A Novel Paradigm to Trigger Apoptosis in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in western societies (1). CLL is characterized by the abnormal accumulation of malignant monoclonal B cells, which has been largely attributed to defective apoptosis programs rather than aberrant proliferation (2–4). Indeed, the vast majority of clonal B cells of CLL are arrested at the G₀-G₁ phase of the cell cycle, with only a very small population that actively proliferates (5). One of the key factors that contribute to malignant B-cell longevity is the misbalance between prosurvival and prodeath molecules (2). Thus, new strategies to reactivate apoptosis may be crucial for the development of molecular targeted therapies in CLL.

Two principal pathways are involved in the initiation of apoptosis [i.e., the death receptor (extrinsic) pathway or the mitochondrial (intrinsic) pathway (6, 7)]. 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, which induces direct cleavage of downstream effector caspases, such as caspase-3 (8). In the mitochondrial pathway, the release of cytochrome c, second mitochondria-derived activator of caspase (Smac)/direct inhibitor of apoptosis (IAP) binding protein with low isoelectric point (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 and via Smac-mediated neutralization of caspase inhibition by IAP proteins (9).

The concept of inducing apoptosis in cancer cells by ligation of death receptors is of special interest for cancer therapy because death receptors are directly linked to the intrinsic death program of the cell (8). The death-inducing ligand TNF-related apoptosis-inducing ligand (TRAIL) is a prime candidate for clinical development because it has been reported to induce apoptosis in a wide spectrum of cancer cell lines with no or minimal toxicity on normal human cells (10). Recombinant TRAIL or fully human monoclonal antibodies that specifically target one of the two agonistic TRAIL receptors are currently evaluated in early clinical trials (10). However, many human tumors, including hematologic malignancies, are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility (8, 10–12). CLL cells have previously been reported to be inherently refractory to TRAIL-induced apoptosis despite the constitutive expression of the apoptosis-inducing TRAIL receptors (13–16). This highlights the demand to develop TRAIL-based combination therapies that counter resistance mechanisms of CLL cells toward TRAIL.

The molecular details that cause resistance of human cancers, including CLL, to TRAIL are still only partially understood (17). Increasing evidence suggests that high levels of IAP proteins may represent a key antiapoptotic mechanism in cancer cells (18, 19). Among the IAP family members, it is especially X-linked IAP (XIAP) that is known for its antiapoptotic function (20). Interestingly, high levels of XIAP were detected in CLL cells, possibly as part of a NF-κB–regulated survival program (20–22). Because XIAP blocks apoptosis at a central point via inhibition of caspases, there is currently much interest to exploit XIAP as a molecular target in human cancers (19). This has led to the development of small- molecule inhibitors or antisense strategies directed against XIAP (18). We previously showed proof-of-concept that Smac peptides that antagonize XIAP can be used to enhance TRAIL-induced killing in vitro and in vivo in a glioblastoma model (23). However, the question whether targeting XIAP is a suitable strategy to prime CLL cells for TRAIL-induced apoptosis has not yet been addressed. Therefore, we investigated the effect of small-molecule XIAP inhibitors on TRAIL-mediated antitumor activity in CLL in the present study.

CLL cell lines (MEC1, JVM2, and JVM3), which were established from the peripheral blood of patients with CLL by EBV transformation (24, 25), were obtained from the German Resource Centre for Biological Material and cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FCS (Life Technologies), 1 mmol/L glutamine (Life Technologies), 1% penicillin/streptavidin (Life Technologies), and 25 mmol/L HEPES (Biochrom). TRAIL was obtained from R&D Systems, Inc., and the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) was obtained from Bachem and used to determine the dependency of apoptosis on caspase activity. XIAP inhibitor 1, XIAP inhibitor 2, and control compound correspond to compounds 2, 11, and 15, respectively, described by Oost and colleagues (26), and XIAP inhibitors 3 and 4 were described by Chao and colleagues (27) and kindly provided by Idun Pharmaceuticals (now Pfizer, Inc.). The control compound, which is a close structural analogue that binds to the BIR3 domain of XIAP with a >100-fold lower affinity (26), was used to correlate the binding affinity of the XIAP inhibitors to the BIR3 domain of XIAP to their ability to induce apoptosis. All chemicals were purchased from Sigma unless indicated otherwise.

