Is Cladribine, a Treatment for Chronic Lymphocytic Leukemia, a DNA Methylation Inhibitor?.

Abstract

Chronic lymphocytic leukemia is a heterogenous B lymphocyte disorder with a variable natural history. Like other Malignancies, key regulatory genes like the tumor suppressor, p16, and the mismatch repair gene, hMLH1, are frequently silenced by DNA methylation in patients with chronic lymphocytic leukemia (CLL). Although focal hypermethylation is found, global genomic DNA is hypomethylated compared to genomic DNA from healthy volunteers. Inhibition of DNA methylation may help CLL cells regain normal cellular function. Few studies have been done evaluating DNA methylation of CLL cells likely in part due to the difficulty in establishing a CLL cell line without altering DNA methylation. In addition, the primary cells do not divide in vitro, making the assessment of effect of a DNA methylation inhibitor very difficult. Of the ten samples tested from patients with CLL, we have not found p16 or hMLH1 expression by western blotting. Since 2-Chlorodeoxyadenosine (Cladribine) is already used clinically for treatment of CLL and may deplete available methyl donors, we asked if Cladribine is effective because of its inhibitory effect on DNA methylation in leukemia cells. To further evaluate the role of DNA methylation in CLL, we assessed the effect of 5-aza-2′-deoxycitidine (Decitabine), a DNA methyltransferase inhibitor compared to Cladribine in HT-29 cells. HT-29 cells do not express p16 and MAGE because of DNA methylation of the promoters. Assessment of global DNA methylation is done using reversed-phase high-performance liquid chromatography. Our results reveal that daily doses of 300nM Cladribine modestly inhibited global DNA methylation by 20+/−14% at 72 hours (n=3), while daily doses of 500nM Decitabine inhibited global DNA methylation by 75+/−9% at 72 hours (n=3). This results in re-expression of MAGE-1, a gene silenced by DNA methylation in all somatic tissues and p16, only in the Decitabine treated cells. We also evaluated the Cdx-2 promoter, an intestinal restricted gene silenced by DNA methylation in HT-29 cells. Decreased methylation of two regions of the Cdx-2 promoter was seen in the cells treated with Decitabine compared to the control cells. Cladribine treated cells had no effect on the Cdx-2, and may actually increase DNA methylation (result of 20 sequences). These results could be explained by the marked growth inhibitory effect of Cladribine at 300nM. We chose to evaluate Cladribine at 300nM because this plasma concentration is obtainable in patients treated with 0.1mg/kg of Cladribine, a dose frequently used in treatment of hairy cell leukemia. Cells with decreased DNA methylation after treatment with Cladribine may have died at the 72 hour time point.