Antiapoptotic Protein Myeloid Cell Leukemia-1 Research Articles

From Inhibition to Degradation: Targeting the Antiapoptotic Protein Myeloid Cell Leukemia 1 (MCL1).

Protein-protein interactions (PPIs) have emerged as significant targets for therapeutic development, owing to their critical nature in diverse biological processes. An ideal PPI-based target is the protein myeloid cell leukemia 1 (MCL1), a critical prosurvival factor in cancers such as multiple myeloma where MCL1 levels directly correlate to disease progression. Current strategies for halting the antiapoptotic properties of MCL1 revolve around inhibiting its sequestration of proapoptotic factors. Existing inhibitors disrupt endogenous regulatory proteins; however, this strategy actually leads to an increase of MCL1 protein levels. Here, we show the development of hetero-bifunctional small molecules capable of selectively targeting MCL1 using a proteolysis targeting chimera (PROTAC) methodology leading to successful degradation. We have confirmed the involvement of the E3 ligase CUL4A-DDB1 cereblon ubiquitination pathway, making these PROTACs a first step toward a new class of antiapoptotic B-cell lymphoma 2 family protein degraders.

Cinobufagin, a bufadienolide from traditional Chinese medicine Bufo bufo gargarizans CANTOR, inhibits PC3 cell growth in vitro and in vivo

Abstract Objective To explore the effects of cinobufagin (CBF), an active component of toad venom (Bufo bufo gargarizans CANTOR), on the proliferation and apoptosis of PC3 human prostate cancer cells in vitro and preliminarily investigate the mechanism of CBF in suppressing tumor cell growth in vivo. Methods The effect of CBF on PC3 cells proliferation was detected using MTT assay. The morphological changes of PC3 cells were observed under an optical microscope. Colony formation assays were used to observe the CBF effect on colony formation by PC3 cells. PC3 cell apoptosis after treatment with CBF for 48 hours was monitored using flow cytometry. Furthermore, the effect of CBF on the expression of myeloid cell leukemia-1 (MCL-1) and related apoptotic proteins was examined using western blotting. A xenograft model was established in BALB/c nude mice to evaluate the effect of CBF on prostate cancer in vivo. Results The MTT assay results illustrated that PC3 cell proliferation was inhibited in vitro by CBF in a concentration- and time-dependent manner. Compared with the control group findings, CBF significantly inhibited the formation of PC3 cells (P = .005). Flow cytometry revealed that after treatment with 50 nM CBF for 48 hours, the apoptotic rate of PC3 cells was 41.97 (5.16)%, indicating that CBF could significantly induce its apoptosis (P = .003). In addition, optical and fluorescence microscopy uncovered remarkable inhibition of cell proliferation accompanied by morphologic changes. The western blotting result indicated that CBF obviously downregulated the expression of the anti-apoptotic protein MCL-1. Most importantly, CBF reduced the carcinogenicity of PC3 xenografts in nude mice. Conclusion CBF can inhibit the growth of PC3 cells both in vitro and in vivo and induce apoptosis of tumor cells. The corresponding mechanism may be correlated with the activation of caspase family proteins via MCL-1.

