The PKM2 activator and molecular glue TP-1454 modulates tumor-immune responses by destabilizing T-regulatory cells

Background: Pyruvate kinase is an important enzyme that catalyzes the last step of glycolysis. The M2 isoform (PKM2) is important for balancing respiration and biosynthesis, which can be achieved by switching between the highly active tetrameric form and the less active dimeric form through allosteric metabolite binding. In addition to its role in metabolic regulation, the dimeric form of PKM2 can translocate to the nucleus, altering transcription to enhance cancer cells’ ability to grow and evade immune detection. Targeting PKM2 activation presents an opportunity to potentially reprogram the tumor-immune microenvironment (TME). TP-1454 is a potent PKM2 activator achieving PKM2 activation with low nanomolar concentration in biochemical assays (AC50 = 10 nM). TP-1454 has an acceptable preclinical safety profile and is currently in a Phase I clinical trial (NCT04328740). Materials and Methods: PKM2 activity was measured using a colorimetric assay. PKM2 tetramerization and Foxp3 protein stability was assayed using western blots. In vivo activity of TP-1454 alone and in combination with checkpoint inhibitors (CPI) was tested in multiple syngeneic mouse models. Flow cytometry was used to conduct immunophenotyping of immune cells in the TME and to test regulatory T cell (Treg) polarization. Treg proliferation was assessed in a Cell Trace Violet Assay. Mass spectrometry was conducted to identify changes in metabolite levels. Foxp3 post-translational modifications were tested using a Foxp3-Flag expressing HEK293 cell line. Results: TP-1454 treatment increased PKM2 activity in vitro and in vivo and showed a dose-dependent increase in PKM2 tetramer formation. Oral administration of TP-1454 resulted in substantial [KK1] [SS2] antitumor activity as a single agent (up to 42% tumor growth inhibition compared to vehicle) and in combination with CPI (100% complete response, 58% increase in mean survival compared to CPI alone) in multiple syngeneic mouse models. In vitro treatment of various immune cell types with TP-1454 resulted in differential PKM2 activation. Oral administration of TP-1454 treatment reduced intratumoral CD4+ Foxp3+ Treg levels as assessed by Flow cytometry immunophenotyping. In vitro treatment with TP-1454 showed a reduction in Treg polarization but not proliferation. Human peripheral blood mononuclear cells (PBMC) treated with TP-1454 showed a decrease in glucosamine-6-phopsphate (G6P), which is reported to stabilize Foxp3 via the O-linked β-N-acetylglucosamine (O-GlcNac) post-translational modification. In vitro treatment of HEK293 cells with TP-1454 reduced O-GlcNac-Foxp3 levels and resulted in faster Foxp3 degradation. Conclusions: These findings suggest a novel mechanism to regulate the TME with a PKM2 activator in preclinical models and should be investigated further in clinical trials. Conflict of interest: Corporate-sponsored Research: Employment