Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1, or Bcl-B

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.     

p53-dependent regulation of Mcl-1 contributes to synergistic cell death by ionizing radiation and the Bcl-2/Bcl-XL inhibitor ABT-737

Treatment with the Bcl-2/Bcl-XL inhibitor ABT-737 is a promising novel strategy to therapeutically induce apoptotic cell death in malignant tumors such as glioblastomas. Although many studies have demonstrated that ABT-737 acts synergistically with chemotherapeutic drugs, the possibility of a combined treatment with ionizing radiation (IR) and ABT-737 has not yet been thoroughly investigated. Similarly, the relationship between p53 function and the pro-apoptotic effects of ABT-737 are still obscure. Here, we demonstrate that IR and ABT-737 synergistically induce apoptosis in glioblastoma cells. The sensitivity to ABT-737-mediated cell death is significantly increased by the IR-dependent accumulation of cells in the G2/M cell cycle phase. Wild type p53 function inhibits the efficacy of a combined IR and ABT-737 treatment via a p21-dependent G1 cell cycle arrest. Moreover, mutant as well as wild type p53 counteract the pro-apoptotic activity of ABT-737 by maintaining the expression levels of the Mcl-1 protein. Thus, p53 regulates the sensitivity to ABT-737 of glioblastoma cells. Our results warrant a further evaluation of a novel combination therapy using IR and ABT-737. The efficacy of such a therapy could be substantially enhanced by Mcl-1-lowering strategies.     

IL-6 rescues the hyporesponsiveness of c-Rel deficient B cells independent of Bcl-xL,Mcl-1, and Bcl-2

The hematopoietically restricted member of the NF-kappaB/Rel family, c-Rel, is essential for B cell survival and proliferation. Here we demonstrate that the production of the interleukins 6, 10, and 15 (IL-6, IL-10, and IL-15) are diminished in c-Rel(-/-) B lymphocytes. In a manner similar to that seen in IL-6(-/-) B cells, resultant STAT activation is reduced in c-Rel(-/-) B cells following B cell receptor (BCR) ligation. Addition of either exogenous IL-6 or IL-10, but not IL-15, partially restores proliferation, and this occurs through enhanced cell survival rather than promoting cell cycle progression. This increase in viability occurs independently of Bcl-xL and Mcl-1 expression though, two survival genes reported to be downstream of IL-6 signaling. Nonetheless, transgenically expressed Bcl-xL, a direct c-Rel target gene in B cells, corrects not only the survival defect of c-Rel deficiency, but also partially ameliorates hypoproliferation. Together IL-6 and Bcl-xL are additive but incomplete in the restoration of proliferation. Known deficits in the induction of several key cell cycle components in c-Rel(-/-)B cells are not corrected upon treatment with exogenous cytokine. Together, these data demonstrate that IL-6 enhances B cell responses by employing multiple survival factors.     

Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

Background and purpose

Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.

Experimental approach

For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.

Key results

The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC₅₀ of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.

Conclusions and implications

Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.     

Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ～60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.     

Bim and the Pro-Survival Bcl-2 Proteins: Opposites Attract,ERK Repels

Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (BimS, BimL and BimEL). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins. Quite how Bim and other BOPs promote apoptosis has been the subject of some debate. Bim was isolated by it’s interaction with pro-survival proteins such as Bcl-2 and it has been suggested that this is key to the ability of Bim to induce apoptosis. However, an alternative model argues that some forms of Bim can bind directly to the pro-apoptotic Bax and Bak proteins to initiate apoptosis. A new study may finally put this debate to rest as it provides strong evidence to suggest that Bim and other BOPs act primarily by binding to pro-survival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The importance of the interaction between Bim and the pro-survival Bcl-2 proteins is underlined by our demonstration that it is regulated by ERK1/2-dependent phosphorylation of BimEL. ERK1/2-dependent dissociation of BimEL from pro-survival proteins is the first step in a process by which the pro-survival ERK1/2 pathway promotes the destruction of this most abundant Bim splice variant. In this review we outline the significance of these new studies to our understanding of how BOPs such as Bim initiate apoptosis and how this process is regulated by growth factor-dependent signalling pathways.     

Granzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737

Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.     

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-X_L is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-X_L may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.     

3-Thiomorpholin-8-oxo-8H-acenaphtho [1,2-b] pyrrole-9-carbonitrile (S1) derivatives as pan-Bcl-2-inhibitors of Bcl-2, Bcl-xL and Mcl-1

Based on the binding mode of our previously discovered dual inhibitor of Bcl-2 and Mcl-1, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3, S1), a library of 9-substituted 3 derivatives was synthesized to further probe the p4 pocket of the two targets. By NMR, structure–activity relationship study, and site-directed mutation, compound 6d (3-(4-aminophenylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-3-phenyl)propylamine) was identified to span p2–p4 pockets of Mcl-1, Bcl-2 and Bcl-x_L, and then exhibited 9- to 35-fold better affinity to the three targets than 3 (IC₅₀ = 10, 20 and 18 nM, respectively), which led to greater activity in induction of apoptosis in multiple cancer cell lines. Different contribution of p4 pocket to binding Bcl-2 and Mcl-1 was also investigated by plotting the potency and the HAC of the derivatives.     

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.     

