which makes the detection an important diagnostic aspect. Thus far, NPM1 mutations can be detected either using molecular techniques or immunocyto- or histochemistry. We reasoned that by the cytoplasmic dislocation, the protein might be accessible also for rapid diagnostics using intracellular flow cytometry. The aim of the present work was to evaluate this procedure in vitro and prospectively in newly diagnosed patients with AML. Therefore, cell lines and in addition bone marrow (BM) or peripheral blood of 134 newly diagnosed AML patients, enrolled in two different treatment protocols, were prospectively investigated by a lyse-wash flow cytometric procedure including an anti-NPM monoclonal antibody labelled with Zenon labelling technology. Bone marrow of 23 healthy donors served as control. Cytoplasmic expression of NPM was measured after fixation and permeabilisation. Mean fluorescence intensity (MFI), assessing the ratio between the intensity of the specific antibody and the isotype matched control, was evaluated. All patients samples were routinely analyzed for NPM1-exon 12 mutations using PCR followed by high resolution fragment analysis. Cell lines with known mutational status (mutant: OCI-AML3 and wt: MV4-11) showed a clear separation (MFI: 44.6 vs. 3.7). In healthy BM the median MFI for the NPM expression was 3.8 ± 1.7. Thus, the threshold MFI for NPM positivity was set to 8.0. In overall in 93% of samples (125/134) both methods showed concordant results – positivity in 33% (44) and negativity in 61% (81) of patients. Thus, the MFI was significantly higher in NPM1c cases than in PCR negative patients and BM donors (MFI: 19.0 vs. 3.3 vs. 3.8; p<0.0001). The NPMc group presented with significantly lower CD34, HLA-DR, MPO, and aberrant CD2 expression (20% vs. 73%, p<0.0001; 73% vs. 90%, p=0.0306; 58% vs. 95%, p<0.0001; 0% vs. 11%, p=0.0242), but a comparable expression of other myeloid and aberrant antigens. FLT3 mutations were detectable in significantly more NPMc patients (52% vs. 11%, p<0.0001). Comparison of NPM1c plus FLT3 mutated patients vs. NPMc but FLT3 wild type patients resulted in a slightly lower NPM antibody expression in the latter group (MFI: 23.5 vs. 16.6, ns), and none of those patients expressed CD34 (32% vs. 0%, p=0.0065). Taking PCR results as standard, discrepant results in flow cytometric NPM detection occurred as 1% (2) false negative and 5% (7) false positive cases after flow cytometric evaluation. Ongoing studies correlate the type of NPM1-mutation with FACS-staining and look for additional mutations outside exon 12 to elucidate the reasons for this discrepancy. In conclusion, flow cytometric measurement of cytoplasmic NPM1 in AML blasts is a rapid and simple screening method resulting in a positive predictive value of 96%. This method will allow early identification of NPM1c patients and could serve as an important laboratory tool, i.e. for potential treatment protocols including specific therapy for NPM1c patients.
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Yi, Sha, Yan Chen, Lu Wen, Benping Zhang, Jing He, Guohui Cui, Lijing Yang, et al. "Deguelin, Selective Silencing of the NPM1 Mutant Protein, Induces Differentiation and Potentiates Apoptosis in Acute Myeloid Leukemia Cells Carrying NPM1 Mutation." Blood 120, no. 21 (November 16, 2012): 2435. http://dx.doi.org/10.1182/blood.v120.21.2435.2435.
Повний текст джерелаАнотація:
Abstract Abstract 2435 Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) gene mutations, leading to aberrant cytoplasmic expression of the nucleolar protein NPM1, accounts for about one-third of adult AML and shows distinctive biological and clinical features. In spite of the relatively good prognosis of NPM1-mutated AML, there are still cases that show poorer outcome, especially those associated with FLT3-ITD mutation and elderly patient population. Therefore new therapeutic strategies need to be explored. Deguelin, a rotenoid isolated from several plant species including Mondulea sericea (Leguminosae), exhibits significant inhibitory effects and induce apoptosis in a variety of cancer cell lines in vitro and in vivo. Researches have been revealed that NPM1 wild type (NPM-wt) and NPM1 mutant protein are potential therapeutic targets for AML cells harboring NPM1 mutations, however, isotype-specific inhibitors remain to be developed. Our previous study found deguelin induced apoptosis on Jurkat cells by disrupting NPM1 expression. Thus, in the present report we investigate the effects and molecular mechanisms of deguelin on AML NPMc+ cells, with focus on the NPM1 protein. Deguelin exerted dose-dependent dural effects on cell line OCI-AML3 carrying NPM1 mutation but not in the OCIM2 cell line (not harboring NPM1 gene mutation). Deguelin strongly induced OCI-AML3 cell apoptosis as shown by annexin V-fluoroisothyocyanate analysis (from 6.8% to 65.9%, 48 h at 32 μM), while apoptosis was minimal in OCIM2 cells (from 2.2% to 11.7%, 48h at 32 μM), which was further confirmed by a western blot assay to evaluate the activation of caspase-3 and the cleavage of PARP. Because of the pivotal role of NPM1 in AML cell survival and proliferation, we explored whether the above effects of deguelin were mediated by interfering with NPM1. Western blot analysis using specific antibodies showed marked downregulation of the leukemic NPM1 mutant protein upon deguelin treatment (0, 4 and 8μM, 48h), while NPM1-wt protein levels reminded unchanged. In addition, western blot and caspase activity assay revealed that the pro-apoptosis effect of deguelin was associated with caspase-6 and −8 activations, which might be caused by NPM mutation protein downregulation. In nontoxic concentration, morphologic features of granulocytic/monocytic differentiation were evident after 8 days treatment with dose of deguelin of 2μM in OCI-AML3 cells whereas no changes were found in OCIM2 cells. A more precise evaluation of cell differentiation by detecting CD11b (from 11.5% to 43.0%, P<0.01) and CD14 (from 1.2% to 11.1%, P<0.05) indicated that the proportion of the differentiation antigens increased in deguelin-treated cells. In accordance with the above results, NPM1 mutant protein was significant downregulated in OCI-AML3 cells even in nontoxic concentration of deguelin (from 0 to 2 μM, at 8 days), accompanied by p53, p21 and the 30kD band of C/EBPα decreasing, while there is no change in the protein level of NPM-wt, p53, p21 and C/EBPα in OCIM2 cells. Moreover, si-NPM-mut was used to further confirm the above results. Si-NPM-mut inhibited OCI-AML3 cell proliferation (inhibition rate 30% vs. control), induced differentiation (CD11b, from 4.2% to 19.2%) and caused significant decrease in the expression of pro-caspase-8, p21, p53 and the 30kD bands of C/EBPα compared with si-NC group, in consistent with the deguelin treatment in nontoxic concentration with long time duration, while it did not affect the levels of pro-caspase-6. Taken together, our results suggest that deguelin is a potent in vitro inhibitor of NPM1 mutation protein which provides the molecular basis for its anti-leukemia activities in NPMc+ AML cells, including suppression of proliferation, induction of apoptosis and differentiation. Disclosures: No relevant conflicts of interest to declare.
