Up-Regulation of Hexokinase II (HK) Alters the Glucose Metabolism and Disrupts the Mitochondrial Potential in Aggressive B-Cell Lymphoma Contributing to Rituximab-Chemotherapy Resistance and Is a Clinically Relevant Target for Future Therapeutic Development
Authors: Gu Juan J , Singh Anil , Mavis Cory , Yanamadala Vivek , Xue Kai , Grau Michael , Lenz Peter , Lenz Georg , Czuczman Myron S. , Hernandez Francisco 
Published: October 2019

Abstract
The results of the prospective multicenter phase III Collaborative trial in relapsed aggressive lymphoma (CORAL) study stressed the need to identify cellular pathways associated with poor clinical outcomes and to develop novel therapeutic strategies for relapsed/refractory aggressive lymphoma. To this end, we generated several pre-clinical models of rituximab resistance and previously reported that the acquirement of rituximab resistant was associated with a deregulation in the glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. Hexokinase (HK) is the first step rate-limiting enzyme of the glycolytic pathway and mediates the phosphorylation of glucose to glucose-6-phosphate (G-6-P), promoting the glycolic pathway. Four isoforms of HK had been characterized. Hexokinase II (HKII), the predominant isoform overexpressed in cancer cells, has a catalytic and a biding domain. The catalytic domain of HKII promotes glycolysis while the binding domain protects tumor cell from mitochondrial outer membrane permeability (MOMP) and inhibits mitochondrial-mediated apoptosis. In our current work, we further study the biological and clinical significance of HKII expression in aggressive B-cell lymphoma. First, we analyzed differences in HKII gene and protein expression in a panel of rituximab-chemotherapy sensitive (RSCL) or resistant (RRCL) diffuse large B-cell lymphoma (DLBCL) and Burkitt’s lymphoma (BL) cell lines. RRCL were found to have higher HKII mRNA and protein levels as determined by qRT-PCR and Western blotting. Subcellular fractionation of the mitochondria and cytoplasm revealed that most of the HKII was localized in the mitochondria in RRCL. Moreover, immunoprecipitation studies demonstrated that HKII was bound to the voltage dependent anion channel (VDAC). Of interest, by targeting HKII using either 1) a competitive substrate inhibitor (2DG), 2) a kinase inhibitor (lonidamine), 3) disruption its interaction with VDAC (HKII/VADC competitive binding peptide assay), or 4) complete silencing of HKII (using HKII siRNA interference or metformin) resulted in a decrease in the mitochondrial membrane potential (MMP), reduction in ATP production, decrease in cell viability and re-sensitization of RRCL to chemotherapy agents. To further assess the contribution of HKII to rituximab/chemotherapy resistance in a more clinically relevant setting, we analyzed gene expression profiling data from 401 patients with DLBCL treated with CHOP (N=181) or R+CHOP (N=220) as front-line therapy. High levels of HKII correlated with a poor prognosis in DLBCL patients. In patients receiving CHOP chemotherapy, high levels of HKII correlated with a shorter overall survival (OS) (P=0.0012). Similarly, high-HKII mRNA levels were associated with a shorter progression free survival (PFS) (P=0.039) and OS (P=0.043) in R-CHOP treated DLBCL. Our data suggest that over-expression of HKII contribute to the acquirement of resistance to rituximab and chemotherapy agents in DLBCL B-cell lymphoma. It appears that HKII binding to VDAC and to a lesser degree HKII catalytic domain, contribute to therapy resistance in lymphoma pre-clinical models. Clinically, higher levels of HKII correlate with a shorter PFS and OS in previously untreated DLBCL in the pre- and post-rituximab era. Targeting HKII appears to be a challenging but attractive strategy to potentiate the anti-tumor activity of current available monoclonal antibodies or chemotherapy agents in aggressive lymphoma. (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)

Disclosures
No relevant conflicts of interest to declare.