Analysis of NPM1 splice variants reveals differential expression patterns of prognostic value in acute myeloid leukemia.

Malgorzata Zajac,Richard F Schlenk,Reddy Chakkarappan Sundaram,Olga Jankowska-Lecka,Konstanze Döhner,Lars Bullinger,Joanna Zaleska,Stephany C Correa,Andrzej Stepulak,Jakub Czapinski,Hartmut Döhner,Matthias Schunn,Grazyna Stasiak,Michal Kielbus,Eliza Glodkowska-Mrowka,Krzysztof Giannopoulos,Anna Dolnik

doi:10.18632/oncotarget.19871

Malgorzata Zajac, Richard F Schlenk + Show 15 more

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https://doi.org/10.18632/oncotarget.19871

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Abstract

Mutations of the nucleophosmin-1 (NPM1) gene in cytogenetically normal (CN) acute myeloid leukemia (AML) identify a group of patients with more favorable prognosis. NPM1 encodes three main alternatively spliced isoforms R1(B23.1), R2(B23.2), and R3(B23.3). The expression of splice variants R1, R2 and R3 were higher in AML patients compared to normal cells of healthy volunteers (HVs), although RNA-seq analysis revealed enhanced R2 expression also in less differentiated cells of HVs as well as in AML cells. The variant R2, which lacks exons 11 and 12 coding for the nucleolar localization domain, might behave similar to the mutant form of NPM1 (NPM1mut). In accordance, in CN-AML high R2 expression was associated with favorable impact on outcome. Moreover, functional studies showed nucleolar localization of the eGFP-NPM1 wildtype and cytoplasmic localization of the eGFP-NPM1 mut protein. While the eGFP-NPM1 R2 splice variant localized predominantly in the nucleoplasm, we also could detect cytoplasmic expression for the R2 variant. These results support a unique biological consequence of R2 overexpression and in part explain our clinical observation, where that high R2 variant expression was associated with a better prognosis in CN-AML patients.

Highlights

Generation sequencing technology has identified many new gene mutations in acute myeloid leukemia (AML) that provide novel insights into the mechanisms of leukemogenesis and that further unravel the molecular heterogeneity, in particular within the group of cytogenetically normal (CN) AML [1, 2]
Sex - no. (%) Male Female Age - yr Median Range Bone marrow blasts Median - % Range - % Data not available – no. (%) Peripheral blood blasts Median - % Range - % Data not available – no. (%) Type of AML - no. (%) Primary AML s-AML t-AML o-AML NPM1 mutations - no. (%) Data not available FLT3-IDT mutations - no. (%) Cytogenetic group - no. (%) normal karyotype complex karyotype inv(16)
While in the first cohort of CN-AML expression of R2 tended to be elevated in NPM1mut compared to NPM1 wildtype status (NPM1wt), in the entire cohort of CN-AML (n=105) we found a similar trend between these groups (1.21 vs 0.82; p=0.13, respectively) (Figure 3B)

Summary

Introduction

Generation sequencing technology has identified many new gene mutations in acute myeloid leukemia (AML) that provide novel insights into the mechanisms of leukemogenesis and that further unravel the molecular heterogeneity, in particular within the group of cytogenetically normal (CN) AML [1, 2]. In addition to genomic abnormalities, aberrant expression levels of several genes have been identified as prognostic markers [3, 4], so as deregulated gene expression involved in CN-AML pathogenesis. MRNA splicing has been reported to be involved in human disease development, and many cancer-related genes have been shown to be regulated by alternative splicing [5]. In myeloid disease several reports have revealed mutations in genes encoding splicing factors, such as SF3B1 [6, 7], and recently a heterogeneous genomic category of AML with mutations in genes encoding chromatin and RNA-splicing regulators, accounting for 18% of patients, could be identified [8]. First analyses of alternative splicing in bone marrow of AML samples revealed a number of significantly spliced genes, which encode e.g. proteins such as NOTCH2, CD13 or FLT3 [9]

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