Gilteritinib Inhibits Acute Myeloid Leukemia Growth via Reduction in Glutamine Uptake and Utilization
Authors: Thomas Megan Zavorka , Jeon Jae Yoon , Shabana Sameh , Buelow Daelynn , Gibson Alice , Anderson Jason , Campbell Moray , Collins Meghan , Lodi Alessia , Sweeney Shannon , Tiziani Stefano , Sparreboom Alex , Baker Sharyn 
Published: April 2020

The tyrosine kinase inhibitor (TKI) gilteritinib has recently been granted FDA approval for the treatment of relapse/refractory FLT3‐ITD+ AML, yet its mechanism of action is not entirely known. Two recent studies investigating metabolic adaptations during treatment with the FLT3 TKI quizartinib in AML have demonstrated reduced glycolysis or impaired glutaminolysis. Although these studies are conflicting, it suggests that TKIs may influence metabolic changes thereby impacting proliferation and cell viability. Many highly proliferative cells require glutamine as a precursor since they depend on the glutaminolysis pathway for generating building blocks and energy for anabolic processes. Here, we hypothesize that gilteritinib reduces cell viability in FLT3‐ITD+ AML by altering glutamine uptake and metabolism.

In our preliminary studies, mice with FLT3‐ITD+ AML (MOLM13 xenograft) were treated with vehicle or gilteritinib (30 mg/kg once daily, five days/week) until leukemic progression. Using an unbiased transcriptomic approach (RNA‐seq) of the leukemic bone marrow, we found that the glutamine transporters (SLC38A1, SLC1A5 and SLC7A5) were significantly downregulated in the gilteritinib‐treated cohort compared to the vehicle cohort (fold change 1.96–4.70, P < 0.0001). Furthermore, expression of metabolic genes was altered in glutamine/aspartate metabolism, glycolysis, and the TCA cycle. Using a resistant xenograft model in which the FLT3‐ITD harbors a mutation in the tyrosine kinase domain at residue D835Y (MOLM13‐RES), SLC38A1 and SLC7A5 were also significantly downregulated after gilteritinib treatment (fold change 0.70–1.28, P < 0.0005), as well as having alterations in the gene expression profile of genes in the aforementioned pathways, although to a lesser extent than MOLM13. In vitro, depleting the cell culture medium of glutamine decreases the cell viability by ~50% in MOLM13 and another FLT3‐ITD+ AML cell line (MV4‐11), indicating that these leukemic cells are addicted to glutamine metabolism as an energy and precursor source. Acute exposure of MOLM13 cells to 100 nM FLT3 TKIs gilteritinib, quizartinib and sorafenib all decrease the transcript of glutamine transporters, with the greatest response from gilteritinib as measured via RT‐PCR (P < 0.01 for all three transporters). In cellular uptake assays, the uptake of glutamine by MOLM13 cells was decreased stepwise to nearly 50% during an 8 h timecourse of 100 nM gilteritinib treatment. This was confirmed using unbiased isotope labeling and tracing in vitro in MOLM13 and MOLM13‐RES cells, which showed reduced glutamine uptake and utilization through the TCA cycle after gilteritinib treatment (15 nM, 24 h). Metabolomics of MOLM13 in vivo samples also revealed alterations in the metabolic pathways. Thus, our data support the premise that gilteritinib downregulates glutamine transporters and alters glutamine metabolism to inhibit cell viability of FLT3‐ITD+ AML.

Support or Funding Information

National Cancer Institute under award R01 CA138744‐08 and the Ohio State University Comprehensive Cancer Center Pelotonia foundation.