Abstract
The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational protein modification that adds GlcNAc to nuclear and cytoplasmic proteins. Increased GlcNAc has been linked to regulatory factors involved in cancer cell growth and survival. However, the biological significance of GlcNAc in diffuse large B-cell lymphoma (DLBCL) is not well defined. This study is the first to show that both the substrate and the endpoint O-GlcNAc transferase (OGT) enzyme of the HBP were highly expressed in DLBCL cell lines and in patient tumors compared with normal B-lymphocytes. Notably, high OGT mRNA levels were associated with poor survival of DLBCL patients. Targeting OGT via small interference RNA in DLBCL cells inhibited activation of GlcNAc, nuclear factor kappa B (NF-κB), and nuclear factor of activated T-cells 1 (NFATc1), as well as cell growth. Depleting both glucose and glutamine in DLBCL cells or treating them with an HBP inhibitor (azaserine) diminished O-GlcNAc protein substrate, inhibited constitutive NF-κB and NFATc1 activation, and induced G0/G1 cell-cycle arrest and apoptosis. Replenishing glucose-and glutamine-deprived DLBCL cells with a synthetic glucose analog (ethylenedicysteine-N-acetylglucosamine [ECG]) reversed these phenotypes. Finally, we showed in both in vitro and in vivo murine models that DLBCL cells easily take up radiolabeled technetium-99m-ECG conjugate. These findings suggest that targeting the HBP has therapeutic relevance for DLBCL and underscores the imaging potential of the glucosamine analog ECG in DLBCL.
Highlights
Diffuse large B-cell lymphoma (DLBCL) is the most frequent non-Hodgkin lymphoma histotype clinically, with approximately 30,000 new cases/year in the United States
O-GlcNAc transferase (OGT) expression is increased in diffuse large B-cell lymphoma (DLBCL) cells, and high OGT mRNA expression is associated with poor prognosis in DLBCL patients
Azaserine treatment induces apoptosis in EJ, MS, and OCI-LY10 DLBCL cells but not in CJ cells (Figure 4B). Azaserine treatment inhibited both p65 and nuclear factor of activated T-cells 1 (NFATc1) protein expression, subsequently leading to the activation apoptotic related proteins, cleaved caspase 3 and PARP (Figure 4C). These results suggest that azaserine can block NF-κB-p65 and NFATc1 in DLBCL cells, which subsequently leads the induction of apoptosis, similar to what was observed in the OGT knockdown and nutrient depletion approaches
Summary
Diffuse large B-cell lymphoma (DLBCL) is the most frequent non-Hodgkin lymphoma histotype clinically, with approximately 30,000 new cases/year in the United States. DLBCL is initially responsive to standard frontline rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) chemoimmunotherapy (~80% partial or complete response), the disease frequently relapses; almost half of all patients with DLBCL are not cured by either chemotherapy or stem cell transplantation and experience relapse or display primary refractory disease with shortened survival [1]. New novel therapeutic approaches are urgently needed for patients with relapsed/refractory (R/R) DLBCL. Because cancer cells preferentially utilize aerobic glycolysis as the major source of energy for growth and survival, this pathway has become a relevant potential therapeutic target in various cancers, including aggressive B-cell lymphomas [2]. Aerobic glycolysis in cancer cells is often defined by excessive cellular glucose uptake, which is readily quantifiable in vitro and in vivo [3, 4]
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