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Abayasekara, Nirmalee, Michelle Levine, Niccolo Bolli, Hong Sun, Matthew Silver, Navid Nia, Paolo Sportoletti, Pier Paolo Pandolfi, Nancy Berliner та Arati Khanna-Gupta. "Upregulation of eIF4E in Nucleophosmin 1 (NPM1) Haploinsufficient Cells Alters CCAAT Enhancer Binding Protein Alpha (C/EBPα) Activity: Implications for MDS and AML". Blood 118, № 21 (18 листопада 2011): 2432. http://dx.doi.org/10.1182/blood.v118.21.2432.2432.
Повний текст джерелаАнотація:
Abstract Abstract 2432 NPM1, is a highly conserved, ubiquitous nucleolar phosphoprotein that belongs to the nucleoplasmin family of nuclear chaperones. NPM1−/− mice die at mid-gestation (E11.5) from anemia, underscoring the gene's role in embryonic development. NPM1 is one of the most frequently mutated genes in AML. Mutations in NPM1 are found in 50% of normal karyotype AML patients, and mutant NPM1 (NPMc+) is aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Furthermore, NPM1 maps to a region on chromosome 5q that is the target of deletions in both de novo and therapy-associated human MDS. NPM1 thus acts as a haploinsufficient tumor suppressor in the hematological compartment, although the mechanism of its contribution to dysmyelopoiesis remains unknown. NPM-1+/− mice develop a hematological syndrome similar to that observed in human MDS, and develop AML over time. The NPM1 deficient model therefore provides a platform to interrogate the molecular basis of MDS. We identified nucleophosmin (NPM1) in a screen for protein binding partners of C/EBPα. C/EBPα is a single exon gene, but is expressed as two isoforms that arise by alternate translation start sites to yield a full length C/EBPα p42 and a truncated dominant negative C/EBPα p30 isoform. Translational control of isoform expression is orchestrated by a conserved upstream open reading frame (uORF) in the 5' untranslated region (5'UTR) and modulated by the translation initiation factors eIF4E and eIF2. We generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. These lines are IL-3-dependent and inducible toward neutrophil maturation with GM-CSF and/ or all- trans retinoic acid (ATRA). Neutrophils derived from MNPM1+/− cells display defective neutrophil-specific gene expression, including a cassette of C/EBPα-dependent genes. These observations led us to postulate that myeloid abnormalities in NPM1 deficiency reflect an aberrant NPM1-C/EBPα axis. We show that NPM1 haploinsufficiency upregulates eIF4E (eukaryotic initiation factor 4E) (but not eIF2), which binds the mRNA-Cap (m7-GTP) as part of the mRNA translation initiation complex, eIF4F. Increased eIF4E is observed in about 30% of all malignancies. Initial increased eIF4E levels in MNPM+/− cells likely reflect transcriptional activation by the oncoprotein c-Myc, protein levels of which are also elevated in MNPM1+/− cells. We propose that increased eIF4E then induces increased C/EBPαp30 translation. C/EBPαp30 is a dominant negative inhibitor of full length C/EBPαp42 activity and disrupts normal neutrophil development. Furthermore, we demonstrate that C/EBPαp30 but not C/EBPαp42, activates the eIF4E promoter. We propose a positive feedback loop, wherein increased C/EBPαp30 induced by eIF4E further increases the expression of eIF4E. Our data suggest that NPM1 deficiency modulates neutrophil-specific gene expression by altering C/EBPα. We propose an aberrant feed-forward mechanism that increases levels of both eIF4E and C/EBPαp30 and likely contributes to MDS associated with NPM1 deficiency. Disclosures: No relevant conflicts of interest to declare.
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Ikeguchi, Mitsunori. "Partial rigid-body dynamics in NPT, NPAT and NP?T ensembles for proteins and membranes." Journal of Computational Chemistry 25, no. 4 (2004): 529–41. http://dx.doi.org/10.1002/jcc.10402.
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Martelli, Maria Paola, Valentina Pettirossi, Christian Thiede, Elisabetta Bonifacio, Mauro Di Ianni, Ilaria Gionfriddo, Debora Cecchini, et al. "Dissecting the Hierarchical Level of Hematopoietic Progenitors' Involvement in AML with NPM1 Gene Mutation and Their Engraftment Potential in Immunocompromised Mice." Blood 114, no. 22 (November 20, 2009): 480. http://dx.doi.org/10.1182/blood.v114.22.480.480.
Повний текст джерелаАнотація:
Abstract Abstract 480 Acute myeloid leukemia with mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features [Falini B et al, Blood 2007;109:874-885] which include negativity for CD34 antigen expression at both immunohistochemistry and gene expression profiling. Flow cytometric analysis shows that, in most NPM1-mutated AML, percentages of CD34+ cells are in the low range (< 5-10%). Detection of NPM1 mutations by molecular techniques and/or immunohistochemistry and Western Blot analysis with specific antibodies provides an important tool for tracking the genetic lesion in leukemic cells at different hierarchical stage. We previously reported involvement by NPM1 gene mutation of the CD34+ cell fraction isolated from patients with NPM1-mutated AML, and, in one case, the involvement, in particular, of the early progenitor CD34+/CD38- [Martelli MP et al, Blood (ASH Annual Meeting Abstracts) 2008;112:307]. Here we expand and confirm our previous observation in 5 cases of CD34-negative NPM1-mutated AML. CD34+/CD38- cells were isolated by either FACS (3 cases, purity >98%) or MACS-sorting (2 cases, purity >92%) and analyzed by molecular analysis and Western Blot with a specific anti-NPM1 mutant antibody, respectively. The presence of either NPM1 gene mutation or mutant protein was demonstrated in all samples analyzed proving the CD34+/CD38- cells belong to the leukemic clone. This cell subpopulation displayed also immunophenotypic features classically associated to leukemic stem cells (LSCs) (CD123+/CD33+/CD90-) in all (16/16) samples analyzed, suggesting they might actually represent the LSCs in NPM1-mutated AML. Indeed, CD34+ cell fraction isolated from NPM1-mutated AML was able to generate leukemia in immunocompromised mice resembling the original patient's disease. However, there is experimental evidence that, at least in some CD34-negative AML, also the CD34- population may contain LSCs. Whether the CD34- cell compartment in NPM1-mutated AML is also able to engraft and outgrow into leukemia in mice remains to be clarified. For this purpose, we assessed the engraftment ability of CD34- cells from 5 NPM1-mutated AML patients. No engraftment was observed in one case. Interestingly, in three patients with myelomonocytic (M4, 2 cases) and myelocytic (M2, 1 case) AML, the CD34- fraction resulted into marrow engraftment by human CD45+/CD33+ myeloid cells that, at morphological and immunohistological grounds, consisted of a mixed population of macrophage cells expressing the CD68 (PG-M1) antigen and mature looking myeloperoxidase (MPO)-positive cells. This pattern possibly reflects short-term engraftment by leukemic cells devoid of self-renewal potential that differentiated into mature elements. However, the neoplastic nature of engrafted cells could be established with certainty only in one case by western blotting detection of NPM1 mutant protein. Immunohistochemistry could not help in these cases to establish the leukemic nature of human cells since terminally differentiated leukemic cells in NPM1-mutated AML show nucleus-restricted NPM1 positivity. In contrast, the pure CD34+ fraction (availabel for comparison in one of these three cases) engrafted as AML with clear blastic morphology and cytoplasmic dislocation of nucleophosmin. In a fourth patient, the highly purified CD34- fraction from relapsed NPM1-mutated AML engrafted in mice with a typical AML picture. These preliminary findings suggest that in general the CD34- fraction from NPM1-mutated AML may have more limited engraftment potential than the CD34+ fraction. Further studies are ongoing to address this issue. Disclosures: Falini: Xenomics: Patents & Royalties.
