NPM1

Gene Summary

Gene:NPM1; nucleophosmin
Aliases: B23, NPM
Location:5q35.1
Summary:This gene encodes a phosphoprotein which moves between the nucleus and the cytoplasm. The gene product is thought to be involved in several processes including regulation of the ARF/p53 pathway. A number of genes are fusion partners have been characterized, in particular the anaplastic lymphoma kinase gene on chromosome 2. Mutations in this gene are associated with acute myeloid leukemia. More than a dozen pseudogenes of this gene have been identified. Alternative splicing results in multiple transcript variants.[provided by RefSeq, Nov 2009]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:nucleophosmin
Source:NCBIAccessed: 16 March, 2017

Ontology:

What does this gene/protein do?
Show (40)

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 16 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • DNA Mutational Analysis
  • Peptides
  • Immunophenotyping
  • Risk Factors
  • ALK
  • Proto-Oncogene Proteins c-kit
  • Residual Disease
  • Neoplasm Proteins
  • Follow-Up Studies
  • Tissue Donors
  • Cytogenetic Analysis
  • Myelodysplastic Syndromes
  • Leukemic Gene Expression Regulation
  • Biomarkers, Tumor
  • Karyotype
  • Karyotyping
  • Adolescents
  • Risk Assessment
  • Flow Cytometry
  • Age Factors
  • Cancer Gene Expression Regulation
  • Antineoplastic Agents
  • Polymerase Chain Reaction
  • Genotype
  • Childhood Cancer
  • Acute Myeloid Leukaemia
  • Cohort Studies
  • Isocitrate Dehydrogenase
  • Non-Hodgkin Lymphoma
  • Chromosome Aberrations
  • DNA (Cytosine-5-)-Methyltransferase
  • RARA
  • Promoter Regions
  • Leukaemia
  • Mutation
  • NPM1
  • CCAAT-Enhancer-Binding Proteins
  • Chromosome 5
  • Hematopoietic Stem Cell Transplantation
  • Gene Expression
  • Gene Expression Profiling
  • Recurrence
  • Disease-Free Survival
  • Tandem Repeat Sequences
  • Bone Marrow
Tag cloud generated 16 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: NPM1 (cancer-related)

Kanazawa Y, Yamashita Y, Fujiwara M, et al.
Two Relapsed Stage III Childhood Anaplastic Large Cell Lymphoma Patients with NPM-ALK Fusion in Bone Marrow from Initial Diagnosis.
Acta Med Okayama. 2016; 70(6):503-506 [PubMed] Related Publications
Childhood anaplastic large cell lymphoma (ALCL) accounts for approx. 10-30% of cases of non-Hodgkin lymphoma, and the ALCL99 study reported 60-75 disease-free survival; however, a relatively high relapse rate was observed (25-30% ). We report 2 patients with Stage III ALCL who relapsed 6-18 months after the end of ALCL99 chemotherapy. A retrospective molecular analysis identified the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) fusion gene in the first diagnostic bone marrow samples taken from both patients. However, antibodies against the ALK protein appeared to be relatively low in the serum of both patients (×100 and ×750). An increase in chemotherapy intensity may be beneficial if Stage III ALCL patients are shown to be NPM-ALK chimera-positive in the first diagnostic bone marrow sample.

Juncà J, Garcia O, Garcia-Caro M, et al.
CD34 expression and the outcome of nucleophosmin 1-mutated acute myeloid leukemia.
Ann Hematol. 2016; 95(12):1949-1954 [PubMed] Related Publications
CD34 positivity has been considered as an adverse prognostic factor in acute myeloid leukemia (AML). Although nucleophosmin 1-mutated (NPM1m) AML is usually CD34 negative, this marker may be expressed at diagnosis or acquired at relapse in a variable number of cases. Our objective was to ascertain if CD34 expression has any influence on the general outcome of this form of acute leukemia. Analysis of clinical outcome (complete remissions, relapses, disease-free survival, and overall survival) was performed depending on the degree of expression of CD34 determined by flow cytometry, in 67 adult patients with NPM1m AML. CD34 expression did not have any influence on the variables analyzed whatever the percentage of blasts expressing this marker. In contrast to other forms of AML, CD34 expression is not an unfavorable prognostic factor in NPM1m AML, neither at diagnosis nor at relapse.

Abdelhamid E, Besbes S, Renneville A, et al.
Minimal Residual Disease assessment of IDH1/2 mutations in Acute Myeloid Leukemia by LNA-RQ-PCR.
Tunis Med. 2016; 94(3):190-7 [PubMed] Related Publications
BACKGROUND: With the growing importance of minimal residual disease (MRD) monitoring and the recent discover of IDH mutations in acute myeloid leukemia (AML), the quantification of this molecular marker provides the possibility to monitor the disease progression and the therapy efficacy.
OBJECTIVE: The aim of this study is to assess the MRD in AML for the first time with IDH1 and IDH2 gene mutations in 15 AML patients.
METHODS: We have screened R132 IDH1, R140 IDH2 and R172 IDH2 mutations by PCR amplification and direct sequencing and we have quantified them for the first time by RQ-PCR using reverse primers modified by an LNA. A good sensitivity has been obtained. MRD rates obtained by LNA-RQ-PCR were used to draw kinetics of the disease evolution during the follow-up.
RESULTS: IDH1/2 Results were compared to NPM1 mutation and WT1 over expression and have showed coherent kinetic between MRD rates in 7/11 cases. For the rest, the direct sequencing and the high resolution melting (HRM) assay have confirmed the quantification Results in diagnosis but not in residual samples.
CONCLUSION: Some optimization will be necessary to improve the mutated allele amplification. The LNA-RQ-PCR might be an easy and less cost method used in a small laboratory for myeloid leukemia MRD assessment using IDH1/2 mutations.

