Nucleophosmin Research Articles

Abstract 1980: Nucleophosmin regulates centrosome amplification and is associated with anti-HER2 resistance in HER2-positive breast cancer cells.

Abstract Aneuploidy is a hallmark of solid tumors, contributing to tumorigenesis and resistance to chemotherapy. Aneuploidy is prevented by two centrosomes directing a bipolar mitotic spindle. Centrosome amplification (CA), the presence of supranumerary centrosomes within a cell, has been detected in both premalignant lesions and breast tumors, supporting the role of this phenomenon in breast cancer initiation and progression. Moreover, CA has been associated with HER2 overexpression. CA is initiated by abnormal signals from oncogenes or altered tumor suppressors that affect regulatory molecules shared by both cell and centrosome cycles. Elucidating the molecular circuitry and functional consequences of impaired centrosome cycle is therefore important for a complete understanding of its role in breast cancer. Nucleophosmin (NPM) is a widely expressed nucleolar phosphoprotein that has several functions, including the early regulation of centrosome duplication. Recent reports from breast cancer patients indicate the presence of NPM antigens in HER2-negative cases and low NPM expression as a prognostic marker of poor survival in the luminal A subtype. We therefore hypothesized that NPM expression and activity might directly impact the CA in HER2-positive breast cancer cells. To test this hypothesis, we have used genetic alteration of total and phospho-NPM to investigate its function in human mammary epithelial cell lines that includes non-tumorigenic and HER2-positive tumorigenic lines. Our initial findings revealed significantly elevated CA percentages and higher expression level of p-NPM (T199) in the HCC1954, JIMT1, and SKBR3 cells (HER2-positive) compared to other subtypes and non-tumorigenic MCF10A cells. This residue is known to be responsible for regulating the function of NPM in centrosome duplication. Conversely, HER2-positive cells displayed lower protein levels of total NPM compared to the other cell lines investigated. Expression of a deletion mutant phospho-NPM (T199A) reduced the percentage of CA in HCC1954 cells. In addition, an HCC1954 derivative with primary resistance to Herceptin showed augmented frequency of CA versus sensitive parental cells. Ongoing experiments involve the inhibition of p-NPM at T199 in JIMT1 and SKBR3 cells as well as the overexpression of a wild type form in all of the three HER2-positive breast cancer cell lines followed by the assessment of CA. In parallel, the non-tumorigenic MCF10A cells have been transfected with a phospho-mimetic NPM (T199D) and will be subjected to similar investigations. Finally, the correlation between p-NPM-driven CA and Herceptin resistance is being studied in all of our HER2-positive cell models of breast cancer. These results suggest that the high NPM expression could have a protective effect against CA and a more aggressive phenotype displayed by HER2-positive breast cancer cells. Citation Format: Mihaela Marina, Harold Ivan Saavedra. Nucleophosmin regulates centrosome amplification and is associated with anti-HER2 resistance in HER2-positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1980. doi:10.1158/1538-7445.AM2013-1980

A novel interaction of nucleophosmin with BCL2-associated X protein regulating death evasion and drug sensitivity in human hepatoma cells

Death evasion is crucial for both carcinogenesis and resistance to anticancer therapies. Recently, we identified nucleophosmin (NPM) as a key factor counteracting death stimuli in human hepatocellular carcinoma (HCC) cells. Here we report the identification of a novel NPM-BCL2-associated X protein (BAX) pathway orchestrating death evasion in human HCC cells. Silencing of NPM expression significantly sensitized HCC cells-particularly those bearing inactivated p53 gene (Huh7, Hep3B, and Mahlavu)-to ultraviolet irradiation, mitomycin C, doxorubicin, cisplatin, sorafenib, and lapatinib. This sensitizing effect was not changed further, as p53 expression had been simultaneously silenced. Following cell stress, NPM and BAX were induced and exported out of the nucleoli and nucleus, respectively. BAX was translocated to cytoplasm in cells with relatively high NPM level, or accumulated in the mitochondria in cells with relatively low NPM level and undergoing apoptosis. Subcellular fractionation revealed that silencing of NPM expression greatly enhanced mitochondrial translocation and oligomerization of BAX in Huh7 and Mahlavu cells. In situ proximity ligation assays and reciprocal co-immunoprecipitation revealed a direct interaction between NPM and BAX in the cytoplasm. Silencing of BAX expression abolished the sensitization effect exerted by silencing of NPM in HCC cells. Clinically, up-regulation of NPM was significantly associated with advanced tumor stage and poor prognosis. By directly blockading BAX mitochondrial translocation and activation, NPM helps human HCC cells evade death induction independently of p53-mediated cell death. Silencing of NPM significantly sensitized HCC cells to anticancer therapies. NPM is a potential cotarget in combination with other therapies for HCC, particularly those that harbor inactivated p53 gene. Our findings are of clinical significance because NPM up-regulation and p53 mutations are usually found in advanced human cancers, including HCC.

