Radiosensitization by a novel Bcl-2 and Bcl-XL inhibitor S44563 in small-cell lung cancer

Radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using the Bcl-2/Bcl-X_L inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 caused SCLC cells to acquire hallmarks of apoptosis. S44563 markedly enhanced the sensitivity of SCLC cells to radiation, as determined by a clonogenic assay. The combination of S44563 and cisplatin-based chemo-radiation showed a significant tumor growth delay and increased overall survival in mouse xenograft models. This positive interaction was greater when S44563 was given after the completion of the radiation, which might be explained by the radiation-induced overexpression of anti-apoptotic proteins secondary to activation of the NF-κB pathway. These data underline the possibility of combining IR and Bcl-2/Bcl-X_L inhibition in the treatment of SCLC as they underscore the importance of administering conventional and targeted therapies in an optimal sequence.Identifying the mechanisms leading to radioresistance including resistance to apoptosis is essential to improve clinical outcome in cancer patients. Disabled apoptosis has been catalogued among the fundamental hallmarks of cancer¹ and the proteins of the Bcl-2 family play a fundamental role in regulating this modality of cell death. The Bcl-2 family comprises both pro- and anti-apoptotic members; the latter (Bcl-2, Bcl-X_L and Mcl-1) are often overexpressed in cancer cells to facilitate the survival of cells that under normal circumstances should have undergone apoptosis.² The molecular interactions between pro- and anti-apoptotic Bcl-2 family members determine cellular sensitivity to multiple lethal triggers, including many standard chemotherapeutic agents and ionizing radiation (IR).^{3, 4} Overexpression of Bcl-2 is known to increase clonogenic survival and inhibit IR-induced apoptosis.^{3, 4} Bcl-X_L expression also shows a strong correlation with resistance to cytotoxic anticancer therapies including IR.^{5, 6}Lung cancer is the leading cause of cancer deaths in western countries.⁷ Small-cell lung cancer (SCLC) accounts for 15% of all lung cancer cases and is distinguished from non-SCLC by its characteristic cytomorphology, rapid proliferation and early dissemination to metastatic sites.⁸ The standard of care to patients with limited-stage SCLC and good performance status is based on a combination of IR and cisplatin-based chemotherapy, resulting in a complete response rate as high as 50–80% coupled to a deceptive 12–20% 5-year survival.⁹ Initially, SCLC is responsive to chemo- and radiotherapy. However, SCLC recurs within the first 12 months.¹⁰ To date, the pathways mediating chemo- and radioresistance in SCLC are largely unknown.Deletion of pro-apoptotic gene and amplification of anti-apoptotic gene are frequently observed in SCLC, especially amplification of the BCL2L1 and BCL2L2 genes.¹¹ At the protein level, increased expression of Bcl-2 has been reported in up to 90% of metastatic SCLC. Bcl-2 overexpression, downregulation of the pro-apoptotic Bcl-2 antagonist Bax and a shift in the Bcl-2/Bax ratio to levels >1 are correlated with lower apoptotic index in tumors¹² and are associated with chemotherapeutic resistance in SCLC cell lines.¹³ In contrast with most solid tumor cell lines, where apoptosis does not appear as a predominant cell death mechanism after IR,¹⁴ overexpression of Bcl-2 can abrogate chemotherapy-induced apoptosis in SCLC cell lines.¹³ Apoptosis may be one of the mechanisms that cause SCLC cells to die in response to radiotherapy.^{15, 16}Recently, a small synthetic compound ABT-737 and its orally bioavailable form ABT-263 (Navitoclax) were shown to efficiently antagonize Bcl-2 and Bcl-X_L by binding to their BH3 receptor domain. ABT737 or its derivatives mediate antitumoral effects in chronic lymphocytic leukemia (CLL) and SCLC in preclinical and early clinical trials.^{17, 18} However, there is no published study that evaluates the combination of new Bcl-2/Bcl-X_L inhibitors, IR and chemo-radiotherapy.     

Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263

Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP₃ receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP₃Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2–IP₃R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2.Lung cancer accounts for 12% of all new cancers worldwide and is a leading cause of cancer-related mortality in the United States.^{1, 2, 3} Although small cell lung cancer (SCLC) comprises only 15% of lung cancer cases,^{2, 3} it has an annual mortality rate approaching that of breast and prostate cancer.⁴ Compared with the more common non-small cell lung cancer (NSCLC), SCLC is more aggressive and associated with rapid development of metastasis.² Moreover, although SCLC is more responsive to chemotherapy and radiation therapy initially, it typically relapses quickly with treatment-resistant disease.² In contrast to dramatic advances in chemotherapy and personalized medicine in other malignancies, the life expectancy of SCLC patients has remained <2 years for decades and is <1 year for patients with extensive disease.^{5, 6} The lethality of SCLC is attributed in part to the development of resistance to standard combination chemotherapies, underscoring the need to develop novel therapeutic approaches based on understanding the molecular and cellular biology of SCLC.^{5, 6}Evasion from apoptosis is a major hallmark of cancer and a prominent factor underlying drug resistance in SCLC.³ Multiple mechanisms contribute to apoptosis resistance in SCLC, including elevated expression of the antiapoptotic Bcl-2 protein³ (Supplementary Figure S1). Tsujimoto and colleagues discovered elevated levels of Bcl-2 mRNA and protein in SCLC cells not long after their identification of Bcl-2 as the protein product of the bcl-2 gene in follicular lymphoma.^{7, 8} Subsequently, immunohistochemistry of 164 primary SCLC samples revealed 76% were positive for Bcl-2, a finding substantiated by microarray detection of increased BCL-2 mRNA levels in 84% of SCLC samples^{9, 10} and by genomic sequencing of circulating SCLC tumor cells.¹¹ Moreover, proteomic profiling documented that Bcl-2 is more highly expressed in SCLC than in NSCLC, reflecting the vastly different biology of these lung cancer subtypes.¹²The major known function of Bcl-2 is to bind and sequester BH3-only proteins such as Bim, preventing these proteins from inducing apoptosis.^{13, 14, 15} Therefore, a major investment has been made in targeting this interaction for cancer treatment. The interaction takes place in a hydrophobic groove on Bcl-2 and the therapeutic strategy for targeting this interaction has been to develop small molecules, BH3-mimetic agents, which bind in the hydrophobic groove and induce apoptosis by displacing the BH3-only proteins. This approach has been reviewed in detail.^{14, 15, 16}Among BH3-mimetic agents advancing through clinical trials for both hematological malignancies^{15, 17} and solid tumors¹⁸ are ABT-737 and its orally bioavailable derivative ABT-263 (Navitoclax). Reported studies of ABT-199, a selective inhibitor of Bcl-2, are at present limited to hematological malignancies.¹⁸ In screening a large number of cancer cell lines, the pioneering work of Oltersdorf et al.¹⁹ demonstrated potent single-agent activity of ABT-737 against cell lines representative of lymphoid malignancies and SCLC. Clinical trials of ABT-263, an orally bioavailable version of ABT-737, achieved overall response rates ranging from as high as 35% in relapsed/refractory chronic lymphocytic leukemia (CLL) and 22% in follicular lymphoma.¹⁷ Reported responses are generally less in solid tumors with the notable exception of SCLC.¹⁸ But even in SCLC, activity of ABT-263 is limited in comparison to hematological malignancies, with 1 of the 39 (3%) of patients achieving a partial response to ABT-263 and 9 of the 37 (23%) achieving stable disease in a phase I clinical trial.²⁰ This experience suggests a need to develop additional ways of targeting Bcl-2 for cancer treatment.A potential alternative therapeutic target for Bcl-2-positive malignancies involves interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP₃R), an IP₃-gated Ca²⁺ channel located on the endoplasmic reticulum (ER). Bcl-2 is located not only on the outer mitochondrial membrane but also on the ER, and at both of these locations, it functions as a potent inhibitor of apoptosis.^{21, 22, 23} ER-localized Bcl-2 interacts with IP₃Rs and inhibits apoptosis by preventing excessive IP₃R-mediated Ca²⁺ transfer from the ER lumen into the cytoplasm and nearby mitochondria.^{24, 25, 26} Notably, regions of Bcl-2 involved in binding BH3-only proteins and IP₃Rs are entirely different. Whereas BH3-only proteins and their BH3-mimetic counterparts bind in a hydrophobic groove composed of BH3 domains 1–3 of Bcl-2,^{13, 14} the BH4 domain of Bcl-2 is necessary for interaction with IP₃Rs.²⁷ To develop a peptide inhibitor of Bcl-2–IP₃R interaction, we identified the Bcl-2-binding region on the IP₃R and developed a small synthetic 20 amino-acid peptide corresponding to this region.²⁸ This peptide, when fused to the cell-penetrating peptide of HIV TAT, binds to the BH4 domain of Bcl-2 and functions as a decoy peptide, inhibiting Bcl-2–IP₃R interaction.^{29, 30} We currently refer to this peptide as BIRD-2 (Bcl-2-IP₃ Receptor Disruptor-2), having formerly named it TAT-IDP_DD/AA.³¹ By disrupting the Bcl-2–IP₃R interaction, BIRD-2 abrogates Bcl-2 control over IP₃R-mediated Ca²⁺ elevation and induces Ca²⁺-mediated apoptosis in primary human CLL cells²⁹ and diffuse large B-cell lymphoma cells.³² Notably, BIRD-2 does not kill normal cells, including human lymphocytes isolated from peripheral blood²⁹ and normal murine embryonic fibroblasts (F Zhong and C Distelhorst, unpublished data).The present investigation was undertaken to determine whether Bcl-2–IP₃R interaction is a potentially useful therapeutic target in SCLC. In support of this concept, we find the majority of SCLC lines tested are sensitive to BIRD-2-induced apoptosis and that BIRD-2 induces apoptosis in several ABT-263-resistant SCLC lines. BIRD-2, we find, lacks generalized cytotoxicity as it does not induce cell death in NSCLC lines or a normal lung epithelial line. On the other hand, we find that BIRD-2 and ABT-263 synergize in killing SCLC cells. These findings for the first time provide preclinical evidence of the potential value of targeting both antiapoptotic mechanisms of Bcl-2 for the treatment of SCLC.     

Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.Colorectal cancer is the third most common cancer worldwide.^{1, 2} Patients with advanced stage colorectal cancer are routinely treated with 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX), or with 5-FU, leucovorin and irinotecan (FOLFIRI), often in combination with targeted agents such as anti-VEGF or anti-EGFR at metastatic disease.^{3, 4, 5, 6} Despite this intensive treatment, therapy is still insufficiently effective and chemotherapy resistance occurs frequently. Although still speculative, it has been suggested that unequal sensitivity to chemotherapy is due to an intratumoral heterogeneity that is orchestrated by cancer stem cells (CSCs) that can self-renew and give rise to more differentiated progeny.^{7, 8} When isolated from patients, CSCs efficiently form tumors upon xenotransplantation into mice which resemble the primary tumor from which they originated.^{9, 10, 11} In addition, many xenotransplantation studies have emphasized the importance of CSCs for tumor growth.^{9, 10, 11, 12} Colon CSCs were originally isolated from primary human colorectal tumor specimens using CD133 as cell surface marker and shown to be highly tumorigenic when compared with the non-CSCs population within a tumor.^{9, 10} Later, other cell surface markers as well as the activity of the Wnt pathway have been used to isolate colon CSCs from tumors.^{12, 13} Spheroid cultures have been established from human primary colorectal tumors that selectively enrich for the growth of colon CSCs,^{11, 12} although it is important to realize that these spheres also contain more differentiated tumor cells.¹² In agreement, we have shown that the Wnt activity reporter that directs the expression of enhanced green fluorescent protein (TOP-GFP) allows for the separation of CSCs from more differentiated progeny in the spheroid cultures.¹²CSCs are suggested to be responsible for tumor recurrence after initial therapy, as they are considered to be selectively resistant to therapy.^{11, 14} Conventional chemotherapy induces, among others, DNA damage and subsequent activation of the mitochondrial cell death pathway, which is regulated by a balance between pro- and anti-apoptotic BCL2 family members.¹⁵ Upon activation of apoptosis, pro-apoptotic BH3 molecules are activated and these may perturb the balance in favor of apoptosis initiated by mitochondrial outer membrane polarization (MOMP), release of cytochrome c and subsequent activation of a caspase cascade.The apoptotic balance of cancer cells can be measured with the use of a functional assay called BH3 profiling.¹⁶ BH3 profiling is a method to determine the apoptotic ‘priming'' level of a cell by exposing mitochondria to standardized amounts of roughly 20-mer peptides derived from the alpha-helical BH3 domains of BH3-only proteins and determining the rate of mitochondrial depolarization. Using this approach, priming was measured in various cancers and compared with normal tissues.^{17, 18} In all cancer types tested, the mitochondrial priming correlated well with the observed clinical response to chemotherapy. That is, cancers that are highly primed are more chemosensitive, whereas chemoresistant tumor cells and normal tissues are poorly primed.^{17, 18} This suggests that increasing mitochondrial priming can potentially increase chemosensitivity, which can be achieved by directly inhibiting the anti-apoptotic BCL2 family members.¹⁸ To this end, small-molecule inhibitors, so-called BH3 mimetics, have been developed. ABT-737 and the highly related ABT-263 both inhibit BCL2, BCLXL and BCLW^{19, 20, 21} and were shown to be effective in killing cancer cells in vitro and in vivo²¹ with a preference for BCL2.^{19, 22} As BCL2 protein expression is often upregulated in hematopoietic cancers, it represents a promising target, which was supported by high efficacy of these BH3 mimetics in animal experiments.²¹ However, in vivo efficacy is limited due to thrombocytopenia, which relates to a dependence of platelets on BCLXL for survival.^{23, 24} To overcome this toxicity, a BCL2-specific compound, ABT-199, was developed.²⁵ Souers et al.²⁵ showed that inhibition of BCL2 using ABT-199 blocks tumor growth of acute lymphoblastic leukemia cells in xenografts. In addition to the single compound effects of ABT-199, combination with rituximab inhibited growth of non-Hodgkin''s lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia tumor cells growth in vivo.²⁵ Moreover, highly effective tumor lysis was observed in all three patients with chronic lymphocytic leukemia that were treated with ABT-199.²⁵ More recently, a BCLXL-specific compound, WEHI-539, was discovered using high-throughput chemical screening.²⁶As the apoptotic balance appears a useful target for the treatment of cancers and CSCs have been suggested to resist therapy selectively, we set out to analyze whether specifically colon CSCs are resistant to therapy and whether this is due to an enhanced anti-apoptotic threshold, specific to CSCs. To study chemosensitivity, we developed a robust single cell-based analysis in which we can measure apoptosis simultaneously in CSCs and their differentiated progeny. Utilizing this system we showed that colon CSCs and not their differentiated progeny are resistant to chemotherapeutic compounds and that this was due to the fact that these cells are less primed to mitochondrial death. Furthermore, inhibition of anti-apoptotic BCLXL molecule with either ABT-737 or WEHI-539, but not ABT-199, breaks this resistance and sensitizes the CSCs to chemotherapy.     

