NPM-ALK Regulated GSK3β Phosphorylation Results in Mcl-1 and CDC25A Accumulation and Enhanced Oncogenic Potential
Authors: McDonnell Scott RP , Hwang Steven R , Basrur Venkatesha , Conlon Kevin P , Fermin Damian , Wey Elizabeth , Murga-Zamalloa Carlos , Zeng Zihua , Zu Youli , Elenitoba-Johnson Kojo SJ , Lim Megan S 
Published: October 2019

Abstract
Abstract 586
Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. ALCLs are frequently characterized by translocations involving the anaplastic lymphoma kinase (ALK) gene. In 70–80% of these cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of ALK with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK tyrosine kinase. NPM-ALK is an oncogenic tyrosine kinase which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules in ALCLs have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β, as a potential signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the phosphorylation of GSK3β at serine-9 (pS9-GSK3β) in a time and dose-dependent manner. Expression of NPM-ALK in 293T cells led to increased phosphorylation of GSK3β at serine-9 compared to kinase-defective K210R mutant NPM-ALK but did not affect total GSK3β levels. Inducible knockdown of ALK in SU-DHL-1 cells also led to decreased phosphorylation of GSK3β at serine-9 without affecting total GSK3β levels. Selective inhibition of PI3Kδ by CAL-101 resulted in decreased phosphorylation of GSK3β at serine-9 in a dose dependent manner without affecting total GSK3β or NPM-ALK activity. Ablation of ALK kinase activity resulted in proteasomal degradation of GSK3β substrates Mcl-1 and CDC25A. This degradation was recovered upon chemical inhibition of GSK3β (GSK3 IX). To address the role of GSK3β in ALCL proliferation, WST-1 assay revealed that ALK inhibition resulted in a decrease in cell viability which was rescued by GSK3β inhibition. ALK inhibition resulted in significant cellular apoptosis as detected by propidium iodide and Annexin V staining followed by flow cytometry. This apoptotic response was rescued by GSK3β inhibition. The effect of GSK3β on cellular oncogenic potential was addressed by colony formation assay using ALCL-derived cell lines. While ALK inhibition resulted in decreased colony numbers, simultaneous ALK and GSK3β inhibition recovered colony numbers compared to ALK inhibition alone. Additionally, stable GSK3β knockdown conferred resistance to growth inhibitory effect of ALK inhibition as determined by colony formation assay. Furthermore, pS9-GSK3β and its known substrate, CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies and showed a significant correlation (p<0.001). Conversely, ALK-ALCL tissue biopsies showed no significant correlation of pS9-GSK3β and CDC25A expression (p<0.2). Our results demonstrate that NPM-ALK signals through PI3K/AKT to phosphorylate GSK3β at serine-9. This phosphorylation inhibits GSK3β kinase activity which results in the accumulation of CDC25A and Mcl-1 thus providing growth advantage and protection from apoptosis. These findings provide support for the role of GSK3β as a novel mediator of NPM-ALK oncogenesis.

Disclosures:
No relevant conflicts of interest to declare.