NPM new interacting partners and influence of NPM mutations on gene regulation

Nucleophosmin (NPM) is a multifunctional protein. NPM mutation is found in one third of all acute myeloid leukemia (AML), and in more than 60% of AML with normal karyotype. It is also a recurrent translocation partner in AML.We got interested in the NPM function as a co‐regulator of gene expression. We hypothesized that the modifications of NPM disrupt gene regulation and thereby, favour leukemia.With a combination of biochemistry and molecular biology techniques, we identified new partners of NPM involved in gene regulation. We found that NPM colocalise with different transcription regulators at specific genes. Based on our results, we propose that the effect of NPM modifications on gene regulation has a significant impact on the capacity of hematopoietic cells to receive extra‐cellular signals which favours abnormal hematopoiesis and potentially, leukemia.

Clinical significance of nucleophosmin/B23 and human epidermal growth factor receptor 2/neu expressions in gastric cancers

The aim of the study was to investigate the expression levels of 'NPM'/nucleophosmin/B23 and human epidermal growth factor receptor 2 (Her-2)/neu in gastric cancer (GC) and corresponding non-malignant tissues, correlation with their clinicopathological parameters and the relationship of nucleophosmin/B23 and Her-2/neu in the occurrence and development of GC. A total of 131 postoperative patients were examined for nucleophosmin/B23 expression by immuno-histochemistry and for Her-2/neu expression by fluorescence in situ hybridization with the median follow-up period of 38 months. The positive expression rates of nucleophosmin (NPM) in neoplastic tissues and adjacent gastric mucosa were 65.6% and 52.7%, respectively. Nucleophosmin/B23 levels were linked to more advanced tumor stages, poor prognosis, and likelihood of recurrence (p < 0.05). The Cox multivariate analysis indicated that the nucleophosmin/B23 expression was an independent indicator for tumor recurrence (p = 0.011). Of the total GC specimens 12.21% were positive for Her-2/neu, but whose expression was of no correlation with patients' survival. Patients who were positive for Her-2/neu also had high NPM expression levels (p = 0.0303). The results suggest that nucleophosmin/B23 is a favorable prognostic indicator for GC. But Her-2/neu has no relationship with the prognosis of GC. The combined clinical significance of nucleophosmin/B23 and Her-2/neu remains to be further investigated.

Proteasome Activity Influences UV-Mediated Subnuclear Localization Changes of NPM

UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress.

Hematopoietic transplantation for acute myeloid leukemia with internal tandem duplication of FLT3 gene (FLT3/ITD)

Patients with acute myeloid leukemia (AML) traditionally classified as having an intermediate cytogenetic risk [mostly cytogenetically normal AML (CN-AML)] really include a significant proportion of cases with a poor outcome. This is based on the molecular findings at diagnosis, mainly the presence of internal tandem duplication in the FMS-like tyrosine kinase 3 gene(s) (FLT3/ITD). Optimal postremission therapy for these high-risk molecular cases is not well established; as the prognosis is adverse hematopoietic cell transplantation (HCT), mainly allogeneic HCT (allo-HCT), is the most widely accepted strategy. As a rule, patients with FLT3/ITD have a poor outcome with conventional chemotherapy alone. Only patients with an associated nucleophosmin 1 (NPM1) mutation and those with a low mutated-to-wild-type allelic ratio of FLT3/ITD have less unfavorable outcome. Most studies show an advantage of allo-HCT in first complete remission (CR1), with higher 3-5 year disease-free survival and lower relapse risk than with chemotherapy or autologous transplantation (auto-HCT). Regarding allo-HCT proceeding early after reaching CR1 seems to improve survival, rather than after several courses of consolidation chemotherapy. Patients with intermediate-risk cytogenetics AML and FLT3/ITD, especially NPM1-wild cases and those NPM1 mutated with a high allelic ratio, should proceed to allo-HCT if possible early after achieving CR1.

