A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF

Abstract

In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14 ARF . Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/ heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14 ARF in a 'dose-dependent tug of war' fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14 ARF ) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14 ARF ) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

“…On the other hand, in the presence of excess of mutant NPM1, all NPM1wt protein was pulled out of the nucleoli into nucleoplasm and cytoplasm, 27 as seen in some patients with NPM1-mutated AML. On the contrary, excess doses of NPM1wt relocated NPM1 leukaemic mutant from cytoplasm to the nucleoli of transfected cells, 27 a pattern never observed in NPM1-mutated AML patients in whom the mutant was always cytoplasmic restricted. 81 This evidence points at a dose-dependent relationship between wild-type and mutated NPM1 proteins, and supports the view that NPM1 leukaemic mutants are 'born to be exported'.…”

“…4,58 Core amino acids in the two physiological aminoterminal NES motifs seem to play a major role in mediating NPM1 homo-and hetero-dimerization, by dictating in a 'dosedependent tug of war' fashion the subcellular distribution of wild-type and mutated NPM1 proteins. 27 In fact, transfected cells showed that, when NPM1wt and mutant proteins were expressed at equimolar doses, a fraction of NPM1wt was consistently retained in the nucleoli (as is most often observed in AML samples). On the other hand, in the presence of excess of mutant NPM1, all NPM1wt protein was pulled out of the nucleoli into nucleoplasm and cytoplasm, 27 as seen in some patients with NPM1-mutated AML.…”

“…27 In fact, transfected cells showed that, when NPM1wt and mutant proteins were expressed at equimolar doses, a fraction of NPM1wt was consistently retained in the nucleoli (as is most often observed in AML samples). On the other hand, in the presence of excess of mutant NPM1, all NPM1wt protein was pulled out of the nucleoli into nucleoplasm and cytoplasm, 27 as seen in some patients with NPM1-mutated AML. On the contrary, excess doses of NPM1wt relocated NPM1 leukaemic mutant from cytoplasm to the nucleoli of transfected cells, 27 a pattern never observed in NPM1-mutated AML patients in whom the mutant was always cytoplasmic restricted.…”

“…1 Core amino acids in its two physiological N-terminal NES motifs (residues 44 and 47; residues 100 and 102) appear to be involved in NPM1 homo-and heterodimerization. 27 It is still unclear how wild-type nucleophosmin oligomerization favours nucleolar targeting. The simplest explanation is that a NPM1 oligomer might result in a 'high NoLS load' that reaches the nucleolar targeting threshold.…”

“…Indeed, artificial NPM1 mutants that contain the nucleolar-binding domain but are unable to form oligomers cannot enter the nucleolus. 27 Thus, it is not surprising that NPM1wt is isolated from cells mainly as oligomers, 28 which are usually hexamers. 29 The NPM1wt oligomer-forming …”