Western blot analysis was performed as described previously (28) using the following antibodies: mouse anti–caspase-8 (ApoTech Corp.), rabbit anti–caspase-3 (Cell Signaling), mouse anti-XIAP (BD), rabbit anti-Smac (MBL), rabbit anti–cellular IAP (cIAP) 2 (Epitomics), goat anti-cIAP1 and rabbit anti-survivin (R&D Systems), or mouse anti–β-actin as loading control (Sigma) followed by goat anti-mouse IgG or goat anti-rabbit IgG or donkey anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). Enhanced chemiluminescence was used for detection (Amersham Biosciences).

Caspase activity was determined in living nonfixed, nonlysed cells as described (29) using the following caspase substrates conjugated to rhodamine R110: N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone-R110 (zDEVD-R110) as caspase-3 substrate and N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone-R110 (zIETD-R110) as caspase-8 substrate (Molecular Probes).

Apoptosis was determined by fluorescence-activated cell sorting (FACS) analysis (FACScan, BD Biosciences) of DNA fragmentation of propidium iodide–stained nuclei or by forward/side scatter analysis as described previously (28, 30) or by Annexin V staining (Roche) according to the manufacturer's instructions. Light scatter analysis is a FACS-based method to detect apoptotic cells (31).

To determine surface receptor expression, cells were incubated with mouse anti-human TRAIL-R1, mouse anti-human TRAIL-R2, mouse anti-human TRAIL-R3, or mouse anti-human TRAIL-R4 (all from ApoTech) monoclonal antibodies for 30 min at 4°C, washed in PBS containing 1% FCS, incubated with rabbit anti-mouse F(ab′)₂ IgG/biotin (BD Biosciences) for 20 min at 4°C in the dark, washed in PBS containing 1% FCS, incubated with streptavidin-phycoerythrin (BD Biosciences) for 20 min at 4°C in the dark, and analyzed by flow cytometry.

A cohort of 27 CLL patients from our institution was analyzed after obtaining informed consent and approval of the Ethical Committee at Ulm University in accordance with the Declaration of Helsinki. Criteria for patient selection were a high lymphocyte count and a percentage of lymphocytes in excess of 80%. Clinical and genetic characterization was performed as previously described and is provided in Table 1 (32–34). Lymphocytes were isolated from blood samples with Biocoll solution and aliquots were frozen viably at −196°C. Primary B cells were isolated from buffy coats of healthy donors over the age of 58 y by Ficoll separation (Biochrom) and magnetic bead isolation using human CD19 Micro Beads (Miltenyi Biotec) and cultured in RPMI 1640 (Biochrom) supplemented with 10% FCS, 1% penicillin/streptomycin, and 1% glutamine.

Statistical significance was assessed by Student's t test or Fisher's exact test, where appropriate, using Winstat (R. Fitch Software) or Statistical Package for the Social Sciences (SPSS GmbH Software) software. Interaction between XIAP inhibitors and TRAIL was analyzed by the combination index (CI) method using CalcuSyn software (Biosoft). A CI of 0.3 to 0.7 indicates synergism and a CI of 0.1 to 0.3 indicates strong synergism.

In a first approach to investigate the role of XIAP in the regulation of apoptosis in CLL, we performed pilot experiments in CLL cell lines. Western blot analysis showed that the IAP proteins XIAP, cIAP1, cIAP2, and survivin as well as the endogenous IAP antagonists Smac/DIABLO and Omi/HtrA2 were all expressed in CLL cell lines (Fig. 1A , left). Next, we examined the effect of small-molecule XIAP inhibitors on apoptosis induction in CLL cell lines using two distinct small-molecule XIAP inhibitors that bind to the BIR3 domain of XIAP with high affinity at nanomolar concentrations (26). As control, we used a close structural analogue that weakly binds to the BIR3 domain of XIAP (26). Treatment with XIAP inhibitors as single agents caused no detectable increase in apoptosis in CLL cell lines (Supplementary Fig. S1), whereas we previously reported that they induce apoptosis in acute lymphoblastic leukemia cells at nanomolar to micromolar concentrations (29).