Acute myeloid leukemia with translocation t(3;5): new molecular insights

Acute myeloid leukemia with translocation t(3;5): new molecular insights

Identification of NPMc+ Acute Myeloid Leukemia in Bone Marrow Smears

The most frequent genetic change currently known in acute myeloid leukemia (AML) is the mutation of the nucleophosmin (NPM) gene. Aberrant cytoplasmic accumulation of NPM protein (NPMc+) is the result of this mutation, and it can be demonstrated by immunohistochemistry for the identification of a favorable subgroup within "AML with normal karyotype" according to the World Health Organization classification. NPM staining pattern was defined in 71 AML and 15 control cases by the use of bone marrow smears in order to overcome limitations observed due to immunohistochemistry. In 13/71 cases (18.3%), clear cytoplasmic staining of the leukemic blast cells was detectable that was comparable with the positivity of mitotic figures physiologically lacking nuclear membrane. The biological and genetic characteristics of the NPMc+ cases determined this way were identical with the previously published results including low CD34 and HLA-DR expression and lack of recurrent karyotype abnormalities. Bone marrow smears are well applicable and therefore a real alternative for the determination of NPM with the highest accuracy for optimal risk stratification in AML.

Expression and Functional Studies on the Noncoding RNA, PRINS

PRINS, a noncoding RNA identified earlier by our research group, contributes to psoriasis susceptibility and cellular stress response. We have now studied the cellular and histological distribution of PRINS by using in situ hybridization and demonstrated variable expressions in different human tissues and a consistent staining pattern in epidermal keratinocytes and in vitro cultured keratinocytes. To identify the cellular function(s) of PRINS, we searched for a direct interacting partner(s) of this stress-induced molecule. In HaCaT and NHEK cell lysates, the protein proved to be nucleophosmin (NPM) protein as a potential physical interactor with PRINS. Immunohistochemical experiments revealed an elevated expression of NPM in the dividing cells of the basal layers of psoriatic involved skin samples as compared with healthy and psoriatic uninvolved samples. Others have previously shown that NPM is a ubiquitously expressed nucleolar phosphoprotein which shuttles to the nucleoplasm after UV-B irradiation in fibroblasts and cancer cells. We detected a similar translocation of NPM in UV-B-irradiated cultured keratinocytes. The gene-specific silencing of PRINS resulted in the retention of NPM in the nucleolus of UV-B-irradiated keratinocytes; suggesting that PRINS may play a role in the NPM-mediated cellular stress response in the skin.

Mutant NPM1 is a Reliable marker of Minimal Residual leukemia in patients with Acute Myeloid Leukemia.