Overcoming MCL1 Resistance in Multiple Myeloma

Disruption of the intrinsic apoptotic pathway by the aberrant expression of the BCL2 family members are frequent events in multiple myeloma (MM). The anti-apoptotic protein myeloid cell leukemia-1 (MCL1) is highly expressed in MM and plays a crucial role in disease progression. Inhibition of MCL1, thus, represents a unique therapeutic opportunity for the control of the disease. Currently, there is no FDA-approved drug with the ability to selectively target MCL1. Because of its pivotal role in MM, MCL1 is considered a high-value therapeutic target in the clinic.In this report, we use a selective small-molecule inhibitor of MCL1, AZD5991, to examine the therapeutic consequences of MCL1 inhibition in MM. AZD5991 treatment resulted in dose-dependent cytotoxicity with EC50 values ranging from 64 and 417 nM at 24 hours for MCL1-sensitive cell lines (H929, MM.1S, RPMI-8226, U266, LP-1 and ANBL6VR). Two cell lines DOX40 and KMS-12-PE remained resistant to MCL1 inhibition. For the MCL1-sensitive MM.1S and H929 cells, the decrease in cell viability upon MCL1 inhibition was due to an increase in apoptosis. AZD5991 treatment led to 40-82% decrease in viability of CD138+ cells isolated from relapsed and refractory MM patients at a dose of 300 nM at 24 hours. These results indicate that AZD5991 has promising single-agent activity, but it would be prudent to study it in combination with other anti-MM therapies.The BM microenvironment enhances tumor cell growth and survival in MM. We found that soluble factors produced during the MM-BMSC interaction reduced the sensitivity of MM cells to AZD5991, and direct MM-BMSC contact blunted the cytotoxic effect of AZD5991. A comprehensive cytokine array analysis revealed an enrichment of a panel of pro-survival cytokines and growth factors, with the cytokines IL-6, IL-8 and GROα/β/γ being among the most highly up-regulated proteins, upon cell-cell contact between MM.1S cells and BMSCs. Enrichment of these cytokines in the BM milieu, at least in part, confer a protective effect on MM cells and endow them with the ability to resist MCL1 inhibition.A shift in the balance of BCL2 family members is often the primary reason for drug resistance. We found that the baseline BCL2 mRNA expression and the BCL2:MCL1 ratios in the MM cell lines examined are negatively correlated to their corresponding sensitivity to AZD5991. In other words, MM cells with a high BCL2 reservoir are more likely to circumvent cell death elicited by MCL1 inhibition. Treatment with AZD5991 alone leads to release of Bim from MCL1 and an increased Bim bound to BCL2. Venetoclax, a BH3 mimetic that selectively binds BCL2, treatment alone releases Bim from BCL2 and results in an increased Bim bound to MCL1. Bim binding to MCL1 and BCL2 was significantly diminished upon co-treatment. The free-floating Bim proteins subsequently activate the intrinsic apoptotic pathway by facilitating cytochrome c release. These results suggested that MM cells switch their survival dependency to BCL2 upon MCL1 inhibition, and that BCL2 blockade could be an effective way to overcome MCL1 resistance in MM.Based on these observations, we combined AZD5991 with Venetoclax in MM cells. A significant decrease in cell viability was observed with the combined therapy compared with both drugs used alone. Isobologram analysis confirmed greater than additive or synergistic effect upon co-treatment. The enhanced cytotoxic effect of the combined therapy retains even when the MM cells are in co-culture with BMSCs. Remarkably, the pro-survival cytokines IL-6, IL-8 and GROα/β/γ, which were expressed at high levels in the co-culture settings, were among the ones being most reduced after the combined therapy.Our results demonstrated that the combined AZD5991/Venetoclax therapy overcomes the inherent MCL1-resistance in MM via two independent mechanisms. First, the concomitant suppression of the anti-apoptotic proteins MCL1 and BCL2 prevent MM cells from escaping apoptosis by releasing Bim to trigger mitochondrial outer membrane potential and the subsequent release of cytochrome c to activate the intrinsic apoptotic pathway. Second, this combined therapy prevents MM cell growth by causing down-regulation of pro-survival cytokines and growth factors in the BM microenvironment. As a proof of concept, our data indicate combining therapeutics that selectively target the anti-apoptotic proteins MCL1 and BCL2 could be an effective therapy for MM patients. DisclosuresCidado:AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment.

Moxetumomab Pasudotox: First Global Approval.

Moxetumomab pasudotox-tdfk (LUMOXITI™), an anti CD22 recombinant immunotoxin, has been developed by MedImmune and its parent company AstraZeneca for the treatment of hairy cell leukaemia. The product, discovered at the National Cancer Institute, is an optimised version of immunotoxin CAT-3888. Moxetumomab pasudotox is composed of the Fv fragment of an anti-CD22 monoclonal antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A, PE38. The Fv portion of moxetumomab pasudotox binds to CD22, a cell surface receptor expressed on a variety of malignant B-cells, thereby delivering the toxin moiety PE38 directly to tumour cells. Once internalised, PE38 catalyses the ADP ribosylation of the diphthamide residue in elongation factor-2 (EF-2), resulting in the rapid fall in levels of the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1), leading to apoptotic cell death. This article summarizes the milestones in the development of moxetumomab pasudotox leading to this first approval for the treatment of adults with relapsed or refractory hairy cell leukaemia who received at least two prior systemic therapies, including treatment with a purine nucleoside analogue. Development of moxetumomab pasudotox for non-Hodgkin’s lymphoma, chronic lymphocytic leukaemia and precursor cell lymphoblastic leukaemia/lymphoma was discontinued.

