DNA Damaging Agents Cause a Sub-Cellular Re-Localisation of Nucleophosmin in an npm1 Mutated Acute Myeloid Leukemia (AML) Cell Line and in Primary AML Cells Carrying an npm1 Mutation

Abstract

Nucleophosmin (NPM) is a ubiquitously expressed phosphoprotein involved in many cellular functions including ribosome biogenesis, p53 activation, cell proliferation and genetic stability and is considered essential for cellular activity and survival. NPM contains a number of motifs which mediate interactions with a range of binding partners as well as influencing its cellular localisation. Primarily, NPM is located to the nucleolus due to a C-terminal nucleolar localisation signal (NoLS), however it is able to translocate through the nucleoplasm into the cytoplasm due to the presence of a bipartite nuclear localisation signal (NLS) and two nuclear export signals (NES).The most commonly mutated gene in Acute Myeloid Leukemia (AML) is npm1, occurring in approximately 35% of all AML patients. All npm1 mutations characterised to date occur within the C-terminal domain and alter the normal nucleo-cytoplasmic shuttling of NPM resulting in an abnormal cytoplasmic localisation of the protein. The mutations are always heterozygous, however mutant NPM oligomerises with wild type NPM resulting in an accumulation of both forms of the protein within the cytoplasm, leading to abnormal cellular functioning.Utilising an AML cell line containing a heterozygous npm1 mutation (OCI-AML3), we have shown that nucleolar localisation of NPM can be restored following short term exposure (4 - 24 hours) of the cells to the DNA damaging agent etoposide. Western blot analysis of the levels of NPM within cytoplasmic and nuclear fractions isolated from OCI-AML3 cells showed a significant decrease in cytoplasmic NPM following treatment relative to untreated cells (p=0.0045; N=3). Further analysis of OCI-AML3 cells using confocal microscopy identified an increase in nucleolar NPM following treatment relative to untreated controls (Figure 1). This post treatment increase of nucleolar NPM was also observed in primary leukemic cells isolated from patients with npm1 mutated AML.Similar results were observed with the DNA damaging agent cytarabine, however the potent anti-leukemic multi-kinase inhibitor, SB1317 had no effect on the sub-cellular localisation of NPM suggesting that the response is specific to DNA damage.In order to identify whether the nucleolar re-localisation of NPM following DNA damage could be abrogated by inhibiting the cellular response to DNA damage, OCI-AML3 cells were co-treated with etoposide and the PARP-1 DNA repair inhibitor, olaparib for 24 hours. Confocal microscope analysis of co-treated cells revealed that the sub-cellular distribution of NPM was predominantly cytoplasmic, with very little NPM-nucleolus co-localisation, i.e. resembling that of untreated cells.In summary, our data show that DNA damage caused by chemotherapeutic drugs results in a sub-cellular re-localisation of NPM in npm1 mutated AML cells, with the post-treatment NPM localisation resembling that of npm1 wildtype cells. In a chemotherapeutic context utilising DNA-damaging agents however, this re-localisation of NPM may not be desirable considering the involvement of NPM in DNA repair pathways. Re-localisation of NPM can be prevented however, by inhibiting the activity of other molecules involved in DNA damage response pathways, specifically PARP-1. Therefore these results suggest that the treatment of npm1 mutated AML with DNA damaging drugs should be considered within this context. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.