Acadesine-Induced Citotoxicy In Chronic Lymphocytic Leukemia Is Independent of Immunoglobulin Mutational Status and Zap70 Expression

The precursor of nucleotide biosynthesis acadesine or 5-aminoimidazole-4-carboxamide (AICA) riboside induces apoptosis in CLL cells, and other lymphoproliferative diseases such as splenic marginal zone lymphoma and mantle cell lymphoma. This effect is selective for B-cells, at least ex vivo, however there is no evidence of whether this cytotoxic effect depends on well known prognostic variables such as ZAP-70 expression or IgV_H mutational status.

To analyse ex vivo the cytotoxic effect of acadesine on peripheral CLL cells and correlate it with prognostic variables.

Cryopreserved cells from 62 CLL patients were incubated ex vivo with acadesine at 0.2, 0.5, and 1 mM for 24 hours. Viability was determined by flow cytometry using Annexin V-FITC and DAPI staining combined with CD19-PE and CD3-PerCP to differentiate cell viability of B- and T-cells. Cells were considered sensitive to the drug when the percentage of acadesine-induced apoptosis was equal to or higher than 15% with respect to the viability of control cells. The mutational status of IgV_H genes was determined by RT-PCR amplification using a set of six V_H family-specific primers (V_H1 through V_H6) along with primers complementary to the constant region (IgM and IgG). Products were directly sequenced from both strands using the Big Dye Terminator Cycle Sequencing Ready Reaction (version 3.1, Applied Biosystems). Sequencing analysis and alignments were performed with use of V-QUEST software and the online international immunogenetics information (IMGT) data library. Samples in which fewer than 2 percent of base pairs differed from those of the consensus sequence have been considered unmutated. ZAP-70 expression was quantified by flow cytometry (cut-off:20%) and cytogenetic alterations associated with CLL (trisomy 12, del13q, del 17p and del11q) were determined by FISH.

After 24h of ex vivo incubation, 0.2 mM acadesine induced a significant cytotoxic effect (> 15%) in 31 out of 62 patients (50%). Higher concentrations, 0.5 mM and 1 mM, induced a significant effect in 91.4% (57 of 62 patients) and 98% (61 of 62 patients), respectively. The viabilities (mean ±SD) of the different culture conditions are shown in the table.

The cytotoxic effect induced by acadesine was analyzed with respect to the IgV_H mutational status and ZAP-70 expression. No significant differences were observed between cases with unmutated IgV_H (n=20) and mutated IgV_H(n=37), or between ZAP-70 positive (n=24) and ZAP-70 negative cases (n=34). Interestingly, 5 cases showing deletion of 17p were sensitive to treatment with acadesine, in agreement with previously published studies showing that acadesine-induced apoptosis is independent of p53. Only one case showing deletion 17p and V_H3-21 usage was not sensitive to acadesine.

Acadesine induces apoptosis in B-cells from CLL regardless of ZAP-70 expression, IgV_H mutational status and 17p status.

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