Abstract 2322: Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer
Authors: Jaggupilli Appalaraju , Ly Stanley , Nguyen Khoa , Borkar Roshan , Yuan Bin , Piyarathna Danthasinghe Waduge , Yan Yuanqing , Piwnica-Worms Helen , Manning Henry Charles , Andreeff Michael , Putluri Nagireddy , Battula Lokesh 
Published: July 2021

Abstract
Nutrient deprivation is one of the hallmark conditions of a rapidly growing tumor. Rapid proliferation of tumor cells leads to increased consumption of metabolites such as glucose and glutamine resulting in metabolic stress. However, phenotypic and functional status of tumor cells under these conditions is not clear. We hypothesize that nutrient deprivation-induced metabolic stress results in a breast cancer stem cell-like (BCSC) phenotype in triple negative breast cancer (TNBC). We found that serum-deprived conditions result in significant upregulation of GD2+ cells compared to serum rich conditions: from 9%±2% to 26%±5% in MDA-MB-231 and 7%±2% to 29%±5% in SUM159 cells. Interestingly, this affect was compensated when the cells were supplemented with serum-rich medium. In addition, a positive correlation was observed between GD2+ cell number and tumor volume in vivo, suggesting metabolic stress in the growing tumors results in GD2+ phenotype in TNBC cells. Next, TNBC cells cultured under glucose deprivation or in the presence of glycolysis inhibitor, 2-deoxy glucose (2DG) showed a 2-fold increase in GD2+ cells compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 expression. Nutrient deprived conditions also showed a significant increase in the expression of stress markers ATF4 and SESN2 demonstrating TNBC cells under metabolic stress. In addition, the above conditions also showed a 2.5-fold upregulation of the mammosphere formation in TNBC cells compared to nutrient rich conditions. Global metabolomics profiling identified glutathione biosynthesis to be most highly upregulated in GD2+ cells. Furthermore, we found that supplementation of glutamine (precursor for glutathione biosynthesis) increases the percentage of GD2+ cells. Carbon and nitrogen tracing using 13C- and 15N-labeled glutamine by mass spectrometry showed its direct contribution to GD2 biosynthesis. Treatment with glutamine transporter (ASCT2) inhibitor V9302 reduced GD2+ cell number by 70%-80% in TNBC cell lines and dramatically inhibited mammosphere and soft agar colony formation, and migration of TNBC cell lines. Mechanistic studies revealed that V9302 inhibits glutathione pathway and induces ROS levels in TNBC cells. Additionally, V9302 treatment inhibited AKT-ERK-mTOR-4E-BP1 pathway and combination of V9302 with paclitaxel synergizes TNBC cell killing. We also identified that, V9302 treatment induced ferroptosis (iron-dependent cell death) in TNBC cells. Finally, V9302 treatment significantly inhibited tumor growth (p&lt;0.0001) in TNBC-PDX model. In conclusion, metabolic stress results in GD2+ BCSC phenotype in TNBC cells and glutamine directly contributes to GD2 biosynthesis. Targeting ASCT2 could complement conventional chemotherapy in TNBC.
Citation Format: Appalaraju Jaggupilli, Stanley Ly, Khoa Nguyen, Roshan Borkar, Bin Yuan, Danthasinghe Waduge Piyarathna, Yuanqing Yan, Helen Piwnica-Worms, Henry Charles Manning, Michael Andreeff, Nagireddy Putluri, Lokesh Battula. Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2322.