Efficacy of Novel Glutaminase Inhibitor CB-839 in Acute Myeloid Leukemia

Abstract

Inhibition of glutaminase (GLS), the principal enzyme in the glutamine utilization pathway that coverts glutamine (Gln) to glutamate (Glu), is an attractive therapeutic approach in many cancers. Gln plays a unique role in the metabolism of proliferating cancer cells, providing building blocks to sustain cell proliferation and regulating redox homeostasis and signal transduction pathways. Recent findings indicate that leukemic cells depend on Gln as a major carbon source for growth and survival [Willems et al., Blood, 2013]. We previously reported that a subset of acute myeloid leukemia (AML) cell lines are sensitive to Gln deprivation as well as inhibition of glutaminase by the small molecule BPTES [Matre et al. ASH 2013 #606]. Here we report the efficacy of CB-839, a novel, potent, orally bioavailable GLS inhibitor currently under clinical investigation, in the Gln-dependent subset of AML.First, expression of GLS gene splice variants glutaminase C (GAC) and kidney glutaminase (KGA) and of the GLS2 gene was determined through analysis of RNA sequencing data from 173 newly diagnosed AML patients in the TCGA dataset. Of the GLS gene splice variants, the expression levels of GAC were much higher than those of KGA; GLS2 was expressed at low levels. Levels of both GAC and KGA mRNA were significantly higher (two-sample Wilcoxon test) in AML patients with complex cytogenetics and monosomal karyotype (n=31) than in those with diploid AML (n=88, p=0.019 and p=0.01); GAC levels were higher in core-binding factor AML (n=14) than in diploid AML (p=0.018). These findings indicate high expression of the GLSGAC splice variant in specific AML subsets.Analysis of a panel of AML cell lines showed that, in a subset of leukemia cells, CB-839 treatment decreased viable cell number and induced apoptosis. In sensitive cell lines (Molm14, OCI-AML3, MV4;11), CB-839 decreased viable cell number with IC50s between 10nM and 100nM and induced significant apoptosis. HL60, MOLM13, KG1α, and OCI-AML2 cells were less sensitive (IC50 100-1000nM) and responded with minor induction of cell death. CB-839 decreased viability by >40% in blasts from 9 of 20 (45%) primary AML samples.GC- or LC-MS metabolic profiling of OCI-AML3 and THP1 cell lines as well as primary patient samples revealed that GLS inhibition by BPTES or CB-839 was accompanied by concomitant decrease in concentration of downstream GLS metabolites such as glutamate, α-ketoglutarate (a-KG), aspartate, fumarate, and malate. Investigation of the effects of CB-839 on mitochondrial OXPHOS by the Seahorse Bioscience XF96 Analyzer showed that CB-839 exposure for 16 h caused a dose-dependent decrease in maximal respiratory capacity in OCI-AML3 cells, indicating reduced availability of the substrates for OXPHOS. Similar results were obtained upon treatment with BPTES and in AML cells stably transduced with GLS shRNA.Gln, through Glu, is a precursor for cellular α-KG, which can undergo further metabolism through the Krebs cycle or be further metabolized to 2-hydroxyglutarate (2-HG) by mutant isocitrate dehydrogenase (IDH). In THP1 cell lines stably transduced with doxycycline-inducible mutant IDH1-R132H or IDH2-R140Q construct, CB-839 exposure for 4 days reduced intracellular 2-HG oncometabolite levels by >50%. This was associated with induction of differentiation marker CD11b and morphological signs of differentiation in CB-839–treated IDH2-R140Q cells [30%±2% increase in CD11b mean fluorescent intensity (p<0.001)] vs untreated cells; but not in IDH2-WT control cells. Further, IDH2-R140Q THP1 cells were significantly more sensitive to 1mM CB-839 than IDH2-WT cells (61% vs 24% reduction in viable cell numbers). In 4 of 6 IDH1- or IDH2-mutated primary samples, reductions in 2-HG (by 24% [p=0.04]; 31% [p=0.016]; 35% [p=0.033], and 43% [p=0.0056]) were observed upon CB-839 exposure. Preliminary data for an IDH2-R140Q-mutated AML primary sample (n=1) indicate that CB-839 promotes CD11b differentiation in primary AML blasts.In summary, these results indicate that GLS is a relevant therapeutic target in AML, warranting future inclusion of GLS inhibitors in the armamentarium of multi-agent therapeutic approaches. In particular, reduction of production of the oncometabolite 2-HG in conjunction with therapeutic blockade of Gln metabolism may serve as a tailored therapeutic strategy in IDH-mutated AML cells. DisclosuresKonopleva:Calithera Biosciences: Research Funding.