Department of Medical Laboratory Science
Contributions
Type: Publication Only
Background
Decitabine (DAC) is a cytidine analogue and hypomethylating agent used in the management of patients with myelodysplastic syndrome (MDS) and increasingly in acute myeloid leukemia (AML). Cellular uptake via the hENT1 transporter and subsequent phosphorylation by deoxycytidine kinase (dCK) produces the active metabolite decitabine triphosphate (DAC-TP). DAC-TP is incorporated into DNA, where it covalently binds to and inactivates DNA methyltransferases (DNMTs), leading to hypomethylation and potential re-expression of silenced tumour suppressor genes. DAC is also known to have a direct cytotoxic effect on leukaemic cells. DAC has been compared extensively with the related compound azacytidine (AZA) clinically. Only 10-20% of intracellular AZA is converted to DAC-TP leading to hypomethylation, compared with 100% DAC-TP from DAC. Trials have shown AZA to be clinically favourable, indicating that a lower intracellular concentration of DAC-TP could be beneficial. Current opinion is that DAC may be useful following appearance of resistance to AZA. Therefore a tool that could assist in predicting individual patient benefit from treatment with DAC is needed, particularly considering associated costs to healthcare services for this drug (~$80,000 pa per patient).
Aims
This study aimed to assess the feasibility of detecting DAC and DAC-TP using a previously validated biosensor sensitive to the cytidine analogue cytosine arabinoside (ara-C) (Alloush et al., Clin Chem. 2010;56(12):1862-70). Biosensor HA-1 is a cdd-deficient, bioluminescent bacterium with inducible expression of human dCK that is specific for toxic analogues of cytidine. Biosensor HA-1 has been formulated into an assay capable of predicting response to treatment within 8-hours and is currently undergoing clinical evaluation for ara-C
Methods
Direct application of DAC to biosensor HA-1 using a clinically relevant dosing range (0.1 - 10 mM) equivalent to 15 - 150 mg/m2 resulted in a dose-dependent increase in light output, with maximum 2.6-fold increase over control. A concurrent decrease in bacterial growth was observed as previously noted with ara-C, confirming intercalation of bacterial DNA with DAC-TP. Direct comparison of DAC with ara-C demonstrated an equivalent response by biosensor HA-1 for both time to peak (5.25 hours) and scale of increase of light output
Results
Subsequently leukaemic cell lines, HL-60 and K562 were exposed to DAC (0 - 10 µM) to assess whether DAC uptake and metabolism to DAC-TP during the initial 1-hour post-dosing would predict cytotoxicity at 72 hours. Assessment of cytotoxicity of DAC performed following daily dosing of DAC (0.1 – 10 mM) on HL-60 and K562 cells over a 72-hour period indicated that HL-60 cells were more susceptible to DAC-induced cytotoxicity than K562 cells. Concurrent assessment of accumulation of parent DAC and DAC-TP using biosensor HA-1 indicated that K562 cells produced more DAC-TP than HL-60 cells in the initial 1-hour post-dosing with DAC (≥1 mM), implying that the cytotoxicity observed may be more related to accumulation of parent DAC than metabolite. Previous in vitro studies have observed that the IC50 concentration of DAC is associated with maximal hypomethylation, indicating that DAC-TP could be responsible for the suppression of proliferation observed
Summary
Studies are on-going in our laboratory to directly assess DAC-TP using biosensor HA-1 and compare with hypomethylation in cell lines and patient blasts to define the intracellular drug kinetics of DAC, and further dissect the role of parent drug and metabolite in DAC-mediated toxicity.
Keyword(s): Bioluminescence, Decitabine, Myelodysplasia
Session topic: Publication Only
Type: Publication Only
Background
Decitabine (DAC) is a cytidine analogue and hypomethylating agent used in the management of patients with myelodysplastic syndrome (MDS) and increasingly in acute myeloid leukemia (AML). Cellular uptake via the hENT1 transporter and subsequent phosphorylation by deoxycytidine kinase (dCK) produces the active metabolite decitabine triphosphate (DAC-TP). DAC-TP is incorporated into DNA, where it covalently binds to and inactivates DNA methyltransferases (DNMTs), leading to hypomethylation and potential re-expression of silenced tumour suppressor genes. DAC is also known to have a direct cytotoxic effect on leukaemic cells. DAC has been compared extensively with the related compound azacytidine (AZA) clinically. Only 10-20% of intracellular AZA is converted to DAC-TP leading to hypomethylation, compared with 100% DAC-TP from DAC. Trials have shown AZA to be clinically favourable, indicating that a lower intracellular concentration of DAC-TP could be beneficial. Current opinion is that DAC may be useful following appearance of resistance to AZA. Therefore a tool that could assist in predicting individual patient benefit from treatment with DAC is needed, particularly considering associated costs to healthcare services for this drug (~$80,000 pa per patient).
Aims
This study aimed to assess the feasibility of detecting DAC and DAC-TP using a previously validated biosensor sensitive to the cytidine analogue cytosine arabinoside (ara-C) (Alloush et al., Clin Chem. 2010;56(12):1862-70). Biosensor HA-1 is a cdd-deficient, bioluminescent bacterium with inducible expression of human dCK that is specific for toxic analogues of cytidine. Biosensor HA-1 has been formulated into an assay capable of predicting response to treatment within 8-hours and is currently undergoing clinical evaluation for ara-C
Methods
Direct application of DAC to biosensor HA-1 using a clinically relevant dosing range (0.1 - 10 mM) equivalent to 15 - 150 mg/m2 resulted in a dose-dependent increase in light output, with maximum 2.6-fold increase over control. A concurrent decrease in bacterial growth was observed as previously noted with ara-C, confirming intercalation of bacterial DNA with DAC-TP. Direct comparison of DAC with ara-C demonstrated an equivalent response by biosensor HA-1 for both time to peak (5.25 hours) and scale of increase of light output
Results
Subsequently leukaemic cell lines, HL-60 and K562 were exposed to DAC (0 - 10 µM) to assess whether DAC uptake and metabolism to DAC-TP during the initial 1-hour post-dosing would predict cytotoxicity at 72 hours. Assessment of cytotoxicity of DAC performed following daily dosing of DAC (0.1 – 10 mM) on HL-60 and K562 cells over a 72-hour period indicated that HL-60 cells were more susceptible to DAC-induced cytotoxicity than K562 cells. Concurrent assessment of accumulation of parent DAC and DAC-TP using biosensor HA-1 indicated that K562 cells produced more DAC-TP than HL-60 cells in the initial 1-hour post-dosing with DAC (≥1 mM), implying that the cytotoxicity observed may be more related to accumulation of parent DAC than metabolite. Previous in vitro studies have observed that the IC50 concentration of DAC is associated with maximal hypomethylation, indicating that DAC-TP could be responsible for the suppression of proliferation observed
Summary
Studies are on-going in our laboratory to directly assess DAC-TP using biosensor HA-1 and compare with hypomethylation in cell lines and patient blasts to define the intracellular drug kinetics of DAC, and further dissect the role of parent drug and metabolite in DAC-mediated toxicity.
Keyword(s): Bioluminescence, Decitabine, Myelodysplasia
Session topic: Publication Only