Abstract

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 has been assigned more than a dozen functions in the cell, including ribosome biogenesis, centrosome duplication and maintenance of genomic stability, stabilization of tumor suppressors p53 and Arf, DNA transcription and response to stress stimuli. 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 NPM-1 have been identified in 60% of cytogenetically normal adult AMLs. Mutant NPM-1, referred to as NPMc+, commonly results from a gain-of-function mutation in the C-terminus of NPM-1 resulting in the generation of a novel nuclear export signal (NES) and consequent mislocalization from the nucleolus and nucleus to the cytoplasm. The role of NPMc+ in contributing to AML however, remains unresolved. Knock-in and transgenic models of NPMc+ in mice demonstrate that NPMc+ expressing animals develop AML after a long latency, suggesting the contribution of collaborating mutations to AML. 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 NPM-1-cargo proteins into the cyoplasm including for example, the tumor suppressor Arf, leading to the activation of the c-MYC oncogene. However, only a few proteins have been shown to be mislocalized by NPMc+ thus far. A second, though not mutually exclusive hypothesis states that reduction in wild type levels of NPM1 in the nucleolus as a result of both heterozygosity as well as mislocalization into the cytoplasm following association with NPMc+, contributes to tumorigenesis. In order to determine the role of myeloid transcription factors in contributing to NPMc+ associated malignancy, we determined the cellular localization of C/EBPα, Gfi1 and PU.1in the OCI/AML3 cell line, which was derived from a CN-AML patient harboring the NPMc+ mutation. While the Gfi1 protein appeared to be transported out of the nucleus to the cytoplasm, the PU.1protein remained within the nucleus of the OCI/AML3 cells. Both proteins remained predominantly in the nucleus in the control HL-60 cells. The fate of the master myeloid regulator C/EBPα in the OCI/AML3 cells was however, unexpected. C/EBPα is a single exon, b-zip 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 the dominant negative p30 isoform. In OCI-AML3 cells, the p40 isoform of C/EBPα was found predominantly in the cytoplasm while the full length p42 and p30 isofoms remained in the nucleus. The p40 isoform of C/EBPα differs from its full length version by 14 amino acids at the N-terminus, and its function has not been well described in the literature. We will present data describing the functional implications of mislocalized Gfi1 as well as the role of p40 C/EBPα isoform in granulopoiesis and their contribution to AML in association with NPMc+. We propose that the depletion of p40 from the nucleus likely deregulates growth and differentiation normally attributed to p42 C/EBPα by perturbing the ratio of the remaining isoforms (p42:p30) in the nucleus, thus blocking p42 activity, thereby contributing to NPMc+ associated leukemogenesis. Our data provide new insights into the mechanism through which mutant NPM-1, a founding mutation in this subset of AML, contributes to leukemogenic progression.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.