No abstract

Evasion of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL), calling for new strategies to bypass resistance. Here, we provide first evidence that small-molecule X-linked inhibitor of apoptosis (XIAP) inhibitors in combination with the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) present a novel approach to trigger apoptosis in CLL, including subgroups with resistant disease or unfavorable prognosis. XIAP, cellular IAP (cIAP) 1, and cIAP2 are expressed at high levels in primary CLL samples. Proof-of-concept studies in CLL cell lines show that subtoxic concentrations of XIAP inhibitors significantly enhance TRAIL-induced apoptosis and also sensitize for CD95-mediated apoptosis. Importantly also in primary CLL samples, XIAP inhibitor acts in concert with TRAIL to trigger apoptosis in 18 of 27 (67%) cases. This XIAP inhibitor-induced and TRAIL-induced apoptosis involves caspase-3 activation and is blocked by the caspase inhibitor zVAD.fmk. The cooperative interaction of XIAP inhibitor and TRAIL is even evident in distinct subgroups of patients with poor prognostic features (i.e., with 17p deletion, TP53 mutation, chemotherapy-refractory disease, or unmutated V H genes). Interestingly, cases with unmutated V H genes were significantly more sensitive to XIAP inhibitor-induced and TRAIL-induced apoptosis compared with V H gene-mutated samples, pointing to a role of B-cell receptor signaling in apoptosis regulation. By showing that XIAP inhibitors in combination with TRAIL present a new strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL, our findings have important implications for the development of apoptosis-based therapies in CLL. [Cancer Res 2009;69(23):8977-86]

Escape of apoptosis may contribute to treatment failure in childhood acute lymphoblastic leukemia (ALL) calling for new approaches to overcome apoptosis resistance. Here, we provide for the first time evidence that small molecule inhibitors that target the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) sensitize ALL cells for CD95-induced apoptosis. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, act synergistically with agonistic anti-CD95 antibodies or MegaFasL, a hexameric form of CD95 ligand, to induce apoptosis in ALL cells. Further, XIAP inhibitors co-operate with MegaFasL to reduce clonogenic survival of ALL cells demonstrating their effect also on long-term survival. In contrast, XIAP inhibitors show little effect on MegaFasL-mediated apoptosis in normal peripheral blood lymphocytes (PBLs), pointing to some tumor selectivity. Molecular studies reveal that XIAP inhibitors enhance CD95-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner. Importantly, XIAP inhibitors sensitize primary leukemic blasts from children with ALL for MegaFasL-induced apoptosis. Thus, small molecule XIAP inhibitors present a promising novel approach to enhance CD95-induced apoptosis in childhood acute leukemia.Acute lymphocytic leukemia (ALL) presents the most frequent malignancy of childhood. 1 Although the overall prognosis for children with ALL is good with a cure rate of 80%, the outcome of patients who relapse remains poor. 1,2 Thus, novel approaches are required to overcome resistance mechanisms in a subgroup of children with ALL. 3 Resistance to apoptosis (programmed cell death) is one of the hallmarks of human cancers including leukemia. 4-7 Apoptosis can be induced via two major pathways, i.e., the death receptor (extrinsic) pathway or the mitochondrial (extrinsic) pathway. 8,9 Ligation of death receptors of the tumor necrosis factor (TNF) receptor superfamily such as CD95 (APO-1/ Fas) by their cognate ligands or agonistic antibodies results in caspase-8 activation at the death inducing signaling complex (DISC) which induces direct cleavage of downstream effector caspases such as caspase-3. 10 In the mitochondrial pathway the release of apoptogenic factors such as cytochrome c, apoptosis inducing factor (AIF), second mitochondria-derived activator of caspase (Smac)/direct IAP binding protein with low pI (DIABLO) or Omi/high temperature requirement protein A2 (HtrA2) from mitochondria into the cytosol results in caspase-3 activation via formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex. 11 The failure of antileukemic therapy has been largely attributed to evasion of apoptosis, because many anticancer treatments including chemo-, radio-or immuno-therapy primarily act by triggering apoptotic cell death in cancer cells. 9 In principle, resistance to apoptosis may be caused by the lack or reduced function of pro-apoptotic molecules or alternatively, by the dominance o...

731 Chronic lymphocytic leukemia (CLL) is characterized by the abnormal accumulation of malignant monoclonal B cells, which has been largely attributed to defective apoptosis rather than aberrant proliferation. This calls for new strategies to re-activate apoptosis programs in CLL in order to develop new therapeutic strategies. “Inhibitor of Apoptosis” (IAP) proteins such as XIAP are aberrantly expressed in many human cancers and block apoptosis at a key node by inhibiting activation of caspases. In the present study, we therefore explored whether targeting XIAP is a suitable strategy to overcome apoptosis resistance of CLL. Here, we provide first evidence that small molecule XIAP inhibitors in combination with the death receptor ligand TRAIL present a novel approach to 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. In addition, XIAP inhibitors sensitize CLL cells for CD95-mediated apoptosis, whereas they have no effect on fludarabine- or chlorambucil-induced apoptosis. This indicates that XIAP inhibitors in particular enhance death receptor-triggered apoptosis in CLL cells. 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, XIAP inhibitors act in concert with TRAIL to trigger apoptosis in 18 of 27 cases (67%). 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 upon treatment with TRAIL. The broad range caspase inhibitor zVAD.fmk completely blocks apoptosis in response to combination treatment with XIAP inhibitor and TRAIL, demonstrating that apoptosis occurs in a caspase-dependent manner. Importantly, the cooperative interaction of XIAP inhibitor and TRAIL is even evident in distinct subgroups of patients with poor prognostic features, including patients with 17p deletion, TP53 mutation, chemotherapy-refractory disease or unmutated VH genes. This suggests that the combination treatment with XIAP inhibitor and TRAIL may present a novel approach to trigger apoptosis in CLL patients that are resistant to other treatment options. 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. This points to a role of B-cell receptor signaling in the regulation of apoptosis in CLL cells. In conclusion, we demonstrate for the first time that XIAP inhibitors in combination with TRAIL present a new strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL. These findings have important implications for the development of novel strategies 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. Disclosures: No relevant conflicts of interest to declare.