BCL-2 is dispensable for thrombopoiesis and platelet survival

Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-X_L and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-X_L for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-X_L. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.Platelets are anucleate blood cells that play essential roles in haemostasis, wound healing and a range of other processes, including inflammation and immunity.¹ They are produced by megakaryocytes, large polyploid cells that develop primarily in the bone marrow, spleen and foetal liver.² Recent work has demonstrated that the survival of megakaryocytes and platelets is governed by the BCL-2 family proteins.³ Both cell types possess a classical BAK/BAX-mediated intrinsic apoptosis pathway that must be restrained in order for them to develop and survive.In platelets, BCL-X_L is the critical pro-survival BCL-2 family member required to keep BAK and BAX in check. The first evidence of this came from Wagner et al.,⁴ who reported severe thrombocytopaenia in mice after MMTV-Cre-mediated deletion of Bclx in the haematopoietic system, skin and various secretory tissues. It has since been shown that megakaryocyte-restricted deletion of Bclx in mice reduces platelet lifespan from ~5 days to ~5 h, with a concomitant decrease in platelet counts to ~2% of wild-type levels.^{5, 6} Pharmacological inhibition of BCL-X_L with the BH3 mimetics ABT-737⁷ or Navitoclax (ABT-263)⁸ (which both also inhibit BCL-2 and BCL-W) triggers BAK/BAX-mediated platelet apoptosis.^{9, 10, 11} As a result, these drugs cause dose-dependent thrombocytopaenia in mice, dogs and humans.^{9, 11, 12, 13, 14} Indeed, thrombocytopaenia is the dose-limiting toxicity for Navitoclax.^{12, 13, 14} This fact provided additional impetus for the development of agents that specifically target BCL-2, beginning with ABT-199,¹⁵ a BCL-2-selective antagonist currently in clinical trials for the treatment of a range of haematological malignancies including chronic lymphocytic leukaemia, non-Hodgkin''s lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma and acute myeloid leukaemia. ABT-199 has already shown very promising anti-tumour activity, with little to no impact on platelet counts.^{15, 16} These data suggest that BCL-2 is dispensable for the development and survival of platelets.In megakaryocytes, BCL-X_L is also critical for survival. Although not absolutely required for their growth and maturation, BCL-X_L is essential for megakaryocytes to proceed safely through pro-platelet formation and platelet shedding.⁵ In addition to BCL-X_L, megakaryocytes also depend on the pro-survival activity of MCL-1. Conditional deletion of Mcl1 alone has no effect on this lineage. In contrast, combined megakaryocyte-specific loss of Bclx and Mcl1 results in the failure of megakaryopoiesis, systemic haemorrhage and embryonic lethality.^{5, 17, 18} These defects are rescued by deletion of Bak and Bax.¹⁸Consistent with the genetic studies, administration of ABT-737 to Mcl1^Pf4Δ/Pf4Δ mice, which lack MCL-1 in megakaryocytes and platelets, induces acute, fulminant BAK/BAX-dependent megakaryocyte apoptosis. Given that, in addition to BCL-X_L, ABT-737 also targets BCL-2,⁷ these data suggested that BCL-2 might also contribute to the development and survival of the megakaryocyte lineage. This is supported by recent studies demonstrating that neonatal human platelets contain increased levels of BCL-2 relative to adult counterparts,¹⁹ and that platelet lifespan is extended in transgenic mice expressing BCL-2 under the control of the pan-haematopoietic Vav promoter.²⁰ In light of these observations, and intense ongoing activity surrounding the development of novel BH3 mimetics,²¹ we set out to elucidate the role of BCL-2 in megakaryocytes and platelets. Mice with a megakaryocyte-specific deletion of Bcl2, either alone or in combination with deletion of Mcl1 or Bclx, were generated. The effect of these mutations, and of BCL-2 or BCL-X_L-selective BH3 mimetics, on the megakaryocyte lineage was assessed.     

Mitochondrial inhibitor sensitizes non-small-cell lung carcinoma cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-XL/p53-mediated amplification mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-X_L overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-X_L downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-X_L and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-X_L and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising cancer therapeutic because it can selectively induce apoptosis in tumor cells in vitro, and most importantly, in vivo with little adverse effect on normal cells.¹ However, a number of cancer cells are resistant to TRAIL, especially highly malignant tumors such as lung cancer.^{2, 3} Lung cancer, especially the non-small-cell lung carcinoma (NSCLC) constitutes a heavy threat to human life. Presently, the morbidity and mortality of NSCLC has markedly increased in the past decade,⁴ which highlights the need for more effective treatment strategies.TRAIL has been shown to interact with five receptors, including the death receptors 4 and 5 (DR4 and DR5), the decoy receptors DcR1 and DcR2, and osteoprotegerin.⁵ Ligation of TRAIL to DR4 or DR5 allows for the recruitment of Fas-associated protein with death domain (FADD), which leads to the formation of death-inducing signaling complex (DISC) and the subsequent activation of caspase-8/10.⁶ The effector caspase-3 is activated by caspase-8, which cleaves numerous regulatory and structural proteins resulting in cell apoptosis. Caspase-8 can also cleave the Bcl-2 inhibitory BH3-domain protein (Bid), which engages the intrinsic apoptotic pathway by binding to Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (BAK). The oligomerization between Bcl-2 and Bax promotes the release of cytochrome c from mitochondria to cytosol, and facilitates the formation of apoptosome and caspase-9 activation.⁷ Like caspase-8, caspase-9 can also activate caspase-3 and initiate cell apoptosis. Besides apoptosis-inducing molecules, several apoptosis-inhibitory proteins also exist and have function even when apoptosis program is initiated. For example, cellular FLICE-like inhibitory protein (c-FLIP) is able to suppress DISC formation and apoptosis induction by sequestering FADD.^{8, 9, 10, 11}Until now, the recognized causes of TRAIL resistance include differential expression of death receptors, constitutively active AKT and NF-κB,^{12, 13} overexpression of c-FLIP and IAPs, mutations in Bax and BAK gene.² Hence, resistance can be overcome by the use of sensitizing agents that modify the deregulated death receptor expression and/or apoptosis signaling pathways in cancer cells.⁵ Many sensitizing agents have been developed in a variety of tumor cell models.² Although the clinical effectiveness of these agents needs further investigation, treatment of TRAIL-resistant tumor cells with sensitizing agents, especially the compounds with low molecular weight, as well as prolonged plasma half-life represents a promising trend for cancer therapy.Mitochondria emerge as intriguing targets for cancer therapy. Metabolic changes affecting mitochondria function inside cancer cells endow these cells with distinctive properties and survival advantage worthy of drug targeting, mitochondria-targeting drugs offer substantial promise as clinical treatment with minimal side effects.^{14, 15, 16} Rotenone is a potent inhibitor of NADH oxidoreductase in complex I, which demonstrates anti-neoplastic activity on a variety of cancer cells.^{17, 18, 19, 20, 21} However, the neurotoxicity of rotenone limits its potential application in cancer therapy. To avoid it, rotenone was effectively used in combination with other chemotherapeutic drugs to kill cancerous cells.²²In our previous investigation, we found that rotenone was able to suppress membrane Na⁺,K⁺-ATPase activity and enhance ouabain-induced cancer cell death.²³ Given these facts, we wonder whether rotenone may also be used as a sensitizing agent that can restore the susceptibility of NSCLC cells toward TRAIL-induced apoptosis, and increase the antitumor efficacy of TRAIL on NSCLC. To test this hypothesis, we initiated this study.     

Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca²⁺ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca²⁺-dependent ROS production.The Bcl-2 proteins are a family of molecules comprised of both pro- and antiapoptotic members essential for the regulation of apoptotic cell death. In the classical paradigm, the antiapoptotic proteins Bcl-2, Bcl-x_L and Mcl-1, inhibit cell death during receipt of apoptotic stimuli by binding and sequestering the proapoptotic members.¹ It is now appreciated, however, that in the absence of apoptotic stimuli, Bcl-2 proteins have numerous non-canonical interactions that influence diverse cellular functions, although the precise mechanisms are poorly understood.² Since antiapoptotic Bcl-2 family members are frequently upregulated in cancer, determining if and how these non-canonical interactions confer survival or other advantages to the cancer cell, will be an important step toward identifying new therapeutic targets. One such interaction is with the outer mitochondrial membrane-localized voltage-dependent anion channel (VDAC), a porin channel with three isoforms that serves as a major diffusion pathway for ions and metabolites,³ and whose gating properties are affected by either Bcl-2 or Bcl-x_L binding.^{4, 5, 6}We recently identified an important role for Bcl-x_L/VDAC interactions in the regulation of mitochondrial [Ca²⁺].⁷ Moving Ca²⁺ from the cytoplasm to the mitochondrial matrix requires transfer across the outer membrane by VDAC^3,8 and across the inner membrane by the Ca²⁺ uniporter.⁹ Our studies showed that Bcl-x_L interacts with VDAC to facilitate Ca²⁺ uptake into the mitochondrial matrix. It is not known if other Bcl-2 family members, particularly Bcl-2 and Mcl-1, which are also known VDAC binding partners impart the same physiological regulation on mitochondrial [Ca²⁺]. Furthermore, the specific physiological consequences and significance of this regulation remain to be determined.Increased production and reduced scavenging of reactive oxygen species (ROS) is frequently observed in cancer cells.¹⁰ While excessive ROS levels are toxic, sub-lethal production serves an important signaling function, particularly in cancers, were ROS promote cell proliferation, migration and invasion.^{11, 12, 13, 14, 15} A primary source of ROS are the mitochondria, and a number of mitochondrial signaling pathways are known to be remodeled and contribute to elevated ROS in cancer cells, including those involved in regulating the electron transport chain (ETC) function and metabolic activity.^{11,16, 17, 18} It is recognized that upregulation of antiapoptotic Bcl-2 proteins are also associated with a pro-oxidant intracellular environment.^{19, 20, 21, 22} Mechanistically, they are thought to act at the level of the mitochondria to affect the respiratory chain and increase production of ROS. Since matrix [Ca²⁺] is an important regulator of mitochondrial metabolism,^23,24 and as such, contributes to the regulation of mitochondrial ROS production,²⁵ we reasoned that antiapoptotic Mcl-1/VDAC interactions could promote ROS generation by facilitating matrix Ca²⁺ uptake.Understanding non-canonical roles of Mcl-1 is an important step toward identifying novel therapeutic targets, particularly in cancers where it is highly expressed, such as in non-small cell lung cancer (NSCLC).^26,27 Therefore, we hypothesized that Mcl-1 binding to VDAC promotes mitochondrial Ca²⁺ uptake and ROS production in NSCLC cells and that this is essential in maintaining the cancer cell phenotype. To test this, we assessed the biochemical interaction between Mcl-1 and VDAC and examined the effects of manipulating Mcl-1 expression levels and Mcl-1/VDAC interactions on mitochondrial Ca²⁺ uptake, ROS generation and NSCLC cell proliferation and migration.     

