Identification of a novel NPM1 mutation in acute myeloid leukemia

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. Supplementary Information The online version contains supplementary material available at 10.1186/s40164-023-00449-4.


To the Editor,
Nucleophosmin (NPM1) is a ubiquitously expressed nucleocytoplasmic shuttling protein, with predominant nucleolar localization.NPM1-mutant acute myeloid leukemia (AML) accounts for 25-35% of adult AML patients, has been defined as a distinct AML entity in the 2022 World Health Organization (WHO) classification and is validated as a marker of measurable residual disease (MRD) [1,2].The majority of NPM1 mutations reported affect exon 12 (classic type A mutation), accounting for 75-80% of adult NPM1-mutated AML cases, which generates an extra nuclear export signal (NES) in the C-terminus leading to aberrant cytoplasmic localization [3].However, accumulating evidence suggests that other than the canonical type A mutation, some novel mutations identified in exons 9 and 11, may also result in NPM1 nucleocytoplasmic shuttling.(Additional file 1: Table S1) [4][5][6][7].Recent large clinical studies reported that AML patients with NPM1 mutations alone had a favorable outcome, compared with NPM1 mutations combined with FLT3-ITD [8].Moreover, the type A mutation was associated with a favorable prognosis, while some newly identified non-type A mutants were associated with poor clinical outcomes [2,9].Thus, identification of novel non-type A mutants is paramount for diagnosis, prognosis, risk stratification, and disease monitoring of potential target populations.

Identification of a novel NPM1 mutation located in exon 5
In this study, somatic mutation analysis was performed on samples from 566 AML patients using the next-generation sequencing (NGS) with myeloid sequencing panel (Additional file 1: Table S2).NPM1 mutations were identified in 109 AML cases, with four mutation types (A, D, Om, and I), which predominately affected exon 12, represented the vast majority of NPM1 mutations: the type A mutation accounted for 78.0% of overall cases, while types D, Om, and I in total accounted for 13.8% (Fig. 1A).Importantly, we identified one AML patient with NPM1 mutation located in exon 5.This patient was a 59-year-old female with de novo AML (Additional file 1: Table S3), with an 18-nucletide in-frame insertion at position 405 (c.405_406insGCC CTG GAA CTG GGG AAC , named NPM1_MutSong) in the middle of exon 5.It generated a new NPM1 mutant protein, which was 6 aa longer than the wildtype one (p.135insALELGN),and contained a leucine-rich NES (Fig. 1B).The mutation was heterozygous for the patient [variant allele fraction (VAF), 11.9%].Notably, different from all NPM1 mutants in exons 9, 11, and 12 reported so far, the newlyidentified exon 5 mutant had an additional leucine-rich NES inserted in the intermediate domain but retained the C-terminal functional NoLS.Similar findings were recently reported on NPM1 mutants in exon 5 but at different positions (Fig. 1C) [10].

The novel NPM1 mutant exhibited aberrant cytoplasmic dislocation
This NPM1 mutant in exon 5 resulted in cytoplasmic localization detected by both confocal microscopy (Fig. 2A) and immunohistochemistry (IHC) (Fig. 2B) in the AML patient's sample.Cytoplasmic dislocation was induced by the extra NES that partially impeded the NoLS driven nucleolar localization.HEK-293 T overexpressing the new GFP-NPM1 exon 5 fusion protein from patient Song (Fig. 2C) indicated the aberrant localization in the cytoplasm and partially in nucleoli (Fig. 2D, left).Moreover, NES-dependent cytoplasmic localization was inhibited by exportin-1 inhibitor leptomycin B (Fig. 2D, right), suggesting that the novel NES generated by the exon 5 mutant was functional and responsible for cytoplasmic localization.
The novel mutant exhibited several similar concomitant clinical features as type A mutation.Compared with commonly co-mutated genes such as DNMT3A and TET2, NPM1 mutations are generally presented at lower VAFs [11].Since mutations acquired later in disease development exhibited lower VAFs than earlier mutations [12], the moderate VAFs suggest that NPM1 mutations may be a late event in leukemogenesis.In our patient, all the mutations showed moderate VAFs.Although our patient did not carry FLT3-ITD mutation, remarkably, she carried the WT1 mutation.While the classic NPM1 mutation alone is reportedly a favorable factor for AML with normal cytogenetics, sole WT1 mutation has been reported to be associated with poor prognosis [13].Interestingly, data from a large cohort of 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 AML patients showed that the coexistence of WT1 and NPM1 mutations significantly altered the positive prognostic impact of NPM1 mutations alone [13], underscoring the importance of more aggressive treatment for this subpopulation.Thus, our patient underwent allo-HSCT after initial remission, but unfortunately, ultimately died of severe pulmonary infection and viral encephalitis.
In this study, we reported a novel NPM1 mutation in exon 5 in a de novo AML patient, which resulted in cytoplasmic dislocation of NPM1 protein.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, identification of our novel • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Fig. 1 A
Fig.1A 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
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