ABSTRACT
An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.
ABSTRACT
Previous osmotic pressure studies of two nonhomologous dihydrofolate reductase (DHFR) enzymes found tighter binding of the nicotinamide adenine dinucleotide phosphate cofactor upon addition of neutral osmolytes. This result is consistent with water release accompanying binding. In contrast, osmotic stress studies found weaker binding of the dihydrofolate (DHF) substrate for both type I and type II DHFRs in the presence of osmolytes; this observation can be explained if dihydrofolate interacts with osmolytes and shifts the equilibrium from the enzyme-bound state toward the unbound substrate. Nuclear magnetic resonance experiments support this hypothesis, finding that osmolytes interact with dihydrofolate. To consider binding without added osmolytes, a high-pressure approach was used. In this study, the type II enzyme, R67 DHFR, was subjected to high hydrostatic pressure (HHP). Both enzyme activity and fluorescence measurements find the protein tolerates pressures up to 200 MPa. Binding of the cofactor to R67 DHFR weakens with increasing pressure, and a positive association volume of 11.4 ± 0.5 cm(3)/mol was measured. Additionally, an activation volume of 3.3 ± 0.5 cm(3)/mol describing k(cat)/K(m(DHF)) was determined from progress curve analysis. Results from these HHP experiments suggest water release accompanies binding of both the cofactor and DHF to R67 DHFR. In an additional set of experiments, isothermal titration calorimetry studies in H2O and D2O find that water reorganization dominates the enthalpy associated with binding of DHF to R67 DHFR·NADP(+), while no obvious effects occur for cofactor binding. The combined results indicate that water plays an active role in ligand binding to R67 DHFR.