Abstract 2484 Background:Mutations in NPM1 (Nucleophosmin-1) have been described in about 35% of adult patients with de novo AML and 45–60 % of AML patients with a diploid karyotype. NPM1 mutations predict for achieving higher complete remission (CR) rates and better outcomes in AML. Few studies have reported on the reliability of mutated NPM1 as a marker for minimal residual disease (MRD) detection in patients with AML. Methods:We conducted a retrospective analysis of patients (n=360) with newly diagnosed AML treated at our institution between 2008 and 2012. The study was approved by the Institutional Review Board. NPM1 mutation status was determined from DNA from unsorted bone marrow (BM) aspirate samples by a PCR-based method at baseline, remission, and relapse. Genomic DNA from bone marrow samples was isolated using the Autopure extractor (QIAGEN/Gentra, Valencia, CA). Mutations in exon 12 of NPM1 were assessed by a DNA-based semi-quantitative polymerase chain reaction capillary electrophoresis (PCR-CE) assay with analytical sensitivity of approximately 2.5%. Results:Data on remission and relapse samples from 360 newly diagnosed and previously untreated patients with AML, with available NPM1 analysis on their BM at the time of initial diagnosis, was collected (see flow chart below). Median age was 60 years (range 21 – 81 years). 262 patients (72%) had de novo AML, and 98 (27%) secondary AML. Cytogenetics was diploid in 137 (38%) patients, t(8;21) in 17 (5%), inversion 16 in 26 (7%), deletion 5 alone in 27 (7.5%), del 7 and 5 in 26 (7%), deletion 7 alone in 21 (5.8%), trisomy 8 in 17 (5%) and miscellaneous in 89 (24.7%) patients, respectively. Overall, 60 (16.6%) patients including 46 of the 137 (33.5%) diploid patients had NPM1 mutation at baseline. Secondary leukemia was more common in the NPM1 wild type (30%) than in the NPM1 mutated (13%) category.When analysed by age, in patients < 60 years (n=175), OS (overall survival), EFS (event free survival) and response rates were significantly superior in NPM1 mutated subgroup (p=0.001, 0.007, 0.02 respectively), while among patients ≥ 60 years (n=185) EFS and response rates were significantly higher in the NPM1 mutated subgroup (p=0.008, 0.03 respectively). Among the patients with diploid cytogenetics who were younger than 60 years (n=60) OS, EFS and CR duration was significantly better in the NPM1 mutated subset (p=0.007, 0.007 and 0.02 respectively), while in those ≥60 years (n=77) there was no statistically significant difference in the outcomes for the NPM1 mutated and wild-type subsets.Among the 60 NPM1 mutated patients 54 (90%) including 41/46 (89%) of those with diploid cytogenetics achieved complete response (CR) or CR without platelet recovery. Thirty nine patients (including 30 with diploid karyotype) had available NPM1 status at the time of CR and all (100%) were negative for NPM1 mutation.Among the patients with mutated NPM1 at baseline who have achieved a NPM1 negative status at CR, 10/39 overall (25%) and 7/30 diploid (23%) patients relapsed. NPM1 status was available for 6 patients overall including 4 with diploid karyotype at the time of relapse. Among them, 5/6 overall (83%) and 3/4 diploid (75%) patients had mutated NPM1, while 1/6 overall (16%) and 1/4 diploid (25%) patients remained NPM1 wild type. This patient relapsed with extramedullary disease (leukemia cutis) without any BM involvement.Among the 300 patients (including 91 with diploid karyotype) with wild type NPM1 at diagnosis, none acquired a mutated NPM1 clone, either at CR or at the time of relapse. Conclusions:These data suggest that mutated NPM1 is a reliable and stable marker for the detection of MRD at the time of CR. Hence, NPM1 mutations can be used to detect MRD and their recurrence may predict pending relapse. Disclosures:Cortes:Celgene: Research Funding. Ravandi:Johnson and Johnson: Honoraria; Celgene: Research Funding.

NPM-RAR Enhances NFκB Transcriptional Activity

Abstract 5117Acute Promyelocytic Leukemia (APL) is characterized by recurrent translocations of 17q21, which result in expression of fusion proteins of the retinoic acid receptor alpha (RARα). We have been studying the t(5;17)(q35;q21) variant of APL, which fuses the N-terminal 117 amino acids of nucleophosmin (NPM) to the C-terminus of RARα. NPM-RAR is capable of binding to retinoic acid response elements (RAREs), and we have previously shown that NPM-RAR can recruit both corepressor and coactivator complexes to retinoic acid target promoters, to modulate transcription in a ligand-dependent fashion, though with a different sensitivity to ligand than RARα. The N-terminal NPM-sequence of NPM-RAR contains a dimerization domain capable of mediating not only homodimerization of the fusion protein, but also heterodimerization with wild-type NPM, to potentially modulate NPM functionality. NPM serves as a molecular chaperone, and binds to a diverse set of proteins. Amongst these are the p50 and p65 subunits of NFκB. NPM has been shown to serve as a coactivator for the NFκB transcriptional complex. We hypothesized that NPM-RAR might interact with NPM to modulate NFκB dependent transcription. We focused on NFκB regulation of the Superoxide Dismutase 2 (MnSOD, SOD2) promoter, since NPM regulation of NFκB mediated transcription has been most extensively studied in this model system. We found that NPM-RAR enhances both basal and TNFα-induced transcription of an NFκB-reporter construct (containing a minimal MnSOD promoter/enhancer) in HepG2 and Hela cells. This effect was not dependent upon translocation of NFκB to the nucleus, indicating that the positive effect on NFκB was mediated downstream of the canonical activation pathway of NFκB. The MnSOD promoter does not contain a consensus RARE, and this effect was not seen in cells transfected with RARα, suggesting that this effect is not mediated through the DNA binding motifs of the NPM-RAR C-terminal RARα sequences. NPM-RAR does not directly interact with NFκB; preliminary experiments using siRNA to down-regulate NPM indicate that NPM-RAR enhancement of MnSOD transcription is dependent upon the presence of wild-type NPM. This leads us to propose a model whereby NPM-RAR binds to the NPM/NFκB transcriptional complex through interaction with NPM, to enhance NFκB mediated transcription. Disclosures:No relevant conflicts of interest to declare.

