Identification of a novel NPM1 mutation in acute myeloid leukemia
Experimental Hematology & Oncology volume 12, Article number: 87 (2023)
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.
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 . 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 . 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_406insGCCCTGGAACTGGGGAAC, 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 newly-identified 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) .
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.
Clinical characteristics of the AML patient with the novel NPM1 mutation
We then summarized the clinical characteristics of the AML patient (Pt. Song) harboring the novel NPM1 exon 5 mutation (NPM1_MutSong) (Additional file 1: Table S3). Besides NPM1 mutation, she also carried WT1 (VAF, 13.1%), IKZF1 (VAF, 17.8%), JAK2 (VAF, 4.6%), and NUP98 (VAF, 18.8%) mutations at the time of diagnosis. The patient was intravenously treated with daunorubicin 60 mg/m2 (days 1–3) plus cytarabine 100 mg/m2, intravenously (days 1–7) for induction, followed by 3 cycles of intermediate-dose cytarabine as consolidation. Then the patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, Pt. Song died of severe pulmonary infection and viral encephalitis 14 months after the initial remission, 7 months after allo-HSCT.
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 . Since mutations acquired later in disease development exhibited lower VAFs than earlier mutations , 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 . Interestingly, data from a large cohort of AML patients showed that the coexistence of WT1 and NPM1 mutations significantly altered the positive prognostic impact of NPM1 mutations alone , 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 NPM1 mutation is beneficial for clinical laboratory diagnosis, genetic risk assessment and MRD monitoring.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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This work was supported in part by the Fundamental Research Funds for the Central Universities (226-2022-00003); 2023 Science and Technology Innovation Plan of Shanghai Science and Technology Commission (23141902700); Natural Science Foundation of Zhejiang Province, China (Y23H080018); Key research and development program of Zhejiang Province, China (No. 2022C03005).
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The study was carried out in accordance with the Declaration of Helsinki and was also approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine.
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: Table S1. Summary of reported rare NPM1 mutants and subcellular localization of mutated NPM1. Table S2. Next generation sequencing (NGS) gene panel. Table S3. Characteristics of the patient with AML carrying the NPM1 exon 5 mutation.
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Yao, Y., Lin, X., Wang, C. et al. Identification of a novel NPM1 mutation in acute myeloid leukemia. Exp Hematol Oncol 12, 87 (2023). https://doi.org/10.1186/s40164-023-00449-4