Inhibition Of Anti-apoptotic Bcl-2 Proteins Research Articles

The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes

Anti-apoptotic proteins such as BCL-XL promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-XL, BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins.

Enhanced Apoptosis and Loss of Cell Viability in Melanoma Cells by Combined Inhibition of ERK and Mcl-1 Is Related to Loss of Mitochondrial Membrane Potential, Caspase Activation and Upregulation of Proapoptotic Bcl-2 Proteins.

Targeting of MAP kinase pathways by BRAF inhibitors has evolved as a key therapy for BRAF-mutated melanoma. However, it cannot be applied for BRAF-WT melanoma, and also, in BRAF-mutated melanoma, tumor relapse often follows after an initial phase of tumor regression. Inhibition of MAP kinase pathways downstream at ERK1/2, or inhibitors of antiapoptotic Bcl-2 proteins, such as Mcl-1, may serve as alternative strategies. As shown here, the BRAF inhibitor vemurafenib and the ERK inhibitor SCH772984 showed only limited efficacy in melanoma cell lines, when applied alone. However, in combination with the Mcl-1 inhibitor S63845, the effects of vemurafenib were strongly enhanced in BRAF-mutated cell lines, and the effects of SCH772984 were enhanced in both BRAF-mutated and BRAF-WT cells. This resulted in up to 90% loss of cell viability and cell proliferation, as well as in induction of apoptosis in up to 60% of cells. The combination of SCH772984/S63845 resulted in caspase activation, processing of poly (ADP-ribose) polymerase (PARP), phosphorylation of histone H2AX, loss of mitochondrial membrane potential, and cytochrome c release. Proving the critical role of caspases, a pan-caspase inhibitor suppressed apoptosis induction, as well as loss of cell viability. As concerning Bcl-2 family proteins, SCH772984 enhanced expression of the proapoptotic Bim and Puma, as well as decreased phosphorylation of Bad. The combination finally resulted in downregulation of antiapoptotic Bcl-2 and enhanced expression of the proapoptotic Noxa. In conclusion, combined inhibition of ERK and Mcl-1 revealed an impressive efficacy both in BRAF-mutated and WT melanoma cells, and may thus represent a new strategy for overcoming drug resistance.

Kinome-wide RNAi screening for mediators of ABT-199 resistance in breast cancer cells identifies Wee1 as a novel therapeutic target

Antiapoptotic and proapoptotic BCL-2 protein family members regulate mitochondrial apoptotic pathway. Small molecule inhibitors of antiapoptotic BCL-2 proteins including BCL-2-specific inhibitor ABT-199 (Venetoclax) are in clinical development. However, the efficiency of ABT-199 as a single agent in solid tumors is limited. We performed a high-throughput RNAi kinome screen targeting 691 kinases to identify potentially targetable kinases to enhance ABT-199 response in breast cancer cells. Our studies identified Wee1 as the primary target kinase to overcome resistance to ABT-199. Depletion of Wee1 by siRNA-mediated knockdown or inhibition of Wee1 by the small molecule Wee1 inhibitor AZD1775 sensitized SKBR3, MDA-MB-468, T47D and CAMA-1 breast cancer cells to ABT-199 along with decreased MCL1. BH3-only proteins PUMA and BIM functionally contribute to apoptosis signaling following co-targeting BCL-2 and Wee1. Suppression of Wee1 function increased mitochondrial cell death priming. Furthermore, we found that Wee1 inhibition altered MCL1 phosphorylation and protein stability, which led to HUWE1-mediated MCL1 degradation. Our findings suggest that Wee1 inhibition can overcome resistance to ABT-199 and provide a rationale for further translational investigation of BCL-2 inhibitor/Wee1 inhibitor combination in breast cancer.