The ratio of Mcl-1 and Noxa determines ABT737 resistance in squamous cell carcinoma of the skin

Tumour progression and therapy resistance in squamous cell carcinoma of the skin (SCC) is strongly associated with resistance to intrinsic mitochondrial apoptosis. We thus investigated the role of various anti-apoptotic Bcl-2 proteins for apoptosis protection in SCC using the BH3 agonist ABT737 that can overcome multidomain Bcl-2 protein protection. Sensitive SCC cells underwent rapid loss of mitochondrial membrane potential (MMP), subsequent apoptosis concomitant with caspase-3 activation and an early release of mitochondria-derived cytochrome c and smac/DIABLO. In contrast, ABT737 resistance in subsets of SCC cells was not explained by XIAP, important for protection from DR-induced apoptosis in SCC. Of note, ABT737 did not prime SCC cells to DR-induced apoptosis. Interestingly, the ratio of Mcl-1 and Noxa determined sensitivity to ABT737: loss of Mcl-1 rendered resistant cells sensitive to ABT737, whereas loss of Noxa promoted resistance in sensitive cells. In line, suppression of Mcl-1 by the pan-Bcl-2 inhibitor Obatoclax or overexpression of Noxa rendered resistant SCC cells sensitive to BH3 mimetics. Our data indicate that targeting of the Mcl-1/Noxa axis is important to overcome resistance to mitochondrial apoptosis in SCC. Therefore, combination treatment of ABT737 or derivatives with Mcl-1 inhibitors, or inducers of Noxa, may represent a novel option of targeted therapy in metastatic SCC of the skin.Apoptosis is an indispensible process to maintain cellular homeostasis, in particular in highly dynamic tissues. Apoptosis can be induced by activation of death receptors (DRs; such as TRAIL-R1/R2 or cluster of differentiation 95 (CD95)) or by intrinsic disturbance of mitochondria.¹ Death ligands (DLs; TNF-related apoptosis-inducing ligand (TRAIL) or CD95L), when bound to their respective DRs, induce apoptosis by activation of procaspase-8 within the death-inducing signalling complex (DISC).² Caspase-8 activation is followed by proteolytic cleavage of caspase-3.³ Extrinsic and intrinsic cell death is negatively controlled by caspase inhibitors such as X-linked inhibitor of apoptosis protein (XIAP)⁴ or by B-cell lymphoma 2 (Bcl-2) proteins that suppress the mitochondria outer membrane permeability (MOMP) by limiting Bax (Bcl-2-associated X protein)/Bak (Bcl-2 homologous antagonist/killer) translocation into the mitochondrial outer membrane.⁵ The extrinsic signalling cascade communicates with the intrinsic death pathway by cleavage of Bid (BH3 interacting-domain death agonist), a pro-apoptotic member of the BH3 (Bcl-2 homology domain 3)-only subfamily of Bcl-2 proteins.¹ Other stimuli such as genotoxic stress allow for translocation and pore formation of pro-apoptotic multidomain Bcl-2 proteins Bax and Bak in the outer mitochondrial membrane.^{6, 7, 8} This process promotes release of mitochondria-derived apoptogenic proteins, in particular cytochrome c,⁹ or smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI).¹⁰ Within the apoptosome,¹¹ active caspase-9 finally leads to activation of caspase-3,¹² and subsequent cell death.Anti-apoptotic multidomain Bcl-2 proteins (Bcl-2, Bcl-2-like protein 2 (Bcl-w), B-cell lymphoma-extra large (Bcl-X_L), induced myeloid leukaemia cell differentiation protein (Mcl-1) and Bcl-2-related protein A1 (A1)) with four Bcl-2 homology domains (BH1, BH2, BH3 and BH4) suppress the pro-apoptotic function of Bax-like proteins such as Bax, Bak and Bok (that contain BH1–BH3 domains) or the BH3-only proteins Bad (Bcl-2-associated death promoter), Bim (Bcl-2-like protein 11), Bid, Noxa (phorbol-12-myristate-13-acetate-induced protein 1) and Puma (p53 upregulated modulator of apoptosis).¹³ Regulation of mitochondria-mediated apoptosis is determined by the balance between pro- and anti-apoptotic Bcl-2 proteins.¹⁴In a variety of cancer types, a decrease of BH3-only protein or upregulation of pro-survival Bcl-2 proteins is associated with poor prognosis.¹⁵ In metastatic squamous cell carcinoma (SCC) of the skin or the so-called ‘head and neck SCC'' (HNSCC), high expression of pro-survival Bcl-2 proteins conferred radio- and chemotherapy resistance.^{16, 17} These findings mark Bcl-2 proteins as regulators of SCC apoptosis and indicate that BH3 mimetics may hold therapeutic potential for metastatic SCC. The BH3 mimetics navitoclax (ABT263) and ABT199 are currently under investigation in clinical studies.^{18, 19, 20} Mechanistically, their lead compound ABT737 suppresses Bcl-2 activity by binding to the hydrophobic groove of Bcl-2, Bcl-w and Bcl-X_L.¹⁸ As ABT263 upregulates Mcl-1, resistance to a number of Bcl-2 inhibitors (ABT737 and ABT263) has been described.²¹ Another compound, Obatoclax, was developed to block all anti-apoptotic Bcl-2 proteins including Mcl-1.²² Obatoclax blocks the interaction of Bim or Bax with Mcl-1.²³ In this report, we have studied the effect of ABT737 for cell death in SCC of the skin and investigated the molecular mechanisms of resistance to different BH3 mimetics.     