姜黄素对晚期宫颈癌患者Bcl-xL、Bcl-2、EphA2及EphrinA1表达的影响

目的:探究姜黄素对宫颈癌晚期放疗患者Bcl-x L、Bcl-2、Eph A2与Ephrin A1表达的影响。方法:收集160例晚期宫颈癌放疗患者的宫颈癌石蜡标本,分为姜黄素组与对照组,均给予放射治疗,姜黄素组放疗期间加服姜黄素片剂,采用免疫组化等实验方法,观察两组病例标本Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达情况,比较两组病理标本化疗前后的凋亡细胞指数(AI)与微血管密度(MVD)。结果:姜黄素组的Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达阳性率分别为8.3%、43.33%、46.67%、15.0%,均显著低于对照组(P0.05);放疗后姜黄素组的AI显著高于对照组(P0.05),MVD显著低于对照组(P0.05)。结论:姜黄素可抑制Bcl-x L、Bcl-2的表达以促进肿瘤细胞凋亡,同时降低Eph A2与Ephrin A1的表达以减少肿瘤微血管形成,有显著的抗肿瘤与放疗增敏作用。     

Distinct Biological Roles for the Notch Ligands Jagged-1 and Jagged-2

Notch signaling is activated in a subset of non-small cell lung cancer cells because of overexpression of Notch3, but the role of Notch ligands has not been fully defined. On the basis of gene expression profiling of a panel of non-small cell lung cancer cell lines, we found that the predominant Notch ligands were JAG1, JAG2, DLL1, and DLL3. Given that Notch ligands reportedly have overlapping receptor binding specificities, we postulated that they have redundant biological roles. Arguing against this hypothesis, we found that JAG1 and JAG2 were differentially regulated; JAG1 expression was dependent upon epidermal growth factor receptor (EGFR) activation in HCC827 cells, which require EGFR for survival, whereas JAG2 expression was EGFR-independent in these cells. Furthermore, HCC827 cells underwent apoptosis following depletion of JAG1 but not JAG2, whereas co-culture experiments revealed that depletion of JAG2, but not JAG1, enhanced the ability of HCC827 cells to chemoattract THP-1 human monocytes. JAG2-depleted HCC827 cells expressed high levels of inflammation-related genes, including interleukin 1 (IL1) and a broad range of IL1-regulated cytokines, which was attenuated by inhibition of IL1 receptor (IL1R). Our findings suggest that JAG1 and JAG2 have distinct biological roles including a previously undiscovered role for JAG2 in regulating the expression of cytokines that can promote antitumor immunity.     

Biochemical and functional comparisons of Mcl-1 and Bcl-2 proteins: evidence for a novel mechanism of regulating Bcl-2 family protein function

Mcl-1 is a recently described homologue of Bcl-2 whose function and biochemical characteristics remain poorly defined. Gene transfer experiments in lnterleukin-3 (IL-3)-dependent myeloid progenitor 32D.3 cells and pro-B-lymphoid FL5.12 cells demonstrated that enforced production of high levels of Mcl-1 protein failed to prolong the survival of cells when cultured in the absence of IL-3, whereas Bcl-2 did delay cell death. Mcl-1 also did not prolong the survival in vitro of 32D.3 cells that had been induced to differentiate into mature neutrophils using Granulocyte-Colony Stimulating Factor (G-CSF), whereas Bcl-2 did. 32D.3 and FL5.12 cells co-transfected with Mcl-1 and Bcl-2 displayed survival kinetics essentially identical to cells transfected with Bcl-2 alone, when cultured in the absence of IL-3, indicating that Mcl-1 neither enhances nor impairs Bcl-2 function. In contrast to the lack of effects of Mcl-1 in 32D.3 and FL5.12 cells, Mcl-1 (like Bcl-2) was able to neutralise Bax-induced cytotoxicity in yeast (S. cerevisiae). Moreover, the recombinant GST-Mcl-1 protein bound specifically to in vitro translated Bax protein, as well as to Bax protein present in detergent lysates prepared from 32D.3 and FL5.12 cells, based on in vitro binding assays. However, Mcl-1 and Bax proteins could not be co-immunoprecipitated from control and transfected 32D.3 and FL5.12 cells, whereas Bcl-2 and Bax were easily co-immunoprecipitated under the same conditions. The findings suggest that while Mcl-1 has the capacity to bind to and neutralise the cell death promoting activity of Bax, other factors such as perhaps additional proteins or undefined post-translational modifications may influence its ability to bind to Bax in vivo and thus affect its function as a cell death blocker.     

HIV-1 Tat targets microtubules to induce apoptosis,a process promoted by the pro-apoptotic Bcl-2 relative Bim

Depletion of CD4(+) T cells is the hallmark of HIV infection and AIDS progression. In addition to the direct killing of the viral-infected cells, HIV infection also leads to increased apoptosis of predominantly uninfected bystander cells. This is mediated in part through the HIV-1 Tat protein, which is secreted by the infected cells and taken up by uninfected cells. Using an affinity-purification approach, a specific and direct interaction of Tat with tubulin and polymerized microtubules has been detected. This interaction does not affect the secretion and uptake of Tat, but is critical for Tat to induce apoptosis. Tat binds tubulin/microtubules through a four-amino-acid subdomain of its conserved core region, leading to the alteration of microtubule dynamics and activation of a mitochondria-dependent apoptotic pathway. Bim, a pro-apoptotic Bcl-2 relative and a transducer of death signals initiated by perturbation of microtubule dynamics, facilitates the Tat-induced apoptosis. Our findings reveal a strategy by which Tat induces apoptosis by targeting the microtubule network. Thus HIV-1 Tat joins a growing list of pathogen-derived proteins that target the cytoskeleton of host cells.