Nucleophosmin-1 Interacts with CCAAT Enhancer Binding Protein Alpha (C/EBPα) to Facilitate Granulocyte Maturation: Implications in MDS and AML.

Abstract 2768 Poster Board II-744 NPM-1, a gene that resides on chromosome 5q, is a highly conserved, ubiquitously expressed nucleolar phosphoprotein that belongs to the nucloeplasmin family of nuclear chaperones. NPM-1 is a multifunctional protein and has been implicated in ribosome biogenesis and the transport of pre-ribosomal particles, maintenance of genomic stability by the control of cellular ploidy, in DNA repair and in the regulation of DNA transcription by controlling chromatin condensation/decondensation. NPM1−/− mice die at mid-gestation (E11.5) due to severe anemia, underscoring the role of this gene in embryonic development. NPM-1 has been shown to be deleted or involved with chromosomal translocations in several hematologic malignancies. Mutant NPM-1 (NPMc+) has been found to be aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Additionally, mutations in the NPM1 gene have been described in 50% of normal karyotype AML patients. This makes NPM-1 one of the most frequently mutated genes in AML. The NPM-1 gene maps to a region on chromosome 5q that is the target of deletions in both denovo and therapy-associated MDS in humans, and is deleted in 50% of MDS associated with 5q deletions. NPM-1+/− mice develop a hematological syndrome similar to that observed in MDS patients. NPM-1 thus appears to act as a haploinsufficient tumor suppressor in the hematological compartment, the molecular basis of which remains unexplored. The NPM-1 deficient model provides an excellent platform to interrogate not only the molecular basis of MDS but also to study AML progression. To further explore the role of NPM1 in myeloid malignancy, we have generated factor-dependent myeloid cell lines from the bone marrow of Npm1 +/+ and Npm1 +/− mice. We demonstrate compromised neutrophil function and neutrophil-specific gene expression in the NPM1 +/− cells similar to that observed in Npm1 +/− mouse bone marrow, and attribute these observations to decreased expression of the master myeloid regulator C/EBPα in the Npm 1+/− cells. We have demonstrated that overexpression of wildtype NPM1 but not mutant NPM1 (NPMc+) upregulates C/EBPα expression, and that overexpression of C/EBPα in Npm1 deficient cells rescues the myeloid phenotype. We further demonstrate by co-immunoprecipitation and oligonucleotide pulldown assays that NPM1 and C/EBPα physically interact. Our observations suggest that as a result of protein-protein interactions at specific cis elements in the promoters of C/EBPα target genes, C/EBPα and NPM1 act in concert to facilitate normal neutrophil gene expression and function. Interestingly, mutations in the C/EBPα gene have also been implicated in normal karyotype AML. We demonstrate for the first time a connection between these two components of the myeloid compartment, dysregulation of which can lead to disruption of the myeloid maturation program. Taken together, our observations may explain the defective myeloid phenotype observed when NPM1 expression is compromised in MDS and AML. We are now poised to examine the effects of knocking down genes such as RPS14 (the pathogenic gene in the 5q- syndrome) or overexpressing ones such as FLT3-ITD (associated with MDS/AML harboring NPM1 mutations) in our factor-dependent NPM1+/− cell line, to determine if these genes cooperate with NPM1 deficiency to render the cells factor independent. This cell system should be a useful model that will allow us to further dissect the role of NPM-1 in MDS and AML at the molecular level. Disclosures: No relevant conflicts of interest to declare.