Phenethyl Isothiocyanate Inhibits In Vivo Growth of Xenograft Tumors of Human Glioblastoma Cells.

Phenethyl isothiocyanate (PEITC) from cruciferous vegetables can inhibit the growth of various human cancer cells. In previous studies, we determined that PEITC inhibited the in vitro growth of human glioblastoma GBM 8401 cells by inducing apoptosis, inhibiting migration and invasion, and altering gene expression. Nevertheless, there are no further in vivo reports disclosing whether PEITC can suppress the growth of glioblastoma. Therefore, in this study we investigate the anti-tumor effects of PEITC in a xenograft model of glioblastoma in nude mice. Thirty nude mice were inoculated subcutaneously with GBM 8401 cells. Mice with one palpable tumor were divided randomly into three groups: control, PEITC-10, and PEITC-20 groups treated with 0.1% dimethyl sulfoxide (DMSO), and 10 and 20 μmole PEITC/100 μL PBS daily by oral gavage, respectively. PEITC significantly decreased tumor weights and volumes of GBM 8401 cells in mice, but did not affect the total body weights of mice. PEITC diminished the levels of anti-apoptotic proteins MCL-1 (myeloid cell leukemia 1) and XIAP (X-linked inhibitor of apoptosis protein) in GBM 8401 cells. PEITC enhanced the levels of caspase-3 and Bax in GBM 8401 cells. The growth of glioblastoma can be suppressed by the biological properties of PEITC in vivo. These effects might support further investigations into the potential use of PEITC as an anticancer drug for glioblastoma.

SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.

B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.

Coxiella burnetii Inhibits Neutrophil Apoptosis by Exploiting Survival Pathways and Antiapoptotic Protein Mcl-1.

Our previous study demonstrated that neutrophils play an important role in host defense against Coxiella burnetii infection in mice. In this study, avirulent strain C. burnetii Nine Mile phase II (NMII) was used to examine if C. burnetii can modulate mouse bone marrow-derived neutrophil apoptosis. The results indicated that NMII can inhibit neutrophil apoptosis. Western blotting demonstrated that caspase-3 cleavage was decreased in NMII-infected neutrophils, while phosphorylated mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase 1 (Erk1) were increased. Additionally, p38, Erk1/2, phosphoinositide 3-kinase (PI3K), or NF-κB inhibitors reduced the ability of NMII to inhibit neutrophil apoptosis. These results suggest that NMII-mediated inhibition of neutrophil apoptosis depends on its ability to activate neutrophil MAPK pathways. Antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) was significantly increased in NMII-infected neutrophils, and an Mcl-1 inhibitor significantly reduced the ability of NMII to inhibit neutrophil apoptosis. Mcl-1 protein stability was enhanced by phosphorylation at Thr-163 by Erk, and the protein levels were regulated by p38, Erk, PI3K, and NF-κB. Furthermore, the observation that a type IV secretion system (T4SS)-deficient dotA mutant showed a significantly reduced ability to inhibit neutrophil apoptosis compared to wild-type (WT) NMII suggests that T4SS-secreted factors may be involved in NMII-induced inhibition of neutrophil apoptosis. Collectively, these results demonstrate that NMII inhibits neutrophil apoptosis through inhibition of caspase-3 cleavage and activation of MAPK survival pathways with subsequent expression and stabilization of antiapoptotic protein Mcl-1, a process that may partially require the T4SS.

PI3K/Akt inhibitor LY294002 potentiates homoharringtonine antimyeloma activity in myeloma cells adhered to stromal cells and in SCID mouse xenograft.

