Mcl-1

Mcl-1 is a Bcl-2 family protein which can act as an apical molecule in apoptosis control, promoting cell survival by interfering at an early stage in a cascade of events leading to release of cytochrome c from mitochondria. Mcl-1 has a short half life and is a highly regulated protein, induced by a wide range of survival signals and also rapidly down regulated during apoptosis. Mcl-1 can also readily be cleaved by caspases during apoptosis to produce a cell death promoting molecule. The multiple levels of control of Mcl-1 expression suggest that Mcl-1 plays a critical role in controlling life and death decisions in response to rapidly changing environmental cues and Mcl-1 is required for embryonic development and the function of the immune system. Expression of Mcl-1 may be useful in informing decision making in the treatment of various cancers, and countering Mcl-1 function may be an attractive therapeutic strategy in malignancy, inflammatory conditions and infectious disease where Mcl-1 may play a major role in suppressing apoptosis.     

Unique biology of Mcl-1: therapeutic opportunities in cancer

Accumulating evidence suggests that Mcl-1 plays a critical pro-survival role in the development and maintenance of both normal and malignant tissues. Regulation of Mcl-1 expression occurs at multiple levels, allowing for either the rapid induction or elimination of the protein in response to different cellular events. This suggests that Mcl-1 can play an early role in response to signals directing either cell survival or cell death. Deregulation of pathways regulating Mcl-1 that result in its over-expression likely contribute to a cell's inability to properly respond to death signals possibly leading to cell immortalization and tumorigenic conversion. Correspondingly, Mcl-1 has been shown to be up-regulated in numerous hematological and solid tumor malignancies. Moreover, this up-regulation appears to be a factor in the resistance of some cancer types to conventional cancer therapies. Mechanisms that abrogate the pro-survival function of Mcl-1 either by diminishing its levels or inactivating its functional BH3 groove have shown promise for the combinational treatment with existing cancer therapies and as single agents in certain malignancies. Here we review the various pathways that regulate Mcl-1 expression and describe agents that are currently under development to modulate Mcl-1 activity for therapeutic benefit in oncology.     

Inhibiting mitochondrial-dependent proteolysis of Mcl-1 promotes resistance to DNA damage

Elimination of Myeloid Leukemia Cell 1 (Mcl-1) is an early event in the onset of cell death following DNA damage and in many settings plays a critical role in dictating the success of chemotherapeutic agents. Following DNA damage, Mcl-1 is rapidly and efficiently targeted to the 26S proteasome through the action of E3 ubiquitin ligases. Tumors having acquired lesions that lead to stabilization of Mcl-1 are highly aggressive and have a poorer prognosis. Herein, we further characterize an additional mechanism of Mcl-1 proteolysis that is proteasome-independent but mitochondrial-dependent. A mitochondrial targeting signal located in the N-terminus of Mcl-1 is essential for targeting Mcl-1 to this alternative degradative avenue. We demonstrate that the Akt/mTORC1 survival pathway protects Mcl-1 from mitochondrial-dependent proteolysis. Disrupting Mcl-1 mitochondrial targeting improves the pro-survival capacity of Mcl-1 both ex vivo and in vivo in the well characterized mouse Eμ-Myc lymphoma model. Our data uncover an important relationship between the mitochondria and the Mcl-1 N-terminus in dictating cell fate following DNA damage.     

The anti-apoptotic Bcl-2 family member Mcl-1 promotes T lymphocyte survival at multiple stages

T lymphocyte development and function are tightly regulated by the intrinsic death pathway through members of the Bcl-2 family. Genetic studies have demonstrated that the Bcl-2 family member Mcl-1 is an important anti-apoptotic protein in the development of multiple cell types including T lymphocytes. However, the expression pattern and anti-apoptotic roles of Mcl-1 in T lymphocytes at different developmental stages remain to be fully determined. In this study, we examined the expression pattern of Mcl-1 in different populations of T cells at the single-cell level and found that Mcl-1 protein is constitutively expressed in all T cell populations and up-regulated upon TCR stimulation. We then investigated the role of Mcl-1 in the survival of these different populations by conditionally deleting Mcl-1 at various T cell stages. Our results show that Mcl-1 is required for the survival of double-negative and single-positive thymocytes as well as naive and activated T cells. Furthermore, we demonstrate that Mcl-1 functions together with Bcl-xL to promote double-positive thymocyte survival. Thus, Mcl-1 is a critical anti-apoptotic factor for the survival of T cells at multiple stages in vivo.     

Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung adenocarcinoma CL1.0 cells. Involvement of phosphatidylinositol 3-kinase/Akt pathway

Cyclooxygenase 2 (COX-2) has been reported to be commonly expressed in advanced stages of human lung adenocarcinoma. In this study, the COX-2 constitutive expression vector was transfected into a human lung adenocarcinoma cell line CL1.0 and several clones were obtained which stably expressed COX-2. These COX-2-overexpressed clones demonstrated remarkable resistance to apoptosis induced by Ultraviolet B (UVB) irradiation, vinblastine B (VBL) cell lymphoma-2 (Bcl-2), or other anti-cancer drugs. To understand how COX-2 prevents apoptosis, the investigators examined the expression level of Bcl-2 family members. Mcl-1, but not other Bcl-2 members, was significantly up-regulated by COX-2 transfection or prostaglandin E(2) (PGE(2)) treatment. Treatment of COX-2-overexpressed cells (cox-2/cl.4) with two specific COX-2 inhibitors, NS-398 and celecoxib, caused an effective reduction of the increased level of Mcl-1. These data suggest that the expression level of Mcl-1 is tightly regulated by COX-2. Moreover, transfection of cox-2/cl.4 cells with antisense Mcl-1 enhanced apoptosis induced by UVB irradiation, revealing that Mcl-1 plays a crucial role in cell survival activity mediated by COX-2. Furthermore, COX-2 transfection or PGE(2) treatment evidently activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Inhibition of the PI3K pathway by LY294002 or wortmannin effectively attenuated the increased level of Mcl-1 induced by COX-2 or PGE(2). Blocking the PI3K activity with a dominant-negative vector, DN-p85, also greatly diminished the level of Mcl-1 and enhanced UVB-elicited cell death in cells transfected by COX-2. In a similar way, LY294002 inhibited cell survival and Mcl-1 level in PGE(2)-treated CL1.0 cells. These findings suggest that COX-2 promotes cell survival by up-regulating the level of Mcl-1 by activating the PI3K/Akt-dependent pathway.     

Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells

Anti-apoptotic Bcl-2 family proteins have been reported to play an important role in apoptotic cell death of human malignancies. The aim of this study was to delineate the mechanism of anti-apoptotic Bcl-2 family proteins in pancreatic cancer (PaCa) cell survival. We first analyzed the endogenous expression and subcellular localization of anti-apoptotic Bcl-2 family proteins in six PaCa cell lines by Western blot. To delineate the functional role of Bcl-2 family proteins, siRNA-mediated knock-down of protein expression was used. Apoptosis was measured by Cell Death ELISA and Hoechst 33258 staining. In the results, the expression of anti-apoptotic Bcl-2 family proteins varied between PaCa cell lines. Mcl-1 knock-down resulted in marked cleavage of PARP and induction of apoptosis. Down-regulation of Bcl-2 or Bcl-xL had a much weaker effect. Simultaneous knock-down of Bcl-xL and Mcl-1 strongly induced apoptosis, but simultaneous knock-down of Bcl-xL/Bcl-2 or Mcl-1/Bcl-2 had no additive effect. The apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was associated with translocation of Bax from the cytosol to the mitochondrial membrane, cytochrome c release, and caspase activation. These results demonstrated that Bcl-xL and Mcl-1 play an important role in pancreatic cancer cell survival. Targeting both Bcl-xL and Mcl-1 may be an intriguing therapeutic strategy in PaCa.     

Selective induction of cell death in melanoma cell lines through targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies.     

ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.     