CLL cells have been described to be inherently resistant to the death receptor ligand TRAIL (13, 14), calling for strategies to enhance the efficacy of TRAIL in CLL. We therefore asked whether XIAP inhibitors could lower the threshold for TRAIL-triggered cell death in CLL cells. To address this question, we first examined expression of TRAIL receptors in CLL cell lines. Surface staining by flow cytometry showed high membrane expression of the agonistic TRAIL-R2 on all CLL cell lines, whereas TRAIL-R1 and the antagonistic TRAIL receptors TRAIL-R3 and TRAIL-R4 were expressed at much lower levels (Fig. 1A,, right). Importantly, nontoxic concentrations of XIAP inhibitors significantly enhanced TRAIL-induced apoptosis in a dose-dependent manner (Fig. 1B). They also significantly increased CD95-mediated apoptosis, whereas they did not alter the susceptibility toward the chemotherapeutic agents fludarabine or chlorambucil (Supplementary Fig. S2). Analysis of DNA fragmentation, one of the biochemical hallmarks of apoptosis, confirmed that XIAP inhibitor 2 in combination with TRAIL enhanced apoptotic cell death (Fig. 1C). Similarly, several structurally distinct XIAP inhibitors cooperated with TRAIL in a dose-dependent manner to trigger apoptosis (Fig. 1D). Monitoring of caspase activation by Western blotting showed that XIAP inhibitor 2 substantially enhanced TRAIL-induced cleavage of caspase-3 into active p17/12 cleavage fragments, whereas it had no detectable effect on the processing of caspase-8 (Fig. 2A). Analysis of caspase activity by enzymatic caspase assay similarly showed that XIAP inhibitor 2 significantly increased TRAIL-induced caspase-3 activity, whereas it did not further augment caspase-8 activity compared with treatment with TRAIL alone (Fig. 2B). These findings are in line with a model that inhibition of XIAP primarily promotes activation of effector caspases such as caspase-3 (20). Addition of the broad-range caspase inhibitor zVAD.fmk almost completely blocked apoptosis in response to the combined treatment with TRAIL and XIAP inhibitor 2 (Fig. 2C). Together, this set of experiments shows that XIAP inhibitors act in concert with TRAIL to induce caspase-3 activation and caspase-dependent apoptosis in CLL cell lines.

In a second approach, we extended our studies to primary CLL cells to validate the results obtained in cell lines. Patients' characteristics are summarized in Table 1. Western blot analysis showed that XIAP, cIAP1, cIAP2, and Smac were all expressed in primary CLL cells (Supplementary Fig. S3). By comparison, survivin was expressed at low or undetectable levels in primary CLL cells compared with the CLL cell line MEC1 (Supplementary Fig. S3), consistent with a previous study of low survivin expression in primary CLL cells (35). Importantly, XIAP inhibitor 2 acted in concert with TRAIL to induce apoptosis in 18 of 27 (67%) primary CLL samples (Fig. 3A; Table 1). Analysis of apoptosis by Annexin V staining yielded similar results compared with forward/side scatter analysis, confirming that apoptotic cell death was induced (compare patient 3 in Fig. 3A and B). Of note, the cooperative interaction of XIAP inhibitor 2 and TRAIL was even evident in patients with an unfavorable prognosis, for example, in all four patients with 17p deletion (patients 3, 14, 16, and 26; Fig. 3A; Table 1), the subgroup with the worst outcome (36). Furthermore, XIAP inhibitor 2 and TRAIL triggered apoptosis in patients with TP53 mutation (patients 3 and 14; Fig. 3A; Table 1), fludarabine-refractory disease (patients 3, 17, and 22; Fig. 3A; Table 1), and, strikingly, in almost all patients with unmutated V_H genes (patients 3, 4, 5, 6, 10, 13, 14, 16, 18, 20, 22, and 26; Fig. 3A; Table 1). Interestingly, cases with unmutated V_H genes were significantly more sensitive to XIAP inhibitor–triggered and TRAIL-triggered apoptosis compared with those with mutated V_H genes (P = 0.04, Fisher's exact test), although these patients bear a worse prognosis (36). Moreover, XIAP inhibitor 2 induced apoptosis also as single agent in several primary CLL samples, whereas treatment with TRAIL alone caused no or minimal apoptosis in CLL samples, with the exception of patient 26 (Fig. 3A). Although the control compound showed some apoptosis-inducing activity alone or in combination with TRAIL, this effect was much weaker compared with XIAP inhibitor 2 (Fig. 3A), consistent with the strongly reduced ability of the control compound to bind to the BIR3 domain of XIAP (26). One patient was studied at an interval of 6 months and no difference in the response to treatment with XIAP inhibitor and TRAIL was observed (Fig. 3A, patients 2a and 2b).

Moreover, we explored whether the interaction of XIAP inhibitor and TRAIL is additive or synergistic. Dose-response experiments and calculation of CI revealed a strong synergism of XIAP inhibitor 2 and TRAIL to induce apoptosis in primary CLL cells (Fig. 3C; Supplementary Fig. S4; CIs of <0.1 for 10 μmol/L XIAP inhibitor and 10 ng/mL TRAIL). To test the broader relevance of our findings, we extended our studies to an additional, structurally slightly different XIAP inhibitor, which also binds to the BIR3 domain of XIAP. XIAP inhibitor 4 similarly cooperated with TRAIL to induce apoptosis in primary CLL cells (Supplementary Fig. S5).