Homoharringtonine (HHT) is a known anti-leukemia drug that inhibits multiple myeloma (MM) cells both in vitro and in vivo. Our prior study demonstrated that the potency of HHT in MM cells was compromised significantly when myeloma cells were co-cultured with BM stromal cells. This study aimed to investigate whether PI3K/Akt inhibitor LY294002 could potentiate the antimyeloma activity of HHT against MM cells adhered to BM stromal cells and in vivo xenograft models. A co-culture system composed of MM cells and human stromal cells was employed to mimic MM cells in bone marrow niche. The inhibitory and pro-apoptotic effect of HHT and LY294002 was determined by CCK-8 assay or flow cytometry. Expression of PI3K/Akt signaling molecules and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) was assessed by western blot analysis and/or reverse transcription real-time quantitative PCR (RT-qPCR). MM xenografts were used to evaluate antitumor effect of combined therapy with HHT and LY294002. Adhesion to BM stromal cells rendered MM cells resistant to HHT whereas silencing Mcl-1 partly reversed the resistance. LY294002 induced apoptosis in MM cells and potentiated the antimyeloma effects of HHT by inhibiting the PI3K/Akt signal pathway which was abnormally activated during adhesion. LY294002 also enhanced the antimyeloma effect of HHT in in vivo xenograft models. These findings suggest that activation of PI3K/Akt signal pathway was responsible for the resistance to HHT in MM cells adhered to stromal cells. LY294002 can potentiate the antimyeloma activity of HHT both in vitro and in vivo, which may represent a new clinical treatment in MM.

Curcumin induces apoptosis and inhibits proliferation in infantile hemangioma endothelial cells via downregulation of MCL-1 and HIF-1α.

Background:Curcumin has been used as an alternative medicine for the treatment of infantile hemangiomas (IHs); however, the mechanism underlying the effectiveness of curcumin in IHs remains largely unclear.Methods:In this study, we isolated primary human hemangioma endothelial cells (HemECs) from fresh surgical specimens of 3 patients. We treated HemECs by curcumin and investigated the alterations in proliferative and apoptotic signaling pathways with cell counting kit-8, flow cytometry, western blotting, immunofluorescence, and real-time polymerase chain reaction.Results and Conclusion:We found that curcumin potently inhibited proliferation in HemECs, achieving low-micromolar IC50 (the half maximal inhibitory concentration) value. We also observed that treatment with curcumin induced apoptosis in HemECs, as evidenced by positively Annexin-V-FITC staining, caspase-3 activation, and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP) in the treated cells. Moreover, we showed that curcumin suppressed the expression of antiapoptotic protein myeloid cell leukemia-1 (MCL-1), hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF).Altogether, our study suggests that the effectiveness of curcumin in IHs may be associated with its potent antiproliferative and apoptotic activities in HemECs.

Protein kinase C-eta regulates Mcl-1 level via ERK1

Protein kinase C (PKC)-eta (PKCη) is a member of the novel category of PKC family. It is overexpressed in breast cancer and was shown to inhibit apoptosis and contribute to chemoresistance. Since the anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance, we investigated if PKCη regulates Mcl-1 level. Silencing of PKCη decreased Mcl-1 in several breast cancer cells, including MCF-7 and T47D cells. PKCη depletion had no effect on MCL1 mRNA but the decrease in Mcl-1 by PKCη knockdown was blocked by proteasomal inhibitors, such as MG132 and lactacystin. Moreover, knockdown of Mule (Mcl-1 ubiquitin ligase) prevented Mcl-1 downregulation caused by PKCη deficiency. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-β, a substrate for Akt, had little effect on Mcl-1 downregulation caused by PKCη silencing. However, knockdown of PKCη but not PKCα, -δ or -ε caused a significant decrease in ERK (extracellular signal-regulated kinase) phosphorylation. Knockdown of ERK1 but not ERK2 decreased Mcl-1 level, and the decrease in Mcl-1 caused by PKCη knockdown was restored by ERK1 overexpression. These results suggest that PKCη utilizes the ERK signaling pathway to protect against ubiquitin-mediated proteasomal degradation of Mcl-1.

Novel post-transcriptional and post-translational regulation of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 determine the fate of breast cancer cells to survive or die.

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is pro-apoptotic protein Bcl-2-related Ovarian Killer (BOK). Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p regulates BOK expression by binding to its 3’-UTR in breast cancers. Interestingly, miR-296-5p also regulates the expression of anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1), which is highly expressed in breast cancers. Our results reveal that Mcl-1 and BOK constitute a regulatory feedback loop as ectopic BOK expression induces Mcl-1, whereas silencing of Mcl-1 results in reduced BOK levels in breast cancer cells. In addition, we show that silencing of Mcl-1 but not BOK reduced the long-term growth of breast cancer cells. Silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1. Depletion of BOK suppressed caspase-3 activation in the presence of paclitaxel and in turn protected cells from paclitaxel-induced apoptosis. Furthermore, we demonstrate that glycogen synthase kinase (GSK3) α/β interacts with BOK and regulates its level post-translationally in breast cancer cells. Taken together, our results suggest that fine tuning of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 may decide the fate of cancer cells to either undergo apoptosis or proliferation.

