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Case Reports
. 2021 Sep 16;12(9):1426.
doi: 10.3390/genes12091426.

A Curious Novel Combination of Nucleophosmin (NPM1) Gene Mutations Leading to Aberrant Cytoplasmic Dislocation of NPM1 in Acute Myeloid Leukemia (AML)

Alessandra Venanzi  1   2 , Roberta Rossi  1 , Giovanni Martino  3   4 , Ombretta Annibali  5 , Giuseppe Avvisati  5 , Maria Grazia Mameli  2 , Paolo Sportoletti  1   2 , Enrico Tiacci  1   2 , Brunangelo Falini  1   2 , Maria Paola Martelli  1   2
Affiliations
Case Reports

A Curious Novel Combination of Nucleophosmin (NPM1) Gene Mutations Leading to Aberrant Cytoplasmic Dislocation of NPM1 in Acute Myeloid Leukemia (AML)

Alessandra Venanzi et al. Genes (Basel). .

Abstract

Nucleophosmin (NPM1) mutations occurring in acute myeloid leukemia (AML) (about 50 so far identified) cluster almost exclusively in exon 12 and lead to common changes at the NPM1 mutants C-terminus, i.e., loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES), at the bases of exportin-1(XPO1)-mediated aberrant cytoplasmic NPM1. Immunohistochemistry (IHC) detects cytoplasmic NPM1 and is predictive of the molecular alteration. Besides IHC and molecular sequencing, Western blotting (WB) with anti-NPM1 mutant specific antibodies is another approach to identify NPM1-mutated AML. Here, we show that among 382 AML cases with NPM1 exon 12 mutations, one was not recognized by WB, and describe the discovery of a novel combination of two mutations involving exon 12. This appeared as a conventional mutation A with the known TCTG nucleotides insertion/duplication accompanied by a second event (i.e., an 8-nucleotide deletion occurring 15 nucleotides downstream of the TCTG insertion), resulting in a new C-terminal protein sequence. Strikingly, the sequence included a functional NES ensuring cytoplasmic relocation of the new mutant supporting the role of cytoplasmic NPM1 as critical in AML leukemogenesis.

Keywords: NPM1; acute myeloid leukemia; exportin-1/XPO1; nuclear export signal (NES); nucleophosmin.

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Conflict of interest statement

B.F. licensed a patent on NPM1 mutants (n. 102004901256449). B.F. and M.P.M. declare honoraria from Rasna Therapeutics, Inc for scientific advisor activities. M.P.M. also declares honoraria/consultancy at scientific advisory board for Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals. O.A. declares honoraria/consultancy at scientific advisory board for Janssen, Novartis, Celgene, Amgen. P.S. declares honoraria/consultancy at scientific advisory board for Abbvie, Janssen, Novartis, AstraZeneca, Incyte. The other authors declare no competing interests.

Figures

Figure 1
Figure 1
Study design and diagnostic discrepancies. Overall, 929 unselected AML patients were studied by multiple diagnostic approaches (i.e., immunohistochemistry, IHC; standard molecular analysis for NPM1 exon 12, and Western blotting with specific anti-NPM1 mutant antibody, WB). 1 indicates comparison between IHC and NPM1 exon 12-molecular analysis. 2 indicates comparison between NPM1 exon 12-molecular analysis and WB. wt: wild type (exon 12 wild type); mut: mutated (exon 12 mutation A and other non-A mutations). Discrepancy 1 indicates NPM1c+ at IHC and NPM1 exon 12 wild-type (reported in [17]). Discrepancy 2 indicates NPM1c+ with mutation in exon 12 (Exon 12 +), but Western blotting (WB) negative with anti-NPM1 mutant antibody, recognizing either NPM1 mutant A and other previously described non-A mutants [20].
Figure 2
Figure 2
(a) Immunohistochemical staining of bone marrow trephine from pt. Rm. HE: hematoxylin-eosin staining; NPM: mouse monoclonal anti-panNPM1, clone 376; CD34: anti-CD34 mAb (to note, blood vessels in tissue sections are used as positive control for CD34 staining). LAT: linker for activation of T cell (marker for megakaryocytes); APAAP technique; hematoxylin counterstaining. Images were collected using an Olympus B61 microscope with a UPlanApo 40×/0.85U and UPlan FI 100×/1.3 NA oil objective for the insets; Camedia 4040, Dp_soft Version 3.2; and Adobe Photoshop CC 2019; (b) WB analysis of total protein extracts from pt. Rm. OCI/AML3: positive control for NPM1 mutant A expression. M: molecular weight marker.
Figure 3
Figure 3
Genomic DNA NPM1 fragment analysis. Electropherograms of genomic DNA fragments of NPM1 exon 12 extracted from PB of the AML patient Rm (upper panel), AML sample with wild type NPM1 (middle panel) and AML sample with NPM1 mutation A (lower panel). Peaks corresponding to WT (wild type), mutant A (Mut A) and mutant Rm (Mut Rm) are indicated. Fragments from Mut Rm are 4 nucleotides shorter (−4 nt) than WT. Fragments from Mut A are 4 nucleotides longer (+4 nt) than WT. The variant allelic frequency (VAF) of the Rm mutated allele is shown.
Figure 4
Figure 4
The new NPM1 mutation Rm disrupts the NoLS and inserts a C-terminal NES. (a) Nucleotide sequences of exon 12 in NPM1 wild-type, mutation A and new composite mutation Rm, and corresponding predicted protein sequences (arrow). The 4 nucleotide insertion TCTG in mutation A and mutation Rm is highlighted in bold black. The 8 nucleotides lost in composite mutation Rm are highlighted in bold red. The C-terminal NES motifs in mutant A and mutant Rm are underlined. (b) Left, NES motif prediction in the new NPM1 mutant Rm. The newly acquired C-terminal NES is highlighted in the red box. Right, representative image of NIH-3T3 overexpressing the new GFP-NPM1 mutant Rm fusion protein. Images were acquired using a Zeiss LSM 800 confocal microscope (Carl Zeiss) with a 488-nm (for eGFP) laser line for excitation, and a 63×/1.4 OIL Plan-Apochromat objective. N: nucleus; C: cytoplasm.
Figure 5
Figure 5
The subcellular localization of the new NPM1 mutant Rm is NES-dependent. NIH-3T3 overexpressing either the GFP-NPM1 mutant A or the new GFP-NPM1 mutant Rm fusion proteins. Untreated: vehicle. LMB: leptomycin B. GFP: green fluorescence protein (of the GFP-NPM1 fusion protein). DNA: indicates stained nuclei (blue). Images were acquired using a Zeiss LSM 800 confocal microscope (Carl Zeiss) with a 488-nm (for eGFP) laser line for excitation, and a 63×/1.4 OIL Plan-Apochromat objective. Asterisk: nucleoli spaces.
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