NPM1 Mutations Are Responsible for Better Response to Induction Therapy in AML Patients through the Inactivation of NF-kB.
Authors: Cilloni Daniela , Messa Francesca , Rosso Valentina , Carturan Sonia , Arruga Francesca , Defilippi Ilaria , Catalano Renata , Chiarenza Annalisa , Morotti Alessandro , Messa Emanuela , Bracco Enrico , Saglio Giuseppe 
Published: April 2020

Abstract
Mutations in NPM1 exon 12 and the resulting shift of NPM1 into the cytoplasm are the most specific and frequent events in acute myeloid leukaemia patients with normal karyotype. Cytoplasmatic NPM is associated with responsiveness to induction chemotherapy, although its role in predicting outcome after remission remains to be defined. The aim of the study was to identify the molecular mechanisms responsible for chemosensitivity in leukemic cells carrying the mutation of NPM1. NF-kB is a transcription factor involved in many intracellular pathways including apoptosis. NF-kB is a heterodimer of p50 and p65 subunits sequestered in the cytoplasm in its inactive form through interaction with inhibitory IKB proteins and activated in the nucleous after degradation of IKB. The activation of NF-kB is responsible for chemoresistance to different drugs including anthracyclines. Based on this assumption, we analyzed the possible cytoplasmatic interaction between the mutated form of NPM1 (NPM+) and NF-kB. The NF-kB DNA binding activity was analyzed in 20 BM samples collected from AML patients carrying the NPM1 mutations and 30 NPM1 wild type samples (NPM1−) using an ELISA method. Immunofluorescence analysis using NPM1 and p65 antibodies was performed to identify the localization of both proteins. Western blot for p65 and NPM1 was used to confirm the protein amount and localization. Finally, co-immunoprecipitation assay was perform to study the interaction of the two proteins. We found a significant lower DNA binding activity in NPM1 mutated cells when compared to the wild type NPM cells (p=0,001). Immunofluorescence analysis confirmed the cytoplasmatic localization of NPM protein in mutated samples and the nuclear localization in wild type samples. In addition, immunofluoresence analysis performed with a monoclonal antibody against p65 subunit shows different pattern of NF-kB localization in NPM1+ when compared to NPM1−. In particular, in NPM1+ cells NF-kB is mainly localized in the cytoplasm in the inactive form and in NPM− cells is mainly nuclear localized. These data were confirmed by Western blot carried out with the same monoclonal antibody against p65 in the nuclear and cytosolic extracts. The interaction of NPM1 and NF-kB was further investigated and demonstrated by co-immunoprecipitation studies. In conclusion, we demonstrated that p65 and NPM1 interact with each other within the cytoplasm and this interaction results in the sequestration of NF-kB within the cytoplasm. The cytosolic localization of the inactive form of NF-kB explains the reduced NF-kB DNA binding activity observed in NPM1+ patients. These data may provide a possible explanation for the chemosensitivity observed in NPM+ patients. Furthermore, since NF-kB is involved in the transcription of many genes which regulate proliferation and differentiation processes, the disruption of NF-kB function may represent one of the mechanism of leukemogenesis induced by NPM1 mutated proteins.