Furthermore, we assessed the effects of XIAP inhibitor and TRAIL on normal B cells that were isolated from healthy donors. Western blot analysis showed similar XIAP protein levels in nonmalignant B cells and CLL cells, whereas cIAP1 protein expression was substantially higher in CLL cells (Fig. 4A). XIAP inhibitors 2 and 4 increased TRAIL-induced apoptosis to some extent in normal B cells, although less than in most CLL samples (Fig. 4B, compare Fig. 3A and C).

Mechanistic studies revealed that XIAP inhibitor 2 acted in concert with TRAIL to trigger cleavage of caspase-3 into active fragments p17 and p12 (Fig. 5A). Additionally, we assessed caspase-3 activity by an enzymatic assay. XIAP inhibitor 2 cooperated with TRAIL to trigger caspase-3 activity in CLL samples that also underwent apoptosis on the combination treatment, whereas no increase in caspase activity was found in unresponsive cases (Fig. 5B; Supplementary Fig. S6), pointing to a link between caspase activation and apoptosis induction. Kinetic studies showed that caspase-3 activity was increased over several hours on treatment with XIAP inhibitor 2 and TRAIL compared with either treatment alone (Fig. 5C). To directly test the requirement of caspases for apoptosis induction, we used the broad-range caspase inhibitor zVAD.fmk. Addition of zVAD.fmk almost completely inhibited apoptosis on treatment with XIAP inhibitor 2 and TRAIL, showing that apoptosis occurred in a caspase-dependent manner (Fig. 5D; Supplementary Fig. S7). Of note, the cooperative effect of XIAP inhibitor 2 and TRAIL to trigger proteolytic activation of caspase-3, enhanced caspase-3 activity, and caspase-dependent apoptosis was even observed in a patient with 17 deletion (patient 16 in Fig. 5A–C), the subgroup with the worst prognosis (36), and occurred irrespective of high Bcl-2 expression in primary CLL cells (Supplementary Fig. S8). Together, this set of experiments shows that the XIAP inhibitor acts in concert with TRAIL to induce caspase-3 activation and caspase-dependent apoptosis in primary human CLL cells even in subgroups with unfavorable prognosis or resistant disease.

Defective apoptosis is an inherent feature of CLL and contributes to the accumulation of malignant monoclonal B cells as well as to treatment resistance (2, 3). Thus, novel approaches are required to restore apoptosis programs in CLL.

Here, we provide for the first time evidence that small-molecule XIAP inhibitors in combination with the death receptor ligand TRAIL present a promising new strategy to trigger apoptosis in CLL, including poor prognostic and chemotherapy-resistant subgroups. This conclusion is supported by data obtained in a large panel of primary CLL samples showing that the XIAP inhibitor cooperates with TRAIL to induce apoptosis in 18 of 27 (67%) primary CLL cases. The mechanistic basis for this cooperative interaction was found to involve increased caspase-3 activation and caspase-dependent cell death. This is consistent with the concept that inhibition of XIAP relieves the XIAP-imposed break on caspase-3 activation, which in turn promotes the effector phase of apoptosis and cell death execution (18, 19).

It is particularly worth to note that the cooperative interaction of XIAP inhibitor and TRAIL was observed in different subgroups of patients with unfavorable prognosis (36), including all four investigated patients with 17p deletion, the subgroup with the worst outcome (31), as well as cases with TP53 mutation, chemotherapy-resistant disease, or unmutated V_H genes. This suggests that the combination of XIAP inhibitor and TRAIL may be effective in at least some resistant forms of CLL. Because TRAIL has previously been described to trigger apoptosis independently of p53 (8), TRAIL in combination with XIAP inhibitors may present an alternative strategy to induce cell death in cases with TP53 mutation instead of DNA-damaging chemotherapeutics, which rely on intact p53 for cell death execution. Interestingly, our study reveals that CLL cases with unmutated V_H genes, a feature linked to poor prognosis (1), were significantly more sensitive to XIAP inhibitor–induced and TRAIL-induced apoptosis compared with those with mutated V_H genes. This finding is in line with the concept that CLL cells with unmutated V_H genes are more prone to respond not only to survival but also to apoptotic stimuli, and points to a role of B-cell receptor signaling in the regulation of apoptosis (1). We observed no differences in Bcl-2 expression or the phosphorylation status of Akt or extracellular signal-regulated kinase between cases with unmutated V_H genes, which responded to XIAP inhibitor and TRAIL, and nonresponsive cases with mutated V_H genes (data not shown). It therefore remains to be determined in future studies which signal tranduction events are involved in the regulation of apoptosis sensitivity in CLL cells with unmutated V_H genes. Nevertheless, the IgVH mutation status may serve as a biomarker to predict the responsiveness of CLL patients toward the combination therapy with XIAP inhibitor and TRAIL, a hypothesis to be confirmed in a larger group of CLL patients.