Mutated Nucleophosmin 1 (NPM1) Is an Immunogenic Target and Patients with NPM1mut Acute Myeloid Leukemia (AML) Showed High Expression of Different Leukemia-Associated Antigens (LAAs)

Abstract 3592Nucleophosmin gene 1 mutations (NPM1mut) are one of the most frequent molecular alterations in AML and distinct immune responses might contribute to the favorable prognosis of AML patients with NPM1mut. Recently, we showed specific T cell responses of CD4+ and CD8+ T cells against epitopes derived from mutated regions of NPM1 (Greiner et al., Blood. 2012 May 16, Epub). In the present study, we investigated clinical parameters and the clinical outcome of NPM1mut AML patients in accordance to their immune responses against different NPM1 epitopes. Moreover, we examined the quantitative expression of different leukemia-associated antigens (LAAs) in NPM1mutAML patients.In ELISpot analysis of 33 healthy volunteers and 27 AML patients, we detected T cell responses of CD4+ and CD8+ T cells against epitopes derived from the mutated region of NPM1. We performed further tetramer assays against the most interesting epitopes and chromium release assays to show the cytotoxicity of peptide-specific T cells. Microarray analysis was performed to analyze the expression of different LAAs in NPM1mut and NPM1wtAML patients.Two epitopes (peptide #1 and #3) derived from NPM1mut induced CD8+ T cell responses. 33% of the NPM1mut AML patients showed immune responses against peptide #1 and 44% against peptide #3. NPM1mut AML patients showed a significantly higher frequency of T cell responses against peptide #3 in contrast to HVs (p=0.046), whereas for peptide #1 the frequency of T cell responses of AML NPM1mut patients and HVs was not significantly different. Specific lysis of pulsed T2 cells but also NPM1mut leukemic blasts was detected in chromium release assays. Therefore, overlapping peptides (OL) were analyzed in ELISpot assays and the peptide called OL8 showed favorable results to activate both CD8+ and CD4+ T cells.We performed survival analysis for these 33 NPM1mut patients analyzed by ELISpot comparing cases with or without specific T cell responses. Our data suggest a trend to a better overall survival (OS) for patients with specific T cell responses against peptide #1 or #3. However, the patient numbers are small and the data have to be interpreted carefully. Analyses with material from larger controlled clinical trials with a high number of patients with NPM1mut AML have to be performed. Our microarray analysis of 30 AML patients showed a high expression of different LAAs like RHAMM, WT-1 and BCL-2 in all subtypes of cells of NPM1mutAML patients, also in leukemic progenitor cells. This demonstrates that NPM1 is an AML subtype suitable for poly-targeted immunotherapeutic trials.Taken together, NPM1mut might constitute an interesting target structure for individualized immunotherapeutic approaches in NPM1mut AML patients. We hypothesize that immune responses to NPM1 mutation may contribute to the favorable prognosis. Disclosures:No relevant conflicts of interest to declare.

