Aberrant Activation of NF-κB and Hypoxia Enhances Glycolytic Enzyme Hexokinase II Expression in B-Cell Lymphoma through Activation of HIF-1

Malignant lymphoma cells tend to use glycolysis for the production of ATP even with a substantial oxygen supply, a process known as aerobic glycolysis that is illustrated by a high uptake of FDG-glucose. Though elevated levels of glycolytic enzymes are responsible for aerobic glycolysis, the precise molecular mechanisms of this process are not well understood. In physiological conditions, the expression of glycolytic enzymes is mainly regulated by hypoxia inducible factor (HIF). Previously, we revealed that the aberrant activation of NF-κB causes the activation of HIF pathways in B-cell lymphoma (Exp.Hematology, 2010 &2013). It is widely accepted that abnormal activation of NF-κB is one of the hallmarks of B-cell lymphoma. Based on these notions, in the present study, we analyzed the possible link between aberrant activation of NF-κB, HIF and enhanced glycolysis in lymphoma cells. We also investigated the possibility that the inhibition of enhanced glycolysis could be a therapeutic target in lymphoma.For this study, we used a series of B-cell lymphoma cell lines. Under basal conditions, these cells display aberrant NF-κB activation and expression of the alpha subunit of HIF-1 (HIF-1α). Of the glycolytic enzymes, hexokinase II (HXKII) levels were specifically elevated in these cells. To confirm that aberrant activation of NF-κB was responsible for the elevation of HIF-1α and HXKII, we treated the cells with several NF-κB inhibitors. The NF-κB inhibitors clearly suppressed the expression of both HIF-1α and HXKII. Inhibition of HIF-1 by panobinostat also led to decreased HXKII levels. Panobinostat also suppressed hypoxia-induced HIF-1α and HXKII elevation. We further analyzed HXKII expression in biopsy specimens from newly diagnosed B-cell lymphoma cases after receiving approval from an institutional review board and acquisition of written informed consent. We analyzed 6 cases of diffuse large B-cell lymphoma (DLBCL), 3 cases of follicular lymphoma (FL) and 1 case of mantle cell lymphoma. HXKII expression was detected in 5 of the 6 DLBCL cases but not in the FL cases. Interestingly, according to Hans classification, the HXKII-positive DLBCL cases were non-GCB types.Finally, we explored the possibility that inhibition of HXKII could enhance the activity of anti-lymphoma chemotherapeutic drugs. We confirmed that hypoxia diminished the anti-lymphoma action of several chemotherapeutic drugs, including cisplatin. To investigate whether inhibition of HXKII could overcome the reduced anti-lymphoma action of these reagents, we treated the cells with either 2-deoxy-glucose or clotrimazole, which are both inhibitors for HXKII. We found that these reagents reversed the anti-lymphoma activity of cisplatin, even during hypoxia. Panobinostat also restored cisplatin-induced apoptosis in B-cell lymphoma cells during hypoxia.In conclusion, our present findings reveal that expression of glycolytic enzyme HXKII was elevated in B-cell lymphoma cells, and two distinct mechanisms, aberrant activation of NF-κB and a hypoxic microenvironment, induce HXKII elevation through activation of HIF-1. This work also suggests that the axis between HIF-1 and HXKII might be an attractive therapeutic target for B-cell lymphomas, especially those with aberrant NF-κB activity.