Clofarabine Induces Hypomethylation of DNA and Expression of Cancer-Testis Antigens

Clofarabine is a second generation purine nucleoside analogue, active in non-dividing cells and in cells with a low proliferation rate. Although clofarabine has been used in patients with refractory AML, ALL and MDS, the precise mechanism of action of clofarabine is unclear. In vitro data suggests that clofarabine induce cytotoxicity through inhibition of DNA synthesis. Clofarabine 5′-triphosphate inhibits both DNA polymerase a and ribonucleotide reductase and causes a depletion of the intracellular deoxynucleotide triphosphate pools and inhibition of elongation of DNA strands during synthesis. Clofarabine also induces apoptosis and disrupts the integrity of mitochondria. Cancer-Testis (CT) antigens are a group of normal testicular proteins aberrantly expressed in tumor cells. They are potential targets for tumor vaccine development because of their limited expression in normal tissues and their in vivo immunogenicity. Changes in genomic methylation associated with malignant transformation of normal cells are thought to be responsible for the aberrant expression of the genes encoding some CT antigens.

Using the LINE-1 PCR approach to amplify the Alu repeats of the long interspersed nucleotide elements of genomic DNA, there were significant decreases in the LINE-1 hypomethylation of the DNA in tumor cell lines following treatment with clofarabine when compared to cells cultured in medium without clofarabine (p < 0.05). With clofarabine concentrations between 1 × 10⁻¹⁰ M to 1 × 10⁻⁷ M, there was a maximum decrease in the LINE-1 hypomethylation of 13% in JVM-2 cells, 14% in RL cells and 21% in Granta 519 cells. DNA hypomethylation was observed in all three cell lines with a clofarabine concentration as low as 1 × 10⁻¹⁰ M. These results, therefore, support the DNA hypomethylating property of low concentration clofarabine. Maximum hypomethylating effects of clofarabine was observed at concentrations of 1 × 10⁻⁷ M for JVM-2 cells, between 1 × 10⁻¹⁰ and 1 × 10⁻⁹ M for RL cells and 1 × 10⁻⁸ M for Granta 519 cells. Above these concentrations, the % of LINE-1 hypomethylation of the DNA declined as the degree of apoptosis, as demonstrated by Annexin V staining, increased.

Clofarabine treatment of these tumor cells resulted in the upregulation of the CT antigens. Before treatment with clofarabine, JVM-2 and Granta 519 cells expressed a very low level of Sp17 gene and undetectable Sp17 protein, and RL cells did not express any Sp17 gene or protein. In all three tumor cells, Sp17 expression was either induced or upregulated at the transcript and protein levels following treatment of the tumor cells with clofarabine. Using real time PCR, we also found a dose response of the mRNA levels encoding Sp17 with increasing concentrations of clofarabine used in the cell culture, up to a clofarabine concentration of 1 × 10⁻⁸ M. However, above this concentration, the levels of mRNA encoding Sp17 declined progressively as apoptosis increased. Similar results were seen with SPAN-Xb expression. Before treatment, transcripts encoding SPAN-Xb were detected in all three cell lines by RT-PCR. At the protein level, RL cells and Granta 519 cells expressed high level and JVM-2 cells low level of SPAN-Xb protein by immunocytochemistry. Following treatment with clofarabine, SPAN-Xb protein expression was greatly upregulated in all three tumor cell lines. These results, therefore, provide the first evidence showing the ability of clofarabine to induce the expression of Sp17 and SPAN-Xb and support the notion that low concentration clofarabine induces DNA hypomethylation, as suggested by the LINE-1 PCR analysis. However, not unlike in Sp17, we also found a dose response of the mRNA levels encoding SPAN-Xb with increasing concentrations of clofarabine used in the cell culture, up to concentrations between 1 × 10⁻⁹ and 1 × 10⁻⁸ M. Above this optimal concentration, SPAN-Xb gene expression declined as apoptosis increased.

In conclusion, clofarabine has a DNA hypomethylating property and is capable of inducing CT antigen expression. Since DNA hypomethylation with clofarabine is most optimal at very low clofarabine concentrations, low dose clofarabine should be used if the mechanism needed for response to treatment is that of DNA hypomethylation. Furthermore, there may be opportunity to administer low dose clofarabine to patients to upregulate the expression of CT antigens to increase the susceptibility of the tumor cells to the cytotoxic effect of antigen-specific cytotoxic T cells prior to specific tumor vaccines targeting CT antigens.