Bogdanic M, Ostojic Kolonic S, Kaic G, et al.
Fine-needle aspiration cytology yield as a basis for morphological, molecular, and cytogenetic diagnosis in alk-positive anaplastic large cell lymphoma with atypical clinical presentation.
Diagn Cytopathol. 2017; 45(1):51-54 [PubMed] Related Publications
ALK positive anaplastic large cell lymphoma is a T-cell lymphoma usually occurring in children and young adults. It frequently involves lymph nodes and extranodal sites and is associated with favorable prognosis. A 20-year old man was admitted for painful mass in the left axilla with overlying skin redness. Clinical presentation and US findings were highly suspicious for sarcoma. Definitive diagnosis was established cytolologically and using ancillary technologies from cytological samples. Fine needle aspiration cytology of tumor mass (lymph node conglomerate and surrounding tissue) show predominance of large, pleomorphic, atypical cells with large nuclei and vacuolised cytoplasm. Atypical cells immunocytochemically were positive for LCA, CD30, CD3, EMA, and ALK; negative for CD15 and CD56. NPM-ALK transcript was detected by reverse transcriptase-polymerase chain reaction (RT-PCT). Molecular analysis of TCRß and TCRγ genes demonstrated clonal TCR genes rearrangement. Complex karyotype with multiple numerical and structural changes was found on conventional cytogenetics. These findings excluded sarcoma and corroborated the diagnosis of ALK positive ALCL. Cutaneous involvement in ALCL can clinically mimic sarcoma, especially in cases with localized disease without B symptoms. In those cases, immunostaining, PCR, and conventional cytogenetics are helpful to exclude sarcoma. Diagn. Cytopathol. 2017;45:51-54. © 2016 Wiley Periodicals, Inc.

Marjanovic I, Kostic J, Stanic B, et al.
Parallel targeted next generation sequencing of childhood and adult acute myeloid leukemia patients reveals uniform genomic profile of the disease.
Tumour Biol. 2016; 37(10):13391-13401 [PubMed] Related Publications
The age-specific differences in the genetic mechanisms of myeloid leukemogenesis have been observed and studied previously. However, NGS technology has provided a possibility to obtain a large amount of mutation data. We analyzed DNA samples from 20 childhood (cAML) and 20 adult AML (aAML) patients, using NGS targeted sequencing. The average coverage of high-quality sequences was 2981 × per amplicon. A total of 412 (207 cAML, 205 aAML) variants in the coding regions were detected; out of which, only 122 (62 cAML and 60 aAML) were potentially protein-changing. Our results confirmed that AML contains small number of genetic alterations (median 3 mutations/patient in both groups). The prevalence of the most frequent single gene AML associated mutations differed in cAML and aAML patient cohorts: IDH1 (0 % cAML, 5 % aAML), IDH2 (0 % cAML, 10 % aAML), NPM1 (10 % cAML, 35 % aAML). Additionally, potentially protein-changing variants were found in tyrosine kinase genes or genes encoding tyrosine kinase associated proteins (JAK3, ABL1, GNAQ, and EGFR) in cAML, while among aAML, the prevalence is directed towards variants in the methylation and histone modifying genes (IDH1, IDH2, and SMARCB1). Besides uniform genomic profile of AML, specific genetic characteristic was exclusively detected in cAML and aAML.

Jo SY, Park SH, Kim IS, et al.
Correlation of NPM1 Type A Mutation Burden With Clinical Status and Outcomes in Acute Myeloid Leukemia Patients With Mutated NPM1 Type A.
Ann Lab Med. 2016; 36(5):399-404 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Nucleophosmin gene (NPM1) mutation may be a good molecular marker for assessing the clinical status and predicting the outcomes in AML patients. We evaluated the applicability of NPM1 type A mutation (NPM1-mutA) quantitation for this purpose.
METHODS: Twenty-seven AML patients with normal karyotype but bearing the mutated NPM1 were enrolled in the study, and real-time quantitative PCR of NPM1-mutA was performed on 93 bone marrow (BM) samples (27 samples at diagnosis and 56 at follow-up). The NPM1-mutA allele burdens (represented as the NPM1-mutA/Abelson gene (ABL) ratio) at diagnosis and at follow-up were compared.
RESULTS: The median NPM1-mutA/ABL ratio was 1.3287 at diagnosis and 0.092 at 28 days after chemotherapy, corresponding to a median log10 reduction of 1.7061. Significant correlations were observed between BM blast counts and NPM1-mutA quantitation results measured at diagnosis (γ=0.5885, P=0.0012) and after chemotherapy (γ=0.5106, P=0.0065). Total 16 patients achieved morphologic complete remission at 28 days after chemotherapy, and 14 (87.5%) patients showed a >3 log10 reduction of the NPM1-mutA/ABL ratio. The NPM1-mutA allele was detected in each of five patients who had relapsed, giving a median increase of 0.91-fold of the NPM1-mutA/ABL ratio at relapse over that at diagnosis.
CONCLUSIONS: The NPM1-mutA quantitation results corresponded to BM assessment results with high stability at relapse, and could predict patient outcomes. Quantitation of the NPM1-mutA burden at follow-up would be useful in the management of AML patients harboring this gene mutation.

