Glutathione S-transferase omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Using genetic tools, including extensive bioinformatics analysis coupled with siRNA, shRNA, proteomics, and CRISPR/Cas9 technologies; and pharmacologic small-molecule inhibitors and degraders, we have validated GSTO1 as an impactful druggable target in oncology. Previously, we identified C1-27 as a potent GSTO1 inhibitor that shows efficacy against cancer cells in both in vitro and in vivo models. We also synthesized and tested the very first GSTO1 PROTAC confirming GSTO1 degradation in vitro. Through transcriptional profiling using Bru-seq and RNA-seq coupled with proteomics, we uncovered novel pharmacodynamic markers and cellular pathways critical for oncogenesis regulated by GSTO1. Taken together, our findings validate GSTO1 as an important drug target for cancer therapeutics and C1-27 as a potent and validated prototype inhibitor. Previously, we solved the crystal structure of C1-27 (IC50 = 31 nM) and other potent inhibitors in complex with GSTO1. Our most recent lead optimization campaign using 6 different co-crystal structures resulted in the most potent GSTO1 inhibitor (IC50 = 0.22 ± 0.02 nM) known to date. Our CRISPR/Cas9 GSTO1 knockout (KO) cell lines do not form tumors or display tumor growth delay in vivo and form smaller 3D spheroids in vitro. Through multi-omics studies in GSTO1 KO cells, we found a strong positive correlation with cell adhesion molecules and interferon response pathways, and a strong negative correlation with Myc transcriptional signature. Importantly, we also identified several clinically used chemotherapies showing significant synthetic lethality with loss or inhibition of GSTO1. We discovered that tissue factor (gene name, F3) transcription and protein expression are downregulated in response to GSTO1 KO and C1-27 treatment, further implicating a role for GSTO1 in the innate immune response. In summary, our results implicate GSTO1 as a therapeutic target in cancer and offer new mechanistic insights into its significant role in cancer progression. Importantly, our results show for the first time that inhibition of GSTO1 can activate immune responses and downregulate F3. We hypothesize that inhibiting GSTO1 will have a two-pronged effect on tumor cells: (A) impair cancer cell survival by reducing Myc transcriptional signature and F3 downregulation, and (B) enhance immune responses through interferon- mediated innate immune sensing of cancers. We further hypothesize that GSTO1 inhibitors will sensitize cancer cells to select chemotherapy and immunotherapy. We will test these hypotheses through the following three specific aims. Aim 1: Elucidate the functions of GSTO1 in enhancing immune response through activation of type-I interferon and reducing tumor cell viability through F3 and My...