Phosphorylation of PKM2 by NPM-ALK Regulates a Metabolic Shift and Increased Oncogenic Potential
Authors: McDonnell Scott RP , Hwang Steven R , Basrur Venkatesha , Conlon Kevin , Fermin Damian , Murga-Zamalloa Carlos , Rolland Delphine C.M. , Thomas Craig J. , Elenitoba-Johnson Kojo SJ , Lim Megan S. 
Published: October 2019

Abstract
Abstract 896
Anaplastic Large Cell Lymphoma (ALCL) is a common form of pediatric non-Hodgkin lymphoma that is often characterized by the chromosomal translocation t(2;5)(p23;q35) and subsequent expression of the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK is a constitutively active tyrosine kinase that functions as a driving oncogenic mutation in ALCL. However, a complete understanding of the signaling mechanisms that contribute to its oncogenesis have not been elucidated. In an effort to identify novel mediators of NPM-ALK signaling, we pursued a phosphoproteomic strategy aimed at the unbiased identification of protein phosphorylation events regulated by NPM-ALK kinase. This analysis resulted in the identification of Pyruvate Kinase M2 (PKM2) as a candidate substrate of NPM-ALK in addition to other metabolic proteins. Phosphorylation of PKM2 is critical for inducing a metabolic shift which leads to accumulation of precursor biomass metabolites required for rapidly dividing cells.
We hypothesized that NPM-ALK regulates an oncogenic metabolic shift through phosphorylation of PKM2. Western blot analysis and in vitro kinase assay confirmed that PKM2 is phosphorylated at the Y105 residue by NPM-ALK. The phosphorylation of PKM2 is inversely correlated with its enzymatic activity using an in vitro activity assay. Stable expression of Flag-PKM2 Y105F or treatment with PKM2 small molecule activators (NCGC00186528) results in increased PKM2 activity and induction of a metabolic switch as measured by decreased lactate (80% ± 6%, p&lt;0.01) and increased ATP production (167% ± 12%, p&lt;0.01). Inhibition of ALK activity using a small molecule inhibitor also resulted in a metabolic switch. Under normoxic conditions, activation of PKM2 resulted in reduced proliferative capacity without an effect on cellular viability. However, PKM2 activation under hypoxia resulted not only in reduced proliferation, but in a significant reduction in viability (51% ± 11%, p&lt;0.01). Furthermore, DEL cells stably expressing WT-PKM2 are resistant to inhibition of ATP synthase (oligomycin), while cells expressing Y105F-PKM2 exhibit reduced proliferation under the same conditions. Similarly, the diminished proliferative capacity by PKM2 activation was intensified by inhibiting ATP synthase. These data support a role for the NPM-ALK – PKM2 axis in the regulation of a metabolic switch that favors proliferation with a decreased dependence on oxidative phosphorylation. Finally, activation of PKM2 and expression of Y105F-PKM2 resulted in reduced tumorigenic capacity as observed by methylcellulose colony formation assay (Y105F 46% ± 7.9% of control, p&lt;0.01, Activator 61% ± 2.2% of control, p&lt;0.01). Taken together, these data identify a novel oncogenic mechanism whereby NPM-ALK induced metabolic shift is mediated through the phosphorylation of PKM2. The results provide rationale for the use of PKM2 activators as therapeutic agents in ALK+ALCL.

Disclosures:
No relevant conflicts of interest to declare.