A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression

Through variation in the cancer cell and their immune infiltrates each tumor represents a unique problem, but therapeutic targets can be found in the shared features. Radiation therapy can change the interaction between the cancer cells and the stroma through release of innate adjuvants including the STING agonist cGAMP. Enpp1 is a phosphodiesterase that can be expressed by cancer cells and can degrade cGAMP. Enpp1 can therefore limit innate adjuvant availability following radiation therapy. We observed that many cancer cells lack Enpp1 expression, but that Enpp1 expression is retained in cells of the tumor stroma and this expression in the tumor stroma limits tumor control by radiation therapy. We demonstrate the efficacy of a novel Enpp1 inhibitor and show that this inhibitor improves tumor control by radiation even where the cancer cells lack Enpp1. We show that the mechanism requires STING and type I IFN receptor expression by non-cancer cells, and is dependent on CD8 T cells as a final effector mechanism of tumor control. This suggests that Enpp1 inhibition may be an effective partner for radiation therapy regardless of whether cancer cells express Enpp1, and identifies a novel therapeutic Enpp1 inhibitor suitable for combination therapies in immuno-oncology. Overall design: Tumors were implanted subcutaneously into the right flank as follows: C57BL/6 2x105 MC38; BALB/c 2x105 CT26. When tumors were approximately 5mm in average diameter at approximately 10-14 days following implantation, mice were randomized to receive treatment with CT-guided radiation using the Small Animal Radiation Research Platform (SARRP) from XStrahl. Dosimetry was performed using Murislice software from XStrahl. The SARRP delivered a single dose of 12Gy to an isocenter within the tumor using a 10mm x 10mm collimator and a 45° beam angle to minimize dose delivery to normal tissues. The Enpp1 inhibitor VIR3 was delivered through oral gavage starting 1 day prior to RT daily. 4d following RT, the tumors were harvested, debulked and immediately flash frozen by rapid immersion in liquid nitrogen. Frozen tumors were crushed, treated with 500µl of RNAlater, then frozen at -80oC. To extract RNA, the samples were thawed on ice, and 500µl of buffer RLT (QIAGEN Cat. No. 79219) was added and samples were homogenized. Following centrifugation, RNA was isolated using a QIAGEN RNeasy Plus Mini Kit (Cat No. 74134), according to the manufacturer's instructions. Quality was checked on nanodrop (ND-1000) and the quantity of RNA was determined on Qubit 4 flourometer. To prepare for sequencing, samples were processed using an Illumina TruSeq® Stranded mRNA Library Prep kit with with Illumina TruSeq RNA Single Indexes Set A and Set B barcoding kits. An Illumina NovaSeq 6000 S1 Reagent Kit v1.5 was used to make RNA libraries along with a NovaSeq XP 2-Lane Kit v1.5. RNA libraries were sequenced on NovaSeq 6000.