Abstract LB323: Inhibition of ENPP1 using small molecule, SR-8541A, enhances the effect of checkpoint inhibition in cancer models
Authors: Weston Alexis S. , Thode Trason , Ng Serina , Ghosh-Halder Tithi , Rheinschmidt Shelby , Adamson Sydney , Gutowsky Katelyn , Durbin Brian , Soldi Raffaella , Kasibhatla Srinivas , Kaadige Mohan , Sharma Sunil 
Published: April 2023

Abstract
Purpose: It has become increasingly clear that the activation of both the innate and adaptive immune systems is vital to provide the best outcomes with immunotherapies. As part of the adaptive immune response, checkpoint inhibitors (CIs) have shown promise in the clinic, but seem to only work in a small subset of cancers with response rates below 20%. It is anticipated that activation of the innate immune response may help sensitize multiple cancer types to adaptive immune therapies. cGAS-STING pathway, which is activated in response to cytosolic DNA, has emerged as a key mechanism to activate innate immunity, primarily through type I interferon (IFN) signaling. Several direct STING agonists have been developed but their performance in the clinic has been dissatisfactory. A key limitation with direct STING agonists is the widespread expression of STING in normal tissues, whereby the hyperactivation of STING can lead to a systemic cytokine storm. Thus, there is a need to identify alternative approaches to activate STING in a controlled manner. ENPP1 is the only known direct negative regulator of the STING pathway that hydrolyzes 2′3′ cGAMP, the direct ligand of STING. Highest levels of 2′3′ cGAMP are found in tumors and recent evidence suggests that 2′3′ cGAMP acts locally, as a paracrine immune transmitter. Therefore, inhibition of ENPP1 may produce superior outcomes by activating STING in the tumor microenvironment. Previously, we reported the development of SR-8541A, a highly selective and potent inhibitor of ENPP1 that activates the STING pathway. Here, we show that the inhibition of ENPP1 with SR-8541A enhances the effect of CIs in breast and colon cancer models.
Methods: Immune infiltration assays were conducted using human breast cancer cell line derived organoids (MDA-MB-231 and MDA-MB-468). Co-cultures of cancer organoids and immune cells (PBMCs) were exposed to SR-8541A +/- CIs (CTLA-4 and/or PD-1) for 48 hours. Confocal Z-Stack imaging, RT-PCR, and MSD cytokine assays were performed to evaluate the effects. In vivo studies were conducted using syngeneic mouse models (CT-26 and EMT-6), which were engrafted subcutaneously and treated with SR-8541A +/- CIs. Tumor growth was monitored over the course of the study. IHC and RT-PCR were conducted on the tumors.
Results: Combination of SR-8541A with CIs showed a significant increase in immune infiltration in both the MDA-MB-231 and MDA-MB-468 organoid models. Corresponding RT-PCR analysis showed activation of IFN signaling (IFN-ß, CXCL10, ISG15). In vivo combination with CIs also exhibited a significant increase in overall efficacy, along with increased levels of CD3+ and CD8+ T-cell infiltration into the tumors, and increased levels of IFN response.
Conclusion: In summary, we show that combination of ENPP1 inhibition with checkpoint inhibition promotes a robust antitumor activity by stimulating both innate and adaptive immune response.
Citation Format: Alexis S. Weston, Trason Thode, Serina Ng, Tithi Ghosh-Halder, Shelby Rheinschmidt, Sydney Adamson, Katelyn Gutowsky, Brian Durbin, Raffaella Soldi, Srinivas Kasibhatla, Mohan Kaadige, Sunil Sharma. Inhibition of ENPP1 using small molecule, SR-8541A, enhances the effect of checkpoint inhibition in cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB323.