Translational Regulation of Gene Expression by the NPM-ALK Fusion Kinase of Anaplastic Large Cell Lymphoma: A Global Evaluation of Differentially Recruited mRNA’s to Polyribosomes.

The NPM-ALK fusion tyrosine kinase generated by the t(2;5)(p23;q35) chromosomal translocation is present in approximately 50% of anaplastic large cell lymphomas. NPM-ALK is known to play a causal role in the generation of hematopoietic neoplasia, and it has multiple potential sites of tyrosine phosphorylation that can bind to signaling effector molecules. We found that expression of NPM-ALK regulates mediators of translational initiation, including mTOR, S6K1, and 4E-BP1. Moreover, other investigators have shown that expression of proteins that mediate the control of translational initiation can be rate-limiting in the pathogenesis of neoplasia. Collectively, these findings stimulated our hypothesis that NPM-ALK promotes neoplasia by altering the partitioning of specific mRNA’s to polysomes and thereby regulating their translation. To evaluate this hypothesis, we assessed the global profile of polysomal mRNA in cells that express the NPM-ALK fusion kinase.

In this study, we generated Ba/F3 murine hematopoietic cells that express kinase-active NPM-ALK or a kinase-deficient NPM-ALK mutant (K210A). Cells were maintained in the presence of the cytokine IL-3 to minimize changes secondary to cell cycle redistribution or early apoptosis. We used these cell lines to compare in a global manner the partitioning of mRNA species to polysomes. We isolated polyribosome preparations using conventional sucrose gradient fractionation, and from this purified polysomal material we extracted mRNA. Similarly, we prepared total mRNA from these cell lines. The global profile of gene expression for both total and polysomal RNA was assessed using Affymetrix mouse genome microarrays. The data generated in this effort were analyzed using the GeneChip robust multichip analysis (GCRMA) method. Significance Analysis of Microarrays (SAM) and Linear Models for Microarray Data (Limma) were employed to identify significantly differentially expressed genes. By culturing each cell line in the presence of IL-3, we found little difference in total cellular mRNA levels. However, we found that expression of kinase-active NPM-ALK markedly altered the partitioning of mRNA’s to the polysomal pool. We identified 286 genes whose mRNA’s exhibited altered polysomal partitioning, including 102 decreased in cells with kinase-active NPM-ALK and 184 with increased polysome association. These translationally regulated mRNA’s encode proteins associated with transcriptional control, post-translational modification of proteins, and hematopoietic neoplasia. Our findings support the assertion that NPM-ALK signaling regulates gene expression in part through translational control. We speculate that concerted dysregulation of transcription and translation by oncogenes might broadly mediate the pathogenesis of hematopoietic neoplasia.