Fellowship Keng Hee Peh: Evaluating The Mechanism Of Glutaminase Inhibition And Artesunate Anti-Cancer Activity In Non-Small Cell Lung Cancer

Abstract Lung cancer is the one of the leading cancers and cause of cancer related deaths globally with non-small cell lung cancer (NSCLC) accounting for 85% of lung cancer diagnoses. Despite clinical benefits of current standard of care treatment options, the 5-year overall survival in patients with NSCLC is approximately 25% and patients with tumors harboring certain genomic mutations confer chemoresistance and poor survival outcomes. Artesunate (ART), an anti-malaria agent, has a favorable toxicity profile and demonstrated promising in vitro anti-cancer activity in several cancer cell lines, including NSCLC. We have also previously demonstrated that KEAP1 was needed for ART anti-cancer activity. However, KEAP1 is a tumor suppressor and function cannot be pharmacologically restored with current strategies. Therefore, we screened ART against compounds targeting pathways regulated by KEAP1 and showed significant synergy between ART and telaglenastat (CB839), a glutaminase (GLS1) inhibitor, in both KEAP1 loss and wild type cell lines. Both drugs have acceptable toxicity profile and synergy occurs at doses that are achievable in humans, presenting an attractive option for further preclinical development. We hypothesize that inhibition of biochemical pathways regulating glutamate sensitizes ART in NSCLC and is independent of KEAP1 loss mutations. Our aims for this proposed grant are the following: Aim 1. Demonstrate extent of ART+CB839 treatment on apoptosis and mitochondrial reactive oxygen species (ROS) production in NSCLC. Our preliminary data demonstrated reduced KEAP1 expression in ART+CB839 in both KEAP1 loss (A549) and wild type (H1299) cell lines. Reduced KEAP1 is associated with increased ROS, and increased ROS leads to apoptosis and cancer cell death. GLS1 inhibition reduces glutamate and potentially α-ketoglutarate (aKG), an essential intermediate for ROS in the mitochondria. Therefore, we hypothesize that cellular apoptosis to ART+CB839 treatment is a result of increased mitochondria ROS production independent of KEAP1 loss. For this aim, • We will utilize ELISA assays to measure glutamate and aKG concentrations in ART+CB839 treated A549 and H1299 cell lines compared to the single agent alone and DMSO as a negative control. • We will utilize MitoSox and Caspase 3/7 assay to measure mitochondria ROS and apoptosis, respectively, and measure relative fluorescence in ART+CB839 treated A549 and H1299 cell lines compared to single agents alone and DMSO as a negative control. This aim will demonstrate the relationship between glutamate, aKG, mitochondria ROS, and apoptosis in ART- CB839 treated NSCLC cell lines. Aim 2. Elucidate mechanism of synergy between GLS1 inhibition and ART in NSCLC. GPT2 is a mitochondria enzyme that catalyzes glutamate production. SLC1A5 is a primary transporter of glutamine, a precursor of glutamate. Relationship between GPT2/SLC1A5 and CB839 have been reported but yet to be fully elucidated nor utilized as clinical biomarkers. KEAP1 loss is known to contribute to glutamate dependence in NSCLC and is sensitized to GLS1 inhibition. However, ART+CB839 synergy is independent of KEAP1, therefore we hypothesize that ART increases glutamate dependence via GPT2 and SLC1A5 upregulation in NSCLC and is sensitized to GLS1 inhibition independent of KEAP1 loss mutation. For this aim, • We will use rtPCR to assess differential mRNA expression of GPT2 and SLC1A5 in ART treated A549 and H1299 cell lines compared to untreated cells. • We will then create siRNA knockout GPT2/SLC1A5 in A549 and H1299 cell lines and assess IC50 of ART+CB839 compared to the single agents alone in knocked out cell lines with DMSO as negative control. This aim will demonstrate whether ART induces glutamate dependence via GPT2 or SLC1A5 in NSCLC independent of KEAP1 loss and serve as a potential biomarker of ART resistance or sensitivity in combination with CB839. Our proposed study for this grant will elucidate the mechanism of synergy of ART+CB839 and identify potential biomarkers that will predict response or sensitivity to the combination. Our strategy will justify ART+CB839 for further preclinical development to serve as a potential frontline treatment option for NSCLC to improve clinical outcomes, including patients with KEAP1 loss mutation.