PF400 ANTI-LEUKEMIA EFFECT OF DISULFIRAM AND ASCORBATE IN ABL TYROSINE KINASE INHIBITOR RESISTANT LEUKEMIA CELLS

Abstract

Background: ABL tyrosine kinase inhibitors (TKIs) such as imatinib improved outcomes for patients with Philadelphia chromosome (Ph)-positive leukemia patients, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Therefore, new approach against ABL TKI resistant cells may improve the outcome of Ph-positive leukemia patients. Aims: Because disulfiram (DSF), which is used to treat alcohol dependence in clinically, has been reported to have anti-tumor effects in various cancers, DSF may be the promising therapeutic value in ABL TKI resistant leukemia cells. Methods: In this study, we established ABL TKI resistant in vitro cell line models. We also investigated whether DSF could suppress Ph-positive leukemia cells including T315I mutation and ABL TKI resistant cells. Results: Drug repositioning and repurposing can be an important part of any drug discovery. In the drug screening system, the alcohol-aversion drug disulfiram, which is already approved for the treatment of alcohol dependence, is selected the candidate drug for ABL TKI resistant cells. Therefore, we examined DSF efficacy by using Ph-positive leukemia cells. We found that DSF treatment for 72 h decreased cell viability in a dose-dependent manner. We also found that DSF inhibited the proliferation of ABL TKI resistant cells (K562 ponatinib-R, K562 nilotinib-R) and T315I mutant Ba/F3 cells. Vitamin C (ascorbate) has also been well documented to reduce the incidence of malignancies in humans. We next investigated the efficacy of DSF and ascorbate against ABL TKI resistant and T315I mutant Ba/F3 cells. DSF and ascorbate treatment induced cellular growth inhibition compared with each drug alone. Most strikingly, cell death started in ABL TKI resistant cell lines within 3 to 6 h of treatment, indicating a first response to DSF and ascorbate lesions, to cell fate decision. Apoptotic cells and cellular cytotoxicity was also increased after DSF and ascorbate treatment. In the Colony Formation method, the number of cell colonies are also reduced. We next investigated the signaling pathway. Nuclear factor-kappaB (NF-kB) is also implicated in cancer development. DSF and ascorbate inhibited constitutive NF-kB activity. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after DSF and ascorbate treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential (Δψm) in cell death induced by DSF and ascorbate on T315I mutant Ba/F3 cells. The in vivo efficacies of DSF and ascorbate were evaluated in a mouse xenograft model. DSF and ascorbate inhibited the growth of T315I mutant Ba/F3 cells in vivo more than the control vehicle did. We also found that co-treatment with DSF and ascorbate increased mouse survival. Combination treatment with DSF and ascorbate was also well tolerated and no animal death in treated mice. Summary/Conclusion: The results of our study indicate that the DSF and ascorbate may be a powerful strategy against ABL TKI resistant cells including T315I mutation and provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.