Evasion of apoptosis may contribute to poor treatment response in pediatric acute lymphoblastic leukemia (ALL), calling for novel treatment strategies. Here, we report that inhibitors of apoptosis (IAPs) at subtoxic concentrations cooperate with various anticancer drugs (that is, AraC, Gemcitabine, Cyclophosphamide, Doxorubicin, Etoposide, Vincristine and Taxol) to induce apoptosis in ALL cells in a synergistic manner as calculated by combination index and to reduce long-term clonogenic survival. Importantly, we identify RIP1 as a critical regulator of this synergism of IAP inhibitors and AraC that mediates the formation of a RIP1/FADD/caspase-8 complex via an autocrine/paracrine loop of tumor necrosis factor-a (TNFa). Knockdown of RIP1 abolishes formation of this complex and subsequent activation of caspase-8 and -3, mitochondrial perturbations and apoptosis. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 or blockage of TNFa by Enbrel inhibits IAP inhibitor-and AraC-triggered interaction of RIP1, FADD and caspase-8 and apoptosis. In contrast to malignant cells, IAP inhibitors and AraC at equimolar concentrations are non-toxic to normal peripheral blood lymphocytes or mesenchymal stromal cells. Thus, our findings provide first evidence that IAP inhibitors present a promising strategy to prime childhood ALL cells for chemotherapy-induced apoptosis in a RIP1-dependent manner. These data have important implications for developing apoptosis-targeted therapies in childhood leukemia.

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3791 Poster Board III-727 Children with high risk acute lymphoblastic leukemia (ALL) do not respond well to current treatments. This failure is, at least in part, due to defects in apoptosis programs. Therefore, new strategies are required that counter apoptosis resistance in order to improve the poor prognosis of high risk pediatric acute leukemia. Increasing evidence suggests that high levels of “Inhibitor of Apoptosis” (IAP) proteins may represent a key antiapoptotic mechanism in cancer cells including acute leukemia. Among the IAP family members, it is especially X-linked inhibitor of apoptosis (XIAP) that is known for its antiapoptotic function. Since XIAP blocks apoptosis at a central point of the apoptotic machinery by inhibiting activation of effector caspases, we explored whether XIAP presents a suitable molecular target for therapeutic intervention in childhood leukemia. Here, we report that neutralizing XIAP by small molecule inhibitors is a novel and effective approach to sensitize childhood acute leukemia cells for chemotherapeutic drugs, which are currently used in clinical protocols for the treatment of children with acute leukemia. Subtoxic concentrations of XIAP inhibitors synergize with various anticancer drugs, for example Cytarabine, Vincristine, Doxorubicin, Etoposide and cyclophosphamide, to induce apoptosis in ALL and also in AML cells. By comparison, no sensitization for chemotherapy-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. In addition, XIAP inhibitors act in concert with anticancer drugs to reduce clonogenic growth of ALL cells demonstrating that they also suppress long-term survival. Analysis of signaling pathways reveals that XIAP inhibitors enhance chemotherapy-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner. Further, XIAP inhibitors cause rapid and profound downregulation of cIAP1 accompanied by activation of NF-κB. Of note, inhibition of RIP1 kinase by necrostatin or caspases by the broad range caspase inhibitor zVAD.fmk also significantly reduces the XIAP inhibitor-mediated sensitization to cytotoxic drugs. Intriguingly, the addition of TNFα blocking antibodies also significantly decreases apoptosis upon combined treatment with XIAP inhibitors and chemotherapeutic drugs, indicating that paracrine/autocrine production of TNFα is involved in this synergistic interaction. In support of this notion, addition of soluble recombinant TNFα further increases apoptosis that is induced by XIAP inhibitors and anticancer drugs. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and act in concert with chemotherapeutic drugs to trigger apoptosis. In contrast to malignant cells, XIAP inhibitors at equimolar concentrations alone or in combination with chemotherapeutics are non-toxic to normal peripheral blood lymphocytes, pointing to a therapeutic window. By demonstrating that XIAP inhibitors present a promising novel approach to enhance the efficacy of chemotherapy in childhood acute leukemia, our findings have important implications for the development of innovative treatment strategies to overcome apoptosis resistance in children with high risk leukemia. This approach could be translated into clinical application in childhood ALL, since IAP inhibitors have recently entered evaluation in early clinical trials, thus underscoring the feasibility to incorporate XIAP inhibitors into chemotherapy protocols. Disclosures: No relevant conflicts of interest to declare.