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Bedekovics, Judit, László Rejtő, Béla Telek, Miklós Udvardy, Anikó Újfalusi, Éva Oláh, Zsuzsa Hevessy, János Kappelmayer, Béla Kajtár, and Gábor Méhes. "Immunohistochemical demonstration of NPMc+ acute myeloid leukemia: biological and clinical features related to cytoplasmic nucleophosmin expression." Orvosi Hetilap 150, no. 22 (May 1, 2009): 1031–35. http://dx.doi.org/10.1556/oh.2009.28623.
Повний текст джерелаАнотація:
A nucleophosmin gén (NPM1) 12-es exonjának mutációja, jelenlegi ismereteink szerint, a leggyakoribb genetikai eltérés akut myeloid leukaemiában. Az eddigi beszámolók alapján kimutatásával a betegség sajátos biológiájú és kedvező lefolyású formája határozható meg, amely a leukaemiák WHO-osztályozásában 2008-tól külön csoportként kerül említésre. A mutáció hatására megváltozik a kódolt fehérje sejten belüli eloszlása, és az NPM fehérje a sejtmag helyett a citoplazmában halmozódik fel (NPMc+). Ez a jelenség, amely a kizárólag mutáns géntermék kapcsán figyelhető meg, szövettani vizsgálat keretében immunhisztokémiával is kimutatható. Jelen tanulmányunkban hazai körülmények között először vizsgáltuk az NPM-mutáció előfordulását felnőttkori AML-ben immunhisztokémia módszerével. A 2005 és 2008 között diagnosztizált összesen 41 eset részletes kiértékelése során 6 esetben (14,6%) észleltünk citoplazmikus reakciót, valamennyi de novo jelentkező és jellegzetes citogenetikai eltérést nem mutató AML-esetek közül került ki (6/23, 26,1%), egy kivételével nőbetegekben. A blasztsejtek alacsony CD34-, c-kit- és HLA-DR-expressziója alapján a NPMc+ AML a nem érintett esetektől jól elkülöníthetőnek bizonyult, ezek a markáns jellegzetességek az eddig közölt külföldi adatokkal teljes mértékben megegyeznek. Tapasztalataink alapján az NPM-mutáció immunhisztokémiai vizsgálata egyszerűen beépíthető a hétköznapi hematológiai diagnosztikai gyakorlatba.
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Wang, You, Sha Yi, Lu Wen, Ben Zhang, Zichu Zhao, Jing Hu, Fei Zhao, et al. "Nontoxic-dose of Deguelin Induce NPMc+ AML Cell Differentiation by Selectively Targeting Mt NPM1/SIRT1 Instead of HDAC1/3." Current Cancer Drug Targets 14, no. 8 (November 24, 2014): 685–99. http://dx.doi.org/10.2174/1568009614666141028123835.
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Oelschlaegel, U., S. Koch, B. Mohr, M. Schaich, B. Falini, G. Ehninger, and C. Thiede. "Rapid flow cytometric detection of aberrant cytoplasmic localization of nucleophosmin (NPMc) indicating mutant NPM1 gene in acute myeloid leukemia." Leukemia 24, no. 10 (August 19, 2010): 1813–16. http://dx.doi.org/10.1038/leu.2010.178.
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Kroeger, Heike, Jaroslav Jelinek, Steven M. Kornblau, Carlos E. Bueso-Ramos, and Jean-Pierre Issa. "Increased DNA Methylation Is Associated with Good Prognosis in AML." Blood 110, no. 11 (November 16, 2007): 595. http://dx.doi.org/10.1182/blood.v110.11.595.595.
Повний текст джерелаАнотація:
DNA methylation of CpG islands at transcriptional start sites (TSS) is an epigenetic modification that leads to permanent gene silencing and plays an important role in cancer development and progression. To better understand its significance in AML, we analyzed DNA methylation in bone marrow cells collected at diagnosis in two subsets of patients with AML distinguished by a long survival duration (group A, 33 patients, median, 90 months [range, 13–249+ months]) or short survival (group B, 32 patients, median, 7 months, [range, 2–12 months]). We quantitatively measured DNA methylation using bisulfite pyrosequencing of CpG islands at TSS of genes frequently methylated in leukemic cell lines: NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We also studied FLT3-ITD and NPM1 mutations as the most frequent genetic changes in leukemia. We compared these results with cytogenetic abnormalities, clinical data, and the AML outcome, and statistically analyzed the data with nonparametric tests and the Cox multivariate analysis. The patients in group A were significantly younger (median, 46 years, [range, 18–78 years]) than those in group B (median, 61 years, [range, 17–77 years]; p=0.02). We noted no significant differences between these groups according to gender, FAB classification, blast count, WBC, Hgb, platelet count, LDH and the frequency of NPM1 mutations and FLT3-ITD. The patients in the longer living group A had significantly lower levels of β2-microglobulin, better cytogenetic classification, and less frequent deletions of chromosome 5 and/or 7 than did those in group B; p<0.01. Remarkably, the patients in group A had significantly higher levels of methylation in multiple studied genes: NOR1, NPM2, OLIG2, HIN1, and SLC26A4, p<0.05. We observed no significant differences in methylation of CDH13, p15, and PGR. The median number of genes simultaneously methylated over the threshold was significantly higher in group A than in group B (five vs. two; p=0.0009). We observed longer survival in FLT3-ITD positive patients who had 4–8 methylated genes compared to those with 1–3 methylated genes, p=0.005. Similarly, the patients with wildtype NPM1 showed a significantly longer survival if they had 4–8 methylated genes compared to those with 1–3 methylated genes, p=0.002. No significant survival differences were seen in terms of the number of methylated genes in patients with FLT3 wild-type or with NPM1 mutations. The multivariate analysis revealed deletions of chromosome 5/7 or complex karyotype, FLT3-ITD, and advanced age as negative prognostic factors, p<0.002. Surprisingly, increased methylation of NOR1, HIN1, SLC26A4 and PGRB, simultaneous methylation of multiple genes and a high methylation z score were positive prognostic factors, p<0.03. We hypothesize that frequent DNA methylation denotes a subset of patients with good prognosis where epigenetics plays an essential role. This “epigenetic leukemia” seems to be more amenable to treatment and may have greater potential for long-term survival or cure compared to AML with less DNA methylation. DNA methylation and Survival DNA methylation and Survival
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Abayasekara, Nirmalee, Czepiel Kathryn, Lee Jaclyn, Anjali Khanna, Nancy Berliner та Arati Khanna-Gupta. "Mutant Nucleophosmin 1(NPMc+) Mislocalizes The p40 Isoform Of The Master Myeloid Transcriptional Regulator C/EBPα To The Cytoplasm: Implications In AML". Blood 122, № 21 (15 листопада 2013): 1417. http://dx.doi.org/10.1182/blood.v122.21.1417.1417.