Papaemmanuil E, Gerstung M, Bullinger L, et al.
Genomic Classification and Prognosis in Acute Myeloid Leukemia.
N Engl J Med. 2016; 374(23):2209-21 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice.
METHODS: We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes.
RESULTS: We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials.
CONCLUSIONS: The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).

Hamadou WS, Bourdon V, Gaildrat P, et al.
Mutational analysis of JAK2, CBL, RUNX1, and NPM1 genes in familial aggregation of hematological malignancies.
Ann Hematol. 2016; 95(7):1043-50 [PubMed] Related Publications
Familial aggregation of hematological malignancies has been reported highlighting inherited genetic predisposition. In this study, we targeted four candidate genes: JAK2 and RUNX1 genes assuring a prominent function in hematological process and CBL and NPM1 as proto-oncogenes. Their disruption was described in several sporadic hematological malignancies. The aim of this study is to determine whether JAK2, CBL, RUNX1, and NPM1 germline genes mutations are involved in familial hematological malignancies. Using direct sequencing, we analyzed JAK2 (exons 12 and 14); CBL (exons 7, 8 and 9); NPM1 (exon 12) and the entire RUNX1 in 88 independent families belonging to Tunisian and French populations. Twenty-one sporadic acute leukemias were included in this study. We reported a heterozygous intronic c.1641 + 6 T > C JAK2 variant (rs182123615) found in two independent familial cases diagnosed with gastric lymphoma and Hodgkin lymphoma. The in silico analysis suggested a potential impact on splicing, but the functional splicing minigene reporter assay on rs182123615 variant showed no aberrant transcripts. In one sporadic acute myeloblastic leukemia, we reported an insertion 846 in. TGTT in exon 12 of NPM1 gene that may impact the normal reading frame. The rs182123615 JAK2 variant was described in several contexts including myeloproliferative neoplasms and congenital erythrocytosis and was supposed to be pathogenic. Through this current study, we established the assessment of pathogenicity of rs182123615 and we classified it rather as rare polymorphism.

Sloan CE, Luskin MR, Boccuti AM, et al.
A Modified Integrated Genetic Model for Risk Prediction in Younger Patients with Acute Myeloid Leukemia.
PLoS One. 2016; 11(4):e0153016 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Although cytogenetics-based prognostication systems are well described in acute myeloid leukemia (AML), overall survival (OS) remains highly variable within risk groups. An integrated genetic prognostic (IGP) model using cytogenetics plus mutations in nine genes was recently proposed for patients ≤60 years to improve classification. This model has not been validated in clinical practice.
METHODS AND FINDINGS: We retrospectively studied 197 patients with newly diagnosed de novo AML. We compared OS curves among the mutational profiles defined by the IGP model. The IGP model assigned patients with intermediate cytogenetics as having favorable, intermediate or unfavorable mutational profiles. The IGP model reassigned 50 of 137 patients with intermediate cytogenetics to favorable or unfavorable mutational profiles. Median OS was 2.8 years among 14 patients with intermediate cytogenetics and favorable mutational profiles (mutant NPM1 and mutant IDH1 or IDH2) and 1.3 years among patients with intermediate mutational profiles. Among patients with intermediate cytogenetics labeled as having unfavorable mutational profiles, median OS was 0.8 years among 24 patients with FLT3-ITD positive AML and high-risk genetic changes (trisomy 8, TET2 and/or DNMT3A) and 1.7 years among 12 patients with FLT3-ITD negative AML and high-risk mutations (TET2, ASXL1 and/or PHF6). OS for patients with intermediate cytogenetics and favorable mutational profiles was similar to OS for patients with favorable cytogenetics (p = 0.697) and different from patients with intermediate cytogenetics and intermediate mutational profiles (p = 0.028). OS among patients with FLT3-ITD positive AML and high-risk genetic changes was similar to patients with unfavorable cytogenetics (p = 0.793) and different from patients with intermediate IGP profile (p = 0.022). Patients with FLT3-ITD negative AML and high-risk mutations, defined as 'unfavorable' in the IGP model, had OS similar to patients with intermediate IGP profile (p = 0.919).
CONCLUSIONS: The IGP model was not completely validated in our cohort. However, mutations in six out of the nine genes can be used to characterize survival (NPMI, IDH1, IDH2, FLT3-ITD, TET2, DNMT3A) and allow for more robust prognostication in the patients who are re-categorized by the IGP model. These mutations should be incorporated into clinical testing for younger patients outside of clinical trials, in order to guide therapy.

Hiemenz MC, Kadauke S, Lieberman DB, et al.
Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing.
PLoS One. 2016; 11(4):e0152851 [PubMed] Free Access to Full Article Related Publications
Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two FLT3 internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.