Activation of p53 Enhances Antileukemia Activity of Bcr-Abl Tyrosine Kinase Inhibitors in a Murine Model of CML

Although CML patients under Bcr-Abl tyrosine kinase inhibitor (TKI) therapy have experienced high response rates and long-time survival, 50% or more patients relapse upon therapy cessation, largely because TKIs do not eliminate CML stem cells. We previously reported that inhibition of antiapoptotic Bcl-2 proteins synergized with TKIs to eradicate CML stem/progenitor cells. The tumor suppressor p53 induces apoptosis in part by modulating Bcl-2 family proteins. Although rarely mutated in CML, p53 activity is antagonized by Bcr-Abl tyrosine kinase-regulated MDM2. We hypothesize that activation of p53 by MDM2 inhibition will synergize with TKIs to eliminate CML cells. Using an inducible transgenic Scl-tTa- BCR-ABL mouse model, we first determined the expression of p53 and its target genes in bone marrow (BM) cells of Tet-on (control) and Tet-off (CML) mice. We found markedly increased RNA levels of p53 and its targets Bax, Noxa, and MDM2 in CML as compared to control mice. Using CyTOF mass cytometry and cytofkid automatic subset detection/clustering based on the expression of cell surface markers, we then determined p53 and its target proteins in bulk and Lin-Sca-1+cKit+ (LSK) populations at single cell levels and found overexpression of p53 and its targets such as Noxa in CML BM cells (CD45+) compared to controls. The differential expression was most pronounced in the LSK populations. For both control and CML mice, LSK cells expressed significantly higher p53 protein than their respective bulk cell population. We next evaluated the sensitivity of these cells to BH3 peptide priming. CML BM cells and more so LSK populations were more sensitive to BH3 peptides, especially to Noxa peptide than cells from control mice (P=0.044 in CD45+ and P=0.0005 in LSK populations) suggesting that CML cells are more primed to apoptosis stimuli. Finally, we tested the antileukemia activity of combined MDM2 and Bcr-Abl inhibition in mice engrafted with BM cells from Scl-tTa- BCR-ABL /GFP mice. Combined inhibition of Bcr-Abl tyrosine kinase (imatinib) and MDM2 (DS-5272) strongly reduced BM leukemic populations and LSK cells, increased Noxa, decreased leukemia burden, and significantly prolonged survival (P=0.0021 vs. control). Median survival days for control, imatinib, DS-5272, and the combination groups were 85, 110, 155, and 248; respectively.Our results demonstrate that CML cells/stem cells have increased p53 signaling and are highly primed to undergo apoptosis. Combined p53 activation and Bcr-Abl tyrosine kinase inhibition targets CML stem/progenitor cells and has the potential to significantly improve response and cure rates of CML. DisclosuresCarter:Daiichi Sankyo: Research Funding; novartis: Research Funding. Seki:Daiichi Sankyo: Employment. Andreeff:Daiichi Sankyo: Consultancy.

Anti-Leukemic Properties of Aplysinopsin Derivative EE-84 Alone and Combined to BH3 Mimetic A-1210477.

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski’s rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.

In vitro and in silico anti-leukemic activity of 2-amino-6-nitro-4-(4-oxo-2-thioxothiazolidin-5-yl)-4H-chromene-3-carbonitrile (ANC) through inhibition of anti-apoptotic Bcl-2 proteins

An array of 4H-chromene derivatives have been reported for anticancer properties but their selectivity and mode of anticancer activity are unexplored. In this context, we have investigated a biologically active synthetically designed 4H-Chromene carbonitrile derivative, 2-amino-6-nitro-4-(4-oxo-2-thioxothiazolidin-5-yl)-4H-chromene-3-carbonitrile (ANC) that is strongly and selectively inhibited Bcl-2 over expressing human leukemic (HL-60 and K562) cells for its interaction and elucidated the mode of action. The interaction of ANC was investigated against the antiapoptotic proteins such as Bcl-2, Bax, Bcl-xL and Bcl-w that were overexpressed in leukemic cells using in silico and fluorescent spectroscopic studies. Fluorescent spectroscopic based interaction studies showed that the derivative had strong interaction with Bcl-xL followed by Bcl-2/Bax and least interaction with Bcl-w. Based on the results, the ANC had strong interactions with antiapoptotic Bcl-2 and Bax proteins than the Bcl-xL and Bcl-w proteins. The in vitro biological validation of ANC treated leukemic cells showed downregulation of Bcl-xL than Bcl-2 but least effect on Bcl-w proteins. Furthermore, the ANC had possible four isomers as RR, RS, SR and SS isomers. Among them, RS isomer of ANC had shown more active that correlated with biological interactions and gene expression studies of ACN with oncoproteins. These results confirmed the induction of apoptosis by RS-ACN isomer through inhibition of antiapoptotic machineries of leukemic cells confirming the antiapoptotic Bcl-2 inhibitory activities. Communicated by Ramaswamy H. Sarma

Inhibition of Anti-Apoptotic Bcl-2 Proteins in Preclinical and Clinical Studies: Current Overview in Cancer.