Conversion of cell-survival activity of Akt into apoptotic death of cancer cells by two mutations on the BIM BH3 domain

Survival and proliferation of cancer cells are often associated with hyperactivity of the serine/threonine kinase, Akt. Herein, we show that prosurvival activity of Akt can be converted into prodeath activity by embedding an Akt recognition sequence in the apoptogenic BH3 domain of human BIM. The recognition sequence was created by introducing two mutations, I155R and E158S, into the core region of the BIM BH3 domain. Although a 21-mer BIM BH3 peptide containing these two mutations bound weakly to BCL-X_L and BCL-2, this peptide with phosphorylation of Ser158 bound to these proteins with a dissociation constant of <10 nM. The crystal structure of the phosphorylated peptide bound to BCL-X_L revealed that the phospho-Ser158 makes favorable interactions with two BCL-X_L residues, which cannot be formed with unphosphorylated Ser158. Remarkably, the designed peptide showed a cytotoxic effect on PTEN-null PC3 tumor cells whose Akt activity is aberrantly high. The cell-killing activity disappeared when the cellular Akt activity was lowered by ectopic PTEN expression. Thus, these results lay a foundation for developing a peptide or protein agent that is dormant in normal cells but is transformed into a potent apoptogenic molecule upon phosphorylation by hyperactivity of Akt in cancer cells.The interplay between the BCL-2 family proteins regulates mitochondrion-mediated apoptotic cell death.^{1, 2} The BCL-2 family proteins are characterized by having at least one BCL-2 homology (BH) domain, and they are classified into three distinct subgroups based on their functional and structural features. One subgroup consists of BAX and BAK, which contain the BH1-BH4 domains and mediate apoptosis by increasing the permeability of the mitochondrial outer membrane (MOM) and thus leading to the release of the apoptogenic factors, such as cytochrome c and Smac/Diablo.^{3, 4, 5, 6} Another subgroup is composed of antiapoptotic proteins, BCL-2, BCL-X_L, BCl-w, MCL-1, A1 and BCL-B, which contain the BH1-BH4 domains that are arranged to form an extended hydrophobic groove known as the BH3-binding groove.⁷ The remaining subgroup is composed of a diverse set of proteins that are unrelated to each other except for the possession of the BH3 domain.⁷ These BH3-only proteins sense and convey apoptotic cell death signals, ultimately leading to the activation of BAX and BAK.^{8, 9} The antiapoptotic BCL-2 subfamily proteins bind the BH3 domain of BAX/BAK and of the BH3-only proteins through their BH3-binding groove.^{10, 11, 12, 13, 14, 15}Biochemical studies have discovered that a number of the BH3-only proteins termed ‘activators'', such as BID and BIM, bind directly to BAX and induce its activation, whereas other BH3-only proteins termed ‘sensitizers'' induce apoptosis by releasing the activators sequestered by the antiapoptotic proteins.^{5, 16, 17} A recent crystallographic study revealed that the BID BH3 peptide binds to the canonical BH3-binding groove of BAX and induces a pronounced conformational change that exposes the BH3 domain of BAX.¹⁸ The activated BAX oligomerizes to induce the permeabilization of the MOM.⁶ The antiapoptotic BCL-2 proteins were suggested to sequester the BH3 domains of both BAX and the activator BH3-only proteins to prevent the BAX oligomerization.¹⁸Apoptosis is attenuated in cancer cells because of the abundance of antiapoptotic BCL-2 proteins and/or prevention of apoptosis induction. Anticancer BH3 peptides have been developed, especially those derived from BIM, which interacts with all of the antiapoptotic proteins with extremely high affinity.^{15, 19} These BH3 peptides exhibit a broad and multimodal targeting of the BCL-2 family proteins.^{20, 21, 22} Promising small molecular anticancer compounds have also been developed that mimic the BH3 peptides and bind to the surface groove of the antiapoptotic proteins.²³ ABT-737 and ABT-263 selectively bind to and lower the amounts of the functional BCL-2, BCL-X_L and BCL-w proteins to induce the apoptotic death of tumor cells that depend especially on the overexpression of the three proteins.^{24, 25} The BH3 peptides and the BH3 mimetics both bear an intrinsic shortcoming in that they inhibit the BCL-2 family proteins not only in cancer cells but also in normal cells as they cannot distinguish cancerous from normal cells.One of the hallmarks of many cancer and tumor cells is the hyperactivation of the serine/threonine (Ser/Thr) protein kinase Akt, which is a key signaling molecule in the cellular survival pathway.²⁶ In many types of cancers, including glioma, prostate cancer and breast cancer, Akt is required to maintain a proliferative state and for progression into a more malignant state in conjunction with genetic mutations.^{26, 27, 28}We set out to develop a molecule that can respond to the hyperactivity of Akt and can lead to the death of cancer cells. Herein, we describe the embedment of the Akt recognition sequence into the BIM BH3 peptide and the cancer cell-specific apoptogenic property of the resulting BIM BH3 peptide variant characterized by X-ray crystallography, calorimetry and cell-based biochemistry.     