TC11, a Novel Phthalimide Derivative, Directly Binds to NPM1 and Induced Apoptosis of High-Risk Myeloma Cells Via Centrosomal Disruption

Abstract 1841 PURPOSE:Despite recent advances in the treatment of multiple myeloma (MM), patients with high-risk chromosomal changes such as del13q, t(4;14) or del17p revealed significantly shorter survival. In addition, bone disease markedly reduces quality of life of the patients with MM. To overcome these problems, we have designed and screened synthetic phthalimides which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purposes of this study are to explore novel drugs which possess anti-tumor activity against high-risk MM cells and to examine the anti-osteoclastogenic activity and to isolate directly binding molecules. METHODS AND RESULTS:Thirty synthetic phthalimides were screened for anti-proliferative effect on KMS34 cells with t(4;14) and deletion of chromosome 17. A phthalimide derivative, 2-(2,6-diisopropylphenyl)- 5-amino- 1H-isoindole- 1,3- dione (TC11) significantly inhibited growth of KMS34 cells as well as other MM cells lines with high-risk chromosomal abnormalities (IC50 to KMS34 cells= 4μM). TC11 increased annexin V fraction and induced apoptosis in a caspase-dependent manner. In vivo anti-myeloma activity was evaluated using KMS34-bearing lcr/SCID mice by intraperitoneal injection of TC11. Twenty mg/kg of TC11 significantly inhibited growth of TC11-derived tumor cells, and apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not suppressed, suggesting low hematopoietic toxicity. In pharmacokinetic study using lcr/SCID mice, the plasma concentrations of TC11 was examined; Cmax=18.1μM, Tmax=1.5hr, T1/2 =4.5hr when 100mg/kg of TC11 was injected, and Cmax=2.1μM Tmax=1.0hr, T1/2 =4.5hr when 20mg/kg was injected. In order to examine efficacy to bone disease, anti-osteoclastogenic activity was examined by adding TC11 to M-CSF/RANK ligand-induced osteoclastogenic culture of mouse bone marrow mononuclear cells. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of 1μM of TC11. It was also found that TC11 inhibited bone resorption by pit assay. We also tried to isolate directly binding proteins to TC11 by our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. We identified nucleophosmin 1 (NPM1) as a TC11-binding molecule. Knockdown assay introducing siRNA for NPM1 into HeLa cells induced emergence of the cells with multipolar spindles, suggesting centrosomal disruption during cell division. Since NPM1 gene localizes at chromosome 5q, anti-MDS effect of TC11 was also examined. TC11 also inhibited growth of MDS-L cells (IC50=7μM). CONCLUSION:A novel phthalimide derivative, TC11, has anti-MM activity in vivo and is a potentially effective drug for high-risk MM with bone lesions. TC11 directly binds to NPM1 and induces apoptosis of MM and MDS cells with low toxicity to normal hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

NPM-RAR Binds to Tradd and Impedes the Extrinsic Apoptotic Pathway

Abstract 5126The t(5;17)(q35;q21) variant of Acute Promyelocytic Leukemia fuses nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). The resultant NPM-RAR fusion protein blocks myeloid differentiation, and leads to a leukemic phenotype similar to that caused by the t(15;17)(q22;q21) PML-RAR fusion. The contribution of the N-terminal 117 amino acids of NPM contained within NPM-RAR has not been well studied. NPM is a molecular chaperone, and binds to a variety of proteins implicated in leukemogenesis. We performed a proteomic analysis to identify NPM-RAR interacting proteins. Vectors encoding NPM-RAR or RARA (as control) fused in frame to calmodulin binding peptide and Protein A were expressed in HEK293 cells, and interacting proteins subjected to tryptic digest. Peptides were analyzed by nanoflow reversed-phase liquid chromatography-mass spectrometry. Amongst other targets, we identified tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD) as a distinct binding partner for NPM-RAR. TRADD did not bind to wild-type RARA or NPM, suggesting that the interaction was unique to the fusion protein. The NPM-RAR/TRADD interaction was verified by reciprocal co-precipitation. Though NPM-RAR localizes primarily in the nucleoplasm, we also found a low level of NPM-RAR/TRADD dimer in the cytoplasm utilizing confocal microscopy. Expression of NPM-RAR in U937 cells impaired TNF activation of caspase 8 and caspase 3. TNF-induced acquisition of Annexin V, generation of sub-G0/G1 nuclear content, and cleavage of PARP were all blunted, indicating that NPM-RAR blocks TNF-induced apoptosis. Our studies identify a novel mechanism through which NPM-RAR impacts leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Detection of an Unbalanced Ratio Between WT1 Isoforms in Acute Myeloid Leukemia and Its Correlation with Molecular Abnormalities.

