Identification of a novel NPM1 mutation in acute myeloid leukemia

Abstract

Nucleophosmin (NPM1) is a widely expressed nucleocytoplasmic shuttling protein with prominent nucleolar localization. It is estimated that 25-35% of adult patients with acute myeloid leukemia (AML) carry NPM1 mutations. The classic NPM1 type A mutation occurs in exon 12, which accounts for 75-80% of adult patients with NPM1-mutated AML. It produces an additional leucine and valine-rich nuclear export signal (NES) at the C-terminus, and causes aberrant cytoplasmic dislocation of NPM1 protein. Notably, emerging evidence indicates that besides the classic type A mutation, rare mutants occurring in other exons may also lead to the imbalance of the nucleocytoplasmic shuttle of NPM1. Identification of novel non-type A mutants is crucial for the diagnosis, prognosis, risk stratification and disease monitoring of potential target populations. Here we reported a novel NPM1 mutation in exon 5 identified from a de novo AML patient. Similar to the classic type A mutation, the exon 5 mutation had the NPM1 mutant bound to exportin-1 and directed the mutant into the cytoplasm by generating an additional NES sequence, resulting in aberrant cytoplasmic dislocation of NPM1 protein, which could be reversed by exportin-1 inhibitor leptomycin B. Our findings strongly support that besides the exon 12 mutation, the exon 5 mutant is another NPM1 "born to be exported" mutant critical for leukemogenesis. Therefore, similar to the classic type A mutation, the identification of our novel NPM1 mutation is beneficial for clinical laboratory diagnosis, genetic risk assessment and MRD monitoring.

Keywords: Aberrant cytoplasmic dislocation; Acute myeloid leukemia; NPM1 mutation; Nuclear export signal; Prognosis.

Fig. 1

A Alignment of genomic sequences of the new NPM1 exon 5 mutant (highlighted) and exon 12 mutants including canonical type A mutant and other rare mutants that identified in our AML patient cohort. The patient numbers and percentages of different mutants were also indicated. B Graphical representation and predicted protein sequences of NPM1 wildtype, type A mutant and the new NPM1 exon 5 mutant (NPM1_MutSong). The new generated amino acids are highlighted in red. C Alignment of predicted protein sequences of NPM1 wildtype, reported exon 5 mutants and the new NPM1 exon 5 mutant. For the new exon 5 mutant, the predicted NES motif is underlined and highlighted

Fig. 2

A Representative images show cellular localization of NPM1 (green) in blasts from typical AML patients, with NPM1 wildtype, NPM1 type A mutation and this new exon 5 mutation (NPM1_MutSong), respectively. DAPI was used for nuclei staining (blue). Fibrillarin was used for nucleolus staining. White arrows indicated the predominate cytoplasmic location of NPM1 in mutation NPM1_MutSong. Images were collected by Nikon eclipse Ti2 confocal microscope; magnification, × 600. B Immunohistochemical staining of bone marrow biopsies from Pt. Song with the new NPM1 exon 5 mutation. Representative images show diffuse infiltration by leukemia blasts (hematoxylin and eosin [HE], left), with aberrant cytoplasmic localization of NPM1 (right, brown). Images were collected by Pannoramic 250 FLASH; magnification, × 630. C Western blot analysis with anti-GFP antibody (left, ~ 27 kD) or anti-NPM1 antibody (right, ~ 38 kD) both recognizing NPM1 proteins in HEK-293T cells overexpressing GFP-NPM1 exon 5 fusion protein (~ 61 kD). D HEK-293T cells overexpressing the new GFP-NPM1 fusion protein show the aberrant cytoplasmic subcellular location, concomitantly, with nucleolar location. Upon treatment with leptomycin B, NPM1 was re-localized in nucleus. Images were collected by Olympus IX71 confocal microscope; magnification, × 200