Cyclic GMP-AMP synthase (cGAS) is an endogenous DNA sensor that synthesizes cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) from ATP and GTP. 2′3′-cGAMP activates the stimulator of interferon genes (STING) pathway, resulting in the production of interferons and pro-inflammatory cytokines. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the phosphodiesterase that negatively regulates the STING pathway by hydrolyzing 2′3′-cGAMP. It has been established that the cGAS–STING pathway plays a major role in inhibiting tumor growth by upregulating T cell response. Herein, we demonstrate that AVA-NP-695, a selective and highly potent ENPP1 inhibitor, apart from the immunomodulatory effect also modulates cancer metastasis by negatively regulating epithelial–mesenchymal transition (EMT). We established that the combined addition of 2′3′-cGAMP and AVA-NP-695 significantly abrogated the transforming growth factor beta (TGF-ꞵ)-induced EMT in MDA-MB-231 cells. Finally, results from the in vivo study showed superior tumor growth inhibition and impact on tumor metastasis of AVA-NP-695 compared to Olaparib and PD-1 in a syngeneic 4T1 breast cancer mouse model. The translation of efficacy from in vitro to in vivo 4T1 tumor model provides a strong rationale for the therapeutic potential of AVA-NP-695 against triple-negative breast cancer (TNBC) as an immunomodulatory and anti-metastatic agent.

Immunotherapy has proved to be a breakthrough in cancer treatment. So far, a bulk of the approved/late-stage cancer immunotherapy are antibody-based. Although these antibody-based drugs have demonstrated great promise, a majority of them are limited due to their access to extracellular targets, lack of oral bioavailability, tumor microenvironment penetration, induction of antibody dependent cytotoxicity etc. In recent times, there has been an increased research focus on the development of small molecule immunomodulators since they have the potential to overcome the aforementioned limitations posed by antibodies. Furthermore, while most biologics based therapeutics that are in clinical use are limited to modulating the adaptive immune system, very few clinically approved therapeutic modalities exist that modulate the innate immune system. The innate immune system, which is the body’s first line of defense, has the ability to turn cold tumors hot and synergize strongly with existing adaptive immune modulators. In preclinical studies, small molecule innate immune modulators have demonstrated synergistic efficacy as combination modalities with current standard-of-care immune checkpoint antibodies. In this review, we highlight the recent advances made by small molecule innate immunomodulators in cancer immunotherapy.

Adenosine deaminase acting on RNA (ADAR1) catalyzes the hydrolytic deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). There are 2 isoforms of ADAR1 (p110 in the nucleus; p150 in cytoplasm) and both modify self dsRNA in coding and non-coding regions. The ADAR1 p150 isoform is expressed from an interferon (IFN)-responsive promoter and has a Z-DNA/Z-RNA binding domain at the N-terminus. Previous reports have provided a strong rationale for the development of ADAR1 p150 inhibitors for cancer immunotherapy. Here, we describe AVA-ADR-001, a potential first-in-class small molecule inhibitor of ADAR1 p150 targeting the Zα domain. AVA-ADR-001 binds specifically to the Zα domain of ADAR1 p150 as confirmed by fluorescence spectroscopy and showed significant interferon induction in THP1 macrophages, which have high ADAR1 p150 expression compared with monocytes. Proteomics and transcriptomics analysis revealed significant upregulation of interferon signaling upon treatment with AVA-ADR − 001. Interestingly, activation of interferon signaling resulted in AVA-ADR-001 induced cell killing in ADAR1-independent cell lines. In addition, treatment with AVA-ADR − 001 resulted in significant activation of PKR, which may explain the decreased cell proliferation. Finally, AVA-ADR-001 showed superior anti-tumor efficacy compared to anti-PD1 in an in vivo tumor efficacy study and has a moderately synergistic effect when combined. Overall, this study reveals that ADAR1 p150 inhibition by AVA-ADR-001 exerts a multipronged impact on anti-tumor efficacy mediated by immune cells, accumulation of interferons and activation of PKR, resulting in protein translation inhibition and cell proliferation arrest.