AbstractAbstract 2500Wilms’ tumor gene 1 (WT1) is a tumor suppressor gene coding for a zinc finger transcriptional factor which was found overexpressed in acute myeloid leukemias (AML). In AML WT1 functions as an oncogene rather than a tumor suppressor. This may depend on the unbalanced expression of its different isoforms derived from alternative splicing in exon 5 (EX5+/−) or the presence of KTS in exon 9 ( KTS+/KTS-). In normal hematopoietic cells the physiological ratio between KTS+/KTS- isoforms is 1:1. KTS are inserted between the third and fourth zinc fingers thus influencing the DNA binding and therefore the transcriptional activity.The aim of the study was to investigate the possibility of an unbalanced ratio between the isoforms thus leading to the disruption of WT1 function. In addition we correlated KTS+ isoform with clinical and biological features. Methods: we analyzed KTS+/KTS- isoforms in 120 AML patients ( 102 BM samples, 18 PB), 63 chronic phase CML patients (48 BM and 15 PB). In addition we evaluated 5 samples of selected blast cells from AML patients. In a subset of 38 adult acute myeloid leukemia (AML) patients we measured all WT1 isoforms (A[EX5 -/KTS -], B[+/−], C[-/+] and D[+/+]. Quantitative PCR was used for the WT1 isoforms detection and measurement. Results:KTS+ isoform is significantly overexpressed with a ratio KTS+/KTS− 2:1 in both AML and CML patients and it is confirmed in blast cell samples. In the subgroup of patients analyzed for the expression pattern of all WT1 isoforms we detected a significant overexpression of isoform D (EX5+/KTS+) (median value 821) while the isoform A (EX5−/KTS−) is the less represented ( median value 303). In this subgroup the expression of KTS+ isoforms (B+D) is higher than KTS- ( A+C) with a median value of 642 compared to 421. The ratio of WT1 isoforms was not significantly different among FAB subgroups or according to cytogenetic risk, FLT3 (fms-like tyrosine kinase receptor-3) internal tandem duplication or exon 17 mutation, NPM (nucleophosmin) gene mutations and BAALC (brain and acute leukemia, cytoplasmic) gene expression. Conclusion:Although WT1 is overexpressed in AML and CML patients it lacks its typical tumor suppressor function. In this study we have demonstrated the overexpression of WT1 KTS+ isoforms. In addition we have previously demonstrated that KTS+ isoform does not have transcriptional activity since it is mainly localized within the cytoplasm. In conclusion, WT1 is overexpressed in AML but the unbalanced ratio between the isoforms may probably influence the transcriptional activity. Any possible correlation between the unbalanced ratio of WT1 isoforms and clinical outcome requires further prospective studies to be established. Disclosures:No relevant conflicts of interest to declare.

An iTRAQ Proteomics Screen Reveals the Effects of the MDM2 Binding Ligand Nutlin-3 on Cellular Proteostasis

Mouse double minute 2 (MDM2) participates in protein synthesis, folding, and ubiquitin-mediated degradation and is therefore a proteostasic hub protein. The MDM2 interactome contains over 100 proteins, yet stratification of dominant MDM2-interacting proteins has not been achieved. 8-plex iTRAQ (nanoLC-MS/MS) of MCF7 cells treated with the MDM2-binding ligand Nutlin-3 identified the most abundant cellular protein changes over early time points; 1,323 unique proteins were identified including 35 with altered steady-state levels within 2 h of Nutlin-3 treatment, identifying a core group of MDM2 related proteins. Six of these proteins were previously identified MDM2 interactors, and the effects of Nutlin-3 on the MDM2-nucleophosmin interaction (NPM) was further validated. This revealed that Nutlin-3 mediates the in vivo conversion of NPM from an oligomer to a monomer as an MDM2-dependent phenomenon, with Nutlin-3 stimulating MDM2 binding to a peptide motif derived from the oligomerization interface of NPM. These data form the first proteomic screen of Nutlin-3 in cells whereby we (i) identify the most abundant MDM2-interacting proteins whose steady-state levels change early after Nutlin-3 treatment; (ii) identify the first protein apart from p53, nucleophosmin (NPM), whose interaction with MDM2 can be stimulated allosterically by Nutlin-3; and (iii) raise the possibility that Nutlin-3 might act as a general agonist of other MDM2 protein-protein interactions.

