Anaplastic large cell lymphoma (ALCL) is a common form of pediatric non-Hodgkin lymphoma. The major genetic event present in 80% of these tumors is the chromosomal translocation t (2;5)(p23;q35) resulting in the aberrant expression of the fusion protein nucleophosmin - anaplastic lymphoma kinase (NPM-ALK). The juxtaposition of these two genes places ALK under the NPM promoter resulting in the ectopic expression in cells that would normally not express this protein. Additionally, the fusion of the oligomerization domain of NPM to the kinase domain of ALK results in the constitutive activation of the tyrosine kinase. The result is the perpetual signaling through growth factor pathways known to drive proliferation and resist apoptosis. In an effort to identify novel signaling mechanisms involved in NPM-ALK mediated oncogenesis, we pursued a mass spectrometric approach aimed at characterizing the changes in the phosphoproteome in response to NPM- ALK activation. Based on these data, we hypothesize that NPM-ALK regulates the metabolism of the ALCL tumor cells to promote rapid proliferation and the Warburg effect. We specifically identified Pyruvate Kinase M2 (PKM2) as a candidate protein of NPM-ALK regulation and metabolic influence.
Aim 1 proposes to investigate the role of NPM-ALK in PKM2 regulation. The phosphoproteomic data was validated to show that NPM-ALK regulates the phosphorylation of PKM2 at Y105 and subsequently negatively regulates PKM2 enzymatic activity. Therefore this aim intends to further validate the role of NPM-ALK regulation of PKM2 by determining whether NPM-ALK directly phosphorylates PKM2 and identify additional phosphorylation sites that may be involved.
Aim 2 will investigate the biological function of PKM2 in ALCL tumor growth. PKM2 activating compounds (obtained from a collaboration with Craig Thomas at the NIH) and phosphorylation mutants (previously developed in our lab) will be utilized to determine how the activity and phosphorylation status of PKM2 contributes ALCL oncogenesis in both in vitro and in vivo systems. Finally, Aim 3 will investigate the metabolic signature of NPM-ALK driven neoplasms. Here, a metabolomics approach will be pursued to characterize the global metabolic changes induced by NPM-ALK activity. These data will be validated and integrated with the phosphoproteomic data in order to map the metabolic signature of NPM-ALK. Through the incorporation of several powerful screening techniques, this proposal will provide both global and focused mechanistic insights into NPM-ALK signaling and oncogenesis. A comprehensive understanding of the mechanisms through which NPM-ALK exerts its oncogenic pressure will lead to future targets for therapeutic intervention of, not only ALK+ALCL, but any tumors containing the oncogenic expression of ALK.
Anaplastic large cell lymphoma (ALCL) is the most common pediatric peripheral T-cell lymphoma. The major genetic abnormality in these tumors is the expression of a protein known as NPM-ALK which contributes to the high mortality and morbidity in patients. The goal of this project is to determine how cells that express NPM- ALK proliferate and survive in order to identify new strategies for treatment.
|McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine et al. (2013) Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma. Blood 122:958-68|