The goal of this proposal is to provide in vivo proof-of-concept for the use of the blood cancer drug asparaginase (ASNase) in hepatocellular carcinoma (HCC) patients who possess the biomarkers for response to this novel biologic. ASNases have a unique mode of action wherein the drug depletes the amino acid asparagine from the blood, and, as a result, cells that rely on blood asparagine are starved and ultimately killed. The current FDA-approved ASNases are of bacterial origin, which makes them immunogenic, and their toxicity-causing glutaminase (GLNase) side activity causes severe drug side effects, which are exacerbated in adults. Therefore, despite the immense potential of ASNases for the treatment of several cancer types, these drugs are predominately confined to the treatment of pediatric acute lymphoblastic leukemia (ALL). To make ASNase therapy an option for adult patients with cancers that depend on blood asparagine, we engineered a human-like ASNase that mitigates the immune response and is highly specific to eliminate the GLNase-related toxicity. Notably, toxicity studies comparing our engineered human-like ASNase to the current standard-of-care bacterial ASNase (Oncaspar) demonstrated the significantly improved safety of our ASNase. Importantly, the improved safety comes with equivalent efficacy for ALL. Together, these developments make it possible to expand ASNase therapy to solid tumors in adult cancer patients. Response to ASNase is dependent on no/low expression of the enzyme that synthesizes asparagine de novo, called asparagine synthetase (ASNS). However, a durable response requires the inability of the cancer cell to upregulate ASNS expression in response to asparagine depletion. The ability to upregulate ASNS expression is determined by the methylation state of the ASNS promoter, where hypermethylation prevents expression and hypomethylation allows for expression. Recent analysis of cancer cell lines and patient samples reveal that many HCC patients possess either the full ASNase-response signature (low ASNS levels, hypermethylated promoter) or partial ASNase- response signature (low ASNS levels, hypomethylated promoter). Tumor cells with the full signature are predicted to strongly respond to ASNase alone, and those with the partial signature are predicted to have a less durable response. In preliminary work, we verified the predictive power of the full and partial ASNase response signatures on several HCC cell lines. To increase the clinical relevance of this observation, in Aim 1 we will collect HCC tumor samples from Veterans (treated at JBVAMC) and from a predominantly minority population (treated at UIC) and determine the predisposition of these patients to possess the ASNase- sensitivity biomarkers. These patient samples will also be used to generate HCC primary cell line, organoid, and PDX models. In Aim 2, using both in vitro and in vivo studies, we will determine the factor(s) that make HCC cell lines sensitive or re...