PROJECT ABSTRACT / SUMMARY: Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, but many patients fail to respond. Expanding the response to ICIs is a major goal in immuno-oncology. We and others recently discovered that blocking exosome biogenesis through the genetic depletion of nSMase2 can overcome resistance to ICIs in multiple mouse models leading to robust antitumor immune response and inhibition of tumor growth. Therefore, we hypothesize that small molecule inhibition of nSMase2 could be a novel therapeutic strategy to promote antitumor immunity. There are no clinically available nSMase2 inhibitors. Current inhibitors have low potency, unknown selectivity, and poor physicochemical properties. Our team recently carried out a human nSMase2 high throughput screening campaign followed by structural optimization of the hits. These efforts led to the identification of PDDC, the first nanomolar potent (IC50=300-600nM), selective, and orally bioavailable nSMase2 inhibitor. PDDC, however, exhibits limitations that hamper its clinical translation including moderate potency, poor solubility, high protein binding, and unexpectedly low exposures in higher species (rat, dog, primate). Despite these limitations, we present preliminary data showing the effectiveness of PDDC in an ICI-resistant mouse model. Here, we propose three aims to build on these findings with the goal of identifying an optimized nSMase2 inhibitor that is effective across multiple mouse and human cancer models and ready for advancement to IND-enabling studies. In AIM 1, we will synthesize PDDC analogs to improve potency, solubility, and pharmacokinetics (PK). Analogs will be tested in vitro for potency, chemical stability, solubility, interspecies metabolic stability, permeability, and selectivity. Compounds meeting prespecified in vitro criteria will advance to PK and tumor target engagement studies in mice. Inhibitors passing mouse criteria will be prioritized for PK in rats and dogs. A predefined Preclinical Target Product Profile will guide our optimization activities. In AIM 2, PDDC and selected optimized analogs which are shown to provide robust inhibition of tumor nSMase2 activity in vivo in Aim 1 will be evaluated for efficacy/tolerability in multiple mouse syngeneic models. We will compare their effectiveness to nSMase2 knockout models as well as test their ability to suppress growth after tumors are well established. We will delve into the mechanism of action by evaluating the impact of the inhibitors alone or together with ICIs on the immune infiltration into the tumors using single-cell analytic tools. In AIM 3, we will evaluate the efficacy/tolerability of the optimized PDDC analogs in human models of cancer. We will evaluate the ability to block tumor exosome production in vivo and again compare small molecule inhibition to genetic knockout in regulating the ability of immune cells to effectively target and kill their cancer cell targets. Successfu...