Targeting Mutant Nucleophosmin 1 (Npm1) Induces Differentiation, Loss Of Survival And Sensitizes Aml Cells To All-Trans Retinoic Acid, Cytarbine And Flt3 Antagonists

Abstract Abstract 733 NPM1 is a nucleolar phosphoprotein that functions as an oligomeric molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML, especially those with the normal karyotype. The mutant (mt) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes mtNPM1 to be localized in the cytoplasm (NPM1c+). NPM1 has an N-terminal conserved, hydrophobic, oligomerization domain, which is common to all isoforms of NPM1 and critical for its chaperone activity. Recently, NSC348884 was identified as a small molecule inhibitor that disrupts wild-type (WT) and mtNPM1 dimer/oligomer formation, inducing apoptosis of cancer cells. Here, we determined the effects of specifically targeting mtNPM1 in cultured and primary AML cells. Treatment with shRNA specific to mtNPM1 or specific to WT and mtNPM1, as compared to the control non-targeted shRNA, induced p53, p21, p27 and CEBPα, but down regulated the levels of HOXA9 and Meis1 in the AML OCI-AML3 cells that heterozygously expressed mt NPM1. This led to an increase in the % of cell cycle G1 phase and a decline in % S phase cells, as well as marked inhibition of the colony growth of OCI-AML3 cells (p < 0.01). Knockdown of mtNPM1 induced myeloid/monocytic differentiation of OCI-AML3, as detected morphologically (∼30% over control) and induction of CD11b by flow cytometry. Knockdown of mtNPM1 also induced the proteins levels of RARα (two-fold) and sensitized OCI-AML3 cells (but not cultured AML cells with wtNPM1) in vitro to all-trans retinoic acid (ATRA) (0.25 to 2.0 μM)-induced p53, p21 and C/EBPα, as well as ATRA-induced differentiation and apoptosis (p < 0.01). Furthermore, mtNPM1 knockdown sensitized OCI-AML3 cells to cytarbine (0.5 to 2.0 μM)-induced apoptosis. Notably, mice bearing OCI-AML3 cells transduced with NPM1 shRNA and showing knockdown of total NPM1 demonstrated significantly less spleen enlargement and dramatically improved survival, as compared to the control mice (p < 0.01). Exposure to NSC348884 (1.0 to 3.0 μM) dose-dependently induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA-induced apoptosis (p < 0.01). NSC348884 treatment (3 μM) significantly disrupted the dimer (∼64 kDa) formation and increased the monomeric NPM1 levels in OCI-AML3 but not in HL-60 cells, as analyzed by native polyacrylamide gel electrophoresis (PAGE). OCI-AML3 cells transfected with FLT3-ITD lentivirus (OCI-AML3/FLT3-ITD cells) exhibited several-fold higher expression of FLT3-ITD, without causing any change in the levels of NPM1. As compared to the vector control cells, OCI-AML3/FLT3-ITD cells, as well as primary AML cells expressing both mtNPM1 and FLT3-ITD, were less sensitive to treatment with NSC348884 and/or ATRA. Notably, combination with NSC348884 (3.0 μM) and PKC412 (100 to 500 nM), a FLT3 antagonist (Novartis Pharmaceuticals), induced more apoptosis than either agent alone against OCI-AML3/FLT3-ITD and primary AML cells expressing both mtNPM1 and FLT3-ITD (p < 0.01). These findings demonstrate that knocking down of the levels and oligomerization of mtNPM1 and WT NPM1 induces differentiation and growth inhibition, as well as sensitizes AML cells with mtNPM1 to ATRA-induced differentiation and apoptosis. Furthermore our findings support the rationale of combining NPM1 antagonist with FLT3 inhibitor against AML with dual expression of mtNPM1 and FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.