The Nucleophosmin-Anaplastic Lymphoma Kinase Fusion Protein Induces c-Myc Expression in Pediatric Anaplastic Large Cell Lymphomas

Abstract

The majority of pediatric anaplastic large cell lymphomas (ALCLs) carry the t(2;5)(p23;q35) chromosomal translocation that juxtaposes the dimerization domain of nucleophosmin with anaplastic lymphoma kinase (ALK). The nucleophosmin-ALK fusion induces constitutive, ligand-independent activation of the ALK tyrosine kinase leading to aberrant activation of cellular signaling pathways. To study the early consequences of ectopic ALK activation, a GyrB-ALK fusion was constructed that allowed regulated dimerization with the addition of coumermycin. Lymphoma is the third most common cancer in children and adolescents in the United States, representing 13% of newly diagnosed malignancies in the pediatric age group.1 Although significant progress has been made in the treatment and outcome of childhood lymphoma, many children still succumb to disease, or suffer acute and long-term toxicity from contemporary multiagent chemotherapy.2-4 A subtype of pediatric anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-positive lymphoma (ALKϩ lymphoma), may serve as an ideal model for the study of the molecular mechanisms of malignant transformation.5-7 ALKϩ lymphoma is characterized by a t(2;5)(p23;q35) chromosomal translocation that creates a chimeric fusion protein consisting of the amino-terminal portion of the nucleolar phosphoprotein, nucleophosmin (NPM), and the cytoplasmic domain of the receptor tyrosine kinase, ALK. 8,9 ALK is a recently described receptor tyrosine kinase in the insulin receptor subfamily whose expression is normally restricted to the central nervous system. 10 -12 NPM is a ubiquitously expressed homohexameric nucleolar phosphoprotein that shuttles ribosomal proteins between the nucleus and cytoplasm. [13][14][15] Essential functions of NPM in the chimera are provision of both a promoter that drives ectopic expression of ALK in lymphoid cells, and a dimerization motif that facilitates trans-phosphorylation and hence activation of signaling. 16The t(2;5) translocation is an essential event in the pathogenesis of ALKϩ lymphoma as NPM-ALK potently transforms both rat fibroblast and murine lymphoid cell lines, and induces lymphoid tumors in mice.17,18 NPM-ALK homodimers autophosphorylate on multiple residues, creating docking sites for several SH2 domaincontaining signaling molecules. 16,19 -21 Formation of this signaling complex leads to changes in gene expression that underlie the malignant phenotype. One goal of our studies is to understand the changes in cellular transcription that the NPM-ALK fusion protein produces.Ligand-induced dimerization is thought to directly induce the activation of receptor tyrosine kinases. [22][23][24] Numerous researchers have generated protein fusions that can be conditionally dimerized in the presence of bivalent compounds such as coumermycin, rapamycin, and FK1012. [25][26][27] To better define the immediate early effects of ALK activation, we used this technology to generate a conditionally dimerizable kinase construct, designed to mimic the activity of...

Identification of differential and functionally active miRNAs in both anaplastic lymphoma kinase (ALK)+and ALK−anaplastic large-cell lymphoma

“…2C). This suggests synergistic regulation of common target proteins and is in agreement with previously published findings demonstrating by immunohistochemistry that myc is only expressed in ALK + ALCL and is absent from ALK − samples (15).…”

“…ALK + ALCL bears the t(2;5) (p23;q35) translocation in greater than 80% of cases, which results in the expression of the chimeric nucleophosmin (NPM)-ALK (1)(2)(3)(4). The consequence of the fusion is constitutive ALK expression, which leads to the activation of many different growth-promoting and antiapoptotic pathways, including PI3K/Akt1/mTOR (5)(6)(7)(8), Jak/Stat (9-11), cJun, JunB (12)(13)(14), and c-myc (12,15). The transforming capacity of the NPM-ALK fusion was shown for the first time in murine chimeras in which lethally irradiated recipients were rescued with NPM-ALK-transduced bone marrow (4,16,17) as well as in several transgenic mouse models that expressed the NPM-ALK fusion protein in hematopoietic cells (14,18,19).…”

Identification of NPM-ALK interacting proteins by tandem mass spectrometry

“…We acknowledge that nonspecific binding is possible when using polyclonal antibody immunoprecipitation as a technique for sample enrichment, thus identifying false protein interactions. However, a careful review of the literature for NPM-ALK interactions revealed that the majority of the published studies utilized polyclonal antibodies in their experimental design (Fujimoto et al, 1996;Bai et al, 1998Bai et al, , 2000Hubinger et al, 1999;Lux et al, 1999;Nieborowska-Skorska et al, 2001;Slupianek et al, 2001;Bonvini et al, 2002;Miyake et al, 2002;Raetz et al, 2002;Zamo et al, 2002;Zhang et al, 2002;Amin et al, 2003;Baba et al, 2003;Khoury et al, 2003;Ruchatz et al, 2003). In this regard, we have chosen to categorize proteins identified using both monoclonal and polyclonal antibodies.…”

Pediatric ALK+ anaplastic large cell lymphoma with t(3;8)(q26.2;q24) translocation and c‐myc rearrangement terminating in a leukemic phase

“…Chromosomal structural alteration of the c-myc gene containing 8q region have also been identified in an ALK+ ALCL cell line [12]. The expression of c-myc RNA is increased with ALK activation, and c-myc protein overexpression is consistently present in pediatric ALK+ ALCLs [11]. These observations suggest that c-myc may be a downstream effector of the ALK signaling cascade and may play a role in lymphomagenesis.…”

“…In a previous study [11], the overexpression of cmyc was demonstrated in pediatric ALK+ but not in ALKÀ ALCL. The overexpression of c-myc was considered a defining characteristic of ALK+ ALCL and deregulation of this oncogene was suggested to play a role in ALK+ ALCL pathogenesis as a downstream target of ALK signaling.…”