Benigno C Valdez, David Murray, Yang Li, Yan Liu, Yago Nieto, Uday Popat and Borje S Andersson
Bisantrene is an anthracene derivative with anti-tumor activity. Unlike the anthracyclines, it has virtually no clinical cardiotoxicity. We hypothesized that its combination with nucleoside analogs and the BCL-2 inhibitor ABT199 would provide improved cytotoxicity in AML cell cultures. Cells were exposed to drugs for 48 hrs and analyzed by MTT assay for cell proliferation, annexin V assay for apoptosis, Western blotting for changes in the status of protein levels and modifications, and flow cytometry for changes in Reactive Oxygen Species (ROS) and Mitochondrial Membrane Potential (MMP). Exposure of OCI-AML3 and MOLM14 cells to bisantrene+nucleoside analog(s) (cytarabine, cladribine, fludarabine/Flu or clofarabine/Clo)+ABT199 resulted in increased cytotoxicity, apoptosis and synergism with combination indices<1. Increased levels of cleaved PARP1 and caspase 3, caspase 3 enzymatic activity, DNA fragmentation and ROS, and decreased MMP with the three- or four-drug combinations, all suggested activation of intrinsic apoptosis. A similarly enhanced activation of apoptosis was observed in leukemia patient-derived cells exposed to (bisantrene+Flu+Clo+ABT199). Exposure of AML cell lines to nucleoside analogs prior to addition of bisantrene exerted a higher level of cytotoxicity than when the reverse sequence was utilized. These results provide a rationale for clinical trials using these drug combinations for conventional (re-)induction therapy or as part of a pretransplant program leading to hematopoietic stem cell transplantation for high-risk AML patients. They also highlight the importance of considering the sequencing of cytotoxic agents when designing combination regimens for the treatment of acute leukemia.
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