Повний текст джерелаАнотація:
Abstract Nucleophosmin 1 (NPM-1) is a highly conserved, ubiquitously expressed nucleolar protein that functions as a molecular chaperone shuttling protein-binding partners between the nucleolus, nucleus and cytoplasm. NPM-1 has been assigned more than a dozen functions in the cell, including ribosome biogenesis, centrosome duplication and maintenance of genomic stability, stabilization of tumor suppressors p53 and Arf, DNA transcription and response to stress stimuli. The NPM-1 gene maps to chromosome 5q35, a region that is the target of deletions in both de novo and therapy-associated MDS in humans. Additionally, heterozygous mutations in NPM-1 have been identified in 60% of cytogenetically normal adult AMLs. Mutant NPM-1, referred to as NPMc+, commonly results from a gain-of-function mutation in the C-terminus of NPM-1 resulting in the generation of a novel nuclear export signal (NES) and consequent mislocalization from the nucleolus and nucleus to the cytoplasm. The role of NPMc+ in contributing to AML however, remains unresolved. Knock-in and transgenic models of NPMc+ in mice demonstrate that NPMc+ expressing animals develop AML after a long latency, suggesting the contribution of collaborating mutations to AML. Two hypotheses to explain the role of NPMc+ in leukemogenesis have been advanced. The first purports that aberrant cytoplasmic mislocalization of NPMc+ also mislocalizes a number of NPM-1-cargo proteins into the cyoplasm including for example, the tumor suppressor Arf, leading to the activation of the c-MYC oncogene. However, only a few proteins have been shown to be mislocalized by NPMc+ thus far. A second, though not mutually exclusive hypothesis states that reduction in wild type levels of NPM1 in the nucleolus as a result of both heterozygosity as well as mislocalization into the cytoplasm following association with NPMc+, contributes to tumorigenesis. In order to determine the role of myeloid transcription factors in contributing to NPMc+ associated malignancy, we determined the cellular localization of C/EBPα, Gfi1 and PU.1in the OCI/AML3 cell line, which was derived from a CN-AML patient harboring the NPMc+ mutation. While the Gfi1 protein appeared to be transported out of the nucleus to the cytoplasm, the PU.1protein remained within the nucleus of the OCI/AML3 cells. Both proteins remained predominantly in the nucleus in the control HL-60 cells. The fate of the master myeloid regulator C/EBPα in the OCI/AML3 cells was however, unexpected. C/EBPα is a single exon, b-zip transcription factor that generates four isoforms derived from separate in-frame AUGs resulting in the translation of a nucleolar p50, a full length p42, a p40 and the dominant negative p30 isoform. In OCI-AML3 cells, the p40 isoform of C/EBPα was found predominantly in the cytoplasm while the full length p42 and p30 isofoms remained in the nucleus. The p40 isoform of C/EBPα differs from its full length version by 14 amino acids at the N-terminus, and its function has not been well described in the literature. We will present data describing the functional implications of mislocalized Gfi1 as well as the role of p40 C/EBPα isoform in granulopoiesis and their contribution to AML in association with NPMc+. We propose that the depletion of p40 from the nucleus likely deregulates growth and differentiation normally attributed to p42 C/EBPα by perturbing the ratio of the remaining isoforms (p42:p30) in the nucleus, thus blocking p42 activity, thereby contributing to NPMc+ associated leukemogenesis. Our data provide new insights into the mechanism through which mutant NPM-1, a founding mutation in this subset of AML, contributes to leukemogenic progression. Disclosures: No relevant conflicts of interest to declare.
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Rawat, Vijay P. S., Silvia Thoene, Vegi M. Naidu, Natalia Arseni, Bernhard Heilmeier, Klaus Metzeler, Konstantin Petropoulos, et al. "Ectopic Expression of CDX2 Perturbs HOX Gene Expression in Murine Progenitors Depending on Its N-Terminal Transactivation Domain and Is Closely Correlated with Deregulated HOX Gene Expression in Human Acute Myeloid Leukemia." Blood 110, no. 11 (November 16, 2007): 2125. http://dx.doi.org/10.1182/blood.v110.11.2125.2125.
Повний текст джерелаАнотація:
Abstract Deregulated homeobox gene expression characterizes more than 60% of all acute myeloid leukemia (AML) patients, particular in patients with normal karyotype (NK). So far it is largely unknown how the aberrant expression of homeobox genes is initiated in the malignant clone. The ParaHox gene Cdx2 was shown to act as positive upstream regulator of Hox genes in embryogenesis. We have previously shown that ectopic Cdx2, which normally is not expressed in hematopoietic cells, can be the key event in the development of AML in mice (Rawat et al., PNAS 2004). In our present study we now demonstrate that ectopic expression of Cdx2 in murine hematopoietic progenitors induced significant up-regulation of Hox genes with leukemogenic potential such as HoxA9, Hoxa10, HoxA5, Hoxa7, Hoxb6, Hoxb8. Deletion of the N-terminal transactivation domain of Cdx2 abrogated its ability to perturb Hox gene expression and eliminated its leukemogenic potential in vivo (n=13), whereas inactivation of the putative Pbx binding site of the protein did not prevent Cdx2 induced leukemogenesis. As we showed that Cdx2 upregulated leukemogenic Hox genes and caused AML in the murine model we analyzed 115 AML patients for a correlation between the expression levels of CDX2 and deregulated HOX gene expression. A total of 71 patients with normal karyotype (AML NPMc+ = 45 cases; NPMc– = 26 cases) was analyzed for CDX2 expression. 89% of the AML NPMc+ cases showed ectopic expression of CDX2 as well as 88% of the cases without the NPM1 mutation. We extended this analysis to 44 patients with abnormal karyotype and detected ectopic CDX2 expression in 64% (28 out of 44) of the cases: 12 of 24 patients with t(8;21), 10 of 10 patients with t(15;17). Importantly, when the expression level of CDX2 was compared between AML cases with normal and abnormal karyotype, there was 14fold higher expression level in the patient group with NK (n=63) compared to the group with aberrant karyotype (n=28) (ØΔCT 7.72 vs. ØΔCT 11.62, respectively; p>0.001). By using oligonucleotide microarray analysis, we confirmed that high Cdx2 expressing AML-NK patients with (n=12) or without NPM1 mutation (n=12) were characterized by aberrant expression of multiple HOXA and B cluster genes such as HOXA10, HOXA9 and HOXB3, HOXB6 in contrast to AML cases expressing the PML-RARA or AML1-ETO fusion gene or normal healthy donors. Three NPMc- cases with normal karyotype showed the same low level of expression of CDX2 (ΔCT 10.55–11.55) as AML cases with aberrant karyotype. Of note, these three cases did not show any perturbation of HOX gene expression and thereby fell into the same cluster as AML cases with t(8;21) or t(15;17) in the microarray data set evaluating HOX gene expression in the different AML subtypes. All AML-NK patients tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression.
