Pre-Clincial Evaluation of a Novel Purine Analogue 8-NH₂-Adenosine in Mantle Cell Lymphoma Indicates Efficacy Via Alterations in RNA/DNA Synthesis and Cellular Bioenergetics

Background: Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) with a prevalence of approximately 15,000 cases in the United States. Although current therapeutics extend longevity, the median survival is 3 to 5 years warranting continued investigation for newer therapeutics. MCL is characterized by cells with enhanced proliferation combined with impaired apoptosis characteristic of indolent lymphomas. Therefore, therapeutic approaches targeting transcription, translation, or cellular bioenergetics may prove to be more effective than therapies targeting DNA replication. In addition, therapeutic strategies that exploit the altered cellular metabolism of tumor cells may be beneficial. Nucleoside analogues have been used extensively in the treatment of hematologic malignancies and are selective for tumor cells. Our laboratories have developed two purine nucleoside analogues i.e. 8-chloro-adenosine (currently in clinical trials) and a congener, 8-amino-adenosine (8-NH₂-Ado), showing high efficacy for multiple myeloma, a slow growing plasma B-cell malignancy. Characterization of the mechanism of toxicity of 8-NH₂-Ado in myeloma shows decreased RNA synthesis preceding decreased DNA synthesis, inactivation of Ser/Thr kinases, and reductions in intracellular ATP and glucose consumption. Based on this pleiotropic profile of cellular pathways involved in the execution of cell death by 8-NH₂-Ado, we sought to determine its efficacy in MCL.

Results: We determined toxicity of 8-NH₂-Ado in a panel of MCL cell lines, including, JeKo-1, Mino and Granta 519. Viability was assessed by Annexin V/Dapi double staining after 24 hours of incubation with increasing concentrations of 8-NH₂-Ado. All three cell lines demonstrated sensitivity to 8-NH₂-Ado with JeKo-1 being the most sensitive (IC50 at 2 uM) followed by Mino and Granta 519. The induction of apoptosis correlated with cleavage of PARP and caspase activation and with decreases in cyclin D1 and Mcl-1 expression. JeKo-1 cells rapidly metabolized 8-NH₂-Ado to 8-NH₂-ATP. After 6 hrs of incubation, the 8-NH₂-ATP intracellular concentration was more than 5 mM, and the ATP concentration was reduced by more than 50%. Additionally after 6 hrs of incubation, the rates of RNA and DNA synthesis were reduced by at least 60% based on [³H]uridine and [³H]thymidine incorporation assays. In an assessment of downstream signaling kinases, p38 and AKT were rapidly de-phosphorylated after 5 hrs of treatment. Because AKT controls cellular glucose consumption, we assessed effects on glucose consumption. In both the JeKo-1 and Granta 519 cells, we observed a similar reduction in glucose consumption; however, baseline glucose consumption in the less sensitive Granta 519 cells was higher.

Conclusions: 8-NH₂-Ado is highly toxic for the MCL cell lines tested. 8-NH₂-Ado decreases Mcl-1 and cyclin D1 expression and decreases phosphorylation of AKT and p38 in both the JeKo-1 and Granta 519 cells. In the JeKo-1 cells, 8-NH₂-Ado is metabolized to 8-NH₂-ATP and decreases RNA/DNA synthesis and intracellular ATP. The early changes in cellular glucose consumption may facilitate 8-NH₂-Ado induction of apoptosis. These pleiotropic features of 8-NH₂-Ado in regulating cellular bioenergetics and induction of apoptosis may be particularly advantageous and warrant further investigation of 8-NH₂- Ado for the treatment of MCL.