The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria

Components of the death receptor-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIP_L), a well-known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca²⁺-release as well as ER-mitochondria tethering was decreased in c-FLIP^−/− mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIP_L and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIP_L emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4.Cellular FLICE inhibitory proteins (c-FLIP) inhibit death receptor (DR)-mediated apoptosis, by preventing caspase-8 activation.¹ Among the three identified c-FLIP splicing forms,^{2, 3} c-FLIP_S,R were described as cytosolic, whereas c-FLIP_L was also observed in the nucleus. A pool of membrane-bound c-FLIP_L was also described⁴ suggesting that caspase-8/c-FLIP_L could re-distribute on stimulation, leading to a more subtle regulation of caspase-8 activity depending on substrates localization.⁵ Furthermore, caspase-8 itself and Fas-Associated Death Domain adaptor protein (FADD) were found or were shown to re-loca5lize in local complexes on ER^{6, 7, 8} and mitochondria,^{9, 10} mediating the exchange of signals between the two organelles.^{11, 12, 13} Several molecular platforms containing both membrane-bound proteins and cytosolic apoptosis modulators have been identified at the ER-mitochondria interface (the so-called mitochondria-associated membranes or MAMs),¹⁴ controlling ER-mitochondria anchorage as well as lipid metabolism, Ca²⁺ signaling and apoptosis.¹⁵ MAMs have been recently described as lipid raft-like domains that orient proteins to promote the ER-mitochondria juxtaposition;¹⁶ consequently, alterations in their composition may profoundly affect the physical and functional inter-organelle crosstalk. Furthermore, as mitochondrial and ER membranes are continuously and concertedly remodeled,¹⁷ it is not surprising that membrane-shaping proteins can also exert a function in regulating the ER-mitochondria coupling.^{12, 18} Different families of ER-shaping proteins control the organization of peripheral ER, which consists of sheet-like cisternae and tubules connected by three-way junctions.¹⁹ Among these, Reticulons (RTN) and Deleted in Polyposis locus 1 (DP1) proteins cause the ER membrane to curve and tubulate,^{20, 21} whereas the GTPases Atlastins (ATL) promote the branching of ER tubules;²² finally, ER sheet-enriched proteins such as the 63-kDa cytoskeleton-linking membrane protein (CLIMP63) control the width of ER cisternae, anchoring the organelle to microtubules and maintaining its spatial distribution.^{23, 24} Along with other components of the extrinsic apoptosis, here we described for the first time the enrichment of c-FLIP_L at ER and ER-mitochondria interface. Furthermore, we observed that ER structure and tethering to mitochondria are impaired in cells lacking c-FLIP. Given the importance of membrane-shaping proteins and MAM complexes in regulating organelles structure and ER-mitochondria juxtaposition, we focused on the mechanism underlying this phenotype and we found that c-FLIP_L deficiency induces the caspase-mediated processing of RTN4, thus affecting organelle shape and coupling to mitochondria. We therefore concluded that c-FLIP_L is a novel regulator of ER morphology and ER-mitochondria crosstalk.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim

Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.Variola virus (VAR), the causative agent of smallpox, is a member of the poxvirus family and belongs to the orthopoxviridae. Despite its successful eradication nearly 30 years ago, VAR remains an ongoing concern because of its potential use as a bioterrorism agent.¹ The threat of intentional use of VAR coupled with the absence of an FDA-approved drug for the prevention or treatment of smallpox infection is cause for considerable interest in the development of small-molecule therapeutics against VAR. Current strategies for dealing with smallpox are based on vaccination using live vaccinia virus (VV),^{2, 3} a closely related member of the orthopoxvirus genus, which shares >90% sequence identity with VAR. Vaccination using live VV, however, can cause serious complications,⁴ underscoring the need for effective anti-viral treatments, particularly since anti-viral treatment may be a more efficacious strategy compared with vaccination.⁵ Recent strategies to target VAR for small-molecule therapeutics included the use of polymerase inhibitors,⁶ notably Cidofovir, inhibitors of extracellular virus formation⁷ and tyrosine kinase inhibitors including Gleevec.^{8, 9} Cidofovir is currently the only approved antiviral drug for the treatment of orthopoxviruses, although it is not approved for smallpox treatment. Other host–virus interactions have been identified that may be suitable drug targets^{10, 11} but currently require further investigation.Several poxvirus members other than VAR have been shown to rely on virulence factors that prevent premature host cell demise via programmed cell death or apoptosis,^{12, 13, 14, 15, 16} thus ensuring survival and proliferation. The B-cell lymphoma 2 (Bcl-2) protein family is a key mediator for maintaining cell survival or to drive apoptosis, thereby removing infected, damaged or unwanted cells,¹⁷ and sequence, structural and functional orthologs of Bcl-2 have been found in a number of poxviruses.¹⁸ Certain viral Bcl-2-like proteins were only identified as family members after their 3D structures were determined, owing to their complete lack of sequence identity to mammalian Bcl-2 proteins. This group of proteins include the myxoma virus M11L¹² and VV F1L¹⁵ and N1L.¹⁹ Myxoma virus M11L was shown to adopt the classical Bcl-2 fold^{20, 21} that utilizes the canonical Bcl-2 homology 3 (BH3)-binding groove to engage BH3 ligands to exert its pro-survival effect. VV F1L also adopts a Bcl-2 fold, but unlike M11L it exists as a domain-swapped dimer,^{22, 23} whereas N1L also adopted a dimeric Bcl-2 fold but with a different dimeric arrangement.^{24, 25}Although F1L from VAR has not previously been investigated, the VV homolog is well characterized. VV F1L has been shown to inhibit the mitochondrial pathway of apoptosis by replacing Mcl-1²⁶ and interacts with the isolated BH3 domains of Bim, Bax and Bak,²³ which are bound in the canonical Bcl-2-binding groove.²² Furthermore, an F1L-deficient VV potently causes Bak/Bax-mediated apoptosis.^{15, 27} Functionally, VV F1L appears to rely primarily on neutralization of Bim in the context of a viral infection.²² Given the close similarity between VAR and VV, VAR may also rely on inhibition of host cell apoptosis for successful infection and proliferation. Disruption of VAR ability to inhibit apoptosis thus may constitute an attractive strategy for small-molecule-based intervention. To investigate this possibility, we performed a biochemical, structural and functional characterization of VAR F1L. Here we report that despite possessing a nearly identical 3D structure and sequence, VAR F1L inhibits apoptosis via a different mechanism compared with its homolog in VV.