Targeting Mutant Nucleophosmin (NPM)1: A Promising Approach To Induce Growth Inhibition and Differentiation in Human AML.

NPM1 is a nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids and is capable of histone and nucleosome assembly. NPM1 shuttles between nucleus and cytoplasm, but is mutated and aberrantly localized to the cytoplasm in 35% of patients with AML. NPM1 is involved in regulating the activity of p53 and p14ARF, such that cells lacking NPM1 show decreased p14ARF. Mutant (m) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES). This causes mNPM1 to remain in the cytoplasm. In AML, mNPM1 has been correlated with the presence of FLT3-ITD and normal karyotype. NPM1 maps to 5q35 locus, which is often deleted in myelodysplastic syndrome. The role of the cytosolic mNPM1 in the pathogenesis of AML is not clear. In the present studies we determined the expression and biologic effects of mNPM1 in cultured and primary AML cells. Mutant nucleophosmin can be readily identified by reverse transcription followed by amplification with PCR primers specific for the mutant sequence. Sub-cellular fractionation and immunoblot analysis and/or immunoflourescent microscopy, as well as RT-PCR to detect the mNPM1 transcripts, confirmed that mNPM1 is localized in the cytosol in the cultured AML OCI-AML3 but is unmutated and nuclear in HL-60 cells. As compared to control siRNA, treatment with siRNA to NPM1 for 72 hours significantly induced p21 expression, inhibited cell proliferation and induced morphologic differentiation of OCI-AML3 cells. NPM1 siRNA did not induce significant apoptosis or loss of cell viability as determined by trypan blue uptake assay. OCI-AML3 cells co-express HoxA9 and Meis1 proteins, which are known to be leukemogenic in mouse myeloid progenitor cells. Notably, treatment with NPM1 siRNA depleted Meis1 and HoxA9 expressions in OCI-AML3 cells. Treatment with leptomycin B (5 nM for 48 hours), an inhibitor of the NES receptor CRM1/exportin-1, shifted the localization of mNPM1 from the cytosol to nucleus and also depleted the levels of Meis1 and HoxA9 in OCI-AML3 cells. Meis1 is known to transactivate FLT3 tyrosine kinase in AML progenitor cells, and a significant association of FLT-3-ITD with the presence mNPM1 has been documented. In the present studies, of the 16 primary AML samples, four that expressed FLT3-ITD also demonstrated expression of mNPM1. Taken together, these findings suggest that abrogation of the levels or reversing the aberrant cytosolic localization of mNPM1 may be a promising anti-leukemia treatment strategy, especially for targeting AML that co-express FLT3-ITD and mNPM1.