Effects of Glutaminase Inhibition on Venetoclax, Ibrutinib, and AZD-5991 Resistance in Chronic Lymphocytic Leukemia Cell Lines

Abstract

Introduction: Bruton tyrosine kinase and BCL-2 inhibitors are widely used in CLL, but resistance problem is highly relevant. One of the resistance mechanisms may be metabolic rewiring followed by activation of oxidative phosphorylation. Glutamine plays a major role in cancer metabolism, and glutamine limitation rather than glucose contributes to lower oxygen uptake and mediates apoptosis of cancer cells. It was shown that glutamine metabolism changes contribute to venetoclax (Chen Z. et al, Blood, 2021; Guièze R. et al. Cancer Cell, 2019) and ibrutinib (Galicia-Vázquez G. et al. Frontiers in Oncology, 2018) resistance. We investigated in vitro effects of glutaminase-1 (GLS-1) enzyme inhibition and its potential synergistic effects with venetoclax, ibrutinib, and MCL-1 inhibitor AZD-5991 on resistant cell lines. CB-839 (telaglenastat) is a potent, reversible, and selective GLS-1 inhibitor with 24 nM IC50 for recombinant human mitochondrial glutaminase C. The compound was tested in the clinic for hematological malignancies and solid tumors. Methods: We have tested CB-839 alone or in combination with venetoclax, ibrutinib, and AZD-5991 on resistant HG-3 and MEC-1 CLL cell lines. Cells were incubated with 0.01 to 100 μM of CB-839 for 72 hours. The inhibition of glutamine, glutamate, and glutathione induced by CB-839 alone was measured using a luminescence assay. DCFDA/ H2DCFDA and MitoSOX Red stainings were utilized to evaluate cellular reactive oxygen species (Total ROS) and mitochondrial superoxide (MitoSOX) accumulation, respectively. Intracellular nucleotides including ATP level were measured using high-pressure liquid chromatography. For drug combinations, MTS assay and Annexin V/Propidium Iodide staining were used to assess cell proliferation and apoptosis, respectively. The combination index (CI) for synergy was calculated using Compusyn software. Results: HG3 and MEC1 cell lines were resistant to venetoclax, ibrutinib, and AZD-5991 single agents. The IC50 values for HG3 are 8.1 μM, 1.3 μM, 9.9 μM, and for MEC1 are 2.9 μM, 1.2 μM, and 1.9 μM for venetoclax, ibrutinib, and AZD-5991, respectively. In these cell lines, CB-839 caused a dose-dependent decrease in GLS-1 activity, measured as intracellular glutamate concentration, and inhibited glutathione synthesis (Figure 1A). CB-839 treated cells showed increased MitoSOX, but not Total ROS accumulation. However, as the endpoint effect, the decrease of cell proliferation rather than apoptosis was detected. CB-839 treatment caused a mild decrease in ATP pool in both cell lines. According to MTS assay, venetoclax demonstrated only the particular synergism with CB-839 (CI <1.0 at all effective doses (ED50, ED75, ED90, ED95) for MEC-1, and at ED50, ED75 for HG-3 cell line). In addition, CB-839 increased pro-apoptotic effect of venetoclax in both cell lines. Notably, that CB-839 did not improve any ibrutinib-mediated effects on cell proliferation and apoptosis in CLL cell lines. The most distinct synergism was observed in CB-839 and AZD-5991 combination group (CI <1.0 at all ED for MEC-1 and HG-3 cell lines) with 5- and 19.8-fold shifts in IC50 (Figure 1B). To extend these results, we are performing these combinations in primary CLL cells that are sensitive or resistant to targeted agents such as ibrutinib or venetoclax. Conclusions: Combinations of CB-839 with other agents targeting multiple mechanisms have the potential to overcome metabolic adaptation. Our findings suggest that CB-839 has limited efficacy in overcoming ibrutinib CLL resistance. However, the synergism with apoptosis inducers such as venetoclax and AZD-5991 require further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal