Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML

Abstract

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AMLassociated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus. IntroductionIn acute myeloid leukemia (AML), a clinically and molecularly heterogeneous disease, 1 recurrent cytogenetic abnormalities help define subgroups with different prognosis, and identify patients who might benefit from targeted therapies. 1 However, almost half adult AMLs display normal karyotype at conventional cytogenetics, 2 and the clinical and molecular features of this large subgroup of patients are still poorly understood. [3][4][5][6][7] We recently observed that about 60% of adult AML with normal karyotype display aberrant cytoplasmic expression of nucleophosmin (NPM). 8 A multifunctional protein [9][10][11][12][13][14][15] that characteristically shuttles between the nucleus and the cytoplasm, 16 NPM is found mainly in the nucleolus, [17][18][19] where it is one of the most abundant of the approximately 700 proteins so far identified by proteomic techniques. 20 Cytoplasmic NPM identifies a distinct subgroup of AML, named NPMc ϩ AML, that accounts for about 35% of all adult AML and is characterized by wide morphologic spectrum, multilineage involvement, high frequency of FLT3-ITD mutations, absence of CD34, and relatively good response to induction therapy. 8 NPMc ϩ AML also has a distinct gene expression profile 21 and carries mutations in exon-12 of the NPM gene 8 that serve as predictor of favorable prognosis in AML with normal karyotype, [22][23][24] and as a marker for monitoring of minimal residual disease. 25 In spite of the close association between the aberrant cytoplasmic expression of NPM and exon-12 NPM mutations, 8 the mechanism underlying cytoplasmic accumulation of NPM in leukemic cells and its interference with wild-type NPM protein remaine...

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

“…First, it was shown that by mutating Trp288 and Trp290 to alanine in the context of the native sequence, the C-terminal domain unfolds 25 and the protein loses its nucleolar localization. 29 This effect was also seen when perturbing the hydrophobic core of the domain by other means, such as by concomitantly mutating Phe268 and Phe276 to alanine. 25 Then, it was shown that a NPM1 variant protein, bearing the terminal sequence of mutant A (Fig.…”

“…22 Furthermore, as the mutated protein is able to oligomerize with the NPM1 protein synthesized from the wild-type allele, through its unchanged N-terminal domain, the majority of wild-type NPM1 is also translocated in the cytosol and only a minimal portion of it is still retained in nucleoli. 29 This is the hallmark of this kind of leukemia that can be detected by immunohistochemistry. 30 Because of its distinctive features, AML with NPM1 mutations has been included as a provisional distinct entity in the 2008 WHO classification of myeloid neoplasms.…”

“…28 The combined effect of the loss of either one or both tryptophan residues and the appearance of a new NES in the variant protein is mandatory to completely alter the shuttling properties of the protein. 29 In AML with NPM1 mutations, the variant NPM1 protein is stably and aberrantly localized in the cytoplasm of leukemic cells (and therefore referred to as NPM1cþ, where cþ stands for cytoplasmic positive). 22 Furthermore, as the mutated protein is able to oligomerize with the NPM1 protein synthesized from the wild-type allele, through its unchanged N-terminal domain, the majority of wild-type NPM1 is also translocated in the cytosol and only a minimal portion of it is still retained in nucleoli.…”

Nucleophosmin: A versatile molecule associated with hematological malignancies

“…(62) Falini et al further confirmed that both alterations are crucial for NPM mutant export from the nucleus to the cytoplasm. (63) Falini et al found that cytoplasmic staining of NPM could define the NPM1 mutation-positive AML cases that had a normal karyotype, NPM1 gene mutations, and responsiveness to induction chemotherapy. (61) Grisendi and Pandolfi noted that NPM staining in cases of AML with aberrant cytoplasmic localization of the protein is mostly cytoplasmic, which suggests that the mutant NPM acts predominantly on the product of the remaining wild-type allele, causing its retention in the cytoplasm by heterodimerization (64) (Fig.…”

“…Structurally, a newly generated NES sequence at the C-terminus is a common feature, although more than 20 different variant NPM1 mutations have been identified. (63) How the acquired NES changes the function of NPM in addition to altering the subcellular localization should be studied. The second question is why only FLT3 mutations frequently accompany the NPM1 mutation.…”