EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

Abstract

The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found in anaplastic large cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remain unclear. We herein demonstrated that NPM-ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM-ALK interacted with EBNA1-binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM-ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G 0 /G 1-phase cell cycle arrest in Ba/F3 cells transformed by NPM-ALK and ALCL patient-derived Ki-JK cells, but not ALCL patient-derived SUDH-L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM-ALK and Ki-JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC-0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, 2.3. Cell culture, retrovirus infection and transfection The IL-3-dependent hematopoietic cell line Ba/F3 cells were cultured in RPMI-1640 medium (Nacalai Tesque) containing 10% heat-inactivated fetal bovine serum (FBS) (BioWest, Nuaille, France), 100 units/ml penicillin (Nacalai Tesque), 100 g/ml streptomycin (Nacalai Tesque), 2 ng/mL IL-3 (PEPROTECH), and 5 g/mL puromycin (InVivoGen). Ki-JK cells and SUDH-L1 cells, derived from NPM-ALK-positive ALCL patients, were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)-High Glucose supplemented with 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. NPM1 −/− /p53 −/− MEF and p53 −/− MEF were kindly gifted from Dr. Pandolfi (Harvard University) and cultured in DMEM-High Glucose (Nacalai Tesque) supplemented with 10% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 55 M 2-mercaptoethanol (Nacalai Tesque). Ba/F3 cells were infected using RetroNectin (Takara Bio Inc., Shiga, Japan) and MEFs were infected using 10 g/ml polybrene as reported previously [16]. HEK293T cells were transfected with plasmids using polyethylenimine (PEI) Max (Polyscience inc. Warrington, PA, USA). 2.4. Fractionation of cells into cytosolic, nucleoplasmic, and nucleolar fractions We conducted fractionation as described previously [22]. Briefly, cells were suspended in mild detergent buffer (20 mM Tris pH7.4, 10 mM KCl, 3 mM MgCl 2 , 0.1% NP-40, and 10% glycerol) and centrifuged at 1,400×g at 4°C for 10 min, and the resulting supernatant was prepared as the cytosolic fraction. Nuclear pellets were then resuspended in 0.25 M sucrose/10 mM MgCl 2 , layered over a cushion of 0.35 M sucrose/0.5 mM MgCl 2 , and centrifuged at 1,400×g at 4°C for 5 min. The resulting nuclear pellet was re...

Proteomics analysis identifies the ribosome associated coiled‐coil domain‐containing protein‐124 as a novel interaction partner of nucleophosmin‐1

“…Mutant forms of Npm1 were previously associated with different types of leukemias and solid cancers originating from a variety of tissues (Subong et al., 1999; Grisendi et al., 2005; Falini et al., 2005; Uchihara et al., 2021; Kelemen, 2022), and causative effects of mutant forms of Npm1 in hematological cancers initially drew our attention to this factor as one of the outstanding candidates in our interactors list. In a previous study initiated with an unbiased proteomics analysis of Npm1 interacting factors, Gu et al.…”

“…The nucleolus is a distinct subnuclear compartment, and other than the synthesis and assembly of ribosomal subunits (Pena et al., 2017; Correll et al., 2019; Klinge and Woolford, 2019), it also comprises proteins associated with DNA damage repair, cellular stress response, and factors linking related molecular pathways to the regulation of cell cycle control (Boulon et al., 2010; Yang et al., 2016; Frottin et al., 2019; Correll et al., 2019; Lafontaine et al., 2021). Mutant forms as well as over‐expression of Npm1 were previously associated with different types of leukemias and solid cancers originating from a variety of tissues (Subong et al., 1999; Grisendi et al., 2005; Falini et al., 2005; La Manna et al., 2021a; 2021b; Uchihara et al., 2021; Kelemen, 2022). In light of our recent studies aiming at elucidating the role of Ccdc124 in oncogenic contexts (Arslan et al., 2021), Npm1 has emerged as a paramount candidate warranting further investigation in the current study.…”

The Leukemic Phase of ALK-Negative Anaplastic Large Cell Lymphoma Is Associated with CD7 Positivity, Complex Karyotype, TP53 Deletion, and a Poor Prognosis

“…Disruption of the p53-MDM2 interaction by nutlin-3a (a small molecule targeting MDM2) activates the p53 pathway resulting in cell-cycle arrest and apoptosis in ALK+ ALCL cells [ 40 ]. A recent study revealed a novel repressive mechanism of p53 activity by EBP2 in NPM-ALK-expressing cells [ 41 ].…”