Targeting Levels, Aberrant Localization or Oligomerization of Mutant Nucleophosmin Induces Differentiation and Loss of Survival of Human AML Cells with Mutant NPM1.

Abstract 2656

Poster Board II-632

NPM1 is a nucleolar phosphoprotein that forms oligomers and functions as a molecular chaperone for both proteins and nucleic acids. NPM1 normally shuttles between the nucleus and cytoplasm but is mutated and aberrantly localized to the cytoplasm in 35% of patients with AML, Mutant (m) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES), which causes mNPM1 to remain in the cytoplasm. In AML, the presence of mNPM1 is often co-detected with internal tandem duplications of FLT-3, which confers a poor prognosis. The effects of the cytosolic mNPM1 on the biology and drug-sensitivity of AML are unclear. In the present studies we determined the expression and biologic effects of mNPM1 in cultured and primary AML cells. Utilizing RT-PCR, immunoblot analysis and immunoflourescence microscopy (with a polyclonal anti-mNPM1 antibody, we confirmed that in the cultured AML OCI-AML3 cells (hetrozygous for NPM1 mutation) mNPM1 protein was localized in the cytosol, while the un-mutated NPM1 was nuclear. In AML HL-60 cells NPM1 was un-mutated and entirely nuclear. As compared to control siRNA, treatment with siRNA to NPM1 for 72 hours significantly induced p21 expression, decreased % of S phase cells in the cell cycle, as well as induced morphologic differentiation of OCI-AML3 but not of HL-60 cells. This was associated with marked induction of CEBPα in OCI-AML3 cells. NPM1 siRNA also attenuated the expression of HOXA9 and MEIS1 (which are known to be leukemogenic), associated with a marked loss of clonogenic survival of OCI-AML3 cells. NPM1 siRNA mediated depletion of Meis1, which is known to transactivate FLT3 tyrosine kinase in AML progenitor cells, was associated with down regulation of FLT-3 expression in OCI-AML3 cells. Importantly, treatment with NPM1 siRNA significantly sensitized OCI-AML3 more than HL-60 cells to all-trans retinoic acid (ATRA, 0.25 to 2.0 uM) and Ara-C (0.5 to 5.0 uM). Treatment with leptomycin B (5 nM for 24 hours), which is an inhibitor of the NES receptor CRM1/exportin-1, shifted the localization of mNPM1 from the cytosol to the nucleus, abrogated the levels of Meis1 and HoxA9 and induced apoptosis of OCI-AML3 but not HL-60 cells. Recently, treatment with NSC348884, a small molecule inhibitor of NPM1 oligomerzation, was shown to induce apoptosis of colon and prostate cancer cells. Our studies determined that NSC348884 (3.0 uM) was highly active in disrupting the oligomerization of mNPM1 and induce apoptosis of 90 % of OCI-AML but only 20% of HL-60 cells. Additionally, treatment with 5.0 uM of NSC34884 induced apoptosis of approximately 75% of primary AML cells with mNPM1, without inducing apoptosis of normal CD34+ progenitor cells. Collectively, these findings suggest that strategies targeting mNPM1 levels or oligomerization, or reversing the aberrant cytosolic localization of mNPM1, would induce relatively selective differentiation and apoptosis, as well as sensitize AML cells with mNPM1 to Ara-C and ATRA.