Intracellular Succinylation of 8-Chloroadenosine and Its Effect on Fumarate Levels

Abstract

8-Chloroadenosine (8-Cl-Ado) is a ribosyl nucleoside analog currently in phase I testing for the treatment of chronic lymphocytic leukemia (CLL). 8-Cl-Ado activity is dependent on adenosine kinase and requires intracellular accumulation of 8-Cl-Ado as mono-, di-, and tri-phosphates. In the current study with four mantle cell lymphoma cell lines, we report a new major metabolic pathway for 8-Cl-Ado intracellular metabolism, the formation of succinyl-8-chloro-adenosine (S-8-Cl-Ado) and its monophosphate (S-8-Cl-AMP). 8-Cl-AMP levels were highly associated with S-8-Cl-AMP levels and reached a steady-state prior to the secondary metabolites, 8-Cl-ATP and S-8-Cl-Ado. Consistent with fumarate as a required substrate for formation of succinyl-8-Cl-adenylate metabolites, the S-8-Cl-adenylate concentrations in multiple cell lines were associated with fumarate loss. The distribution of metabolites was also altered using the energy metabolism modifiers, metformin and oligomycin. The rates of succinyl-8-Cl-adenylate metabolism were enhanced by increasing the intracellular fumarate concentrations after metformin co-treatment. In addition, the S-8-Cl-AMP concentrations were increased after acute inhibition of ATP synthase by oligomycin. We conclude that 8-Cl-Ado metabolism not only affects intracellular purine metabolism; 8-Cl-Ado conversion to succinyl analogs ties its metabolism to the citric acid cycle by reduction of the fumarate pool. 8-Chloroadenosine (8-Cl-Ado)3 is a purine nucleoside analog with reported preclinical activity in multiple hematological malignancies (1-3). Currently 2, 3) or by incorporating into the poly(A)tail of mRNA transcripts, which is required for functionality and stability of the transcripts (9). Subsequent apoptosis is promoted by reduced expression of short-lived survival proteins, such as Mcl-1 in CLL and MET in multiple myeloma (4, 6). In addition, intracellular 8-Cl-ADP competes with ADP for mitochondrial ATP synthase and consequently reduces intracellular ATP concentrations (10). 8-Cl-Ado phosphorylated metabolites may also inhibit ribonucleotide reductase and reduce dATP levels causing an imbalance of the dNTP pool (1).As previously described, the mechanisms of action for 8-ClAdo have focused on phosphorylated metabolites, in particular 8-Cl-ATP, as the primary active intracellular metabolites. However, as with normal adenosine metabolism, 8-Cl-Ado may be a precursor not just for its mono-, di-, and tri-phosphate nucleotides but for multiple metabolic conjugates including nicotinamide adenine dinucleotide (NAD), S-adenosyl methionine (SAM), and flavin adenine dinucleotide (FAD). In the current study, we identified two new major 8-Cl-Ado metabolites, but the metabolites were not the downstream products we predicted. Instead, 8-Cl-Ado was metabolized to adenosine precursor molecules, succinyl-conjugated metabolites: succinyl-8-chloro-adenosine (S-8-Cl-Ado) and succinyl-8-chloro-AMP (S-8-Cl-AMP). Whereas the loss of ATP has already been associated with 8-Cl-ATP formation (1, 2, 5), this new...

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