Defects in apoptosis contribute to poor outcome in high risk pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. XIAP, a member of “Inhibitor of Apoptosis” (IAP) proteins, presents a promising target, since it is expressed at high levels in acute leukemia. Therefore, we investigated the effect of small molecule XIAP inhibitors alone and in combination with TRAIL or chemotherapeutics in ALL cell lines, primary leukemic blasts, normal peripheral blood lymphocytes and in a mouse model of pediatric ALL. Here, we report that XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, cooperate with TRAIL to induce apoptosis in ALL cells in a highly synergistic manner as calculated by combination index. Also, XIAP inhibitors act in concert with TRAIL to reduce clonogenic growth of ALL cells demonstrating that they suppress longterm survival. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent mitochondrial amplification loop. Intriguingly, XIAP inhibitors overcome Bcl-2-mediated resistance to TRAIL by promoting Bcl-2 cleavage, Bak conformational change and caspase-3-driven mitochondrial perturbations. Thus, the combination of XIAP inhibitors and TRAIL can even break Bcl-2-imposed resistance, a defect in the apoptotic pathway that frequently occurs in acute leukemia and predicts poor prognosis. Moreover, XIAP inhibitors sensitize ALL cells for chemotherapy, e.g. cytarabine, doxorubicin, etoposide and 6-mercaptopurine. Intriguingly, this chemosensitization is inhibited by a TNFalpha-neutralizing antibody, demonstrating that it depends on XIAP inhibitor-triggered production of TNFalpha. Notably, XIAP inhibitors potently trigger cell death in leukemic blasts from children with ALL ex vivo and also sensitize primary leukemia cells for TRAIL- or chemotherapy-induced cell death. In contrast to malignant cells, XIAP inhibitors and TRAIL at equimolar concentrations are non-toxic to normal peripheral blood lymphocytes despite expression of the apoptosis-inducing TRAIL receptors on the cell surface, pointing to a therapeutic window. Most importantly, XIAP inhibitors exert potent anti-leukemic activity in vivo in a mouse model of pediatric ALL engrafted in NOD/SCID mice. These findings provide first evidence that XIAP inhibitors present a novel strategy to trigger apoptosis in childhood leukemia and to prime acute leukemia cells for TRAIL- or chemotherapy-induced cell death. These data have important clinical implications for the development of apoptosis targeted therapies for childhood leukemia. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2911.

Evasion of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL), calling for new concepts to overcome apoptosis resistance. We therefore investigated whether targeting the antiapoptotic protein XIAP by small molecule inhibitors would sensitize for apoptosis. Here, we provide first evidence that XIAP inhibitors in combination with the death receptor ligand TRAIL present a new strategy to synergistically trigger apoptosis in CLL even in subgroups with resistant disease. Analysis of apoptosis regulatory proteins reveals that XIAP, cIAP1 and cIAP2 are expressed at high levels in primary CLL samples. Proofs of concept studies in CLL cell lines demonstrate that subtoxic concentrations of several distinct XIAP inhibitors significantly enhance TRAIL-induced apoptosis. By comparison, no sensitization for death receptor-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. Importantly also in primary CLL samples, subtoxic concentrations of XIAP inhibitors act in concert with TRAIL to trigger apoptosis in 18 of 27 cases (67%), whereas primary CLL cells are resistant to treatment with TRAIL alone. Analysis of combination index reveals that this interaction of XIAP inhibitor and TRAIL is highly synergistic. Mechanistic studies in primary CLL cells show that the addition of XIAP inhibitor profoundly enhances TRAIL-induced cleavage of caspase-3 into active fragments and significantly increases caspase-3 enzymatic activity. The broad range caspase inhibitor zVAD. fmk completely blocks apoptosis in response to combination treatment with XIAP inhibitor and TRAIL, pointing to caspase-dependent apoptosis. Intriguingly, the cooperative interaction of XIAP inhibitor and TRAIL is even evident in several distinct subgroups of CLL patients with poor prognostic features, including patients with 17p deletion, TP53 mutation, chemotherapy-refractory disease or unmutated VH genes. Interestingly, we found that cases with unmutated VH genes are significantly more sensitive to XIAP inhibitor- and TRAIL-induced apoptosis compared to VH gene mutated samples, pointing to a role of B-cell receptor signaling in the regulation of apoptosis in CLL cells. In conclusion, we provide first evidence that XIAP inhibitors in combination with TRAIL present a novel strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL. These findings have important implications for the development of innovative approaches to overcome the intrinsic resistance to apoptosis in CLL. Since IAP inhibitors as well as TRAIL receptor agonists as single agents are currently already under evaluation in early clinical trials, it is feasible that such combination protocols of XIAP inhibitors and TRAIL could be translated into clinical application in CLL. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4508.