No abstract

Mutations leading to aberrant cytoplasmic localization of Nucleophosmin 1 (NPM1) have been recently identified as the most frequent genetic alteration in acute myelogenous leukemia. However, the oncogenic potential of this nucleophosmin mutant (NPMc +) has never been established, which casts doubt on its role in leukemogenesis. By performing classical transformation assays, we find that NPMc +, but not wild-type NPM, cooperates specifically with adenovirus E1A to transform primary mouse embryonic fibroblasts in soft agar. We demonstrate that NPMc + blocks the p19 Arf (Arf) induction elicited by E1A. Surprisingly, however, we find that NPMc + induces cellular senescence and that E1A is able to overcome this response. We propose a model whereby the NPMc + pro-senescence activity needs to be evaded for oncogenic transformation, even though NPMc + can concomitantly blunt the Arf/p53 pathway. These findings identify for the first time NPMc + as an oncogene and shed new unexpected light on its mechanism of action.

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1–RARα fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML −/− and p53 −/− compound mutants lends support to a model by which the RARα and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

A key oncogenic force in acute promyelocytic leukemia (APL) is the ability of the promyelocytic leukemia-retinoic acid receptor α (PML-RARA) oncoprotein to recruit transcriptional repressors and DNA methyltransferases at retinoic acid-responsive elements. Pharmacological doses of retinoic acid relieve transcriptional repression inducing terminal differentiation/apoptosis of the leukemic blasts. APL blasts often harbor additional recurrent chromosomal abnormalities, and significantly, APL prevalence is increased in Latino populations. These observations suggest that multiple genetic and environmental/dietary factors are likely implicated in APL. We tested whether dietary or targeted chemopreventive strategies relieving PML-RARA transcriptional repression would be effective in a transgenic mouse model. Surprisingly, we found that 1) treatment with a demethylating agent, 5-azacytidine, results in a striking acceleration of APL; 2) a high fat, low folate/choline-containing diet resulted in a substantial but nonsignificant APL acceleration; and 3) all-trans retinoic acid (ATRA) is ineffective in preventing leukemia and results in ATRA-resistant APL. Our findings have important clinical implications because ATRA is a drug of choice for APL treatment and indicate that global demethylation, whether through dietary manipulations or through the use of a pharmacologic agent such as 5-azacytidine, may have unintended and detrimental consequences in chemopreventive regimens.

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.