One frequently reported change to acute myeloid leukemia (AML) cells, as they become resistant to arabinoside cytarabine (Ara-C), is the downregulation of deoxycytidine kinase (Dck) (Veuger et al, Blood, 2000, Shi et al, Pharmacogenetics, 2004, Cai et al, Cancer Res, 2008, Song et al, Int J Oncol, 2009, Rathe et al, Leukemia, 2010). Dck is the rate limiting enzyme in the phosphorylation and activation of Ara-C. Transcriptome deep sequencing (RNA-seq) was performed on the transcriptome of two Ara-C resistant murine leukemia cell lines (B117H and B140H), as well their Ara-C sensitive parental cell lines (B117P and B140P). Previously published microarray analysis identified the downregulation of Dck as the primary common change in the B117H and B140H cell lines, when compared to their parental cell lines (Rathe et al, Leukemia, 2010). Analysis of the RNA-seq results identified an alternatively spliced form of Dck in the B117H cell line secondary to a genomic deletion/rearrangement, as well as a loss of heterozygosity. Indeed, knockdowns of Dck in the parental cell lines demonstrated that downregulation of Dck can result in a significant increase in the IC50 of Ara-C, consistent with the increase in Ara-C resistance displayed by the B117H and B140H cells. The RNA-seq analysis showed a number of significant expression changes that did not appear on the microarray analysis. These changes include the significant upregulation of specific isoforms of deoxyguanosine kinase (Dguok) and protein phosphatase 2 (formerly 2A), regulatory subunit A (PR65), beta isoform (Ppp2r1b). It has already been shown that downregulation of Dguok results in the upregulation of Dck (Franco et al, Exp Cell Res, 2007), thereby demonstrating an important balance between these two molecules. Ppp2r1b is a putative deactivator of Dck (Smal et al, Biochem Pharmacol, 2004), which may assist in the prevention of Ara-C metabolic activation and thereby contribute to resistance. This effort demonstrates the power of using RNA-seq transcriptome analysis to identify drug targets for the treatment of AML and suggests multiple genetic/epigenetic mechanisms contribute to alterations in Dck activity during the acquisition of Ara-C resistance.
Largaespada:NeoClone Biotechnology, Inc.: Co-founder, Consultancy, Equity Ownership; Discovery Genomics Inc.: Co-founder, Consultancy, Equity Ownership.
Asterisk with author names denotes non-ASH members.