The dynamic interplay between pro-death and pro-survival Bcl-2 family proteins is responsible for a cell’s fate. Due to the recognized relevance of this family in cancer progression and response to therapy, different efforts have made in recent years in order to develop small molecules able to target anti-apoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1. The limitations of the first Bcl-2 family targeted drugs, regarding on-target and off-target toxicities, have been overcome with the development of venetoclax (ABT-199), the first BH3 mimetic inhibitor approved by the FDA. The purpose of this review is to discuss the state-of-the-art in the development of drugs targeting Bcl-2 anti-apoptotic proteins and to highlight the potential of their application as single agents or in combination for improving anti-cancer therapy, focusing in particular on solid tumors.

Mesothelioma Cells Depend on the Antiapoptotic Protein Bcl-xL for Survival and Are Sensitized to Ionizing Radiation by BH3-Mimetics

The incidence of mesothelioma continues to rise and prognosis remains dismal owing to resistance to conventional therapies and few novel treatment options. Failure to activate apoptotic cell death is a resistance mechanism that may be overcome by inhibition of antiapoptotic Bcl-2 proteins using BH3-mimetic drugs. We investigated the role of antiapoptotic proteins in the radioresistance of mesothelioma, identifying clinically relevant targets for radiosensitization and evaluating the activity of BH3-mimetics alone and in combination with radiation therapy in preclinical models. Mesothelioma cell lines 211H, H2052, and H226 exposed to BH3-mimetics demonstrated Bcl-xL dependence that correlated with protein expression and was confirmed by genetic knockdown. The Bcl-xL inhibitor A1331852 exhibited cytotoxic (EC50, 0.13-1.42 μmol/L) and radiosensitizing activities (sensitizer enhancement ratios, 1.3-1.8). Cytotoxicity was associated with induction of mitochondrial outer membrane permeabilization and caspase-3/7 activation. Efficacy was maintained in a 3-dimensional model in which combination therapy completely eradicated mesothelioma spheroids. Clinical applicability was confirmed by immunohistochemical analysis of Bcl-2 proteins in patient samples and radiosensitizing activity of A1331852 in primary patient-derived mesothelioma cells. Mesothelioma cells exhibit addiction to the antiapoptotic protein Bcl-xL, and their intrinsic radioresistance can be overcome by small molecule inhibition of this novel therapeutic target.

Molecular dynamic simulations on an inhibitor of anti-apoptotic Bcl-2 proteins for insights into its interaction mechanism for anti-cancer activity

Inhibition of normal cellular apoptosis or programed cell death is the hallmark of all cancers. Apoptotic dysregulation can result in numerous pathological conditions, such as cancers, autoimmune disorders, and neurodegeneration. Members of the BCL-2 family of proteins regulate the process of apoptosis by its promotion or inhibition and overexpression of the pro-survival anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) has been associated with tumor maintenance, growth and progression Small molecules and peptides which bind the BH3 binding groove of these proteins have been explored in the recent times for their anticancer potential. The first anticancer agents targeting this family of proteins were aimed primarily toward inhibition of Bcl-2. An elevated level of Mcl-1, despite Bcl-2 inhibition, continues to be a cause for resistance in most cancers. However, in silico exploration of Mcl-1 specific drugs and their associated mechanisms have not been clearly elucidated. In order to understand the same, we have carried out docking and molecular dynamic simulations on ABT-263 (Navitoclax), an orally active inhibitor of Bcl-2, Bcl-xL, and Bcl-w proteins; Obatoclax, a pan-Bcl-2 inhibitor as well as Maritoclax, an Mcl-1 specific inhibitor. Docking studies revealed that binding to the hydrophobic grooves is a prerequisite for action on the BCL protein and the binding mechanism and chemical space utilization dictates stability as well as specificity of the inhibitor molecular dynamic simulations showed that on binding, the α-helices of these proteins exhibited less fluctuations than loop regions, also hydrophobic contacts and hydrogen bonding were observed to be the predominant interactions in the drug-receptor complexes.Communicated by Ramaswamy H. Sarma

Discovery of novel inhibitors of anti-apoptotic Bcl-2 proteins derived from Bim BH3 domain

The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2 family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5 (KD=9.48nmol/L for Bcl-2) and SM-6 (KD=0.08nmol/L for Bcl-xL), which exhibited improved binding affinity compared with the lead Bim (KD=16.90nmol/L for Bcl-2 and 22.2nmol/L for Bcl-xL, respectively). These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.