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Payne, Elspeth M., Clemens Grabher, Niccolo Bolli, John Kanki, Brunangelo Falini, and A. Thomas Look. "Function of Nucleophosmin in Zebrafish Hematopoiesis." Blood 110, no. 11 (November 16, 2007): 2644. http://dx.doi.org/10.1182/blood.v110.11.2644.2644.
Повний текст джерелаАнотація:
Abstract Nucleophosmin (NPM1) is a multi-functional ubiquitous phosphoprotein that shuttles between the nucleolus and cytoplasm. Located on chromosome 5q35 NPM1 is involved in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) both as a chimeric fusion partner and as a putative key haploinsufficient tumor suppressor. Heterozygous NPMc+ mutations have been identified in 30% of AML, usually with normal karyotype. Such mutations create new nuclear export signals and disrupt the normal nucleolar localization signal, resulting in re-localization of both mutant and heterodimeric wild-type (WT) NPM protein from the nucleolus to the cytoplasm. The zebrafish is a model organism ideally suited to genotypic and phenotypic analysis of myelopoiesis and leukemogenesis, with a proven track record for facilitating the discovery of novel pathogenetic pathways. Using in silico analysis we identified two homologues (a common finding in zebrafish due to genome duplication during piscine evolution) of the human nucleophosmin gene in zebrafish. These two genes have been designated znpm1a and znpm1b. znpm1a is annotated by the National Center for Biotechnology (NCBI) while znpm1b is a known protein-coding region located via blast search of the human NPM1 amino acid sequence at www.ensembl.org/Danio_rerio/index.html. Whilst znpm1b exhibits slightly less identity to human NPM1 (47%) than znpm1a (64%) it demonstrates clear synteny with human chromosome 5q35 and mouse chromosome 11 (Figure 1). We confirmed expression of both znpm1a and znpm1b in embryonic tissue and adult hematopoietic tissue of the major lineages by RT-PCR of Green Fluorescent Protein (GFP)-sorted cells in pu.1-GFP transgenic zebrafish embryos and in adult zebrafish kidney cells (sorted by forward and side scatter charactersistics). Morpholinos (stable, synthesized antisense oligonucleotides that specifically block gene expression when injected into embryos at the one-cell stage) were designed to inhibit znpm1a or 1b and injected into zebrafish embryos at the 1–4 cells stage to assess the effect of knockdown of znpm1a and 1b alone and in combination on hematopoiesis. Whole-mount in situ hybridization of 48 hours post-fertilization(hpf) injected embryos demonstrated a 50% reduction in the expression of myeloperoxidase (mpo) and a similar reduction in alpha globin (α-globin) expression as markers of myelo- and erythropoiesis. To investigate the mechanism of the reduction in hematopoietic cells we injected the znpm1a and 1b morpholinos into zebrafish carrying mutated p53 and observed partial rescue of the hematopoietic phenotype suggesting that loss of npm1 in zebrafish activates p53 dependent cell cycle arrest, senescence or cell death. Thus zebrafish npm1 proteins are required for normal hematopoiesis consistent with the role for NPM1 as a tumor suppressor in AML/MDS with loss of all or part of chromosome 5. Future studies using this model will address which pathways are disrupted by the loss of npm1 and thus may contribute to the pathogenesis of human AML/MDS and facilitate identification of potential therapeutic targets. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11.
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Martelli, Maria Paola, Lorenzo Brunetti, Luca De Carolis, Elisabetta Agliani, Laura Berchicci, Elisabetta Bonifacio, Valentina Pettirossi, et al. "Acute Myeloid Leukemia with Mutated NPM1 Presenting with Life-Threatening, Either Arterial or Venous, Thromboembolism: a Report of 4 Cases." Blood 114, no. 22 (November 20, 2009): 4135. http://dx.doi.org/10.1182/blood.v114.22.4135.4135.
Повний текст джерелаАнотація:
Abstract Abstract 4135 Acute myeloid leukemia (AML) expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features. AML with mutated NPM1 usually presents with a high white blood cell count; the bone marrow biopsy is usually markedly hypercellular and leukemic cells frequently show myelomonocytic or monocytic features, with dysplasia and involvement of two or more cell lineages in about 25% of cases. Lack, or low expression, of CD34 in over 90% of cases is the most distinctive immunophenotypic feature of NPM1-mutated AML and is independent of leukemic cell maturation. NPM1 gene mutation without concomitant FLT3-ITD identify a subgroup of AML patients with a favorable prognosis and has been associated with an approximately 50-60% probability of survival at 5 years in younger patients. Here we report 4 out of 41 (10%) patients, admitted at our Hospital in the last year, with new-diagnosed AML with mutated NPM1 presenting with life-threatening thromboembolic (either arterial or venous) events. The main characteristics of these patients are summarized in Table 1. The patients had neither personal nor family history concerning thromboembolism. Hyperleukocytosis was a common feature of the vast majority of NPM1-mutated AML patients at diagnosis. Immunophenotypic analysis did not show a peculiar phenotype in these patients. Table 1 Characteristics of patients with NPM1-mutated AML and thrombosis. Case report no Age Sex (M/F) FAB subtype WBC/mmc Type of thrombosis Site of thrombosis 1 41 F M1 14970 arterial Anterior interventricular branch of left coronary artery 2 56 M M4 93990 arterial external iliac and femoral (right limb) 3 63 M M2 113000 deep venous great saphenous veins (bilateral) 4 73 F M4 190000 deep venous iliac and femoral In two patients (cases 1 and 2), the arterial thromboembolic event (acute myocardial infarction and acute ischemia of right lower limb, respectively) presented about one month before diagnosis of leukemia. In the other 2 patients (cases 3 and 4), deep venous thromboembolism was concomitant with the diagnosis of leukemia. One patient (case 4), who could not initiate chemotherapy for severe concomitant renal failure, died few days after diagnosis. The other patients recovered from the acute event and upon diagnosis of leukemia were promptly treated with standard polychemotherapy which allowed to obtain complete hematological remission associated with complete resolution of the thromboembolic event. The clinical course after chemotherapeutic treatment of the patients outlines the importance and life saving role of early chemotherapy even under adverse circumstances. The pathogenesis of thromboembolic disease in hematological malignancies is complex and multifactorial: tumor cell-derived procoagulant, fibrinolytic or proteolytic factors and inflammatory cytokines affect clotting activation. Other important factors include infectious complications and hyperleukocytosis. However, large vessel thrombosis is a very rare clinical presentation. Our report of severe thromboembolic events at presentation in AML with mutated NPM1 suggests some still unidentified biological features of this leukemia which we are currently investigating. Disclosures: No relevant conflicts of interest to declare.
