Despite small improvements to standard of care protocols, the overall survival of patients with ovarian cancer (OvCa) has not significantly changed for several decades. There is a clear unmet clinical need to develop new therapies for this highly aggressive disease ranking 5th in cancer deaths in women, with the 3rd highest mortality to incidence ratio of all cancers. Aldehyde dehydrogenase-1A enzymes (ALDH1A) represent a therapeutic target for OvCa. ALDH1A enzymes are upregulated in ovarian cancer initiating cells where they mediate the biosynthesis of retinoic acid (RA), to regulate numerous cellular processes. We recently demonstrated that inhibition of ALDH1A (ALDHi) can have a dual effect: it can directly target tumor cells to slow proliferation and act on immune cells to enhance proliferative response of both dendritic cells (DC) and CD8+ T cells. Our overarching hypothesis is that ALDHi can act as an immune modulator (via individual RA receptors RAR/RXR and NR4A1 co-receptor) and can be used to enhance immunotherapeutic approaches in OvCa. To test this hypothesis, our five-year project R01CA238315, the parent grant to this Research Supplement to Promote Diversity in Health-Related Research, proposes three specific aims (SA) SA1: To identify the RA receptors and downstream factors that drive ALDHi induced necrosis in tumor cells and to determine whether ALDHi-induced CIC necroptosis is inflammatory cell death. SA2: Assess the impact of ALDHi on host DC and T cells. SA3: To determine the ability of ALDHi to enhance immunotherapy in OvCa. The current, one-year project builds on our new findings showing that, in addition to their immune promoting function in DCs and CD8 T cells, ALDHi also lower the frequency of tumor associated macrophages (TAM), a critically important immune cell subset with immune suppressive, tumor-promoting function. The goal of the current one-year proposal is to extend our studies (originally focused on DC and T cells) to (1) identify the mechanisms of ALDHi induced effects on TAM and (2) elucidate the downstream consequences of these ALDHi effects on modulating the tumor microenvironment and enhancing immune therapy response. To achieve this, we will use primary macrophages derived ex vivo from blood (human) or bone marrow precursors (mice) and matured under M1/M2 inducing conditions in the presence or absence of ALDHi. NR4A1 involvement will be dissected using bone marrow precursors from NR4A1 reporter mice and target specificity will be confirmed in NR4A1 null mice. The immune modulatory roles of TAM will be further dissected in vivo, in NR4A1 mice challenged orthotopically with ovarian tumors and treated with ALDHi or vehicle control. IMPACT: This project is in line with the NIH mission to promote diversity among investigators doing research in health-related sciences. The studies proposed here will define new roles for a ALDHi in immunotherapy in OvCa. Given ALDH is broadly linked with therapeutic resistance in can...