In acute myeloid leukaemia (AML), NPM1 gene mutations occur almost exclusively at exon 12.1 Therefore, we read with great interest the recent letter by Pitiot et al.2 on a novel type of mutation occurring at exon 11 of the NPM1 gene, which generates a 274-amino-acid NPM leukaemic mutant truncated at its C-terminal end. This mutation is very similar to the one we previously described.3 Specifically, we first identified a truncated form of NPM protein generated by a mutation at NPM1 exon 11, which localizes aberrantly in the cytoplasm of leukaemic cells due to the concerted action of two abnormalities: loss of tryptophan residues 288 and 290 (which impairs the mutated NPM protein capability to bind nucleoli) and the new Rev-type NES motif (VFLI) at the mutant C terminus.3 We concluded that a common molecular theme4 underlies the exaggerated nuclear export and cytoplasmic accumulation of NPM mutants in NPMc+ AML carrying mutations either at exon 12 or 11.
In their paper, Pitiot et al.2 claim that, despite the bulk of the mutated NPM protein is aberrantly localized in the cytoplasm, some of it is localized in the nucleoli. On the basis of this finding, they speculated that ‘in this case the wild-type protein would not be sequestered in the cytoplasm when forming heterodimers with the mutant protein, as has been suggested to occur with the described exon 12-NPM1 mutants’.2
We disagree with this interpretation for several reasons. First, to label their patient's leukaemic cells, the authors used a polyclonal antibody against NPM that recognizes both wild-type and mutated NPM proteins. Consequently, the relative contribution of the wild-type and C-terminal-truncated NPM proteins to nucleolar and cytoplasmic positivity cannot be established. Most importantly, site-directed mutagenesis and transfection studies showing the inability of our patient's NPM-truncated protein to bind nucleoli3 strongly suggest that the NPM nucleolar positivity reported by Pitiot et al. in patient's leukaemic cells was most likely due to the wild-type NPM rather than the NPM mutant. This finding does not imply, as suggested by the Pitiot et al., that in cells harbouring exon 11 NPM1 mutation, the wild-type NPM would not be sequestered in the cytoplasm. In fact, immunohistochemical slides of hundreds of AML samples with antibodies that react specifically against either wild-type or mutated NPM proteins have clearly shown that leukaemic cells carrying all types of NPM1 mutations (including mutation A), display not only aberrant cytoplasmic NPM (due to the presence in that site of both NPM mutant and dislocated wild-type NPM), but also consistent nucleolar NPM positivity,5 because a fraction of the wild-type NPM protein is always retained in nucleoli. Pitiot's finding that leukaemic cells carrying mutation A (unlike those with exon 11 NPM1 mutations) show no nucleolar NPM staining is also in conflict with the above studies and could well be due to technical reasons, such as sample processing and/or differences in antibody reactivity. Finally, the authors' concerns that in the previous studies, immunohistochemical determinations may have failed to detect the truncated NPM mutant alteration is contradicted by the immunohistochemical findings of aberrant cytoplasmic expression of exon 11 NPM mutant in our patient.3
Pitiot et al. do not provide an explanation for aberrant NPM expression in their patient's leukaemic cells. Our previous report3 provides a rationale for this finding. Although in their patient, the inserted eight nucleotide sequence2 is different to what we found,3 both mutations produce similar truncated proteins at C terminus: VEAKFARLSIM2 vs VEAKFRRLSIM.3 Most importantly, looking at the protein sequence, we found that the truncated NPM mutant described by Pitiot et al. carries at the C terminus the same NES motif (VLFI) we had already identified in our patient.3 This NES motif is yet another on the list of NES motifs that have been observed in all NPM leukaemic mutants identified to date (Table 1). Thus, the molecular variant of the exon 11 NPM1 mutation reported by Pitiot et al.2 carries the same alterations (lack of tryptophans and creation of a new C-terminal NES motif) that we have already shown as underlying aberrant NPM cytoplasmic accumulation in NPMc+ AML.4
Notably, all NPM leukaemic mutants (including those generated by NPM1 mutations involving exons other than 12) aberrantly localize in the cytoplasm.5 More recently, we also found that the rare NPM leukaemic mutants, retaining tryptophan residue 288, carry a NES motif, which is stronger than that of NPM mutants lacking both tryptophan residues (288 and 290).6 On the basis of these observations, we proposed that NPM1 mutations in AML act to achieve maximum nuclear export and cytoplasmic accumulation of NPM.6 As NPM mutants appear to be ‘born to be exported’, NPM1 mutations could be potentially critical for leukaemogenesis. The case reported by Pitiot et al.2 is most interesting since it reinforces this concept.
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Spectral and Kinetic Study of 3-Methylquinoxalin-2-ones Photoreduced by Amino Acids: N-Phenylglycine Radical Chain Reactions and N-Acetyltryptophan Decarboxylation
The Journal of Physical Chemistry A (2016)