| NPM-ALK is a fusion tyrosine kinase that drives oncogenesis in a subset of anaplastic large cell lymphoma. Based on the role of NPM-ALK in cancer initiation and progression, we pursued a mass spectrometry-based phosphoproteomic approach aimed at the unbiased identification of novel mediators of NPM-ALK signaling. The analysis of cell lines under differing NPM-ALK activation conditions revealed a significant number of proteins that regulate cellular metabolism to be affected by NPM-ALK signaling. We therefore, pursued an untargeted metabolomic screen aimed at the identification of specific metabolites and metabolic pathways that are regulated by NPM-ALK. This analysis revealed significant alterations in pathways implicated in the Warburg effect and biomass production, including glycolysis, the pentose phosphate pathway, pyrimidine metabolism, etc. Metabolic flux analysis revealed an NPM-ALK-driven up-regulation of these pathways and down-regulation of energy production. We hypothesized that PKM2 and GSK3beta, both identified in the phosphoproteomic study, mediate the metabolic changes. Biochemical studies revealed that NPM-ALK directly phosphorylated PKM2, decreasing its enzymatic activity and driving a metabolic shift away from energy production and toward biomass production. Chemical activation of PKM2 or expression of a mutant PKM2 resulted in a reversal of this metabolic shift and decreased tumorigenesis. The phosphoproteomic analysis identified another candidate mediator of NPM-ALK signaling, GSK3beta. Through the PI3K/AKT pathway, NPM-ALK regulated pS9-GSK3beta, inhibited its activity and provided proteasomal protection for Mcl-1, CDC25A and GYS. This pathway increased proliferation and survival, while increasing glycogen production as a form of metabolic regulation. Studies described in this dissertation globally characterize the ALK driven phosphoproteome and metabolome while providing mechanistic discoveries of two novel NPM-ALK driven pathways. This work provides significant advances to our understanding of oncogenesis and will lead to advances in targeted therapies for ALK driven neoplasms. |