Clonal heterogeneity in patients with cytogenetically normal acute myeloid leukemia with NPM1 mutations

The nucleophosmin 1 (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 60% of adult cytogenetically normal AML (CN-AML). To date, these mutations have only been detected in cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In our study, NPM1 mutations were analyzed using the highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) method on fluorescence-activated cell-sorted (FACS) purified different circulating mature cell populations in patients with NPM1-mutated CN-AML. As expected, NPM1 mutations were found in myeloid blood cells, including CD14+ monocytes and CD66b+ granulocytes. However, we were also able to detect NPM1 mutations in CD19+ B cells and CD3−14−16+56+ natural killer (NK) cells, albeit at lower levels. Surprisingly, mutations were also detected in CD3+ T cells from all analyzed patients. Our data demonstrate that NPM1-mutated CN-AML originates in an early stem cell with both lymphoid and myeloid differentiation potential.

Flow cytometry and immune disorders

cytometry (FCM) is the natural technical tool to mea-sure cells of the immune system in health and disease. By itsability to rapidly measure millions of individual cells it allowscollection of statistically reliable information from rare cellsubsets. Due to the opportunity to multiplex analysis—by thecombination of manifold markers even on the single celllevel—unequivocal identification of complex cell phenotypesis enabled. Standardization and high level of quality controlallows overall control of the preanalytical and analytical pro-cess and by standardized reporting, the reproducibility byother laboratories. This is an extremely important issue inclinical diagnosis, where right or wrong may decide the fate ofa patient. Several improvements are on their way to improvespecificity by optimized marker combinations and novel tech-nologies.Human peripheral blood monocytes can be subclassifiedby phenotypic markers into three functionally differentsubsets (1). These subsets play different roles in pathologicaldisorders and thus are relevant for diagnosis and immunomo-nitoring. One of the main obstacles in their unequivocal iden-tification is that typical phenotypic cell surface markers suchas CD14 are downregulated in one subset and others (CD16and HLA-DR) are shared by other leukocytes, for exampleNatural Killer and B cells. Furthermore, HLA-DR is downre-gulated in septic patients obscuring identification of activatedmonocytes. Recently, a single platform multicolor assay wasintroduced to quantify these monocyte subpopulations (1).Now, Abeles and colleagues from Imperial College in Londonand Oxford, United Kingdom (this issue, page 823) reducedthe number of required markers for clinical diagnosis for theidentification of various human monocyte subsets to three inorder to analyze peripheral blood monocytes in individualswith acute liver failure. This new critically evaluated panelrelies on the backbone of the above three surface markers anda careful gating strategy allowing the identification of allmonocyte subtypes unequivocally in healthy and severely illindividuals.Several genetic disorders also affect the development andpopulation composition of peripheral blood leukocytes. Manyof these congenital immune defects may lead to severeimmune deficiency, recurrent infections, cancer formation orautoimmune diseases. Pia˛tosa and colleagues from Warsaw,Poland, and Rotterdam, The Netherlands (this issue, page835) looked more closely at the immune system of individualswith Nijmegen Breakage Syndrome who have a preference forlymphoma formation, among others. Blood samples ofinfants, toddlers, children and adults were analyzed regardingabsolute count of B-cell maturation stages by four-colorimmunophenotyping by a single platform assay. The authorsconfirmed the already know reduction in the number of circu-lating B-cells but in addition also reported an arrest in B-celldevelopment leading to an impaired immunoglobulin classshift, low antibody levels and thereby reduced immune defensecapability.The prevalence for Diabetes Mellitus of type 2 (DM2)alarmingly increases worldwide. Today, the disease not onlyaffects the population in the northern hemisphere but alsospreads into India and Asian countries. Furthermore, not onlyadults but also increasingly children are affected. This ismostly due to the elevation in childhood obesity. Accompany-

Hepatocyte odd protein shuttling (HOPS) is a bridging protein in the nucleophosmin-p19Arf network

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19(Arf), directs localization of p19(Arf) in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19(Arf), resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19(Arf) and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19(Arf), providing new mechanistic insight into how NPM and p19(Arf) will oppose tumor cell proliferation.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.