Role of the Nucleophosmin (NPM) Portion of the Non-Hodgkin’s Lymphoma-Associated NPM-Anaplastic Lymphoma Kinase Fusion Protein in Oncogenesis

Abstract

The NPM-ALK fusion gene, formed by the t(2;5)(p23;q35) translocation in non-Hodgkin's lymphoma, encodes a 75-kDa hybrid protein that contains the amino-terminal 117 amino acid residues of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase ALK (anaplastic lymphoma kinase). Here, we demonstrate the transforming ability of NPM-ALK and show that oncogenesis by the chimeric protein requires the activation of its kinase function as a result of oligomerization mediated by the NPM segment. Sedimentation gradient experiments revealed that NPM-ALK forms in vivo multimeric complexes of approximately 200 kDa or greater that also contain normal NPM. Cell fractionation studies of the t(2;5) translocation-containing lymphoma cell line SUP-M2 showed NPM-ALK to be localized within both the cytoplasmic and nuclear compartments. Immunostaining performed with both polyclonal and monoclonal anti-ALK antibodies confirmed the dual location of the oncoprotein and also indicated that NPM-ALK is abundant within both the nucleoplasm and the nucleolus. An intact NPM segment is absolutely required for NPM-ALK-mediated oncogenesis, as indicated by our observation that three different NPM-ALK mutant proteins lacking nonoverlapping portions of the NPM segment were each unable to form complexes, lacked kinase activity in vivo, and failed to transform cells. However, NPM could be functionally replaced in the fusion protein with the portion of the unrelated translocated promoter region (TPR) protein that activates the TPR-MET fusion kinase by mediating dimerization through its leucine zipper motif. This engineered TPR-ALK hybrid protein, which transformed cells almost as efficiently as NPM-ALK, was localized solely within the cytoplasm of cells. These data indicate that the nuclear and nucleolar localization of NPM-ALK, which probably occur because of transport via the shuttling activity of NPM, is not required for oncogenesis. Further, the activation of the truncated ALK protein by a completely heterologous oligomerization domain suggests that the functionally important role of the NPM segment of NPM-ALK in transformation is restricted to the formation of kinase-active oligomers and does not involve the alteration of normal NPM functions.The oncogenic activation of protein tyrosine kinases (PTKs) through structural alterations produced by chromosomal rearrangements has been frequently associated with human cancer (52, 57). These alterations often produce chimeric genes encoding novel amino-terminal sequences derived from an unrelated locus fused to the kinase catalytic domain of the PTK. The juxtaposition of these novel sequences with truncated receptor tyrosine kinases (RTKs) is believed to produce conformational changes normally generated by ligand binding, resulting in constitutive activation of the RTK catalytic domain and an unregulated mitogenic signal (12,55). A shared feature of the various amino-terminal fusion partners of the truncated PTKs that have been characteri...

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein

“…These results imply that NPM-MLF1 activates a novel signaling pathway of apoptotic induction. Deletion of N-terminal 83 amino acids, which are required for dimerization of NPM (Fujimoto et al, 1996;Bischof et al, 1997), only partially impaired the apoptosisinducing activity of the fusion protein, while mutants lacking nuclear localization signal completely lost the ability. This can be explained by the fact that MLF1 itself has the ability to dimerize (N Yoneda-Kato, unpublished observation).…”

“…The region of NPM retained in the NPM-MLF1 fusion protein contains most of the identi®able functional domains of NPM, including a potential Cys-X 5 -His-X 4 -His metal binding motif, one of two nuclear localization signals (NLSs), and most of the acidic amino acid clusters (Yoneda-Kato et al, 1996), while NPM-ALK and NPM-RARa contain 58 less NPMderived amino acids (Morris et al, 1994;Redner et al, 1996), suggesting that functional modi®cation of MLF1 by fusion to NPM is di erent from that for ALK and RARa. All three fusion proteins, however, retain the N-terminal domain of NPM, which are required for dimerization of the protein (Fujimoto et al, 1996;Bischof et al, 1997). The ALK and RARa genes encode a tyrosine kinase and a transcription factor, respectively, whereas the function of MLF1 remains unknown because of no signi®cant homology with any previously identi®ed proteins, suggesting that NPM-MLF1 acts through a novel pathway to contribute to myeloid neoplasia.…”

Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA

“…The NPM/ ALK chimeric protein is constitutively expressed and activated through autophosphorylation (Shiota et al, 1994;Morris et al, 1997). NPM/ALK is highly oncogenic as documented both in vitro (Fujimoto et al, 1996;Bischof et al, 1997) and in vivo (Kuefer et al, 1997;Chiarle et al, 2003). NPM/ALK executes its oncogenicity by activating a number of signal transduction proteins, including signal transducer and activator of transcription 3 (STAT3) (Zhang et al, 2002;Chiarle et al, 2008;Li and Morris, 2008).…”

Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway

“…The t(2;5)(p23;q35) translocation that occurs in >80% of ALK þ TCL fuses a distal portion of the ALK gene with the proximal part of the nucleophosmin (NPM) gene (Morris et al, 1994;Shiota et al, 1994). The chimeric NPM/ALK protein is constitutively active due to autophosphorylation and promotes malignant cell transformation as shown both in vitro (Fujimoto et al, 1996;Bischof et al, 1997) and in vivo (Kuefer et al, 1997) by phosphorylating and activating signaling transmitters and pathways, such as phospholipase C (PLC)gphosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), Stat3, Stat5 and mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) (Wasik, 2002;Marzec et al, 2007).…”