Cui F, Zan X, Li Y, et al.
Grifola frondosa Glycoprotein GFG-3a Arrests S phase, Alters Proteome, and Induces Apoptosis in Human Gastric Cancer Cells.
Nutr Cancer. 2016; 68(2):267-79 [PubMed] Related Publications
GFG-3a is a novel glycoprotein previously purified from the fermented mycelia of Grifola frondosa with novel sugar compositions and protein sequencing. The present study aims to investigate its effects on the cell cycle, differential proteins expression, and apoptosis of human gastric cancer SGC-7901 cells. Our findings revealed that GFG-3a induced the cell apoptosis and arrested cell cycle at S phase. GFG-3a treatment resulted in the differential expression of 21 proteins in SGC-7901 cells by upregulating 10 proteins including RBBP4 associated with cell cycle arrest and downregulating 11 proteins including RUVBL1, NPM, HSP90AB1, and GRP78 involved in apoptosis and stress response. qRT-PCR and Western blot analysis also suggested that GFG-3a could increase the expressions of Caspase-8/-3, p53, Bax, and Bad while decrease the expressions of Bcl2, Bcl-xl, PI3K, and Akt1. These results indicated that the stress response, p53-dependent mitochondrial-mediated, Caspase-8/-3-dependent, and PI3k/Akt pathways were involved in the GFG-3a-induced apoptosis process in SGC-7901 cells. These findings might provide a basis to prevent or treat human gastric cancer with GFG-3a and understand the tumor-inhibitory molecular mechanisms of mushroom glycoproteins.

Nichol JN, Galbraith MD, Kleinman CL, et al.
NPM and BRG1 Mediate Transcriptional Resistance to Retinoic Acid in Acute Promyelocytic Leukemia.
Cell Rep. 2016; 14(12):2938-49 [PubMed] Article available free on PMC after 29/03/2017 Related Publications
Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA)-sensitive acute promyelocytic leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrant interaction among PML/RARA, nucleophosmin (NPM), and topoisomerase II beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML); therefore, our model may be applicable to other more common leukemias driven by NPM.

Nardi V, Hasserjian RP
Genetic Testing in Acute Myeloid Leukemia and Myelodysplastic Syndromes.
Surg Pathol Clin. 2016; 9(1):143-63 [PubMed] Related Publications
Cytogenetic analysis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is essential for disease diagnosis, classification, prognostic stratification, and treatment guidance. Molecular genetic analysis of CEBPA, NPM1, and FLT3 is already standard of care in patients with AML, and mutations in several additional genes are assuming increasing importance. Mutational analysis of certain genes, such as SF3B1, is also becoming an important tool to distinguish subsets of MDS that have different biologic behaviors. It is still uncertain how to optimally combine karyotype with mutation data in diagnosis and risk-stratification of AML and MDS, particularly in cases with multiple mutations and/or several mutationally distinct subclones.

Hua X, Hyland PL, Huang J, et al.
MEGSA: A Powerful and Flexible Framework for Analyzing Mutual Exclusivity of Tumor Mutations.
Am J Hum Genet. 2016; 98(3):442-55 [PubMed] Article available free on PMC after 29/03/2017 Related Publications
The central challenges in tumor sequencing studies is to identify driver genes and pathways, investigate their functional relationships, and nominate drug targets. The efficiency of these analyses, particularly for infrequently mutated genes, is compromised when subjects carry different combinations of driver mutations. Mutual exclusivity analysis helps address these challenges. To identify mutually exclusive gene sets (MEGS), we developed a powerful and flexible analytic framework based on a likelihood ratio test and a model selection procedure. Extensive simulations demonstrated that our method outperformed existing methods for both statistical power and the capability of identifying the exact MEGS, particularly for highly imbalanced MEGS. Our method can be used for de novo discovery, for pathway-guided searches, or for expanding established small MEGS. We applied our method to the whole-exome sequencing data for 13 cancer types from The Cancer Genome Atlas (TCGA). We identified multiple previously unreported non-pairwise MEGS in multiple cancer types. For acute myeloid leukemia, we identified a MEGS with five genes (FLT3, IDH2, NRAS, KIT, and TP53) and a MEGS (NPM1, TP53, and RUNX1) whose mutation status was strongly associated with survival (p = 6.7 × 10(-4)). For breast cancer, we identified a significant MEGS consisting of TP53 and four infrequently mutated genes (ARID1A, AKT1, MED23, and TBL1XR1), providing support for their role as cancer drivers.

Marcinkowska-Swojak M, Handschuh L, Wojciechowski P, et al.
Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification.
Mutat Res. 2016; 786:14-26 [PubMed] Related Publications
The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The presented method is simple, reliable and cost-effective. It can be easily introduced into clinical practice or developed to target both less-frequent mutations in the NPM1 gene and other cancer-related genes.

