Small-Molecule XIAP Inhibitors Derepress Downstream Effector Caspases and Induce Apoptosis of Leukemia Cell Lines and Patient Samples.

XIAP is a potent inhibitor of caspases 3, 7, and 9 and its over-expression renders malignant cells resistant to chemotherapy. Through an enzymatic derepression assay, we identified chemical XIAP antagonists, including 1396-12, based on a polyphenylurea pharmacophore (Cancer Cell, 1:25–35;2004). This compound binds and inhibits the caspase 3/7 inhibitory BIR2 domain of XIAP. Given the potential therapeutic utility of IAP inhibitors, we tested this XIAP antagonist in leukemia cell lines and primary patient samples. The XIAP antagonist 1396-12, but not the structurally related control compound, directly induced apoptosis in leukemia cell lines at low micromolar concentrations and sensitized leukemia cells to cytarabine. 1396-12 activated downstream caspases 3/7 prior to the activation of upstream caspases 8 and 9, and independent of Bcl-2 or caspase-8, consistent with the inhibition of the BIR2 domain of XIAP. To evaluate this XIAP antagonist as a potential novel therapy for acute myeloid leukemia (AML), primary AML blasts (n= 27), normal bone marrow mononuclear cells (n =1), or normal mobilized peripheral blood stem cells (PBSC) (n =6) were treated with increasing concentrations of 1396-12. Apoptosis was measured 24 hours after treatment by Annexin V staining. Median LD₅₀ among the AML patient samples tested was 6 μM (range: 2μM to >40μM). The XIAP antagonist 1396-12 induced apoptosis of primary AML samples with a LD₅₀ ≥ 10μM in 16 of 27 (60%) samples and with a LD₅₀ >40μM in 7 of 27 (26%) samples. In contrast, 1396-12 was less toxic to the normal PBSC or marrow with a LD₅₀>40μM in all normal samples tested. As a comparison, the inactive control compound was not toxic to any of the AML or normal samples at concentrations up to 40μM. In addition to the short-term cytotoxicity assays, the effects of 1396-12 on AML and normal samples were evaluated in colony formation assays. The XIAP antagonist inhibited clonogenic survival in 4 AML samples tested with a mean LD₅₀ of 4 ± 0.8μM. Treatment with 1396-12 also reduced colony formation by 2 normal PBSC samples with LD₅₀’s of 8.5 ± 0.3μM and 5.6 ± 0.4μM. In the normal PBSC samples, both BFU-E and CFU-GM lineages were equally reduced after treatment with the XIAP antagonist. Treatment with the control compound did not reduce colony growth in the AML or normal samples. Among the primary AML samples, response to the XIAP inhibitors correlated with XIAP protein levels. Low to absent levels of XIAP were associated with a higher probability of resistance to treatment with XIAP inhibitors (p =0.04, by logistic regression analysis). In conclusion, polyphenylurea-based XIAP antagonists directly induce apoptosis in leukemia cells and patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents. These antagonists can be used as biological tools to understand the role of IAPs in normal and malignant hematopoietic cells. They may also serve as lead compounds for the development of useful therapies for the treatment of leukemia and other malignancies, but their potential hematologic toxicity will have to be carefully evaluated in phase I clinical trials.