Second Mitochondria-Derived Activator of Caspases Mimetic Compounds (Smac) Enhance Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) Pro-Apoptotic Effect On Human Myeloid Leukemia Cell Lines.

Abstract 2756

Poster Board II-732

The Inhibitor of Apoptosis Proteins (IAP) are important regulators of programmed cell death. Among them, XIAP, which is characterized by 3 tandem BIR domains selectively blocking caspases 3, 7 and 9, is the most potent and is over-expressed in several hematological malignancies. Its activity is antagonized by Second Mitochondria-derived Activator of Caspases (Smac) and also by small molecules mimicking Smac able to induce apoptosis in tumor cells, alone or in combination with other drugs.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a pro-apoptotic cytokine, is capable of triggering programmed death in cancer cells, where it synergizes with chemotherapeutic agents and/or radiation by several malignant cell-type specific mechanisms, and it is currently under evaluation in clinical trials for its strong pro-apoptotic activity.

Here we describe the pro-apoptotic effect of newly synthesized monovalent and bivalent Smac-mimetic compounds tested for cytotoxicity on a cohort of human leukemic cell lines over-expressing XIAP (HL60, K562 and Jurkat) as well as on normal CD34⁺ hematopoietic progenitor cells, alone or in combination with TRAIL. The Smac-mimetics, designed and produced by the University of Milan Center for biomolecular Interdisciplinary Studies and Industrial applications, were dissolved in dimethylsulfoxide (DMSO) to obtain a 10 mM solution and stored at −20°C. Drug stocks were diluted with phosphate buffer (PBS) prior to their use. The cells were treated with 0.1 nM – 50 μM doses for up to 72 hours. TRAIL was kindly provided by Prof. Henning Walczak (Imperial College, London, UK). In combined treatments, Smac-mimetics and TRAIL were used simultaneously. The cytotoxic effect was evaluated by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect on fresh human CD34⁺ hematopoietic normal progenitor cells selected from healthy donors' peripheral blood stem cells (PBSC) was assayed by myeloid colony (CFU-GM) formation. Apoptosis was determined by flow cytometric analysis of DNA fragmentation and by Western blot analysis of caspases activation and PARP cleavage.

The combination of some of our Smac-mimetics (both monomers and dimers) with TRAIL highly enhanced the inhibition of proliferation of the chronic myelogenous leukemia cell line K562, which was shown to be resistant to TRAIL single agent. In particular, a synergistic effect was observed in combined treatment with Smac012 10 μM and TRAIL 50 ng/ml (R Kern index = 6.2). Our Smac-mimetics, particularly the dimeric ones, were capable of inducing apoptosis, as demonstrated by DNA fragmentation and accumulation of cleaved PARP, caspase 8 and caspase 3, especially when administrated in combination with TRAIL. No cytotoxic effect was observed on normal CD34⁺ progenitor cells by Smac mimetics at doses ranging from 40 μM to 70 μM, even normal controls when were treated simultaneously with TRAIL.

As many hematological malignancies are resistant to TRAIL alone, thus limiting its therapeutic effectiveness, the observed strong synergistic effect with Smac mimetic compounds not affecting the normal hematopoietic progenitor cell compartment might be of great consequence for the development of innovative potent pro-apoptotic drug combinations in myeloid leukemia treatment.