BORN TO BE EXPORTED: C-TERMINUS NUCLEAR EXPORT SIGNALS OF DIFFERENT STRENGTH ENSURE CYTOPLASMIC ACCUMULATION OF NUCLEOPHOSMIN LEUKEMIC MUTANTS

Nucleophosmin (NPM1) gene mutations are the most common genetic lesion in Acute Myeloid Leukemia (AML) (Falini, NEJM, 352:254, 2005) and specifically identify a distinct molecular and clinico-pathological entity (Falini, Blood, 109:874, 2007). NPM1 mutations result in aberrant NPM cytoplasmic dislocation due to two indispensable alterations at C-terminus of NPM protein: 1) disruption of both tryptophans (W) 288 and 290 (or 290 only) that constitute the Nucleolar Localization Signal; 2) creation of a new carboxy-terminal Nuclear Export Signal (NES), with 6 molecular variations observed to date. We previously showed that there is correlation between the new NES sequence and the number of mutated tryptophan(s). In fact, the most common NES motif (LxxxVxxVxL) always associates with loss of both W288 and W290 (e.g. NPM mutant A), while leukemic mutants retaining W288 always carry rare NES variant sequences (e.g. LxxxLxxVxL in NPM mutant E). These findings suggest diverse sequences of NPM mutant NES motifs function differently. Hereby, we provide evidence of how C-terminus alterations functionally cooperate to delocalize NPM mutants to cytoplasm. We first investigated whether different NPM leukemic mutants differ in their ability to be exported in the cytoplasm. NIH 3T3 cells were transfected with eGFP-tagged NPM mutants A and E. After incubation with low doses of Leptomycin B (a specific inhibitor of Crm1, the protein responsible for NES-mediated nuclear export), NPM mutant A was almost completely nuclear whilst NPM mutant E was still markedly cytoplasmic. This demonstrate that NPM mutant E is less sensitive to Crm1 inhibition than NPM mutant A. To directly measure the export efficiency of each of the six different NPM C-terminal NESs so far identified, we isolated and cloned them into a pREV(1.4)-eGFP plasmid expressing a mutagenized REV protein lacking its NES but retaining its Nuclear Localization Signal, an assay that allows to measure the export efficiency of various NES sequences (Henderson, Exp Cell Res 256:213, 2000). The REV(1.4) fusion protein containing the most common NPM mutant NES LxxxVxxVxL (never found with W288) was nuclear in the majority of transfected cells (indicating a functional NES with weak activity), whilst all variant NESs were mostly cytoplasmic (indicating a stronger activity). These findings prove that NPM mutants carry NES motifs with different nuclear export efficiency. NPM subcellular localization is dictated by opposing balance of forces (tryptophans and NES), and no NPM mutant from AML leukemic patients has ever been found to contain the weak LxxxVxxVxL NES in the presence of W288. We therefore investigated the consequence of artificially combining these two features on NPM subcellular traffic in NIH 3T3 cells. Notably, these artificial NPM mutants were not exported efficiently into cytoplasm, since the force (W288) driving mutants towards the nucleolus overwhelmed the force (NES motif) exporting them into cytoplasm. These findings show that NPM leukemic mutants must carry a strong NES motif if W288 is retained in order to ensure efficient cytoplasmic accumulation. This reveals a mutational selective pressure toward efficient NPM nuclear export and points to this event as critical for leukemogenesis and therefore as a potential therapeutic target.