Xian J, Shao H, Chen X, et al.
Nucleophosmin Mutants Promote Adhesion, Migration and Invasion of Human Leukemia THP-1 Cells through MMPs Up-regulation via Ras/ERK MAPK Signaling.
Int J Biol Sci. 2016; 12(2):144-55 [PubMed] Article available free on PMC after 29/03/2017 Related Publications
Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a unique subgroup in the new classification of myeloid neoplasm, and the AML patients with mutated NPM1 frequently present extramedullary infiltration, but how NPM1 mutants regulate this process remains elusive. In this study, we found that overexpression of type A NPM1 gene mutation (NPM1-mA) enhanced the adhesive, migratory and invasive potential in THP-1 AML cells lacking mutated NPM1. NPM1-mA had up-regulated expression and gelatinolytic matrix metalloprotease-2 (MMP-2)/MMP-9 activity, as assessed by real-time PCR, western blotting and gelatin zymography. Following immunoprecipitation analysis to identify the interaction of NPM1-mA with K-Ras, we focused on the effect of NPM1-mA overexpression on the Ras/Mitogen-activated protein kinase (MAPK) signaling axis and showed that NPM1-mA increased the MEK and ERK phosphorylation levels, as evaluated by western blotting. Notably, a specific inhibitor of the ERK/MAPK pathway (PD98059), but not p38/MAPK, JNK/MAPK or PI3-K/AKT inhibitors, markedly decreased the cell invasion numbers in a transwell assay. Further experiments demonstrated that blocking the ERK/MAPK pathway by PD98059 resulted in reduced MMP-2/9 protein levels and MMP-9 activity. Additionally, NPM1-mA overexpression had down-regulated gene expression and protein production of tissue inhibitor of MMP-2 (TIMP-2) in THP-1 cells. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that MMP-2 was overexpressed in AML patient samples with NPM1 mutated and high MMP-2 expression associated with leukemic skin infiltration. Taken together, our results reveal that NPM1 mutations contribute to the invasive potential of AML cells through MMPs up-regulation via Ras/ERK MAPK signaling pathway activation and offer novel insights into the potential role of NPM1 mutations in leukemogenesis.

Gill H, Leung AY, Kwong YL
Molecularly targeted therapy in acute myeloid leukemia.
Future Oncol. 2016; 12(6):827-38 [PubMed] Related Publications
Acute myeloid leukemia (AML) is molecularly heterogeneous. Formerly categorized cytogenetically and molecularly, AML may be classified by genomic and epigenomic analyses. These genetic lesions provide therapeutic targets. Genes targeted currently include mutated FLT3, NPM1 and KIT with drugs entering Phase III trials. Complete remission can be achieved in relapsed/refractory AML, albeit mostly transient. Mutated epigenetic modifiers, including DNMT3A, IDH1/2 and TET2, can be targeted by small molecule inhibitors, hypomethylating agents and histone deacetylase inhibitors. Other agents include cellular signaling pathway inhibitors and monoclonal antibodies against myeloid-associated antigens. Combinatorial strategies appear logical, mostly involving smaller molecular inhibitors partnering with hypomethylating agents. Currently limited to relapsed/refractory AML, targeted therapies are increasingly tested in frontline treatment with or without standard chemotherapy.

Ivey A, Hills RK, Simpson MA, et al.
Assessment of Minimal Residual Disease in Standard-Risk AML.
N Engl J Med. 2016; 374(5):422-33 [PubMed] Related Publications
BACKGROUND: Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission.
METHODS: We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction.
RESULTS: Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status.
CONCLUSIONS: The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).

Malcolm TI, Villarese P, Fairbairn CJ, et al.
Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress.
Nat Commun. 2016; 7:10087 [PubMed] Article available free on PMC after 29/03/2017 Related Publications
Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM-ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy.

Shiba N, Ohki K, Kobayashi T, et al.
High PRDM16 expression identifies a prognostic subgroup of pediatric acute myeloid leukaemia correlated to FLT3-ITD, KMT2A-PTD, and NUP98-NSD1: the results of the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 trial.
Br J Haematol. 2016; 172(4):581-91 [PubMed] Related Publications
Recent reports described the NUP98-NSD1 fusion as an adverse prognostic marker for acute myeloid leukaemia (AML) and PRDM16 (also known as MEL1) as the representative overexpressed gene in patients harbouring NUP98-NSD1 fusion. PRDM16 gene expression levels were measured via real-time polymerase chain reaction in 369 paediatric patients with de novo AML, of whom 84 (23%) exhibited PRDM16 overexpression (PRDM16/ABL1 ratio ≥0·010). The frequencies of patients with high or low PRDM16 expression differed widely with respect to each genetic alteration, as follows: t(8;21), 4% vs. 96%, P < 0·001; inv(16), 0% vs. 100%, P < 0·001; KMT2A (also termed MLL)- partial tandem duplication, 100% vs. 0%, P < 0·001; NUP98-NSD1, 100% vs. 0%, P < 0·001. The overall survival (OS) and event-free survival (EFS) among PRDM16-overexpressing patients were significantly worse than in patients with low PRDM16 expression (3-year OS: 51% vs. 81%, P < 0·001, 3-year EFS: 32% vs. 64%, P < 0·001) irrespective of other cytogenetic alterations except for NPM1. PRDM16 gene expression was particularly useful for stratifying FLT3-internal tandem duplication-positive AML patients (3-year OS: high = 30% vs. low = 70%, P < 0·001). PRDM16 overexpression was highly recurrent in de novo paediatric AML patients with high/intermediate-risk cytogenetic profiles and was independently associated with an adverse outcome.