Mcl-1 is essential for the survival of synovial fibroblasts in rheumatoid arthritis

Mcl-1 is a Bcl-2-family, antiapoptotic molecule that is critical for the survival of T and B lymphocytes and macrophages; however, its role in nonhemopoietic cells remains to be fully elucidated. The current study focuses on the role of Mcl-1 in rheumatoid arthritis (RA). Mcl-1 was strongly expressed in the synovial lining and was increased in the sublining fibroblasts of patients with RA, compared with control synovial tissue. The expression of Mcl-1 in sublining fibroblasts correlated with the degree of inflammation and TNF-alpha, and IL-1beta treatment of cultured synovial fibroblasts resulted in the increased expression of Mcl-1 at the mRNA and protein levels. Mcl-1 was critical for the survival of RA synovial fibroblasts, because the forced reduction of Mcl-1 using a Mcl-1 antisense-expressing adenoviral vector induced apoptotic cell death, which was mediated through Bax, Bak, and Bim. These observations document a critical role for Mcl-1 in protecting against apoptosis in RA and suggest that Mc1-1 is a potential therapeutic target in this disease.     

Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell responses

Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4⁺ and CD8⁺ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4⁺ and CD8⁺ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

Sodium salicylate promotes neutrophil apoptosis by stimulating caspase-dependent turnover of Mcl-1

Mcl-1 is an antiapoptotic member of the Bcl-2 family of proteins that plays a central role in cell survival of neutrophils and other cells. The protein is unusual among family members in that it has a very short half-life of 2-3 h. In this report, we show that sodium salicylate (at 10 mM) greatly enhances the rate at which neutrophils undergo apoptosis and, in parallel, greatly accelerates the turnover rate of Mcl-1, decreasing its half-life to only 90 min. Whereas constitutive and GM-CSF-modified Mcl-1 turnover is regulated by the proteasome, the accelerated sodium salicylate-induced Mcl-1 turnover is mediated largely via caspases. Sodium salicylate resulted in rapid activation of caspase-3, -8, -9, and -10, and salicylate-accelerated Mcl-1 turnover was partly blocked by caspase inhibitors. Sodium salicylate also induced dramatic changes in the activities of members of the MAPK family implicated in Mcl-1 turnover and apoptosis. For example, sodium salicylate blocked GM-CSF-stimulated Erk and Akt activation, but resulted in rapid and sustained activation of p38-MAPK, an event mimicked by okadaic acid that also accelerates Mcl-1 turnover and neutrophil apoptosis. These data thus shed important new insights into the dynamic and highly regulated control of neutrophil apoptosis that is effected by modification in the rate of Mcl-1 turnover.     

Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis

Venetoclax plus cytarabine therapy is approved for elderly acute myeloid leukemia (AML) patients and needs further improvement. We studied the mechanisms of venetoclax plus cytarabine treatment and searched for a third agent to enhance their effects. Cytarabine induces S phase arrest-mediated DNA damage with activation of DNA replication checkpoint kinase 1 (Chk1) through phosphorylation, while venetoclax induces B cell lymphoma 2 (Bcl-2)-interacting mediator of cell death (Bim)-mediated apoptotic DNA damage. Myeloid cell leukemia-1 (Mcl-1) plays negative roles in both events by sequestering Bim and accelerating Chk1 phosphorylation. Venetoclax releases Bim from Bcl-2 with increased Bim binding to Mcl-1. Artesunate, an antimalaria drug, induces Noxa to replace Bim from Mcl-1 and induces synergistic apoptosis with venetoclax accompanied with Mcl-1 reduction. Silencing Mcl-1 or adding venetoclax/artesunate diminishes the cytarabine resistance pathway p-Chk1. The triple combination exhibits S phase arrest with enhanced DNA damage, improves AML colony formation inhibition, and prolongs survival of two mice xenograft models compared to the venetoclax/cytarabine dual combination. Artesunate serves as a bridge for venetoclax and cytarabine combination by Noxa and Bim-mediated apoptosis and Mcl-1 reduction. We provide a new triple combination for AML treatment by targeting the Noxa/Mcl-1/Bim axis to reverse Mcl-1/p-Chk1 resistance of cytarabine therapy.Subject terms: Acute myeloid leukaemia, Pharmacodynamics     

