NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis

Abstract

Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70–80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric 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 have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS9-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS9-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS9-GSK3β and 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 did not show significant correlation of pS9-GSK3β and CDC25A expression (P < 0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S9-GSK3β by PI3K/AKT. The subsequent inhibition of GSK3β activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3β as a mediator of NPM-ALK oncogenesis.

G-CSF ameliorates neuronal apoptosis through GSK-3β inhibition in neonatal hypoxia–ischemia in rats

“…Activated GSK-3β is phosphorylated at tyrosine-216, which upregulates pro-apoptotic and downregulates pro-survival signaling pathways (Krafft et al, 2012). Active p-Akt inhibits GSK-3β activity by phosphorylating serine-9 (McDonnell et al, 2012). Since GSK-3β activity is regulated by PI3K/Akt signaling (Pap and Cooper, 1998), thus we examined if G-CSFR stimulation by G-CSF attenuates apoptosis via the PI3K/Akt pathway and downstream GSK-3β inhibition.…”

Genetics of anaplastic large cell lymphoma

“…A recent mass spectrometry-based phosphoproteomic screen has identified glycogen synthase kinase (GSK) 3β as a novel mediator of NPM-ALK signaling [20]. NPM-ALK was shown to phosphorylate S(9)-GSK3β via the PI3K/AKT signaling pathway, resulting in inhibition of GSK3β activity and accumulation of oncogenic CDC25A and MCL1.…”

Conversion of the LIMA1 tumour suppressor into an oncogenic LMO-like protein by API2–MALT1 in MALT lymphoma

“…Colony formation assay was carried out in 1% methylcellulose-based media and assessed according to the manufacturer's protocol (Stemcell Technologies, Vancouver, British Columbia, Canada). After 3 weeks, colonies were stained with iodonitrotetrazolium chloride overnight and counted 69 .…”

“…Cell growth curves were generated by directly counting cells or cell aggregates or by using the WST-1 assay (Roche Applied Science, Indianapolis, IN, USA) and/or CellTiter-Glo Luminescent Cell Viability assay (Promega) performed according to the manufacturer's protocol 69 . Cells were counted using trypan blue exclusion assay.…”