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Wallner, Britta, and Roswitha Schweindl. "Wundunterdrucktherapie (NPWT)." CNE.fortbildung 12, no. 04 (August 1, 2019): 14–16. http://dx.doi.org/10.1055/a-0959-4787.
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Falini, Brunangelo, Niccolo Bolli, Jing Shan, Maria Paola Martelli, Ildo Nicoletti, Arcangelo Liso, Laura Pasqualucci, et al. "Mechanism of Altered Nucleo-Cytoplasmic Traffic of Nucleophosmin in Acute Myelogenous Leukemia Carrying Exon-12 NPM Mutations (NPMc+ AML)." Blood 106, no. 11 (November 16, 2005): 4396. http://dx.doi.org/10.1182/blood.v106.11.4396.4396.
Повний текст джерелаАнотація:
Abstract We recently found that about one-third of adult AML (60% of all AML with normal karyotype) display aberrant cytoplasmic expression of nucleophosmin (NPM) which is due to mutations occurring at the exon-12 of NPM gene (Falini B, et al., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype..N Engl J Med2005; 352: 254–266). Hereby, we clarify the molecular mechanism underlying cytoplasmic NPM accumulation that yet remained to be elucidated. AML-associated mutated NPM alleles encode abnormal NPM proteins (25 mutants so far identified) which have acquired at the C-terminus a nuclear export signal-(NES) motif and lost at least one of tryptophan residues 288 and 290 which determine nucleolar localization. Both alterations are crucial for mutant NPM export from nucleus to cytoplasm. In fact, the cytoplasmic NPM accumulation is blocked by leptomycin-B and ratjadones which are specific inhibitors of exportin-1/CRM1, and by re-insertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. Thus, for cytoplasmic accumulation of NPM to occur, the NES motif and tryptophan mutations must act in concert. Possibly, when NPM mutans enter the nucleus by virtue of their nuclear localization signals (NLS), their capability to bind nucleoli must be hindered at least partially to become a CRM-1 target. Specific antibodies anti-NPM mutant proteins showed that the mutants localized exclusively in the cytoplasm and recruited in that site the wild-type NPM protein which is physiologically located in the nucleoli. These findings suggest that the NPM mutants may interfere with the functions of wild-type NPM and possibly contribute to leukemogenesis. Immunostaining of 393 AML cases using anti-NPM monoclonal antibodies predicted the presence of NPM exon-12 mutations in all 191 NPM-cytoplasmic positive cases. This finding is consistent with the fact that, despite genomic heterogeneity, all NPM mutants contain a NES motif which, in the presence of altered tryptophans, promotes their rapid export from the nucleus to the cytoplasm. The immunohistochemical test is diagnostically relevant since it can be used as simple first-step procedure in molecular-genetic characterization of AML and as a surrogate for mutational analysis in selected cases. These findings are also clinically relevant since cytoplasmic NPM/NPM mutations are predictors of good response to induction therapy and favourable prognosis in AML with a normal karyotype.
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Ghasemi, Reza, Struthers B. Heidi, and David H. Spencer. "Homozygous Loss of a Critical CTCF Recognition Sequence in the HOXA gene Cluster Eliminates CTCF Binding but Does Not Appreciably Reduce HOXA9 expression in NPM1 Mutated AML Cells." Blood 132, Supplement 1 (November 29, 2018): 2613. http://dx.doi.org/10.1182/blood-2018-99-119000.
Повний текст джерелаАнотація:
Abstract HOX genes encode homeodomain transcription factors that play a central role in hematopoietic stem/progenitor cell (HSPCs) self-renewal and the development of acute myeloid leukemia (AML). HOXA and HOXB genes display canonical expression patterns in primary AML samples that correlate with specific recurrent mutations, including expression of only HOXA genes in AMLs with MLL rearrangements and both HOXA and HOXB gene expression in normal karyotype AMLs with mutations in NPM1 (i.e., the NPMc mutation). Although regulation of HOXA genes in MLL-rearranged AML cells has been studied extensively, the regulatory mechanisms that govern HOXA expression in other AML types remains unclear. The chromatin organizing factor CTCF contributes to HOXA gene regulation by controlling chromatin looping and three-dimensional structure in a variety of cell types, including AML cells, and a recent study demonstrated that expression of HOXA9 in MLL-rearranged AML cell lines is dependent on a critical CTCF-binding site between HOXA7 and HOXA9 (CBS7/9). However, whether this site is required for HOXA gene expression in AMLs with NPMc remains unknown. The AML cell line OCI-AML3 contains the NPMc mutation (in addition to a DNMT3AR882C mutant allele) and expresses both HOXA and HOXB genes. We therefore used this cell line to investigate the role of CTCF in regulating HOXA expression in AML cells with this canonical stem cell expression pattern. ChIP-seq for CTCF via 'ChIPmentation' identified eight CTCF binding sites across the HOXA cluster in OCI-AML3 cells, including CBS7/9, which is consistent with previous data in AML cells. These sites were identical to those identified in MLL-rearranged AML cell lines, suggesting that CTCF binding is conserved between these AML types. Similar to previous work, ChIP-seq for histone methylation marks associated with active (H3K4 trimethylation) and repressed (H3K27 trimeylation) chromatin demonstrated distinct chromatin domains at the HOXA locus on either side of CBS7/9, indicating that CTCF acts as a barrier between these domains. Using a computational prediction tool for CTCF binding, we identified two potential CTCF-binding sequences at the CBS7/9 locus, including one bidirectional major site (CBS7/9-A; 20 bp) with a high binding score, separated by only 6 bp from a secondary unidirectional site (CBS7/9-B; 9 bp). To define the precise sequence important for CTCF binding in OCI-AML3 cells, we used CRISPR-Cas9 to generate a library of mutant cells with deletions of different sizes at the CBS7/9 locus, and then performed ChIP-seq on the mutant cells to measure CTCF binding. We found that mutants with deletions at CBS7/9-A were depleted relative to wild-type cells. Further analysis demonstrated that deletions as small as 10 bp affecting the CBS7/9-A site were sufficient to reduce CTCF binding to background levels. CRISPR-Cas9 generated deletions in the MLL-rearranged THP-1 cell line that spanned the same sequence also eliminated CTCF ChIP-seq signal, indicating that it is equally important for CTCF binding in MLL-rearranged and NPM1-mutant AML cells. Our collection of mutant OCI-AML3 cell lines included five with biallelic deletions of the CBS7/9-A site, which completely eliminated all CTCF binding at this locus, but did not affect cellular proliferation or survival. Surprisingly, preliminary analysis of expression in the five homozygous mutant OCI-AML3 cells demonstrated that HOXA9 gene expression by RT-qPCR remained largely intact compared to wild-type OCI-AML3 cells, and was not statistically different from three OCI-AML3 cell lines with a heterozygous deletion of the CBS7/9 site. In summary, we have mapped the precise recognition sequence for a critical CTCF binding site in the HOXA locus in human AML cells. Deletion of both alleles of this regulatory sequence in NPM1-mutated OCI-AML3 cells does not affect cell viability and appears to have only modest effects on expression of the adjacent HOXA9 gene. This is in contrast to a recent report suggesting that homozygous deletion of this CTCF binding site is critical for maintaining HOXA gene expression and may be required for cell viability in MLL-rearranged AML cell lines. These differences suggest that maintenance of HOXA expression in NPM1-mutated vs. MLL-rearranged AML cells may occur through distinct regulatory mechanisms. Disclosures No relevant conflicts of interest to declare.
