Meike Vogler

Bcl-2 inhibitors: small molecules with a big impact on cancer therapy

Despite tremendous advances over the last 15 years in understanding fundamental mechanisms of apoptosis, this has failed to translate into improved cancer therapy for patients. However, there may now be light at the end of this long tunnel. Antiapoptotic Bcl-2 family members may be divided into two subclasses, one comprising Bcl-2, Bcl-XL and Bcl-w and the other Mcl-1 and Bcl2A1. Neutralization of both subclasses is required for apoptosis induction. Solution of the structure of antiapoptotic Bcl-2 family proteins has led to the design of novel small molecule inhibitors. Although many such molecules have been synthesized, rigorous verification of their specificity has often been lacking. Further studies have revealed that many putative Bcl-2 inhibitors are not specific and have other cellular targets, resulting in non-mechanism based toxicity. Two notable exceptions are ABT-737 and a related orally active derivative, ABT-263, which bind with high affinity to Bcl-2, Bcl-XL and Bcl-w and may prove to be useful tools for mechanistic studies. ABT-263 is in early clinical trials in lymphoid malignancies, small-cell lung cancer and chronic lymphocytic leukemia, and some patients have shown promising results. In in vitro studies, primary cells from patients with various B-cell malignancies are exquisitely sensitive to ABT-737, exhibiting novel morphological features of apoptosis including marked outer mitochondrial membrane rupture.

Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia

ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-X(L). ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC(50) approximately 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737-induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-X(L) and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737-related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.

Different forms of cell death induced by putative BCL2 inhibitors.

Several inhibitors of BCL2 proteins have been identified that induce apoptosis in a variety of tumor cells, indicating their potential in cancer therapy. We investigated the specificity of six putative BCL2 inhibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine and ABT-737). Using cells deficient either for Bax/Bak or caspase-9, we found that only ABT-737 specifically targeted BCL2 proteins and induced apoptosis by activation of caspase-9, as only ABT-737 induced apoptosis was completely inhibited in cells deficient for Bax/Bak or caspase-9. Our data show that only ABT-737 is a specific BCL2 inhibitor and all other compounds investigated were not specific for BCL2 proteins. Furthermore, investigations of the effects of these compounds in primary chronic lymphocytic leukemic cells showed that all compounds induced certain biochemical hallmarks of apoptosis, such as release of cytochrome c and caspase cleavage. However, they all caused strikingly different ultrastructural changes. ABT-737 induced all the characteristic ultrastructural changes of apoptosis together with early rupture of the outer mitochondrial membrane, whereas obatoclax, chlelerythrine and gossypol induced pronounced mitochondrial swelling with formation of phospholipid inclusions. Therefore, we conclude that biochemical measurements used earlier to define apoptosis like mitochondrial release of cytochrome c and caspase cleavage, are insufficient to distinguish between classic apoptosis and other forms of cell death.

Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL and cooperates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo.

Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.

Small Molecule XIAP Inhibitors Enhance TRAIL-Induced Apoptosis and Antitumor Activity in Preclinical Models of Pancreatic Carcinoma

Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer.

Regulation of TRAIL-induced apoptosis by XIAP in pancreatic carcinoma cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapy because of its relative tumor selectivity. However, many cancers including pancreatic cancer remain resistant towards TRAIL. To develop TRAIL for cancer therapy of pancreatic carcinoma, it will therefore be pivotal to elucidate the molecular mechanisms of TRAIL resistance. Here, we identify X-linked inhibitor of apoptosis (XIAP) as a regulator of TRAIL sensitivity in pancreatic carcinoma cells. Full activation of effector caspases, loss of mitochondrial membrane potential and cytochrome c release following TRAIL treatment were markedly impaired in pancreatic carcinoma cell lines, which poorly responded to TRAIL (PaTuII, PancTu1, ASPC1, DanG), compared to TRAIL-sensitive Colo357 pancreatic carcinoma cells. Stable downregulation of XIAP by RNA interference significantly reduced survival and enhanced TRAIL-induced apoptosis in pancreatic carcinoma cells. Also, downregulation of XIAP significantly increased CD95-induced cell death. Importantly, knockdown of XIAP strongly inhibited clonogenicity of pancreatic cancer cells treated with TRAIL indicating that XIAP promotes clonogenic survival of pancreatic carcinoma cells. Thus, our findings for the first time indicate that targeting XIAP represents a promising strategy to enhance the antitumor activity of TRAIL in pancreatic cancer, which has important clinical implications.