Gou H, Zhou J, Ye Y, et al.
The prevalence and clinical profiles of FLT3-ITD, FLT3-TKD, NPM1, C-KIT, DNMT3A, and CEBPA mutations in a cohort of patients with de novo acute myeloid leukemia from southwest China.
Tumour Biol. 2016; 37(6):7357-70 [PubMed] Related Publications
While a substantial amount of data on gene mutations related to acute myeloid leukemia (AML) prognosis from western and other populations have been reported, these studies largely describe one or two genes. Additionally, in southwest China, only insufficient data exist regarding FLT3-ITD, FLT3-TKD, NPM1, C-KIT, DNMT3A, and CEBPA mutations have been widely used in clinical settings. Therefore, a comprehensive study about these mutations of clinical importance in the prognosis of AML in western China is necessary. In a cohort of 255 patients with de novo AML, we retrospectively analyzed the prevalence of the six gene mutations, and then we assessed the results in conjunction with clinical characteristics and treatment responses. As for the frequencies of these mutations, the NPM1 mutation occurred most frequently (17.7 %; 42/237), followed by the CEBPA mutation (15.0 %; 19/127) and the FLT3-ITD mutation (10.2 %; 25/244). The frequencies of the FLT3-TKD, DNMT3A, and C-KIT mutations were 3.7 % (9/234), 4.0 % (9/225) and 4.2 % (10/238), respectively. These mutations were closely related to clinical characteristics including FAB classification, gender and age, hemogram, blasts (%), fusion genes, and immunophenotypes. Additionally, a higher complete remission (CR) rate was found in NPM1-mutated patients. The occurrence of these mutations is variable among different countries and regions worldwide, which may provide clues to the etiology of AML. Besides, we identified new clinical characteristics that advance our understanding of these mutations and further clarify the involvement of these mutations in the development of leukemia.

Rastogi P, Naseem S, Varma N, Varma S
Nucleophosmin mutation in de-novo acute myeloid leukemia.
Asia Pac J Clin Oncol. 2016; 12(1):77-85 [PubMed] Related Publications
OBJECTIVE: Acute myeloid leukemia (AML) with mutated nucleophosmin gene (NPM1) has distinctive clinical, hematological and molecular features, and is included as a provisional entity in 2008 World Health Organization classification. In this study, we analyzed the frequency and features of AML with mutated NPM1 in Indian patients.
METHODS: One-hundred consecutive patients of de-novo AML were evaluated for NPM1 mutation and their features were compared with unmutated NPM1 patients.
RESULTS: AML with mutated NPM1 was seen in 21% cases. There was female preponderance with median age of 51 years. Distinguishing Features in mutated group were less bleeding manifestations and bone pains; more lymphadenopathy; higher median total leukocyte and platelet count; less frequency of pancytopenia and more preserved megakaryocytes. Morphologically, cup-shaped nuclei in peripheral blood blasts correlated with NPM1 mutation (p <0.01), but not bone marrow blasts. Among the French-American-British subtypes, NPM1 mutation was seen in M1, M4 and M2 subtypes but not in M0 and M3. Immunophenotypically, there was statistically significant negativity for CD34, strong association with monocytic markers (especially CD11c), CD123 was seen at higher frequency and higher mean fluorescence intensity (MFI) values for CD33 were observed in mutated cases.
CONCLUSIONS: Important findings in this study that have not been highlighted in detail in previous studies in NPM1-mutated cases include less bleeding manifestations and bone pains, lower frequency of pancytopenia and more preserved magakaryocytes, higher CD123 expression and higher MFI values for CD33. Presence of blasts with cup-shaped nuclei correlated with NPM1 mutation.

Azari-Yam A, Bagheri SD, Tavakkoly-Bazzaz J, et al.
NPM1 Mutation Detection in Acute Myeloid Leukemia: A Method Comparison Study.
Genet Test Mol Biomarkers. 2016; 20(2):63-6 [PubMed] Related Publications
BACKGROUND: Mutations in the nucleophosmin (NPM1) gene have been used as molecular biomarkers for prognostication of patients with adult acute myeloid leukemia (AML).
METHODS: We designed a rapid and sensitive method using the allele-specific-refractory mutation system-polymerase chain reaction (ARMS-PCR) to detect the most common mutations of NPM1 gene, which are mostly four base pair insertions and compared its efficacy with direct sequencing and capillary electrophoresis which served as the gold standards.
RESULTS: The incidence of mutation was 22% (33% of patients with normal karyotypes had mutation compared with 16% of patients with abnormal karyotypes) based on the results obtained with capillary electrophoresis analysis and direct sequencing. All of the specimens determined to be mutation-positive by the gold standard tests were also positive by the ARMS-PCR method. Significantly, the ARMS-PCR test also helped determine the mutation status of an extra set of patients who had low call rates on capillary electrophoresis and appeared normal on direct sequencing.
DISCUSSION: The low mutation rate in some patients hindered its detection in the gold standard assays because of the interference of the mutation signal by high background noise. The low sensitivity of the gold standard assays for detecting low copy number mutations rates thus increase their risk of producing false negative results that adversely affects prognostication and therapy. Our results suggest that the mutation detection rate of the ARMS-PCR assay is better than existing tests. This is most probably because of the fact that in an ARMS-PCR-based method, the mutated variant is specifically amplified, based on a mutation-specific primer.
CONCLUSIONS: We conclude that the high sensitivity of the ARMS-based method together with its rapidity and low expense should make it a suitable choice for clinical laboratories.

