Abstract 5010: Nucleophosmin (NPM) is univerally deregulated in acute promyelocytic leukemia

Abstract

Abstract NPM is a multifunctional nucleolar phosphoprotein with roles in ribosome biogenesis, centrosome duplication, p53 response and DNA repair, and is commonly mutated in Acute myelogenous leukemia (AML). NPM is juxtaposed with retinoic acid receptor α (RARα[[Unsupported Character - [[Unsupported Character - ]]]]) in Acute Promyelocytic Leukemia (APL), raising the possibility that NPM functions are disrupted in this leukemia. We sought to determine the extent of NPM deregulation in the U937-NPM-RARα+ cell line model, and to extend this analysis more generally to APL. Immunofluorescent microscopy (IF) demonstrated abnormal distribution of NPM in patient-derived APL cells carrying NPM-RARα or PML-RARα, as well as cell lines expressing X-RARα: NPM was distributed into abnormally large aggregates within the nucleus and/or throughout the cytoplasm. NPM distribution reverted to normal after 48 hr of treatment with 1.0 μM all-trans retinoic acid (ATRA). NPM protein levels were also significantly increased in X-RARα+ cell lines. This may by due to an alteration at the protein level, as NPM mRNA expression was unaffected by X-RARα, and no mutations were detected in the NPM locus. Indeed, we found that NPM protein half-life was increased in U937 cells expressing X-RARα. These data suggested a potential disruption in nucleolar architecture in APL cells. Alterations in size and number of nucleolar organizing regions (NORs) in NB4 and U937-X-RARα cells were evident, when compared to U937 controls. Defects in nucleolar organization may lead to altered ribosome biogenesis, protein synthesis, as well as cell size and proliferation. Ribosomal RNA precursor expression was found to be increased significantly in U937-NPM-RARα cells, compared to controls. Relative cell volume, assessed by a flow cytometric assay, was moderately increased, by approximately 10% in NPM-RARα+ cells, while cell proliferation rates were significantly increased, and doubling time decreased, compared to U937 controls. Analysis of protein synthesis rates in U937-NPM-RARα+ cells indicated that the incorporation of a fluorescent Methionine analogue was twice that in control U937 cells. Finally, in order to determine the applicability of our in vitro results to APL patients, we analyzed pre-rRNA and 18S rRNA expression in bone marrow RNA extracted at diagnosis from 16 APL patients (10 BCR1/2 and 6 BCR3), in order to determine whether elevated nucleolar function was found in APL. 10/16 APLs had elevated 18S rRNA levels (>1.5-fold compared to normal BM); 8/16 had similarly elevated pre-rRNA levels. Overall, pre-rRNA levels were elevated an average of 2-fold compared to normal controls, while 18S rRNA levels were elevated an average 1.8-fold (p < 0.05 for both comparisons). We therefore present the first evidence that NPM may be universally deregulated in APL, leading to defective NPM function within the leukemic cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5010.