ABSTRACT Compelling evidence suggests that careful and therapeutically relevant activation of the STING (STimulator of INterferon Genes) pathway is necessary to elicit potent anti-cancer innate immune responses. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the STING pathway's only known direct negative regulator expressed in many tumor types, and, when it is overexpressed, tumors show limited efficacy to front-line therapies. Such as, in triple-negative breast cancer (TNBC), high ENPP1 expression is associated with drug resistance and poor prognosis. If a safe and efficacious ENPP1 inhibitor were available, it would have widespread utility for multiple cancer types and, if used in combination with other cancer therapies, may enhance their performance. Towards this end, we have developed an orally bioavailable potent small-molecule inhibitor of ENPP1 called SR-8541A. It inhibits hENPP1 activity with an IC50 of 3.6 nM (Ki=1.9 nM) and demonstrates robust selectivity. We have established that it activates the STING pathway, promotes immune cell infiltration, and inhibits cancer spheroid growth. Furthermore, in syngeneic tumor mouse models, SR-8541A demonstrates a synergistic effect with radiation, and a preliminary study also shows synergy with checkpoint inhibitors. To date, we have completed preclinical development activities on SR-8541A that include API development and manufacturing, stability, pharmacokinetics, tolerability, and preliminary toxicology (mouse, rat, dog). The overall goal of this Direct to Phase II SBIR application is to complete non-GLP and GLP preclinical studies for our lead molecule SR-8541A with TNBC as our initial focus. In Aim 1, we will evaluate the efficacy of SR-8541A in combination with FDA-approved drug regimens (e.g., cisplatin, anti-mCTLA-4, anti-mPD-1, PARP inhibitor) in 4T-1 and EMT-6 breast cancer mouse models. In Aim 2, we will conduct IND enabling GLP toxicology study in dogs as the rat GLP study is complete. In Aim 3, we will develop and manufacture cGMP clinical-grade tablets necessary to conduct a Phase 1 clinical trial. Direct to Phase II SBIR success will result in the completion of the required preclinical studies to seek IND acceptance for a first-in-human Phase I clinical trial and to engage with private-sector investors in funding clinical trials in TNBC. If SR-8541A is approved for patient use, it would be the first-in-class molecule to modulate the innate immune system, expanding the benefits of immunotherapy to more patients.