Garcia JS, Huang M, Medeiros BC, Mitchell BS
Selective Toxicity of Investigational Ixazomib for Human Leukemia Cells Expressing Mutant Cytoplasmic NPM1: Role of Reactive Oxygen Species.
Clin Cancer Res. 2016; 22(8):1978-88 [PubMed] Related Publications
PURPOSE: This study was performed to determine whether the investigational proteasome inhibitor ixazomib demonstrated selective antineoplastic activity against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1 gene and to gain a better understanding of its mechanisms of action.
EXPERIMENTAL DESIGN: The cytotoxic effects of ixazomib treatment were analyzed in human acute myelogenous leukemia (AML) cell lines and primary AML samples expressing wild-type or mutated NPM1 (NPMc(+)). The potential roles of oxidative stress in mediating cytotoxic activity were determined using flow cytometry, enzyme-based assays, and Western blots.
RESULTS: Apoptosis induced by ixazomib was abrogated by knockdown of NPM1/NPMc(+)expression using an inducible shRNA construct and enhanced by NPMc(+)overexpression. Cytotoxicity was associated with superoxide generation and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells expressing NPMc(+)had significantly reduced levels of intracellular glutathione and NADPH associated with reduced antioxidant responses to drug treatment. Treatment of 3 patients with relapsed NPMc(+)AML resulted in an antileukemic effect in 1 patient as demonstrated by a marked reduction of leukemic blasts in the peripheral blood. Efficacy was associated with superoxide generation, reduced glutathione levels, and reduced mRNA and protein expression of antioxidant effectors in responding cells.
CONCLUSIONS: In this study, a direct association was observed between NPMc(+)expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. These data suggest that intracellular determinants of antioxidant responses may be good predictors of therapeutic response to ixazomib.

Falini B, Martelli MP
Impact of genomics in the clinical management of patients with cytogenetically normal acute myeloid leukemia.
Best Pract Res Clin Haematol. 2015 Jun-Sep; 28(2-3):90-7 [PubMed] Related Publications
Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease. Cytogenetics and FISH have contributed to the stratification of AML patients into favorable, intermediate, and unfavorable risk categories. However, until recently, the prognostic stratification and treatment decision for the intermediate risk category, mostly comprising AML patients with normal cytogenetics (CN-AML), has been difficult due to the scarce knowledge of the molecular alterations underlying this large AML subgroup (which accounts for about 50% of all adult AML). During the past decade, the discovery of numerous mutations associated with CN-AML has resulted in significant advances in the AML field. Here, we review the biological characteristics of the most common mutations underlying CN-AML and outline their clinical impact in the following settings: (i) definition of new molecular leukemia entities in the WHO classification; (ii) risk stratification of CN-AML patients according to mutational profile; and (iii) monitoring of minimal residual disease by specific quantitative molecular assays.

Welsh KJ, Nedelcu E, Wahed A, et al.
Bioinformatics Analysis to Determine Prognostic Mutations of 72 de novo Acute Myeloid Leukemia Cases from the Cancer Genome Atlas (TCGA) with 23 Most Common Mutations and no Abnormal Cytogenetics.
Ann Clin Lab Sci. 2015; 45(5):515-21 [PubMed] Related Publications
OBJECTIVES: Up to 40% of acute myeloid leukemia (AML) patients have normal cytogenetics (CN-AML) but they may have gene mutations. An important issue in the treatment of CN-AML is how gene mutation patterns may help with patient management. The Cancer Genome Atlas (TCGA) database has data from 200 cases of de novo AML including cytogenetics, gene mutations, and survival duration (prognosis).
METHODS: Cases with the most common mutations and no cytogenetic abnormalities were selected from the TCGA. Unsupervised neural network analysis was performed to group them into clusters according to their pattern of mutations and survival.
RESULTS: 72 cases of CN-AML with the 23 most common mutations were obtained from TCGA. Clustering was found to be based on 6 mutations, with the following prognostic groups: (a) good: NPM1, CEBPA, or TET2, (b) intermediate: NPM1/DNMT3A, or other mutations, (c) poor: RUNX1, FLT3-ITD, FLT3-ITD/NPM1, or FLT3-ITD/CEBPA. Some discrepancy between our results and those from previous studies is most likely due to inclusion of AML cases transformed from myeloproliferative neoplasms or myelodysplastic syndrome in previous studies.
CONCLUSIONS: This study provides further molecular characterization and prognostic data most specific for the de novo subgroup of CN-AML patients.

Ponti D, Bastianelli D, Rosa P, et al.
The expression of B23 and EGR1 proteins is functionally linked in tumor cells under stress conditions.
BMC Cell Biol. 2015; 16:27 [PubMed] Article available free on PMC after 29/03/2017 Related Publications
BACKGROUND: The nucleolus is a multi-domain enriched with proteins involved in ribosome biogenesis, cell cycle and apoptosis control, viral replication and differentiation of stem cells. Several authors have suggested a role for the nucleolus also in malignant transformation. We have recently demonstrated that under specific circumstances the transcriptional factor EGR1 is shuttled to the nucleolus where it functions as a negative regulator of RNA polymerase I. Since this activity is hampered in ARF -/- cells, and ARF transcription is regulated by EGR1 while the turnover of ARF protein is under the control of B23, we speculated that some sort of cooperation between EGR1 and B23 might also exist.
RESULTS: In this work we identified a canonical EGR1 binding site on the B23 promoter through experiments of transactivation and in vitro DNA binding assay. We then found that the levels of B23 expression are directly correlated with those of EGR1, and that this correlation applies to several cellular types and to different stress conditions. Furthermore, we showed that EGR1 stability and accumulation within the nucleolus is in turn regulated by B23 through proteasome involvement, similarly to ARF turnover.
CONCLUSION: Our results highlight EGR1 as a regulator of B23 expression actively playing within the newly discovered nucleolar B23-ARF-EGR1 network.

