Haploinsufficiency of NPM1 in AML Derived NPM1 Mutant (NPMc+) Expressing Cells Contributes to Aberrant C/EBPα Activity

• Published in: Blood 2014-12, Vol.124 (21), p.2188-2188
• Main Authors: Czepiel, Kathryn, Nichol, Kelly, Tippy, Mason, Abayasekara, Nirmalee, Lee, Jaclyn, Wajswol, Ethan, Khanna, Anjali, Berliner, Nancy, Khanna-Gupta, Arati
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V124.21.2188.2188

Nucleophosmin 1 (NPM-1) is a highly conserved, ubiquitously expressed nucleolar protein that functions as a molecular chaperone shuttling protein-binding partners between the nucleolus, nucleus and cytoplasm. NPM-1 and has been assigned more than a dozen functions in the cell, including ribosome biogenesis and centrosome duplication. The NPM-1 gene maps to chromosome 5q35, a region that is the target of deletions in both de novo and therapy-associated MDS in humans. Additionally, heterozygous mutations in the NPM-1 gene have been identified in 60% of cytogenetically normal adult AMLs. Mutant NPM-1, (NPMc+), commonly results in the generation of a novel nuclear export signal (NES) leading to cellular mislocalization of NPMc+ from the nucleolus and nucleus to the cytoplasm. The role of NPMc+ in contributing to AML however remains unresolved to date. Two hypotheses to explain the role of NPMc+ in leukemogenesis have been advanced. The first purports that aberrant cytoplasmic mislocalization of NPMc+ also mislocalizes a number of NPM1-cargo proteins into the cytoplasm including for example, the tumor suppressor Arf, leading to the activation of the c-MYC oncogene, thereby contributing to leukeomogenesis. A second hypothesis states that reduction in wild type levels of NPM-1 in the nucleolus as a result of both heterozygosity as well as mislocalization into the cytoplasm following association with NPMc+, contributes to tumorigenesis. To address these hypotheses we generated a series of IL-3-dependent cell lines from the bone marrow of NPM1+/+ and NPM+/-mice. In addition, we stably transduced an NPMc+ expression vector into the NPM+/- cells resulting in the NPM+/-c+ line, thereby providing cellular models for both NPM1 haploinsufficieny as well as mutant NPM-1 associated AML. We then sought to examine the role of the master myeloid transcription factor C/EBPα in contributing to NPM-1-associated AML. C/EBPα is a single exon, bzip transcription factor that generates four isoforms derived from separate in-frame AUGs resulting in the translation of a nucleolar p50, a full length p42, a p40 and a dominant negative p30 isoform. We found that in NPM+/-c+ and OCI-AML3 cells (derived from a CN-AML patient harboring the NPMc+ mutation), only the p40 isoform of C/EBPα migrated to the cytoplasm while the full length p42 isofom remained in the nucleus. The p40 isoform of C/EBPα lacks the first 14 N-terminal amino acids when compared to the full length p42 isoform, and its fu