High expression of XIAP has previously been reported in CLL, possibly as part of a NF-κB–regulated survival program (20–22). In addition, XIAP has recently been implicated in apoptosis resistance that is imposed by constitutively activated Notch signaling in CLL cells (37), further indicating that XIAP may be a relevant target in CLL. However, the functional relevance of XIAP in the resistance of CLL toward TRAIL has not yet been explored. Thus, the present study is the first report that inhibition of XIAP by small molecules sensitizes CLL cells to TRAIL-induced apoptosis. Evasion of TRAIL-mediated apoptosis in CLL has previously been linked to low TRAIL receptor expression in one study (13), whereas constitutive expression of both TRAIL-R1 and TRAIL-R2 and no correlation between agonistic TRAIL receptors and the sensitivity of CLL cells to TRAIL were observed in other studies (14, 15). Further, high levels of the antiapoptotic death effector domain–containing proteins c-FLIP and PED were reported to interfere with activation of caspase-8 at the receptor signaling complex in CLL (13, 15, 16). Moreover, impaired mitochondrial signaling because of high expression levels of Bcl-2 has been implicated in apoptosis resistance in CLL (38, 39). To this end, it is important to note that the combination of XIAP inhibitor and TRAIL triggered apoptosis in primary CLL cells irrespective of high Bcl-2 levels, a characteristic feature of CLL (38, 39). These findings are in line with our recent results that XIAP inhibitors and TRAIL bypass Bcl-2–mediated resistance to TRAIL by directly triggering the effector phase of apoptosis (40).

Of note, our results also show that apoptosis sensitization by XIAP inhibitors is not restricted to TRAIL and extends to CD95 as another death receptor system. This suggests that the neutralization of XIAP more broadly enhances the susceptibility of CLL cells toward death receptor–mediated apoptosis. This notion is further supported by a report showing that phenylurea-based compounds that target the BIR2 domain of XIAP enhance the sensitivity of CD40-stimulated CLL cells to CD95-triggered apoptosis (41). Compared with death receptor stimulation via CD95 or TRAIL receptors, we observed no sensitization for apoptosis on treatment with commonly used chemotherapeutic agents such as fludarabine or chlorambucil. Along these lines, Silva and colleagues reported that the expression of XIAP, cIAP1, or cIAP2 in CLL cells did not correlate with sensitivity to fludarabine-induced apoptosis (42). Although the underlying mechanisms of this differential sensitization for death receptor–mediated versus chemotherapy-mediated cell death have not yet been identified, these findings indicate that inhibition of XIAP may be particularly promising to lower the threshold for death receptor–mediated apoptosis in CLL cells, which may also have implications for immune-mediated tumor control. Compared with CLL cells, nonmalignant B cells showed a lower susceptibility to the combined treatment with XIAP inhibitors and TRAIL. The molecular basis for this differential sensitivity is subject to future investigations.

Beyond CLL, XIAP inhibitors have recently been reported by our group and other investigators to act in concert with TRAIL to trigger apoptosis in childhood acute leukemia, pancreatic, colon, or breast carcinoma cells (29, 43, 44). In addition, low–molecular weight Smac mimetics displayed antitumor activities as single agents in multiple myeloma and several solid cancers (45–49). These results highlight the therapeutic potential of small-molecule XIAP inhibitors to promote apoptosis in cancer cells. Because IAP inhibitors and TRAIL receptor agonists are currently under evaluation in early clinical trials (10, 18), it is crucial to identify those cancers that may benefit from these new treatment options. As the present study indicates that the combination of XIAP inhibitors and TRAIL presents a new approach to bypass apoptosis resistance in CLL, our findings have important implications for the development of apoptosis-targeted therapies in CLL.

No potential conflicts of interest were disclosed.

Grant support: Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, European Union (ApopTrain, APO-SYS), IAP6/18 (S. Fulda), and Else-Kröner-Fresenius Stiftung (S. Stilgenbauer, T. Zenz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank C. Hulford and M. Luzzio (Pfizer, Inc., Groton, CT) for providing XIAP inhibitors.