Abstract 5380: Synergistic effects of clofarabine and decitabine in acute myeloid leukemia
Authors: Thudium Karen E. , Den Haese Jason , Karpf Adam , Fetterly Gerald , Wetzler Meir 
Published: June 2012

Abstract
Clofarabine (CLO) is a purine nucleoside analog with promising efficacy in acute myeloid leukemia (AML). Its mechanism of action involves inhibition of the ribonucleotide reductase (RR) that converts nucleotide diphosphates to the corresponding deoxynucleotide diphosphates. Expression of the small RR subunit, p53R2 (product of RRM2B), demonstrated that a shorter protein is associated with CLO resistance. We have also shown that decitabine (DEC), another active drug in AML, induces p53R2 mRNA and protein expression and therefore asked herein whether the combination will be synergistic in a human AML cell line, HEL. To understand the role of p53R2 in this interaction, we knocked it down (KD) with shRNAmir. HEL and HEL-p53R2KD cells were exposed to CLO and DEC for 24,48,72, and 96-hr. Cells were stained with trypan blue for cell viability. The Hill model was fit to each concentration-response curve for each drug. After fitting and determination of 50% inhibition concentration (IC50), five combination ratios of the IC50 (CLO:DEC; 1:1, 1:4, 4:1, 1.5:3.5; 3.5:1.5) were characterized. In addition, western blot analysis was performed for both cell lines to determine levels of P53R2 expression for the same concentration range and exposure time to each drug, and in combination. Bands in western blot were quantitated using densitometry. HSC70 was used as a house keeping gene. At 24 hr, the IC50 values for CLO and DEC in HEL cells were 500 nM. In p53R2KD cells, the IC50 values for CLO and DEC were 700 nM and 120 nM. As exposure time increased to 48, 72, or 96 hr, the potency increased 10-15-fold for clofarabine, while only 2-4-fold for decitabine. At 48 hr, IC50 values were less than 50 nM for clofarabine in both HEL and p53R2KD cells, while it was 145 and 215 nM for decitabine. For the combination treatment, a pharmacodynamic (PD) interaction model was developed to assess the degree of synergy between CLO and DEC on cell viability. A PD parameter, labeled as ψ, characterized the extent of the interaction. When ψ &gt;=1, the interaction between the two drugs may be additive, while ψ&lt;1 represents synergy and ψ&gt;1 indicates antagonism. Following 24 hr of drug exposure of CLO and DEC at the 50% of the IC50 values for each drug combination, ψ was estimated to be 1.00 (CI 0.850-1.22) for P53R2KD and &gt;1 for HEL cells. In addition, western blot analyses performed at 24-hr following combined drug exposure revealed that the upper band did not change as a function of drug concentration, while the lower band showed a concentration-dependent increase in p53R2 protein expression, especially in p53R2KD.
The PD model demonstrated a synergistic effect for CLO and DEC in the AML cell line HEL; knocking down p53R2 decreased this synergism, attesting to p53R2's role in both drugs’ mechanism of action. The increase in the lower band supports its role as part of clofarabine resistance. A clinical trial combining CLO and DEC in AML is warranted.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2011-5380