Abstract 577: ENPP1 inhibitor ZX-8177 in combination with chemotherapy or radiation exhibits synergistic anti-tumor effects via STING-mediated response

2’3’-cGAMP serves as a second messenger binding to the stimulator of interferon genes (STING) to induce the STING-dependent interferon response, triggering downstream antiviral and anti-tumor mechanism. Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) hydrolyzes the extracellular 2’3’-cGAMP and attenuates the immune stimulatory tumor microenvironment, and thereby facilitates tumor progression. Inhibition of ENPP1 activity in tumors that produce a significant amount of 2’3’-cGAMP, potentially activates the anti-tumor immune response. We previously presented a selective ENPP1 inhibitor ZX-8177 with high potency in biochemical and MDA-MB-231 cell-based enzymatic assays (IC50 values are 9.5 nM and 11 nM, respectively). In multiple syngeneic mouse models, ZX-8177 alone (2mg/kg, i.p., BID) exhibited 37-60% tumor growth inhibition (TGI) and significantly increased NK cells, T cells (Granzyme B+CD8+), and M1/M2 macrophages ratio in the tumor microenvironment. Radiation and many chemotherapy regimens can result in DNA-damaging and activation of innate immunity. In this report, we further assessed the synergistic effect on tumor inhibition in combination of ZX-8177 with chemotherapies or radiation in multiple in vivo mouse models. Synergistic anti-tumor efficacy was observed in CT-26 model with the combination of Mitomycin C (0.5 mg/kg, BIW) and ZX-8177 (25 mg/kg, oral, BID), and in 4T1 and Pan02 models with the combination of Cisplatin (5 mg/kg, BIW) and ZX-8177 (25 mg/kg, oral, BID). Flow cytometry analysis demonstrated the increase of tumor infiltrating NK cells in the ZX-8177 treated tumor tissues. Meanwhile, tumor tissues showed a higher level of 2’3’-cGAMP and the increased gene expression of type I interferon (IFN) related genes, i.e. CCL5 and CXCL10. In Pan02 syngeneic mouse model, ZX-8177 (25 mg/kg, oral, BID) plus radiation at 20 Gy displayed an enhanced anti-tumor efficacy compared with radiation only treatment (100% TGI vs. 78.5% TGI, p < 0.005). Two of five mice achieved tumor free in the combination group at the end of treatment. Increased level of tumor p-STING, 2’3’-cGAMP, IFN-related genes (i.e., IFN-r, IFN-β, and CXCL10), and cytotoxic T cells-related markers (i.e., Perforin, Granzyme A and B) was observed in the combination group, correlated with the synergistic effect on tumor growth. These in vivo studies demonstrated the synergistic inhibition of tumor growth in combination of ZX-8177 with radiation or chemotherapies through activati