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Cilloni, D., F. Messa, V. Rosso, F. Arruga, I. Defilippi, S. Carturan, R. Catalano та ін. "Increase sensitivity to chemotherapeutical agents and cytoplasmatic interaction between NPM leukemic mutant and NF-κB in AML carrying NPM1 mutations". Leukemia 22, № 6 (10 квітня 2008): 1234–40. http://dx.doi.org/10.1038/leu.2008.68.
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Van Helden, G. J. "NPM is dood, leve NPM!" Maandblad Voor Accountancy en Bedrijfseconomie 80, no. 11 (November 1, 2006): 528–29. http://dx.doi.org/10.5117/mab.80.12803.
Повний текст джерелаАнотація:
New Public Management (NPM) staat voor de aan het bedrijfsleven ontleende technieken en stijlen waarmee overheidsorganisaties beter moeten gaan functioneren. NPM kent verschillende verschijningsvormen, zoals de ontvlechting van beleid en beleidsuitvoering, uitbesteding/privatisering, de vorming van min of meer zelfstandige eenheden binnen organisaties, de vervanging van input control door output control en allerhande vormen van prestatiemanagement en -verantwoording.
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Zhang, Xia, Zichu Zhao, Sha Yi, Lu Wen, Jing He, Jingyu Hu, Jun Ruan, Jun Fang, and Yan Chen. "Deguelin Induced Differentiation of NPM1-Mutant AML In Vivo and in Vitro." Blood 128, no. 22 (December 2, 2016): 4030. http://dx.doi.org/10.1182/blood.v128.22.4030.4030.
Повний текст джерелаАнотація:
Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy due to abnormal proliferation of hematopoietic progenitor cells, with difficulty in treatment and high mortality. Nucleophosmin 1 (NPM1), a restricted nucleolar localization protein, shuttles between nucleus and cytoplasm. NPM1-mutant protein, aberrant cytoplasmic dislocation of nucleophosmin, occurs in about one third of AML. Deguelin exhibits significant inhibitory effects and induces apoptosis in a variety of cancer cell lines in vitro and in vivo. Our previous researches showed deguelin induced differentiation of NPMc+ AML cells potentially by targeting NPM1-mutant protein. Thus, we extend our investigations of deguelin-induced differentiation activity to an AML xenograft mouse model and clinical samples. After sub-lethal whole body radiation, OCI/AML3 cells were injected intravenously in NOD/SCID mice. 7 days later, either deguelin or vehicle (2%DMSO) was injected intraperitoneally every day. Kaplan-Meier estimates showed 1mg/kg/day deguelin extended the longest survival of mice. The percentage of CD11b+ cells was 17.73%±3.03% in control group, 41.33%±13.22% and 76.15%±10.25% after deguelin-treated for 7 days and 14 days (P < 0.05). Thus, we used 14 days in the following experiments. The percent of CD114+ cells in the vehicle- and deguelin-treated group were 13.29%±10.79% and 47.36%±21.66%, respectively (P < 0.05). The percentage of CD14+ cells in the vehicle- and deguelin-treated group were 22.86%±15.54% and 83.49%±8.06%, respectively (P < 0.05). Wright's Giemsa staining of mouse bone marrow cells showed differentiated. The ratios of differentiated cells were 36.92%±4.99% and 77.01%±1.98% in control and deguelin group (P < 0.01). Median OS of vehicle-treated group was 23 days, while that of deguelin-treated group was 55 days (HR, 0.5818, 95% confidence interval [CI], 0.1623-1.001). Survival of deguelin-treated mice was significantly longer compared with mice injected with vehicle (P=0.0002). IHC and IF staining for mutated NPM1 protein showed bone marrow biopsy densities significantly reduced in deguelin-treated mice which was further confirmed by western blot assay (P < 0.05). We analyzed the toxicity of deguelin (1mg/kg/day for 14 days) to healthy NOD/SCID mice. They had steady movement and no rigidity posture treated with deguelin. HE staining indicated no pathological changes in striatum, substantia nigra pars compacta (SNpc), liver, kidney and heart in deguelin-treated mice. Striatum and SNpc, subjected to immunostaining with anti-tyrosine hydroxylase (TH) antibody, showed deguelin did not alter TH immunoreactivity in striatum and SNpc in vivo (P >0.05). Plasmatic levels of liver enzymes (AST and ALT), renal function (Scr and BUN) and cardiac enzymes (CK-MB and cTnT) in deguelin-treated group had not statistical difference comparing with control group (P>0.05). Thus, deguelin did not cause neurotoxicity, hepatotoxicity, nephrotoxicity and cardiotoxicity to mice. We collected 82 cases of patients with acute leukemia. 24 cases out of 68 AML patients carried mutated NPM1 gene (20 cases of type A; 1 case of type B; 2 cases of type D and 1 case of type I). Bone marrow mononuclear cells (BMMCs) of 6 AML patients were analyzed by FACS analysis. We analyzed patient (pt.) 1 to 4 with NPM1 mutation. FACS showed deguelin increased the percentage of CD11b+ and CD14+ cells of pt.1 in a dose-dependent manner. The percent of CD11b+ and CD14+ cells were 55% and 57.9% separately at 16 nM. Deguelin increased CD14 but CD11b expression of BMMCs of pt.2 in the concentration of 8 nM, 12 nM, 16 nM and 20 nM. The highest percentage of CD14+ cells was 85.5%. The expression of CD14 in cells from pt.3 increased after treating with 16 nM or 20 nM and the highest percentage of CD14+ cells was 49.4%. Deguelin induced differentiation of BMMCs from pt.4. The maximum percent of CD11b+ cells was 42.5% at 16 nM. Moreover, FACS analysis showed deguelin did not promote differentiation in BMMCs from pt.5 and 6 without NPM1 mutation. These results demonstrated deguelin induced differentiation of BMMCs cells from AML patients with NPM1 mutation in vitro. Taken together, mutated NPM1 protein may be one of targets of deguelin, which provides molecular basis for inducing differentiation in NPM1-mutant AML cells. Disclosures No relevant conflicts of interest to declare.
