PF228 DIAGNOSTIC VALUE OF PU.1 LOCALIZATION IN ACUTE MYELOID LEUKEMIA WITH MUTATED NPM1
Authors: Pianigiani G. , Betti C. , Rossi R. , Bigerna B. , Brunetti L. 
Published: June 2019

Background:NPM1 is among the most frequently mutated genes in acute myeloid leukemia (AML), with one third of newly diagnosed cases carrying NPM1 mutations. These mutations are heterozygous small insertions that result in the generation of a novel C‐terminal nuclear export signal. In contrast with the nucleolar localization of the wild type NPM1, mutant NPM1 (NPM1c) is aberrantly localized to the cytoplasm of leukemic cells. It has been recently proposed that NPM1c directly binds the myeloid transcription factor PU.1, relocating it from the nucleus to the cytoplasm (Gu et al., JCI 2018). Therefore, we sought to investigate whether PU.1 subcellular localization could be used to predict NPM1 mutations in AML.Aims:This study aimed to determine the impact of PU.1 subcellular localization in the diagnosis of AML with mutated NPM1.Methods:We analysed 39 bone marrow biopsies from patients with AML either at diagnosis or at relapse (22 NPM1‐mutated and 17 NPM1‐wild type). Samples were studied by immunohistochemistry (IHC) using two different commercially available monoclonal antibodies against PU.1 (ab1 and ab2) and total NPM1 (tNPM1). We also evaluated PU.1 localization in 4 AML cell lines (2 NPM1‐mutated ‐ i.e. OCI‐AML3 and IMS‐M2 ‐ and 2 NPM1‐wild type ‐ i.e. OCI‐AML2 and HNT34) through western blot (WB) of nuclear and cytoplasmic fractions and immunofluorescence (IF). The mutational status of the NPM1 gene was determined by PCR and capillary electrophoresis in all patients and cell lines. An homemade antibody specific for NPM1c (Martelli et al., Leukemia 2008) was used to confirm the presence of NPM1 mutation in the cell lines in WB.Results:As expected, all NPM1‐wild type cases showed nuclear localization of NPM1, while all NPM1‐mutated cases displayed simultaneous nuclear and cytoplasmic localization. In all NPM1‐wild type cases PU.1 localization was nuclear (Figure 1A), with 3/17 cases showing also weak cytoplasmic staining. Surprisingly, all NPM1‐mutated cases showed nuclear localization of PU.1 (Figure 1A), with 4/22 cases displaying also weak cytoplasmic staining. To validate these results, we stained 6 NPM1‐wild type and 6 NPM1‐mutated biopsies also with a second anti‐PU.1 antibody. All NPM1‐wild type and NPM1‐mutated cases had nuclear localization of PU.1, with respectively 2/6 and 1/6 cases with faint cytoplasmic staining, supporting our previous observastions. To further investigate PU.1 localization in NPM1‐mutated AML, we performed IF in two NPM1‐mutated and 2 NPM1‐wild type AML cell lines. All cell lines, studied with two different PU.1 antibodies showed strong PU.1 staining in the nuclei, but no cytoplasmic PU.1 was detected in any of the four cell lines. Finally, to confirm IF data, we performed WB analysis in the same AML cell lines. PU.1 was clearly detected in the nuclear fraction of all cell lines, independently of NPM1 mutational status, while no band was clearly visible in any of the cytoplasmic fractions (Figure 1B ‐ ab1). These results were confirmed with a second anti‐PU.1 antibody (Figure 1B ‐ ab2).Summary/Conclusion:In summary, we did not observe differences in PU.1 localization between NPM1‐wild type and NPM1‐mutated AML cases. No case showed strong PU.1 staining in the cytoplasm, with cell line data supporting our observations. We conclude that PU.1 localization should not be used to predict NPM1 mutations in AML. Our data suggest that further studies are needed to determine the amount of PU.1 undergoing NPM1c‐mediated relocation to the cytoplasm and to establish the biological relevance of PU.1 abnormal localization in AML with mutated NPM1.image