Role of Glutamine in Metabolic and Epigenetic Reprogramming in AML

Glutamine (Gln) was shown to play a role in generation of oncometabolite 2-hydroxyglutarate (2-HG) in tumors with high GLS (glutaminase) expression, whereby IDH2 (Isocitrate Dehydrogenase 2) enzyme catalyzes carboxylating reduction of glutamine-derived α-ketoglutarate (α-KG) to isocitrate and noncarboxylating reduction to 2-HG (Wise PNAS 2011). 2-HG in turn is known to inhibit α-KG dependent dioxygenases that mediate epigenetic events, including DNA and histone demethylation (Licht Cancer Cell 2010). A recent report demonstrated that hypoxia induces production of the L-enantiomer of 2-HG (L-2HG), through enzymatic reduction of α-KG by lactate dehydrogenase A (LDHA) (Intlekofer Cell Metabolism 2015). We have previously demonstrated that leukemic bone marrow microenvironment is highly hypoxic (Benito PLoS One 2011). Further, our unpublished data indicate upregulation of GLS protein and increase production of total 2-HG in AML (acute myeloid leukemia) cells cultured under hypoxia. We therefore propose a link between hypoxia, Gln metabolism, and epigenetic regulation in AML. Since increased methylation (and decreased hydroxymethylation) is seen in AML, we hypothesize that GLS inhibition can abrogate these changes via reduction of 2-HG levels.

First, we examined effects of hypoxia and selective GLS inhibitor CB-839 (Calithera Biosciences) on cellular growth of AML cells with wild type IDH (OCI-AML3 and HL-60), cultured alone or co-cultured with bone marrow derived stromal cells (MSC). The culture of untreated OCI-AML3 alone in normoxic and hypoxic conditions caused a decrease in viability from 96 ± 2.5% to 84 ± 4.1% respectively, while the treatment with CB-839 (1 mM) for 6 days decreased viability in OCI-AML3 cells from 94 ± 0.23% to 71 ± 2.3% respectively (P=0.015). While MSC co-cultures improved survival of floating AML cells, the attached cells that were in direct contact with MSC were more affected under hypoxic conditions, having a viability of 64 ± 8.7% at the end of the experiment. These data indicate that GLS inhibitor is more effective under hypoxic conditions mimicking leukemic BM microenvironment.

Hypoxia selectively induced the production of L-2HG (measured by liquid chromatography-tandem mass spectrometry) under hypoxic conditions (>40 fold) in OCI-AML3 cells, both with and without MSC co-culture. This increase in L-2HG was partially inhibited by co-treating OCI-AML3 cells with GLS inhibitor CB-839 (reduction of 1.7-fold in media only and 1.3-fold in MSC co-culture). Determination of hydroxymethylation (hmc) levels using HELP-GT assay demonstrated a significant increase in hmc in cells treated with CB-839. Of importance, genes that were differentially hydroxymethylated after CB-839 treatment belonged to important functional categories with cancer being the dominant pathway affected by these changes.

Under hypoxia, glucose metabolism is known to be directed towards anaerobic glycolysis, with increased pyruvate-lactate enzymatic conversion by LDHA. To characterize the role of Gln and GLS on these processes within leukemia microenvironment, we performed nuclear magnetic resonance imaging with hyperpolarized pyruvate in NSG (NOD scid gamma) mice engrafted with GFP/luc-labeled OCI-AML3 cells. Inhibition of GLS in vivofollowing exposure of mice with 200 mg/kg dose of CB-839 showed a decrease in lactate conversion rate within leukemic bone marrow (femur area) (0.31 + 0.03  (pre) to 0.20 + 0.04 (post) P < 0.05), possibly  due to the reduction of the level of NADH from decreased flux of Gln in the TCA cycle.

In summary, our results indicate that Gln and GLS contribute towards hypoxia-induced production of L-2HG and critical epigenetic changes in AML; as well as playing a role in enhanced production of lactate from pyruvate. These findings suggest a major importance of Gln in metabolic and epigenetic reprogramming of microenvironment.