Establishment of Novel Leukemic Cell Lines Resistant to Clofarabine by Dual Mechanisms of Decreased Active Metabolite and Increased Antiapoptotic Factor
Authors: Shigemi Hiroko , Yamauchi Takahiro , Ueda Takanori 
Published: October 2019

Abstract
Abstract 1356
Clofarabine(2-Chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine,2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, CAFdA) is a relatively new purine nucleoside analog. Upon administration, CAFdA is incorporated into leukemic cells by human Equilibrative Nucleoside Transporters (hENT) 1 and 2, and human Concentrative Nucleoside Transporter (hCNT) 3. Inside the cell, the agent is phosphorylated to CAFdA monophosphate by cytosolic deoxycytidine kinase (dCK) and mitochondrial deoxyguanosine kinase (dGK), and then to an intracellular active metabolite CAFdA triphosphate (CAFdATP). CAFdATP inhibits ribonucleotide reductase and is incorporated into DNA, thereby terminating DNA synthesis as an antimetabolite. Moreover, CAFdA induces apoptosis via direct mitochondrial damage. Clinical studies suggest that CAFdA is effective against both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). CAFdA therapy should be optimized based on the mechanistic understanding, because pharmacological determinants that correlate to the drug sensitivity may predict clinical efficacy of CAFdA as biological surrogate markers. Here, we have established two novel leukemic cell line variants that were resistant to CAFdA, and elucidated the mechanism of the drug resistance. The study focused on factors that were involved in the intracellular CAFdATP production and in the induction of apoptosis.
To develop resistant variants, HL-60 cells were treated with escalating concentrations of CAFdA with the initial concentration at 1/100 of the concentration that inhibited 50% cell growth. After 7 months of the repeated passage, one cell line resistant to CAFdA (HL/CAFdA20) was cloned by the limiting dilution method. A part of this clone was further maintained with the drug for the subsequent 4 months to develop another variant (HL/CAFdA80). The 2 variants were 20- and 80-fold more CAFdA-resistant than HL-60 cells, respectively. They were cross-resistant to similar nucleoside analogs such as cladribine, gemcitabine, and cytarabine. Compared with HL-60 cell line, mRNA levels of the transporters (hENT1, hENT2, hCNT3) and protein levels of kinases (dCK, dGK), and the subsequent production of intracellular CAFdATP were all reduced in both CAFdA-resistant variants. Real time RT-PCR demonstrated that mRNA levels of hENT1, hENT2, and hCNT3 were 53.9%, 41.8%, 18.3% in HL/CAFdA20 cells, and 30.8%, 41.6%, 31.5% in HL/CAFdA80 cells, respectively, compared to the parental cells. The values of the initial uptake of CAFdA into the cell at 40 seconds after administration of tritiated CAFdA are 0.2 pmol/107cells in HL/CAFdA20 cells, and 0.1 pmol/107cells in HL/CAFdA80 cells, compared with 0.6 pmol/107 cells in parental HL60 cells. Western blotting revealed that protein levels of kinases were also reduced in these resistant variants with the greater reduction in HL/CAFdA80 cells. The subsequent production of CAFdATP after 4-h incubation with 10 μM CAFdA was 20 pmpl/107 cells in HL/CAFdA20 cells, 3 pmol/107cells in HL/CAFdA80 cells, and 63 pmol/107cells in HL-60 cells. The decreased CAFdATP production led to the attenuated incorporation of the drug into both mitochondrial and nuclear DNA. Concerning apoptosis, antiapoptotic Bcl2 protein overexpressed in the 2 resistant variants. The two variants were resistant to mitochondria-related apoptosis induced by CAFdA, in part due to the enhanced Bcl2 expression. A Bcl2 inhibitor ABT737 synergized the cytotoxic effect and the growth inhibition effect of CAFdA in both variants and HL-60, but the synergism was more profound in the resistant cell lines. The Combination Index values were 0.27 in HL/CAFdA20 cells, and 0.21 in HL/CAFdA80 cells, compared with 0.63 in HL-60 cells. This suggested the contribution of the enhanced Bcl2 protein to the mechanism of drug resistance.
In conclusion, the mechanism of cellular resistance to CAFdA in the 2 variants was multifactorial, but primarily includes the reduced CAFdATP production and the increased antiapoptotic factor. It is noted that the decreased dGK level and Bcl2 overexpression were not reported previously in the context of CAFdA resistance. We suggest combination of CAFdA and ABT737 might be effective to CAFdA resistant and refractory leukemia. (This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. No.23501307)

Disclosures:
No relevant conflicts of interest to declare.