The proteasome inhibitor MG132 potentiates TRAIL receptor agonist-induced apoptosis by stabilizing tBid and Bik in human head and neck squamous cell carcinoma cells

Head and neck squamous cell carcinoma (HNSCC) is often resistant to conventional chemotherapy and thus requires novel treatment regimens. Here, we investigated the effects of the proteasome inhibitor MG132 in combination with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAIL receptor 1 (DR4)-specific monoclonal antibody, AY4, on sensitization of TRAIL- and AY4-resistant human HNSCC cell lines. Combination treatment of HNSCC cells synergistically induced apoptotic cell death accompanied by caspase-8, caspase-9, and caspase-3 activation and Bid cleavage into truncated Bid (tBid). Generation and accumulation of tBid through the cooperative action of MG132 with TRAIL or AY4 and Bik accumulation through MG132-mediated proteasome inhibition are critical to the synergistic apoptosis. In HNSCC cells, Bak was constrained by Mcl-1 and Bcl-X(L), but not by Bcl-2. Conversely, Bax did not interact with Mcl-1, Bcl-X(L), or Bcl-2. Importantly, tBid plays a major role in Bax activation, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-X(L), pointing to the synergistic mechanism of the combination treatment. In addition, knockdown of both Mcl-1 and Bcl-X(L) significantly sensitized HNSCC cells to TRAIL and AY4 as a single agent, suggesting that Bak constraint by Mcl-1 and Bcl-X(L) is an important resistance mechanism of TRAIL receptor-mediated apoptotic cell death. Our results provide a novel molecular mechanism for the potent synergy between MG132 proteasome inhibitor and TRAIL receptor agonists in HNSCC cells, suggesting that the combination of these agents may offer a new therapeutic strategy for HNSCC treatment.     

Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.     

Importance of Bcl-2-family proteins in murine hematopoietic progenitor and early B cells

Mitochondrial apoptosis regulates survival and development of hematopoietic cells. Prominent roles of some Bcl-2-family members in this regulation have been established, for instance for pro-apoptotic Bim and anti-apoptotic Mcl-1. Additional, mostly smaller roles are known for other Bcl-2-members but it has been extremely difficult to obtain a comprehensive picture of the regulation of mitochondrial apoptosis in hematopoietic cells by Bcl-2-family proteins. We here use a system of mouse ‘conditionally immortalized’ lymphoid-primed hematopoietic progenitor (LMPP) cells that can be differentiated in vitro to pro-B cells, to analyze the importance of these proteins in cell survival. We established cells deficient in Bim, Noxa, Bim/Noxa, Bim/Puma, Bim/Bmf, Bax, Bak or Bax/Bak and use specific inhibitors of Bcl-2, Bcl-X_L and Mcl-1 to assess their importance. In progenitor (LMPP) cells, we found an important role of Noxa, alone and together with Bim. Cell death induced by inhibition of Bcl-2 and Bcl-X_L entirely depended on Bim and could be implemented by Bax and by Bak. Inhibition of Mcl-1 caused apoptosis that was independent of Bim but strongly depended on Noxa and was completely prevented by the absence of Bax; small amounts of anti-apoptotic proteins were co-immunoprecipitated with Bim. During differentiation to pro-B cells, substantial changes in the expression of Bcl-2-family proteins were seen, and Bcl-2, Bcl-X_L and Mcl-1 were all partially in complexes with Bim. In differentiated cells, Noxa appeared to have lost all importance while the loss of Bim and Puma provided protection. The results strongly suggest that the main role of Bim in these hematopoietic cells is the neutralization of Mcl-1, identify a number of likely molecular events during the maintenance of survival and the induction of apoptosis in mouse hematopoietic progenitor cells, and provide data on the regulation of expression and importance of these proteins during differentiation along the B cell lineage.Subject terms: Apoptosis, Immune cell death