AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response

Abstract

Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.

Principles and functions of condensate modifying drugs

“…The proteins ejected from the nucleolus reside in two distinct pools: 1) a cytoplasmic pool, generated by Crm1/XPO1-dependent nuclear export ( Falini et al, 2006 ), and 2) a chromatin-bound pool, generated by direct recruitment via Crm1/XPO1 to HOX loci ( Brunetti et al, 2018 ). Consequently, this condensatopathy is associated with a gain of function in the nucleus (reactivation of silenced HOX genes) ( Brunetti et al, 2018 ) and loss of nucleolar function (nucleolar stress response) ( Holoubek et al, 2021 ), leading to cellular transformation.…”

“…Small molecule localizer c-mods such as Avrainvillamide ( Andresen et al, 2016 ) and Selinexor (KPT-330) ( Table 1 , shown and plotted in Figure 2A ) ( Gu et al, 2016 ; Brunetti et al, 2018 ; Holoubek et al, 2021 ), rescued NPM1 mislocalization, returning it to the nucleus and nucleolus, as well as restoring aspects of the gain and/or loss of function (reviewed in ( Falini et al, 2006 )). KPT-330 inhibits the interaction between mutant NPM1 and Crm1/XPO1 ( Andresen et al, 2016 ; Gu et al, 2016 ; Brunetti et al, 2018 ).…”

Cytoplasmic localization of Mdm2 in cells expressing mutated NPM is mediated by p53

“…Consequently, multiple NPMmut-interacting partners are targeted to the cytoplasm [3,4]. Among these, tumour suppressor p53 has recently been proven to interact with both wt and mutated NPM and is consequently found in the cytoplasm of NPMmut-expressing cells [5]. This probably causes impaired p53 signalization and delayed/ inhibited p53-dependent apoptosis [6].…”

“…Drugs preventing the cytoplasmic p53 localization may, therefore, help to cure AML with NPM mutation. In our previous work, we tested specific effect of the putative NPM-oligomerization inhibitor, NSC348884, as well as the inhibitor of the nuclear exporter Crm1, Selinexor, exerted on apoptosis of the cells with NPM mutation [5,7]. However, NSC348884 was found ineffective as the NPM-oligomerization inhibitor.…”

“…In cells with NPM mutation, the signalization pathways based on the NPM-p53-Mdm2 axis are deregulated by the cytoplasmic delocalization of p53 [5], which might contribute to the NPMmut-induced leukemogenesis. Impairment of the complex stability by proper inhibitors could, therefore, release p53 for the induction of apoptosis and restoration of the Mdm2 nuclear localization.…”