Abstract 1185: Translational study of the enhancement of cytarabine toxicity to AML blasts by a differentiation agent combination

Abstract

Abstract Cytarabine (AraC) is an important component of AML therapy, but like other DNA damaging therapeutic agents it is rarely curative. Exploratory studies suggest that therapy outcomes may be improved by combining AraC with other compounds. In this context, we have recently reported that the addition of a vitamin D-based differentiating agent combination following AraC damage to AML cells in established culture, increases the cell kill. Here we present data obtained with AML blasts cells freshly obtained from patients with this disease which demonstrate that HL60 and U937 cell lines provide a good model to supplement studies of the mechanisms underlying this potential therapeutic regimen. Importantly, in contrast to malignant cells, mononuclear cells isolated from normal human bone marrow do not show the enhancement of AraC-induced cell death by combinations of differentiation agents. The cells were exposed to AraC at concentrations which produced approximately 30-40% cell kill, followed by a combination of 100 nM 1alfa-hydroxyvitamin D2 (D2) and 10 uM carnosic acid (CA), which together serve as a powerful differentiating agent combination (D2/CA) for AML cells, but are minimally toxic alone. Increased cell death, measured by Annexin V/Propidium Iodide, compared to AraC alone, was selective for malignant cells, occurred by both apoptosis and necrosis, and was associated with increased levels of vitamin D receptor (VDR), DNA damage, and higher levels of DNA damage response marker P-H2AX. The knock-down of VDR with siRNA markedly reduced the potentiating effect of D2/CA on the AraC- induced cell death and the associated cellular changes. Since several caspases showed increased expression, and a caspase 3-specific inhibitor markedly reduced the enhancement of cell death, we measured the levels of the pro-apoptotic member of the Bcl family, Bim, which prominently increased following the addition of D2/CA to cells previously exposed to AraC. The increased Bim expression occurred at both mRNA and protein levels. Consistent with a role in the enhancement of cell death in this system, knock down of Bim by siRNA abrogated the effect of adding D2/CA to AraC-treated cells. Thus, our data indicate that in AML cells with pre-existing DNA damage activated VDR can interact with apoptotic pathways, and rather than promoting differentiation, leads to increased leukemia cell kill. (Supported by grant R01CA044722-25 from the National Cancer Institute, NIH, and grant 10A049 from American Institute for Cancer Research, both to GPS; JSH received support from the Nellie B. Smith Endowment at the University of Missouri and the Ellis Fischel Cancer Research Fund at the University of Missouri). Citation Format: Xuening Wang, Jonathan Harrison, George P. Studzinski. Translational study of the enhancement of cytarabine toxicity to AML blasts by a differentiation agent combination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1185.