The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.     

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.     

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

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

Methylseleninic acid potentiates multiple types of cancer cells to ABT-737-induced apoptosis by targeting Mcl-1 and Bad

ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins, holds great promise to complement current cancer therapies. However many types of solid cancer cells are resistant to ABT-737. One practical approach to improve its therapeutic efficacy is to combine with the agents that can overcome such resistance to restore the sensitivity. In the present study, a second-generation selenium compound methylseleninic acid (MSeA) synergistically sensitized MDA-MB-231 human breast cancer cells, HT-29 human colon cancer cells and DU145 human prostate cancer cells to apoptosis induction by ABT-737, as evidenced by greater than additive enhancement of Annexin V/FITC positive (apoptotic) cells and activation of multiple caspases and PARP cleavage. Mechanistic investigation demonstrated that MSeA significantly decreased basal Mcl-1 expression and ABT-737-induced Mcl-1 expression. Knocking down of Mcl-1 with RNAi approach supported the functional significance of this molecular target. More importantly, we identified inactivation of Bad by phosphorylation on ser-136 and ser-112 as a novel mechanism involved in ABT-737 resistance, which can be overcome by combining with MSeA. In addition, we found that expression of Bax was required for the efficient execution of synergistic sensitization. Our findings, for the first time, provide a strong mechanistic rationale for developing MSeA as a novel sensitizing agent of ABT-737.     

Molecular dynamics study of small molecule inhibitors of the Bcl-2 family

We carried out docking and molecular dynamics simulations on ABT-737 and obatoclax, which are inhibitors of the Bcl-2 family of proteins. We modeled the binding mode of ABT-737 with Bcl-x(L) , Bcl-2, and Mcl-1 and examined their dynamical behavior. We found that the binding of the chlorobiphenyl end of ABT-737 was quite stable across all three proteins. However, the phenylpiperazine linker group was dramatically more mobile in Mcl-1 compared to either Bcl-x(L) or Bcl-2. The S-phenyl group at the p4 binding site was well-anchored in Bcl-x(L) and Bcl-2 but was somewhat more mobile in Mcl-1 although the phenyl ring itself on average stayed close to the p4 binding site in Mcl-1. This greater mobility is likely due to the greater openness of the p3 and p4 binding sites on Mcl-1. The calculated binding free energies were consistent with the much weaker binding affinity of ABT-737 for Mcl-1. Obatoclax was predicted to bind at the p1 and p2 binding sites of Mcl-1 and the binding mode was quite stable during the molecular dynamics simulation with Mcl-1 wrapping around the molecule. The modeled binding mode suggests that obatoclax is able to inhibit all three proteins because it makes use of the p1 and p2 binding sites alone, which is a fairly narrow groove in all three proteins unlike the p4 binding site, which is much broader in Mcl-1.     

Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-X_L, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.     

RNA Silencing of Mcl-1 Enhances ABT-737-Mediated Apoptosis in Melanoma: Role for a Caspase-8-Dependent Pathway

Background

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma''s striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance.

Methodology/Principal Findings

Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity.

Conclusions/Significance

Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.     

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.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.     

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.     

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.     

Design, synthesis, and activity evaluation of broad-spectrum small-molecule inhibitors of anti-apoptotic Bcl-2 family proteins: characteristics of broad-spectrum protein binding and its effects on anti-tumor activity

On the basis of the comparison of the structure of the Bim BH3: Bcl-x(L) complex and that of the ABT-737: Bcl-x(L) complex, a series of class A compounds were designed. These compounds had the basic skeleton of ABT-737 and the h2 residues of Bim BH3. These residues had shown themselves to be relevant to Bim BH3's broad-spectrum binding properties in saturation mutagenesis assays. Unlike ABT-737, which is a selective inhibitor of anti-apoptotic members of the Bcl-2 protein family, the class A compounds showed broad-spectrum binding activity to target proteins similar to those of Bim BH3 peptide. Then class B compounds were synthesized by modifying the structure of the most effective class A compound, A-4. Most of these class B compounds showed better binding affinity to the target proteins than the class A compounds had. They also showed themselves more effective than ABT-737 at inhibiting growth in multiple tumor cell lines known to express Bcl-x(L), Bcl-2, and Mcl-1 proteins at high levels. Compounds B-11 and B-12 had the strongest anti-tumor activity of any compounds we produced. This study suggests that it is feasible to design small-molecule inhibitors based on the structure of Bim BH3, which shows broad-spectrum binding to Bcl-x(L), Bcl-2, and Mcl-1 proteins. Our results also suggest that the broad-spectrum properties of small-molecule inhibitors binding to target proteins play a critical role in inhibiting the growth of many tumor cells. Finally, our study provides a series of lead compounds that merit further research into anti-cancer therapeutics.     

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-x_L, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.     

Platinum compounds sensitize ovarian carcinoma cells to ABT-737 by modulation of the Mcl-1/Noxa axis

Ovarian cancer is the leading cause of death from gynecological cancer. The anti-apoptotic protein Bcl-x_L is frequently overexpressed in ovarian carcinoma which correlates with chemotherapy resistance. It has been demonstrated that Bcl-x_L cooperates with another anti-apoptotic protein, Mcl-1, to protect ovarian cancer cells against apoptosis, and that their concomitant inhibition induces massive cell death. Here, we examined the interest of ABT-737, a potent BH3-mimetic molecule targeting Bcl-x_L, both alone and in combination with Mcl-1 modulators, in ovarian cancer cell lines. As a single agent, ABT-737 was ineffective at promoting cell death in the four cell lines we tested in vitro. However, the specific inhibition of Mcl-1 by siRNA dramatically increased the sensitivity of chemoresistant cells to ABT-737. Platinum compounds also sensitize to ABT-737 by dose-dependently decreasing Mcl-1 expression or by increasing the expression of pro-apoptotic BH3-only proteins Noxa and, to a lower extent, Bim. Furthermore, we demonstrated that Noxa accumulation was involved in apoptosis occurring in response to the combination of ABT-737 and platinum compounds, since cells were protected from apoptosis by its silencing. Moreover, the combination was also highly cytotoxic ex vivo in sliced SKOV3 tumor nodes. However we observed in these slices a strong basal expression of Noxa and apoptotic cell death in response to ABT-737 alone. Therefore, we have revealed that the modulation of the Mcl-1/Noxa axis by platinum compounds results in a strong sensitization of chemoresistant ovarian carcinoma cells to ABT-737, which could constitute a promising therapeutic in these cancers.     

Noxa/Bcl-2 protein interactions contribute to bortezomib resistance in human lymphoid cells

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K(D)) of 3.4 nm for Mcl-1, 70 nm for Bcl-x(L), and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x(L) antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.     

Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival

Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine). Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L), Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.     

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.