PTBP1 Interacts with PKM2 in an NPM-ALK-Dependent Manner and Contributes to the Pathogenesis of Anaplastic Large Cell Lymphoma (ALCL)

Introduction: Pyruvate kinase (PKM2) is a glycolytic enzyme that plays an important role in cancer metabolism. Our previous work demonstrated that NPM-ALK phosphorylates PKM2 at Y105 and that this regulates altered metabolism that promoted ALK-mediated lymphomagenesis. There is emerging evidence that PKM2 may contribute to oncogenesis independent of its role in cell metabolism. We hypothesized that identification of novel PKM2 interactors via mass spectrometry could provide additional insights on its expanding functional role in cancer. Methods: In order to identify novel proteins that interact with PKM2, we generated an ALCL-derived cell line (DEL) stably transduced to express FLAG-tagged WT PKM2. We isolated the FLAG-tagged PKM2-immunocomplex and subjected it to proteomic analysis by using liquid-chromatography tandem mass spectrometry (LC-MS/MS). A subset of the proteins unique to the PKM2 bait as compared to control was selected for subsequent studies including reciprocal immunoprecipitations (IP), western blot analysis and functional experiments. Results: Mass spectrometry identified 63 proteins that interacted with WT PKM2 including -catenin and FBP-6, both known interactors of PKM2. Among these we also noted the polypyrimidine tract-binding protein (PTBP1), a protein with a role in RNA stability as a candidate interactor of PKM2. Reciprocal immunoprecipitations confirmed the interaction between endogenous PKM2 and PTBP1 in two ALCL-derived cell lines. In order to determine if phosphorylation of PKM2 at Y105 was necessary for its interaction with PTBP1, FLAG-WT-PKM2 and FLAG-Y105F-PKM2 were used for IP and western blot analysis which confirmed that the PTBP1 interaction occurred with WT-PKM2 and not Y105F-PKM2. Similarly, the interaction of PTBP1 with PKM2 was significantly decreased in the presence of a selective ALK inhibitor. To determine whether the interaction of PTBP1 with pY105-PKM2 occurred in distinct subcellular compartments, we carried out subcellular fractionation of ALCL-derived cell lines and evaluated the interaction of PKM2 with PTBP1. Forward and reciprocal immunoprecipitations demonstrated that the interaction of PTBP1 and PKM2 and occurs primarily in the nucleus and selective to pY105-PKM2. Analysis of subcellular fractions after selective inhibition of ALK by crizotinib also showed that the expression of nuclear pY105 PKM2 was abolished. Based on the previous observation that PKM2 acts as a protein kinase and phosphorylates STAT3, we assessed the role of pY105-PKM2 on STAT3 phosphorylation in DEL cells stably expressing either FLAG-WT PKM2 WT or Y105F-PKM2. Western blot analysis demonstrated that nuclear expression of active (pY705)-STAT3 was decreased in cells expressing Y105F-PKM2 relative to WT-PKM2. Stable knockdown by lenti-viral transduction of PTBP1 shRNA in DEL cells demonstrated a marked decrease in expression of pY105-PKM2 and Y705-STAT3 without affecting total STAT3 protein. Moreover, knockdown of PTBP1 led to decreased ALCL cell proliferation and colony formation in soft agar relative to control. Conclusion: These data demonstrate that PTBP1 is a novel PKM2 interacting protein and phosphorylation of PKM2 at Y105 by NPM-ALK regulates the interaction. The interaction of PTBP1 occurs preferentially with pY105-PKM2 within the nucleus and regulates the phosphorylation of Y705-STAT3. PTBP1 promotes oncogenesis in ALCL by regulating the nuclear localization of Y105-PKM2 and phosphorylation of Y705-STAT3. Our studies provide evidence for a novel role of PTBP1 in mediating oncogenesis in NPM-ALK expressing ALCLs. Disclosures No relevant conflicts of interest to declare.