Cysteine 288 Regulates NPMc+ Cytoplasmic Localization and Sensitizes Leukemic Cells to Bortezomib-Induced Apoptosis Through a Redox-Sensitive Mechanism

Abstract 532

Increasing evidence points to NPM1 mutations in exon 12 of the NPM1 gene (NPMc+) as a founder genetic event that defines a distinct leukemia entity accounting for approximately one-third of all AML. Unlike wild type NPM1, which resides predominantly in the nucleoli, NPMc+ mutants localize aberrantly in the leukemic-cell cytoplasm due to mutations at the C-terminus of NPM1. We have found that expression of NPMc+ in K562 and 32D cells sensitizes these cells to Bortezomib-induced apoptosis. Conversely, inducible small interfering RNA (shRNA)-induced knockdown of NPM1 in OCI-AML3 cell line, an AML cell line carrying the NPMc+ mutation, and in K562 cells over-expressing NPMc+ markedly reduced the ability of Bortezomib to induce apoptosis in both cell lines. Bortezomib-induced apoptosis in both OCI-AML3 and K562 cells was reversed with N-acetyl-1-cysteine (NAC), a ROS scavenger, suggesting that activation of ROS pathway plays an essential role in Bortezomib-induced apoptosis. Further studies showed that the cytoplasmic localization of NPMc+ in OCI-AML3 and 32D cells expressing NPMc+ increased in response to ROS activators such as diamide, hydrogen peroxide, and Bortezomib, but was reduced by NAC. These results prompted us to explore ROS-sensitive elements that might differentiate NPMc+ from wt-NPM1. We discovered that tryptophan 288 in wt-NPM1 is mutated to cysteine 288 in the majority of NPMc+ C-terminal mutants identified to date. Strikingly, mutagenesis of C288 to alanine re-localized NPMc+ completely to nucleolus, whereas mutagenesis of C21 and C104, the only two cysteines that exist in wt-NPM1, failed to alter the cytoplasmic localization of NPMc+. Unexpectedly, we also found that NPM1 oligomerization is disrupted in C21A+C104A mutants of both wt-NPM1 and NPMc+, indicating that C21 and C104 are essential for the oligomerization of NPM1. In addition, the proportion of cytoplasmic-localized endogenous wt-NPM1 also increased in response to ROS generation in K562 cells over-expressing NPMc+ or C21A+C104A-NPMc+, indicating that C288-NPMc+ partially relocates wt-NPM1 to the cytoplasm upon ROS activation through disulfide bond formation between C288-NPMc+ and C21 or C104 of wt-NPM1. Finally, we established the importance of C288-NPM1c+ as a sensitizing factor in Bortezomib-induced apoptosis by demonstrating that the sensitizing effects of NPMc+ to Bortezomib was significantly attenuated in K562 and 32D cells expressing C288A-NPMc+. In contrast, expression of C21A+C104A-NPMc+ sensitized K562 and 32D cells to Bortezomib-induced apoptosis to the same extent as NPMc+. We conclude that the mutation of tryptophan 288 to cysteine that is present in the great majority of NPM1c+ leukemic cells is the essential event that mediates the cytoplasmic localization of NPMc+ and that sensitizes leukemia cells to Bortezomib-induced apoptosis regardless of the oligomerization status of NPMc+. We also conclude that the intracellular redox level is an additional major factor that regulates the distribution of NPMc+ in the cytoplasm as opposed to the nucleolus in AML cells.