Targeting glutamine metabolism and redox state for leukemia therapy

Abstract

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional genotoxic chemotherapy and a diagnosis of AML is usually fatal.More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. Here, we show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating acute lymphoblastic leukemia cells in vitro and in vivo. Thus, targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia. Key Points• Glutaminase inhibition commonly impairs glutathione metabolism and induces mitochondrial oxidative stress in acute myeloid leukemia cells • A glutaminase inhibitor synergizes with pro-oxidant drugs in inducing apoptosis and eliminating leukemia cells in vitro and in vivo Homoharringtonine (HHT; omacetaxine mepesuccinate), arsenic trioxide (ATO), cell-permeable glutathione reduced ethyl ester (GSH-MEE) and dimethyl 2oxoglutarate (α-ketoglutarate) were purchased from Millipore Sigma. Metabolic tracing experimentsCells were seeded at 3 x 10 5 /ml (replicates of 3) and treated with vehicle (DMSO) or CB-839 at 500 nM for 8 h, followed by incubation in glutamine-free RPMI 1640 supplemented with 13 C 5 , 15 N 2 -labeled L-glutamine (Cambridge Isotope Laboratories) for up to 12 h, in the presence of vehicle or drug. Flash frozen cell pellets (~ 1 x 10 6 cells) or supernatants (50 µl) were extracted and subjected to analysis by ultra-high pressure liquid chromatography and mass spectrometry (UHPLC/MS) as was previously described 20 . Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of 13 C and 15 N isotopes were performed using MAVEN (Princeton University, Princeton, NJ) and manually validated. Cell viability assaysCells were seeded at 0.5-1.0 x10 5 /ml in triplicate wells of 48-well tissue culture plates. Where indicated, the cells were treated with drug for a period of 48-72 h.After treatment, a sample of cells from each well was stained with PI (10 µg/ml) and viable cells (PI -) were counted with a flow cytometer (Millipore Guava

Glutamine uptake and utilization of human mesenchymal glioblastoma in orthotopic mouse model

“…Our results are in agreement with numerous studies showing either distinct metabolic fuel choice and dependency based on distinct molecular signature [ 15 ] or glutamine uptake in GBM in vivo using PET imaging based on 18 F-Fluoroglutamine [ 16 ]. Furthermore, several reports have shown that many tumors rely on glutamine to fuel TCA cycle in vivo, in agreement with glutamine dependency observed in corresponding in vitro models [ 17 – 19 ]. Reliance of mesenchymal GBM on glutamine metabolism in vivo is reinforced by the inhibition of key nodes in glutamine metabolism which retards tumor growth in our preclinical models.…”

B-cell lymphoma-2 inhibition and resistance in acute myeloid leukemia

“…To sustain high proliferative advantage, LSCs may adapt a metabolic preference for glutamine to drive biomass. This so-called glutamine addiction has been demonstrated in multiple studies and represents a potential target for anti-leukemic therapy[ 20 - 23 ].…”

VAS3947 Induces UPR-Mediated Apoptosis through Cysteine Thiol Alkylation in AML Cell Lines

“…We focused on IRE1α and PERK, well-known participants in inducing apoptosis. Indeed, PERK has been shown to inhibit the translation initiation factor 2α (eukaryotic initiation factor 2 alpha (eIF2α) by phosphorylation, thereby, reducing protein synthesis and overload [ 23 , 24 , 25 ]. In accordance, our results revealed rapid phosphorylation of eIF2α in MV-4-11 cells within 15 min following VAS3947 exposure ( Figure 7 A).…”

“…Indeed, the authors showed that the survival of ROS low LSCs is dependent on the availability of cysteine. The combination of arsenic trioxide (ATO) and homoharringtonine (HHT), drugs that perturb the mitochondrial redox state, with CB-839, a drug that blocks glutamine metabolism and consequently GSH production, efficiently induced cell death of AML cell lines, primary AML patient-derived samples, and in vivo mice models of AML [ 25 ]. Additionally, other studies showed that glutathione peroxidases (GPX)-1 and GPX-3, two antioxidant enzymes requiring GSH to detoxify hydrogen peroxide, are highly involved in AML development [ 24 , 26 ].…”