Depletion of NUP98: A Common Leukemogenetic Mechanism in AML?

Abstract

The nuclear pore complex (NPC) is a protein assembly that contains several distinct subcomplexes including a 98kDa protein -NUP98- that mediates nucleo-cytoplasmic transport of proteins and RNAs. The NUP98 gene is located on chromosome 11p15.5, and has been reported to be fused with 17 different partners as a result of translocations in AML and MDS. We have observed earlier that introduction of the NUP98-HOXA9 fusion gene into normal cord blood CD34+ cells provided a selective growth advantage at the stem cell level together with alterations in differentiation. To further investigate the function of NUP98 in leukemogenesis, we transduced CD34+ cells with a lentivector expressing a small, interfering RNA targeting NUP98. By Western blot, >80% reduction in NUP98 protein levels was observed in both cell lines and CD34+ cells. We evaluated the NUP98 siRNA phenotype in clonogenic assays and cytokine-stimulated suspension cultures and compared this to control cells and CD34+ cells transduced with NUP98-HOXA9. In suspension cultures of NUP98 siRNA-transduced CD34+ cells stimulated with Flt3 and c-Kit ligands and thrombopoietin, cellular expansion significantly exceeded that observed with control siRNA but was less than that observed with NUP98-HOXA9. In clonogenic assay, both NUP98 siRNA and NUP98-HOXA9-transduced CD34+ cells produced more myeloid colonies and fewer erythroid colonies, with particular impairment in formation of large multifocal BFU-e, compared to the control.The enhanced proliferative potential of CD34+ cells with depleted or disrupted NUP98 might be caused by intracellular signaling changes. In an Affymetrics array analysis of CD34+ cells transduced with NUP98 siRNA or control siRNA, several candidate genes, such as PBX2 and DYRK1 were up-regulated, while genes involved in apoptosis such as caspase 2 were down-regulated.Our initial experiments led us to screen for NUP98 disruption in 12 AML patients (1 including with 11p15 rearrangement). Using allelic markers for the NUP98 region, we found a loss of heterozygosity in 6 out of 12 patients with overt AML compared to controls, suggesting that disruption of NUP98 is a frequent event in leukemogenesis.To determine the localization of NUP98 fusions within the cell, we performed immunostaining of CD34+ cells expressing siNUP98 or NUP98 fusions (NUP98-HOXA9, and NUP98-PMX1) using the NUP98 antibody that detects both fusion and wild-type protein. The staining showed that CD34+ cells transduced with NUP98 fusion proteins showed impaired assembly of the NPC with formation of distinct NUP98 co-localization bodies both within the nucleoplasm and cytoplasm. This data indicates that normal NUP98 function is essential for the formation of properly functioning NPCs and that NUP98 depletion by siRNA or LOH, or dysfunction due to expression of NUP98 fusion protein impairs NUP98 function.In summary, NUP98 depletion causes molecular and cellular changes that contribute to the pathogenesis of leukemia. The frequent loss of heterozygosity for NUP98 in AML further supports this finding.