Holmberg Olausson K, Elsir T, Moazemi Goudarzi K, et al.
NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape.
Sci Rep. 2015; 5:16495 [PubMed] Article available free on PMC after 29/03/2017 Related Publications
Glioblastoma (grade IV glioma) is the most common and aggressive adult brain tumor. A better understanding of the biology of glioblastoma cells is crucial to identify molecular targets stimulating cell death. NPM1 (nucleophosmin) is a multifunctional chaperone that plays an important role in cancer development. Herein, NPM1 was analyzed by immunohistochemistry in human astrocytic gliomas. NPM1 was detected in all tumors but with a significantly higher staining intensity in grade IV than in low grade tumors. Depletion of NPM1 had only modest effects on the viability of U251MG, U1242MG, and U343MGa Cl2:6 glioma cells, despite alterations in nucleolar morphology. Glioma cell cultures depleted of NPM1 exposed to micromolar levels of actinomycin D were more prone to cell death (apoptosis) compared to cultures retaining NPM1. We had previously found that NPM1 binds to linker histone H1.5. Here we could show that silencing of H1.5 triggered glioma cell apoptosis as evidenced by a marked increase in both the numbers of cleaved caspase-3(+) cells and in the amounts of cleaved PARP. Enforced expression of NPM1 suppressed apoptosis in H1.5 depleted glioma cells. Although our studies would suggest little effectiveness of targeting NPM1 alone there could be potential using it as a combination treatment.

Ho AD, Schetelig J, Bochtler T, et al.
Allogeneic Stem Cell Transplantation Improves Survival in Patients with Acute Myeloid Leukemia Characterized by a High Allelic Ratio of Mutant FLT3-ITD.
Biol Blood Marrow Transplant. 2016; 22(3):462-9 [PubMed] Related Publications
Allogeneic hematopoietic cell transplantation (alloHCT) as a postremission therapy in patients with FLT3-ITD-positive intermediate-risk acute myeloid leukemia (AML) remains controversial. FLT3-ITD mutations are heterogeneous with respect to allelic ratio, location, and length of the insertion, with a high mutant-to-wild-type ratio consistently associated with inferior prognosis. We retrospectively analyzed the role of alloHCT in first remission in relationship to the allelic ratio and presence or absence of nucleophosmin 1 mutations (NPM1) in the Study Alliance Leukemia AML2003 trial. FLT3-ITD mutations were detected in 209 patients and concomitant NPM1 mutations in 148 patients. Applying a predefined cutoff ratio of .8, AML was grouped into high- and low-ratio FLT3-ITD AML (HR(FLT3-ITD) and LR(FLT3-ITD)). Sixty-one patients (29%) were transplanted in first remission. Overall survival (OS) (HR, .3; 95% CI, .16 to .7; P = .004) and event-free survival (EFS) (HR, .4; 95% CI, .16 to .9; P = .02) were significantly increased in patients with HR(FLT3-ITD) AML who received alloHCT as consolidation treatment compared with patients who received consolidation chemotherapy. Patients with LR(FLT3-ITD) AML and wild-type NPM1 who received alloHCT in first remission had increased OS (HR, .3; 95% CI, .1 to .8; P = .02) and EFS (HR, .2; 95% CI, .1 to .8; P = .02), whereas alloHCT in first remission did not have a significant impact on OS and EFS in patients with LR(FLT3-ITD) AML and concomitant NPM1 mutation. In conclusion, our results provide additional evidence that alloHCT in first remission improves EFS and OS in patients with HR(FLT3-ITD) AML and in patients with LR(FLT3-ITD) AML and wild-type NPM1.

Ching RH, Lau EY, Ling PM, et al.
Phosphorylation of nucleophosmin at threonine 234/237 is associated with HCC metastasis.
Oncotarget. 2015; 6(41):43483-95 [PubMed] Article available free on PMC after 29/03/2017 Related Publications
Hepatocellular carcinoma (HCC) is frequently complicated by the occurrence of intrahepatic and extrahepatic metastases, leading to poor prognosis. To improve the prognosis for HCC patients, there is an urgent need to understand the molecular mechanisms of metastasis in HCC. Since protein Serine/Threonine phosphorylation emerges to be an important posttranslational modification critical in signaling process associated with cell proliferation, survival and metastasis, we employed a pair of primary tumor-derived and corresponding lung-metastatic counterparts (PLC/PRF/5-PT and PLC/PRF/5-LM) and aimed to identify these changes using CelluSpot Serine/Threonine kinase peptide array. Upon analysis, we found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr234/237) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Similar observation was observed in another match primary and their metastatic counterparts (MHCC-97L and MHCC-97H). By immunohistochemical staining, p-NPM-Thr234/237 was consistently found to be preferentially expressed in metastatic HCCs when compared with primary HCC in 28 HCC cases (p < 0.0001). By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1). Wild-type NPM was found to physically interact with a metastatic gene, ROCK2, and defective in Thr234/237 phosphorylation decreased its binding affinity, resulting in decrease in ROCK2 mediated signaling pathway. Identification of CDK1/p-NPM/ROCK2 signaling pathway provides a novel target for molecular therapy against HCC metastasis.

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