Elucidating the Oncogenic Potential of NPMc+ In Vitro and In Vivo.

Abstract

Nucleophosmin (NPM) is a nucleolar phosphoprotein that plays a key role in ribosome biogenesis, control of genomic stability and stabilization of tumor suppressors, such as ARF (Grisendi et al. Nature Review Cancer, 2006). We have recently shown that NPM can act as a tumor suppressor through the generation and characterization of an NPM hypomorphic series in vivo (Grisendi et al. Nature, 2005). NPM is one of the most frequent targets of genetic alterations in hematopoietic tumors, as about 60% of all adult Acute Myelogenous Leukemia with a normal karyotype harbor C-terminal frameshift mutations (NPMc+). As a consequence, NPMc+ aberrantly localizes to the cytoplasm of leukemic blasts. It has been shown that NPMc+ can act as a dominant negative NPM mutant sequestering endogenous NPM to the cytosol. However, to date this mutant has never been shown to behave as an oncogene, which casts doubt on its role in leukemogenesis. To assess the oncogenic potential of NPMc+, we carried out soft agar and low-density seeding assays using primary mouse embryonic fibroblasts (MEFs) expressing NPMc+ in combination with a battery of oncogenes. Interestingly, we found that NPMc+, but not wild type NPM, cooperate with adenovirus E1A to transform primary MEFs in soft agar. NPMc+/E1A also consistently showed high efficiency at forming foci in low-density seeding assays comparable to RasV12/E1A. However, NPMc+ does not transform alone or in combination with RasV12 or c-Myc and does not transform p53−/−, p21−/− or Arf−/− primary MEFs. This suggests that two complementary growth signals from NPMc+ and E1A can cooperate to evade cellular senescence and apoptosis. These data are consistent with a model by which NPMc+, through the inhibition of NPM function, can block the ARF response elicited by E1A. In support of these observations, we found that in primary cells from our NPM hypomorhic series Arf level and localization are dramatically affected by the progressive reduction in Npm dose. To elucidate in vivo the crosstalk between NPM and ARF, genetic crosses using Npm+/− and Arf+/− mice are currently ongoing and data from this analysis will also be presented.