Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH 2 -terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis. [Cancer Res 2007;67(13):6230-7]

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.

As we observed marked cross-resistance between calicheamicin and anthracyclines, combining these compounds may be less likely to result in increased clinical efficacy. Based on our in vitro data, we suggest combining GO with cytarabine and/or L-asparaginase. Currently, most trials combine GO with cytarabine and an anthracycline. No clinical trial is studying the combination of GO with cytarabine and L-asparaginase.The interpatient differences in calicheamicin sensitivity are the largest differences in drug sensitivity we have ever observed in pediatric AML, suggesting that it is likely that primary calicheamicin resistance plays a role in the response to GO. This needs to be validated in future clinical trials in which in vitro and in vivo response to GO/calicheamicin can be compared. In conclusion, when analyzing resistance to GO, primary resistance to calicheamicin should be considered as an important mechanism. AcknowledgementsThis work was partially funded by ZonMW AGIKO Grant 920-03-374 (BFG). Calicheamicin was provided free of charge by Wyeth Pharmaceuticals. BFG performed the experiments, analyzed the data and wrote the paper; CMZ designed the research and wrote the paper; SJHV performed the experiments, analyzed the data and edited the paper; AHL performed the experiments and edited the paper; UC, KH, DR and BESG provided the leukemic samples and clinical data and edited the paper; JC analyzed the data and edited the paper; GJLK designed the research and edited the manuscript. Conflict of interestThe authors state no conflict of interest.

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