Immunotherapy resistant ALL cells exhibit selective dependence on glutamine and specific transporters for growth and survival

Abstract Introduction: Acute lymphoblastic leukemia (ALL) needs newer therapeutic options, especially in patients that fail targeted immunotherapies such as CD19 CART and blinatumomab. Prior research has shown that resistant cells exhibit metabolic adaptations that enable cell proliferation and survival in hostile microenvironments. In this study, we found glutamine utilization as a metabolic adaptation of CD19 immunotherapy resistant ALL and demonstrated the therapeutic potential of targeting glutamine pathways in overcoming resistance. Methods: To model acquired resistance to CD19 targeted immunotherapy, we developed an ex vivo model of ALL resistance to CD19 based immunotherapy (Aminov et al, JCI 2024). CD19 immunotherapy resistant and parental cells were utilized to determine metabolic adaptations and evaluate sensitivity to targeted therapeutic interventions. Results: We developed an ex vivo ALL model of CD19 immunotherapy resistance by chronic exposure of ALL (NALM6 and REH) cells to an anti-CD19 immunotoxin. Resistant cells were characterized by reduced expression of CD19 and CD22 and a more stem like phenotype. We observed that the resistant ALL cells showed increased expression of both glutaminase enzyme and the glutamine transporter SLC38A1 compared to parental cells. Treatment with the clinically useful glutaminase inhibitor CB-839 produced a selective decrease in viability in resistant cells when compared to parental cells (p<0.001). Glutaminase inhibition also restored sensitivity to L-asparaginase in resistant cells. We subsequently developed cell lines with knockdown of SLC38A1 using CRISPR-Cas9. SLC38A1 knockdown in resistant cells led to a significant decrease in cell growth of CD19 resistant cells compared to parental (p<0.0001). This was replicated in multiple ALL cell lines. Metabolic profiling with Seahorse XF assay revealed increased oxygen consumption rate and extracellular acidification rate in resistant cells compared to parental indicating metabolic adaptation. Metabolic labeling studies revealed increased glutamine flux into the tricarboxylic acid (TCA) cycle in resistant cells compared to parental cells. We further observed that glutamine is differentially utilized: for glutathione biosynthesis in parental cells, and predominantly for TCA cycle anaplerosis in resistant cells. Treatment with the glutaminase inhibitor CB-839 sensitized resistant cells, consistent with their enhanced reliance on glutamine for energy production. Conclusions: CD19 immunotherapy resistant ALL cells exhibit increased expression of both glutaminase and specific glutamine transporter SLC38A1. Our findings suggest that CD19 immunotherapy resistant cells are dependent on glutamine as a source of cellular energy. Further, our data suggests that glutamine is selectively utilized for cellular energy in resistant cells, while it is shunted toward additional pathways in parental cells, including the formation of asparagine and glutathione. This evidence supports the glutamine pathway as a novel targetable metabolic adaptation, particularly in resistant ALL.