Deletion of Glutathione S-Transferase Omega 1 Activates Type I Interferon Genes and Downregulates Tissue Factor

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 CRISPR/Cas9 technologies, we have validated GSTO1 as an impactful target in oncology. Through transcriptional profiling using Bru-seq and RNA-seq coupled with proteomics, we uncovered novel pharmacodynamic markers and cellular pathways regulated by GSTO1. Our CRISPR/Cas9 GSTO1 knockout (KO) cell lines do not form tumors or display 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 drugs 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. Associated with poor patient survival and promotion of tumor progression in multiple cancers, F3 is a known risk factor for metastasis. F3 transcription is under the regulation of IL-1β, whose secretion is decreased upon GSTO1 inhibition, suggesting that IL-1β links GSTO1 expression and F3 transcription. In summary, our results implicate GSTO1 as a therapeutic target in cancer and offer new mechanistic insights into its significant role in cancer progression. Cells were lysed with TRIzol® Reagent (ThermoFisher Scientific, Waltham, MA) at room temperature. RNA was further purified with DirectZol kit (Zymo Research, Irvine, CA). RNA quality was assessed using the TapeStation (Agilent Technologies, Santa Clara, CA). Samples with RINs (RNA Integrity Numbers) of 8 or greater were prepared with TruSeq Stranded mRNA Library Prep (Illumina) per the supplier’s protocol with 1μg of RNA and 12 cycles of PCR amplification. Libraries were checked for size on the TapeStation and quantified using the Kapa Biosystems library quantification kit (Illumina). The libraries were barcoded, pooled and sequenced using 50bp paired-end 50bp (U-87 MG, University of Michigan DNA Sequencing Core, MI) and single-end 50bp (HCT116, University of Michgian DNA Sequencing Core, MI) sequencing. Reads were mapped to GRCh38 using STAR v2.5.2 and gene quantifications were calculated using Cufflinks v2.2.1 to quantify refGene annotations. Gene read counts calculated using featureCounts v1.6.1 were used to evaluate differential expression using DESeq2 v1.18.1. Protein coding genes were considered significantly differentially expressed with a mean FPKM > 0.5 and absolute fold change > 1.5 and FDR adjusted p-value < 0.05. All gene readouts where required to be mappable to both an HGNC and Entrez identifier to be considered for gene set enrichment analyses.