Regulation of anti-apoptotic Bcl-2 family protein Mcl-1 by S6 kinase 2.

The anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance. We have previously shown that knockdown of the 40S ribosomal protein S6 kinase-2 (S6K2), which acts downstream of the mechanistic target of rapamycin complex 1 (mTORC1), enhanced breast cancer cell death by apoptotic stimuli. The increase in cell death by S6K2 depletion was partly due to inactivation of Akt. In the present study, we investigated if S6K2 regulates Mcl-1, which acts downstream of Akt. Silencing of S6K2 but not S6K1 in T47D cells decreased Mcl-1 level, and potentiated apoptosis induced by TRAIL and doxorubicin. Knockdown of S6K2 also decreased the level of anti-apoptotic Bcl-xl. Depletion of the tumor suppressor protein PDCD4 (programmed cell death 4), which regulates translation of several anti-apoptotic proteins, reversed downregulation of Bcl-xl but not Mcl-1 and failed to reverse the effect of S6K2 knockdown on potentiation of doxorubicin-induced apoptosis. Downregulation of Mcl-1 by S6K2 knockdown was partly restored by the proteasome inhibitor MG132. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-β, a substrate for Akt, had little effect on Mcl-1 downregulation caused by S6K2 deficiency. Silencing of S6K2 increased the level of c-Jun N-terminal kinase (JNK) and knockdown of JNK1 increased basal Mcl-1 level and partly reversed the effect of S6K2 knockdown on Mcl-1 downregulation. JNK1 knockdown also had a modest effect in attenuating the increase in doxorubicin-induced apoptosis caused by S6K2 deficiency. These results suggest that S6K2 regulates apoptosis via multiple mechanisms, and involves both Akt and JNK.

Prostaglandin E1 reduces the keratinocyte toxicity of sorafenib by maintaining signal transducer and activator of transcription 3 (STAT3) activity and enhancing the cAMP response element binding protein (CREB) activity

Hand-foot skin reaction (HFSR) is a common side effect of multiple tyrosine kinase inhibitors (mTKIs). HFSR can necessitate dose reductions or interruption of therapy owing to its negative effect on the quality of life. Therefore, effective use of mTKIs requires measures to prevent HFSR. We evaluated the effect of prostaglandin E1 (PGE1) on HFSR, because PGE1 is already used to treat bed sores and skin ulcers and has established angiogenic and antiproliferative effects in keratinocytes. We found that the pathogenesis of sorafenib-induced HFSR is characterized by a decrease in levels of a phosphorylated signal transducer and activator of transcription 3 (STAT3). We investigated the effect of PGE1 on the sorafenib-mediated reduction in phosphorylated STAT3 levels in HaCaT human epidermal keratinocytes. In cells treated with sorafenib, phosphorylated STAT3 levels decreased in a concentration-dependent manner, and this effect was blocked in cells treated with sorafenib and PGE1. Furthermore, the expression of phosphorylated STAT3, the antiapoptotic proteins myeloid cell leukemia-1 (Mcl-1) and survivin decreased in cells pretreated with an inhibitor of cAMP response element binding protein (CREB). Cell viability increased in cells treated with sorafenib and PGE1 compared with that in cells treated with sorafenib alone, and these effects were not observed in STAT3 knockdown HaCaT cells. Collectively, these findings indicate that PGE1 blocks the inhibitory effects of sorafenib on cell growth by maintaining the activity of STAT3 and enhancing the CREB activity. Therefore, PGE1 might represent an effective treatment for the prevention of sorafenib-induced HFSR.

Myeloid cell leukaemia 1 has a vital role in retinoic acid-mediated protection of Toll-like receptor 9-stimulated B cells from spontaneous and DNA damage-induced apoptosis.

Vitamin A is an essential anti-infective agent with pleiotropic effects on cells of the immune system. The goal of the present study was to unravel the impact of the vitamin A metabolite retinoic acid (RA) on B-cell survival related both to normal B-cell homeostasis and to the detrimental effects imposed by DNA-damaging agents. By combining RA with Toll-like receptor 9 (TLR9) ligands, we show that RA prevents spontaneous, irradiation- and doxorubicin-induced apoptosis of human B cells in an RA receptor-dependent manner. RA-mediated survival involved up-regulation of the anti-apoptotic protein myeloid cell leukemia 1 (MCL1) at the transcriptional level, and knock down of MCL1 by small interfering RNA partially reversed the effects of RA. To ensure that the combination of TLR9-ligands and RA would not promote the survival of malignant B cells, the combined effects of stimulation with RA and TLR9 ligands was assessed on cells from patients with B-cell malignancies. In contrast to the effects on normal B cells, the combination of TLR9 stimulation and RA neither enhanced the MCL1 levels nor inhibited the death of malignant B cells challenged by DNA-damaging agents. Taken together, the present results reveal a vital role of MCL1 in RA-mediated survival of normal B cells. Moreover, the findings suggest that RA in combination with TLR9 ligands might be useful adjuvants in the treatment of B-cell malignancies by selectively protecting normal and not malignant B cells from DNA-damage-induced cell death.

Abstract 375: MCL-1 Regulates Mitochondrial Dynamics and Mitophagy

The anti-apoptotic BCL-2 family protein Myeloid Cell Leukemia-1 (MCL-1) is essential for maintaining mitochondrial integrity and cardiac function in the adult heart. Recently, we reported that cardiac-specific deletion of MCL-1 in mice leads to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Surprisingly, MCL-1 ablation does not result in apoptotic cell death, but rather cells show signs of mitochondrial deterioration and necrotic cell death. This suggests that in addition to its anti-apoptotic role, MCL-1 has unidentified roles in maintaining mitochondrial function in cardiac myocytes. MCL-1 exists in two distinct locations in myocytes: one form is localized to the outer mitochondrial membrane (MCL-1 OM ) and a shorter cleaved form exists in the mitochondrial matrix (MCL-1 Matrix ). Interestingly, overexpression of MCL-1 WT or MCL-1 OM , but not MCL-1 Matrix , leads to translocation of Drp1 to mitochondria, which correlates with fission and perinuclear aggregation of mitochondria. We also found that Drp1 co-immunoprecipitates with MCL-1 in heart lysates and that MCL-1-deficient hearts lack mitochondrial Drp1. Additionally, the interaction between Drp1 and MCL-1 and mitochondrial fission increase in response to serum starvation in neonatal myocytes. This suggests that MCL-1 OM recruits Drp1 to mitochondria to induce fission. Since there is a strong link between mitochondrial fission and degradation, we examined the effect of MCL-1 on mitophagy. Parkin is an E3 ubiquitin ligase that plays an important role in mitophagy. MEFs, which lack detectable Parkin, do not efficiently clear their depolarized mitochondria in response to FCCP treatment. However, overexpression of Parkin or MCL-1 increased the efficiency of mitophagy in MEFs in response to FCCP treatment. MCL-1-mediated mitophagy is abrogated in autophagy-deficient Atg5 -/- MEFs, confirming that the clearance occurs via enhanced autophagy. Thus, our data suggest that MCL-1 OM has dual actions in coordinating Drp1-mediated fission and mitophagy, which allows for more efficient removal of damaged mitochondria.

High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition.

The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression.

Role of microRNA-29b in myelodysplastic syndromes during transformation to overt leukaemia

Chromosome 7q32 is a frequently deleted region in myelodysplastic syndromes (MDSs) and encodes the microRNAs (miRNAs) miR-29a/miR-29b. Both miR-29s down-regulate the anti-apoptotic protein myeloid cell leukaemia 1 (MCL-1) in acute myeloid leukaemia. Thus, to investigate the role of miR-29s in the transformation of MDS to overt leukaemia (OL), we analysed the relationship between miR-29 expression and MCL-1 expression. MiR-29b expression was down-regulated in refractory anaemia and OL bone marrow as compared to that in control bone marrow. MCL-1 expression level in OL was significantly higher than that in refractory anaemia with excess blasts and a negative correlation was observed between miR-29b and MCL-1 messenger RNA expression levels in OL samples. Immunohistochemical analysis showed that the MCL-1 positive rate among MDS bone marrow CD34 positive cells significantly increased during transformation to OL. Additionally, MCL-1 positive cells were negative for cleaved caspase 3, which indicated that these cells avoided apoptosis. Reduced miR-29b expression in MDS bone marrow cells might trigger transformation to OL via overexpression of MCL-1 in blastic cells.