Design, synthesis, and activity evaluation of selective inhibitors of anti-apoptotic Bcl-2 proteins: The effects on the selectivity of the P1 pockets in the active sites

The anti-apoptotic Bcl-2 proteins are attractive targets for anti-cancer drug development, and the discovery of their selective inhibitors has become a research focus. In this Letter, obvious differences in the P1 pocket of the active site between Bcl-2, Bcl-xL, and Mcl-1 proteins were proposed by the structural comparison of these proteins. As a result, the groups in their inhibitors binding to the P1 pockets may have significant effect on the selectivity for these proteins. Based on this hypothesis, five types of derivatives of the lead compound B-1 were designed, and several highly selective inhibitors of Bcl-xL (E-1) or Mcl-1 proteins (G) were found. The selective inhibitors of Mcl-1 protein found in this Letter provide new structural types for the development of novel antitumor agents.

705 - Inhibition of anti-apoptotic Bcl-2 proteins by ABT-263 sensitizes hypoxic cancer cells to ionizing radiation

705 - Inhibition of anti-apoptotic Bcl-2 proteins by ABT-263 sensitizes hypoxic cancer cells to ionizing radiation

A Novel Bioavailable BH3 Mimetic Efficiently Inhibits Colon Cancer via Cascade Effects of Mitochondria.

Gossypol and its analogs, through their ability to bind to and inactivate BH3 domain-containing antiapoptotic proteins, have been shown to inhibit the growth of various human cancer cells in culture and xenograft models. Here, we evaluated the antitumor efficacy of a novel gossypol derivative and BH3 mimetic ch282-5 (2-aminoethanesulfonic acid sodium-gossypolone) in colon cancer models. Several innovative combination strategies were also explored and elaborated. Ch282-5 was synthesized by modifying the active aldehyde groups and R groups of gossypol according to a computer-aided drug design program. The stability of ch282-5 was examined by high-performance liquid chromatography, and cytotoxic effects of ch282-5 on colon cancer cells were assessed by MTS assay. Activation of mitochondrial apoptotic pathway by ch282-5 was evidenced with a series of molecular biology techniques. In vivo antitumor activity of ch282-5 and its combination with chloroquine, rapamycin, oxaliplatin, and ABT-263 was also evaluated in colon cancer xenograft models and experimental liver metastasis models. Ch282-5 showed antiproliferative and pro-cell death activity against colon cancer cells both in vitro and in vivo, and the response to the drug correlated with inhibition of antiapoptotic Bcl-2 proteins, induction of mitochondria-dependent apoptotic pathway, and disruption of mitophagy and mTOR pathway. Ch282-5 also suppressed liver metastasis produced by intrasplenic injection of colon cancer cells. Furthermore, ch282-5 could potentiate the effectiveness of oxaliplatin and rescue ABT-263 efficacy by downregulation of Mcl-1 and elevation of platelet number. These findings provide a rational basis for clinical investigation of this highly promising BH3 mimetic in colon cancer.

Inhibition of MARCH5 ubiquitin ligase abrogates MCL1-dependent resistance to BH3 mimetics via NOXA.

BH3 mimetic compounds induce tumor cell death through targeted inhibition of anti-apoptotic BCL2 proteins. Resistance to one such compound, ABT-737, is due to increased levels of anti-apoptotic MCL1. Using chemical and genetic approaches, we show that resistance to ABT-737 is abrogated by inhibition of the mitochondrial RING E3 ligase, MARCH5. Mechanistically, this is due to increased expression of pro-apoptotic BCL2 family member, NOXA, and is associated with MARCH5 regulation of MCL1 ubiquitylation and stability in a NOXA-dependent manner. MARCH5 expression contributed to an 8-gene signature that correlates with sensitivity to the preclinical BH3 mimetic, navitoclax. Furthermore, we observed a synthetic lethal interaction between MCL1 and MARCH5 in MCL1-dependent breast cancer cells. Our data uncover a novel level at which the BCL2 family is regulated; furthermore, they suggest targeting MARCH5-dependent signaling will be an effective strategy for treatment of BH3 mimetic-resistant tumors, even in the presence of high MCL1.

