Altered Glucose Metabolism In Rituximab-Chemotherapy Resistant Cells Is a Potential Target For Future Therapeutic Development

Although treatment of lymphoma has greatly improved over recent years, acquired rituximab-chemotherapy resistance is an emerging clinical challenge for physicians treating relapsed/refractory diffuse large B-cell lymphoma and other aggressive histological subtypes. The discovery and characterization of cellular pathways up-regulated or down-regulated in rituximab-chemotherapy resistant B-cell lymphoma cells can potentially identify prognostic biomarkers and/or the development of novel therapeutic strategies. Differences in cellular metabolism exist between normal and malignant cells. Cancer cells are known to have an increase in aerobic glycolysis as a source of adenosine triphosphate (ATP) generation (a.k.a. Warburg effect). Inhibition of glucose uptake or its metabolism has been shown to induce cytotoxicity in solid tumor cancer cells. Therefore, we evaluated differences in the glucose metabolism between rituximab-sensitive (RSCL) or rituximab-resistant cell lines (RRCL) representing aggressive subtypes of B-cell lymphoma. Differences in the baseline cell proliferation rate, ATP generation, lactic acid dehydrogenase (LDH) activity, glucose uptake and lactic acid production between RSCL and RRCL were determined. We then blocked glucose uptake in RSCL and RRCL by exposing these cells to 2-deoxy-D-glucose (2-DG), a glycolytic inhibitor, and measured changes in cell viability, cell cycle distribution, mitochondrial potential and induction of apoptosis using the MTT assay and flow cytometry (propidium iodine [PI], DiOC6, and PI/AnnexinV staining, respectively. Finally, we evaluated the cytotoxic effects of 2-DG in combination with multiple chemotherapy agents in RSCL and RRCL. Significant differences in metabolic rate and glucose metabolism were identified. RRCL had a lower proliferation rate, but alternatively had a higher: 1) intracellular concentrations of ATP, 2) LDH activity, 3) production of Lactate, and 4) glucose uptake/consumption. Together these data suggest that RRCL are more dependent on glucose metabolism for generation of energy (i.e. ATP). In vitro exposure of RRCL, and to a lesser degree of RSCL to 2-DG, resulted in a dose- and time-dependent cell death. As expected, in vitro exposure of RRCL to 2-DG resulted in a decrease in: 1) glucose uptake, 2) lactate production, 3) ATP production and subsequent depletion of ATP intracellular reserves. In addition, 2-DG induced greater G1-phase arrest in RRCL. The baseline mitochondrial potential was higher in RRCL than RSCL and correlated with resistance to multiple chemotherapy agents commonly used in the treatment of B-cell lymphoma. Caspase inhibition did not rescue RRCL to the cytotoxic effects of 2-DG, suggesting the execution of as caspase-independent cell death pathway (i.e. autophagy or senescence). In addition, 2-DG exhibited additive/synergistic effects when combined with doxorubicin in RRCL. In summary, our data suggests that the acquirement of rituximab-chemotherapy resistance in RRCL is associated with deregulation in the mitochondrial potential leading to chemotherapy resistance and an altered cellular metabolism that is primarily dependent on glucose uptake and glycolysis to generate ATP. Developing therapeutic strategies that selectively inhibit glucose uptake or glucose metabolism in cancer cells could prove to be a promising strategy for the treatment of relapsed/refractory lymphoid malignancies in the future. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund)