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

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.

Keywords: AML; NPM1; splicing variants.

Figure 1. RNAseq analysis of R2 expression in 10 AML transcriptomes and 3 HVs bone marrow samples

Ten cell fractions of cells from normal bone marrow were obtained: myeloblasts (from n=2 healthy donors), promyelocytes (n=2), metamyelocytes (n=3) and neutrophils (n=3). P-value refers to all analyzed subfractions of cells. The analysis was performed with ANOVA test.

Figure 2. (A) Conventional PCR and Western blot analysis of NPM1 isoforms in cell lines

cDNA from the cell lines NB4, KG1, HEK293, K562, OCI-AML3, ME-1 and Kasumi-1 was analyzed by conventional PCR and protein lysates of the corresponding cell lines were analyzed on Western blot. β-actin was used as a loading control. Conventional PCR shows the expression of R1, R2 and R3 NPM1 isoforms in the cell lines, which could be confirmed by Western blot analysis. (B) Western blot analysis of NPM1 expression in 4 AML patients.

Figure 3

(A) Enhanced expression of NPM1 splice variant R2 was observed in 201 AML patients compared to HVs. (B) Expression levels of NPM1 splice variant R2 within CN-AML groups with NPM1 mutations (NPM1mut) and without its mutations (NPM1wt). No difference was seen between those groups of patients. (C) OS in 105 CN-AML patients divided according to the expression levels of R2 splice variant. (D) OS of the total 201 AML patients divided according to the expression levels of R2. (E) OS in CN-AML patients divided into four groups according to the expression levels of R2 and FLT3-ITD mutational status: R2high/noFLT3-ITD R2low/noFLT3-ITD, R2low/FLT3-ITD, R2high/FLT3-ITD and CEBPAdm cases. (F) OS in CN-AML patients divided into four groups according to the NPM1 and FLT3-ITD mutational status: NPM1mut/noFLT3-ITD, NPM1wt/noFLT3-ITD, NPM1wt/FLT3-ITD, NPM1mut/FLT3-ITD with additional group of CEBPAdm.

Figure 4. NPM1 wt, R2 and mut show different subcellular localization in AML patient and HEK293 cells

(A) Confocal analysis of AML patient cells transiently transfected with expression vectors for eGFP-NPM1 wt, eGFP-NPM1 R2 or eGFP-NPM1 mut. Transfected cells expressed eGFP-NPM1 fusion proteins (green fluorescence). (B) Confocal analysis of the stable HEK293 cell lines expressing NPM1 variants (NPM1 wt, NPM1 R2 and NPM1 mut) fused with fluorescent GFP tag. Nuclei were stained with DAPI. Images were collected with Nicon Ti confocal microscope.