Blockade of BCL-2 proteins efficiently induces apoptosis in progenitor cells of high-risk myelodysplastic syndromes patients.

Deregulated apoptosis is an identifying feature of myelodysplastic syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-XL/-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/secondary acute myeloid leukemia (sAML) patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34(+) cells and the decreased colony-forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by the ABT-737 or ABT-199 treatment and implies that BH3 mimetics might delay disease progression in higher-risk MDS or sAML patients.

A phase I/II study of the pan Bcl-2 inhibitor obatoclax mesylate plus bortezomib for relapsed or refractory mantle cell lymphoma

Obatoclax, a BH3 mimetic inhibitor of anti-apoptotic Bcl-2 proteins, demonstrates synergy with bortezomib in preclinical models of mantle cell lymphoma (MCL). This phase I/II study assessed obatoclax plus bortezomib in patients with relapsed/refractory MCL. Twenty-three patients received obatoclax 30 or 45 mg plus bortezomib 1.0 or 1.3 mg/m2, administered intravenously on days 1, 4, 8 and 11 of a 21-day cycle. In phase I, the combination was feasible at all doses. Obatoclax 45 mg plus bortezomib 1.3 mg/m2 was selected for phase II study. Common adverse events were somnolence (87%), fatigue (61%) and euphoric mood (57%), all primarily grade 1/2. Grade 3/4 events included thrombocytopenia (21%), anemia (13%) and fatigue (13%). Objective responses occurred in 4/13 (31%) evaluable patients (three complete and one partial response). Six patients (46%) had stable disease lasting ≥ 8 weeks. Obatoclax plus bortezomib was feasible, but the synergy demonstrated in preclinical models was not confirmed.

Synthetic lethality by co-targeting mitochondrial apoptosis and PI3K/Akt/mTOR signaling

Small-molecule inhibitors that antagonize anti-apoptotic Bcl-2 proteins such as BH3 mimetics are currently considered as promising cancer therapeutics to engage the mitochondrial pathway of apoptosis in cancer cells. However, BH3 mimetics may be effective as monotherapy only in cancers that critically depend on anti-apoptotic Bcl-2 proteins for their survival. Since most cancers have evolved multiple strategies to evade programmed cell death, concomitant targeting of several signaling transduction pathways becomes more and more relevant. The current review highlights the potential of combined inhibition of anti-apoptotic Bcl-2 proteins together with the PI3K/Akt/mTOR signaling cascade to trigger apoptosis in cancer cells.

OBATOCLAX and ABT-737 Induce ER Stress Responses in Human Melanoma Cells that Limit Induction of Apoptosis

Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.

Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes

Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death.

BAK activation is necessary and sufficient to drive ceramide synthase-dependent ceramide accumulation following inhibition of BCL2-like proteins

Determining mechanistic details about how drugs kill cancer cells is critical for predicting which cancers will respond to given therapeutic regimens and for identifying effective combinations of drugs that more potently kill cancer cells while sparing normal cells. The BCL2 family of proteins and bioactive sphingolipids are intricately linked during apoptotic cell death. In fact, many chemotherapeutic drugs are known to cause accumulation of the pro-apoptotic sphingolipid ceramide; however, the mechanism by which this occurs is not completely understood. In the present study we demonstrate that direct inhibition of anti-apoptotic BCL2 proteins with ABT-263 is sufficient to induce C(16)-ceramide synthesis in multiple cell lines, including human leukaemia and myeloma cells. ABT-263 activates CerS (ceramide synthase) activity only in cells expressing BAK or in cells capable of activating BAK. Importantly, recombinant BAK is sufficient to increase in vitro CerS activity in microsomes purified from Bak-KO (knockout) cells and activated BAK more potently activates CerS than inactive BAK. Likewise, ABT-263 addition to wild-type, but not Bak-deficient, microsomes increases CerS in vitro activity. Furthermore, we present a feed-forward model by which BAK activation of CerS by chemotherapeutic drugs leads to elevated ceramide levels that result in synergistic channel formation by ceramide (or one of its metabolites) and BAX/BAK.