BCL2/BCL-X(L) inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation.

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

A novel paradigm for rapid ABT-737-induced apoptosis involving outer mitochondrial membrane rupture in primary leukemia and lymphoma cells

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.

Evaluation and critical assessment of putative MCL-1 inhibitors

High levels of BCL-2 family proteins are implicated in a failed/ineffective apoptotic programme, often resulting in diseases, including cancer. Owing to their potential as drug targets in cancer therapy, several inhibitors of BCL-2 family proteins have been developed. These primarily target specific members of the BCL-2 family, particularly BCL-2 and BCL-XL but are ineffective against MCL-1. Major efforts have been invested in developing inhibitors of MCL-1, which is commonly amplified in human tumours and associated with tumour relapse and chemoresistance. In this report, the specificity of several BCL-2 family inhibitors (ABT-263, UCB-1350883, apogossypol and BH3I-1) was investigated and compared with putative MCL-1 inhibitors designed to exhibit improved or selective binding affinities for MCL-1 (TW-37, BI97C1, BI97C10, BI112D1, compounds 6 and 7, and MCL-1 inhibitor molecule (MIM-1)). ABT-263, BI97C1, BI112D1, MIM-1 and TW-37 exhibited specificity in inducing apoptosis in a Bax/Bak- and caspase-9-dependent manner, whereas the other agents showed no killing activity, or little or no specificity. Of these inhibitors, only ABT-263 and UCB-1350883 induced apoptosis in a BCL-2- or BCL-XL-dependent system. In cells that depend on MCL-1 for survival, ABT-263 and TW-37 induced extensive apoptosis, suggesting that at high concentrations these inhibitors have the propensity to inhibit MCL-1 in a cellular context. TW-37 induced apoptosis, assessed by chromatin condensation, caspase processing and phosphatidylserine externalisation, in a BAK-dependent manner and in cells that require MCL-1 for survival. TW-37-mediated apoptosis was also partly dependent on NOXA, suggesting that derivatives of TW-37, if engineered to exhibit better selectivity and efficacy at low nanomolar concentrations, may provide useful lead compounds for further synthetic programmes. Expanded medicinal chemistry iteration, as performed for the ABT series, may likewise improve the potency and specificity of the evaluated MCL-1 inhibitors.

Sensitization for γ-Irradiation–Induced Apoptosis by Second Mitochondria-Derived Activator of Caspase

Resistance to current treatment regimens, such as radiation therapy, remains a major concern in oncology and may be caused by defects in apoptosis programs. Because inhibitor of apoptosis proteins (IAPs), which are expressed at high levels in many tumors, block apoptosis at the core of the apoptotic machinery by inhibiting caspases, therapeutic modulation of IAPs could target a key control point in resistance. Here, we report for the first time that full-length or mature second mitochondria-derived activator of caspase (Smac), an inhibitor of IAPs, significantly enhanced gamma-irradiation-induced apoptosis and reduced clonogenic survival in neuroblastoma, glioblastoma, or pancreatic carcinoma cells. Notably, Smac had no effect on DNA damage/DNA repair, activation of nuclear factor-kappaB, up-regulation of p53 and p21 proteins, or cell cycle arrest following gamma-irradiation, indicating that Smac did not alter the initial damage and/or cellular stress response. Smac enhanced activation of caspase-2, caspase-3, caspase-8, and caspase-9, loss of mitochondrial membrane potential, and cytochrome c release on gamma-irradiation. Inhibition of caspases also blocked gamma-irradiation-induced mitochondrial perturbations, indicating that Smac facilitated caspase activation, which in turn triggered a mitochondrial amplification loop. Interestingly, mitochondrial perturbations were completely blocked by the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or the relatively selective caspase-2 inhibitor N-benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone, whereas caspase-8 or caspase-3 inhibitors only inhibited the increased drop of mitochondrial membrane potential provided by Smac, suggesting that caspase-2 was acting upstream of mitochondria after gamma-irradiation. In conclusion, our findings provide evidence that targeting IAPs (e.g., by Smac agonists) is a promising strategy to enhance radiosensitivity in human cancers.