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Téot, Luc. "NPWT for debridement?" Revue Francophone de Cicatrisation 1, no. 1 (January 2017): 68–69. http://dx.doi.org/10.1016/s2468-9114(17)30179-2.
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van Thiel, Sandra. "NPM is dood, lang leve NPM!" Beleid en Maatschappij 42, no. 1 (March 2015): 64–66. http://dx.doi.org/10.5553/benm/138900692015042001009.
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Martelli, M. P., N. Manes, V. Pettirossi, A. Liso, R. Pacini, R. Mannucci, T. Zei, et al. "Absence of nucleophosmin leukaemic mutants in B and T cells from AML with NPM1 mutations: implications for the cell of origin of NPMc+ AML." Leukemia 22, no. 1 (July 19, 2007): 195–98. http://dx.doi.org/10.1038/sj.leu.2404857.
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Sportoletti, Paolo, Silvia Grisendi, Samia M. Majid, Ke Cheng, John G. Clohessy, Agnes Viale, Julie Teruya-Feldstein, and Pier Paolo Pandolfi. "Npm1 is a haploinsufficient suppressor of myeloid and lymphoid malignancies in the mouse." Blood 111, no. 7 (April 1, 2008): 3859–62. http://dx.doi.org/10.1182/blood-2007-06-098251.
Повний текст джерелаАнотація:
Abstract Nucleophosmin (NPM1) gene has been heavily implicated in cancer pathogenesis both as a putative proto-oncogene and tumor suppressor gene. NPM1 is the most frequently mutated gene in acute myeloid leukemia (AML), while deletion of 5q, where NPM1 maps, is frequent in patients with myelodysplastic syndromes (MDS). We have previously shown that mice heterozygous for Npm1 (Npm1+/−) develop a hematologic syndrome with features of human MDS. Here we analyzed Npm1+/− mutants to determine their susceptibility to cancer. Npm1+/− mice displayed a greater propensity to develop malignancies compared with Npm1+/+ mice. The Npm1+/− cohort frequently developed hematologic malignancies of both myeloid and lymphoid origin with myeloid malignancies displaying the highest incidence. Malignant cells retained the wild-type allele with normal localization and expression of Npm1 at the protein level, suggesting that complete Npm1 loss is not a prerequisite for tumorigenesis. Our results conclusively demonstrate that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment.
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Falini, B., B. Bigerna, A. Pucciarini, E. Tiacci, C. Mecucci, S. W. Morris, N. Bolli, et al. "Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): A comparison with NPMc+ AML." Leukemia 20, no. 2 (December 8, 2005): 368–71. http://dx.doi.org/10.1038/sj.leu.2404068.
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Pratcorona, Marta, Montserrat Torrebadell, Neus Villamor, Maria Rozman, Mireia Camós, Ana Carrió, Dolors Costa, Marina Díaz Beyáa, Emili Montserrat, and Jordi Esteve. "Assessment of CEBPA Mutations Might Contribute to a Better Prognostic Assignment in Patients with Intermediate-Risk Cytogenetics Acute Myeloid Leukemia (AML)." Blood 114, no. 22 (November 20, 2009): 2611. http://dx.doi.org/10.1182/blood.v114.22.2611.2611.
Повний текст джерелаАнотація:
Abstract Abstract 2611 Poster Board II-587 The prognostic heterogeneity of patients with intermediate-risk cytogenetics AML (AML-IR) is mostly clarified by determination of mutations of NPM1 gene (NPMmut) and internal-tandem duplication of FLT3 gene (FLT3-ITD). Nonetheless, other genetic lesions described in this population might contribute to a better prognostic categorization. In this context, we analyzed the presence of CEBPA mutations and associated features in patients with AML-IR lacking both NPMmut and FLT3-ITD. Overall, 136 patients (51% female; median age: 53, range: 17=74) diagnosed with de novo AML-IR (MRC definition) in our institution between 1994 and 2008 who received standard AML chemotherapy were included in the analysis. CEBPA mutations (CEBPAmut) were investigated by whole gene sequencing using 4 primer pairs according to previously reported methods (Fröhling et al, 2004). Sixty-five patients (48%) harbored NPM1 mutations, 30 of them having concomitant FLT3-ITD. Among NPM1 wild-type patients (NPMwt), FLT3-ITD, CEBPA and MLL mutations were detected in 18, 11, and 5 patients, respectively. Regarding cases with CEBPAmut, biallelic mutations were found in 8 patients, including 6 cases with combined mutations of N-terminal and bZIP domains and two homozygous mutations, whereas a single mutation in bZIP domain was found in the three remaining patients. As compared to patients with wild-type CEBPA (CEBPAwt), those with CEBPAmut were younger (29 vs. 53, p=0.027), and showed a trend to male predominance (73 vs. 46%, p=0.09) and lower WBC count at presentation (16 vs. 33.5 × 109/L, p=.095). Of note, CD7 antigen was aberrantly expressed in virtually all CEBPAmut cases (10/11), compared to only 21% of CEBPAwt patients (p<0.001). Moreover, an abnormal karyotype was observed in 4 patients with CEBPAmut. In the overall series, complete response rate (CR), survival (OS), and relapse incidence (RI) were 83%, 39±4% (5-yr), and 50±5% (5-yr), respectively. Independent favorable factors for survival were younger age (<median; RR: 1.9, 95% CI: 1.2-3, p=0.004), low WBC count at diagnosis (< median; RR: 1.7, 95% CI: 1.1–2.6, p=0.025) and NPMmut/FLT3-ITDneg status (RR: 2.4, 95% CI: 1.3–4.2, p=0.003). Remarkably, patients with CEBPAmut showed a favorable outcome, with a trend for a more prolonged survival, compared to patients with a high-risk NPM/FLT3 status (5-year OS in pts <60 years: 74±16% vs. 33±6%, p=0.087). Based on these results, patients were grouped in a favorable (i.e., either NPMmut /FLT3-ITDneg or CEBPAmut, FAV) or unfavorable molecular category (i.e., those with FLT3-ITD, double NPMwt and CEBPAwt configuration, or MLL abnormalities, UNFAV); these two groups had independent prognostic impact on OS (RR: 2.3, 95% CI: 1.3–4, p=0.001; see figure), RI (RR: 2.2, 95% CI: 1.2–4.3, p=0.016), and leukemia-free survival (RR: 2.2, 95% CI: 1.3–3.7, p=0.002). Importantly, the outcome of patients in the FAV group did not differ according to post-remission treatment (autologous vs. allogeneic stem-cell transplantation, SCT), whereas relapse risk was significantly higher in patients with unfavorable markers who received autologous SCT (60±13% vs. 18±12%, p=0.03). In summary, the assessment of CEBPA mutations, especially in patients lacking NPMmut and FLT3-ITD and expressing CD7 antigen, may refine the molecular prediction of prognosis and guide therapeutic strategy in patients with intermediate-risk AML. Disclosures: No relevant conflicts of interest to declare.