Ovarian cancer (OvCa) is a deadly disease ranking 5th in cancer deaths in women, with the 3rd highest mortality to incidence ratio of all cancers. With patient overall survival not significantly changed for several decades, there is a clear unmet clinical need to develop new therapies for OvCa. Aldehyde dehydrogenase-1A enzymes (ALDH1A) represent a therapeutic target for OvCa. ALDH1A enzymes are upregulated in ovarian cancer initiating cells (CIC) and mediate the biosynthesis of retinoic acid (RA), to regulate numerous cellular processes. We recently identified a novel ALDH1A family inhibitor (ALDHi). Our preliminary data indicate that ALDHi treatment of cancer cells and immune cells have opposite effects, both of which could promote anti- tumor immunity. ALDHi treatment of CIC induces an RA and transcription- dependent necroptotic cell death, potentially related to altered function of the RA transcription partner NR4A1. This ALDHi-induced CIC death is associated with the release of Damage Associated Molecular Patterns (DAMPs) and other inflammatory mediators. In contrast, ALDHi treatment is associated with enhanced proliferative response of both dendritic cells (DC) and CD8+ T cells. Our overarching hypothesis is that ALDHi can act as an immune modulator and can be used to enhance immunotherapeutic approaches in OvCa. To test our hypotheses, we propose the following specific aims: SA1: To identify the RA receptors and downstream factors that drive ALDHi induced CIC necrosis and to determine whether ALDHi-induced CIC necroptosis is inflammatory cell death. We propose to test the role of individual RA receptors RAR/RXR and NR4A1 co-receptor in ALDHi-mediated CIC death and evaluate the impact of ALDHi/NR4A1 on CIC release of inflammatory mediators and this stimulation of an anti- tumor T cell response. Next, we will, in SA2: Assess the impact of ALDHi on host DC and T cells. As RA can regulate the differentiation of T cells and DC, we will use reporter mice to evaluate the role of ALDHi on disruption of RA/NR4A1 signaling and DC and T cell biology in vitro and in vivo. We will evaluate the capacity of DC to differentiate into IL-12 producing DC1, and CD4 T helper cell subsets differentiation with focus on the Th17/Treg trans-differentiation. Finally, our preliminary data suggest ALDHi may enhance anti- tumor immunity both via actions on the tumor cell and immune cells. We therefore propose SA3: To determine the ability of ALDHi to enhance immunotherapy in OvCa. To test this hypothesis, we will use several immune competent models of OvCa to determine the ability of ALDHi to enhance checkpoint inhibitor therapy and antitumor vaccines. IMPACT: While immune therapy has demonstrated significant benefit in many cancers, the impact in OvCa has been limited. The studies proposed here will define the role for a novel therapeutic ALDHi in immunotherapy in OvCa. Given ALDH is broadly linked with therapeutic resistance in cancer, these studies will have far-re...