Abstract B09: MCL-1 inhibition improves ABT199 efficacy for BCL-2-dependent neuroblastoma

Abstract Introduction: The gene encoding anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in a large subset of all neuroblastoma patients and plays an important oncogenic role. Previous studies with BCL-2 inhibitor ABT263 showed favorable antitumor activity for BCL-2-dependent neuroblastoma cells, but unfortunately the clinical use of ABT263 was associated with dose-limiting thrombocytopenia due to the concomitant inhibition of anti-apoptotic protein BCL-extra large (BCL-XL). Therefore, the more specific BCL-2 inhibitor ABT199 was developed. In this study, we explored the preclinical therapeutic potential of ABT199 for neuroblastoma treatment and strategies to prevent ABT199 resistance. Experimental design: IC50 and LC50 values of ABT199 were determined for 24 neuroblastoma cell lines to study predictive biomarkers for sensitivity to ABT199. In vitro target inhibition and apoptosis were studied in predictive biomarker-positive and -negative cell lines. The in vivo efficacy of ABT199 was subsequently investigated in a BCL-2-dependent neuroblastoma xenograft model and compared with the in vivo efficacy of ABT263. Next, in vitro and in vivo effects of ABT199 on other BCL-2 family members were studied by Western Blot analysis and Affymetrix mRNA profiling. Lastly, combined effects of ABT199 and MCL-1 knockdown using shRNA or inhibition with the selective inhibitor A-1210477 were studied in vitro. Results: The sensitivity of neuroblastoma cell lines to ABT199 was best predicted by protein levels of BCL-2 and BCL-2-like protein 11 (BIM)/BCL-2 complex. ABT199 killed neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels more potently than low expressing cell lines with average LC50 values of 0.17 versus 15.46 μmol/L, respectively. In vitro effects on cell viability nicely correlated with target-specific effects on BIM displacement from BCL-2 and downstream effects on apoptotic markers cytochrome c and cleaved PARP. Target-specific effects and effects on apoptotic markers were dose-dependent, indicating that BIM displacement from BCL-2, cytochrome c release from the mitochondria and cleaved PARP can be utilized as pharmacodynamics biomarkers of ABT199 efficacy. Oral treatment of female NMRI homozygous (nu/nu) mice with KCNR neuroblastoma xenografts expressing high BCL-2 levels with 100 mg/kg/d ABT199 for three consecutive weeks resulted in significant tumor growth inhibition. However, similar treatment with ABT263 demonstrated superior antitumor activity over ABT199 (i.e., complete tumor regression). The superior in vivo activity of ABT263 was most probably the result of the simultaneous inhibition of multiple anti-apoptotic BCL-2 family proteins by ABT263, since maximum BIM displacement from BCL-2 was observed after ABT199 treatment. ABT199 furthermore induced a strong apoptotic response, as was shown by the remarkable increase in cleaved caspase-3 levels. We showed in vitro and in vivo that neuroblastoma cells might survive ABT199 treatment due to NOXA-mediated upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and sequestration of released BIM by MCL-1. In vitro knockdown of MCL-1 sensitized high BCL-2-expressing neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Results were confirmed with the selective MCL-1 inhibitor A-1210477. Conclusions: Taken together, the results presented in this study strongly suggest that children with neuroblastoma tumors expressing high levels of BCL-2 and BIM/BCL-2 complex might benefit from combined treatment with ABT199 and inhibitors of MCL-1. Citation Format: Laurel T. Bate-Eya, Ilona J.M. den Hartog, Ida van der Ploeg, Linda Schild, Jan Koster, Rogier Versteeg, Huib N. Caron, Jan J. Molenaar, M. Emmy M. Dolman. MCL-1 inhibition improves ABT199 efficacy for BCL-2-dependent neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B09.

Leishmania donovani Exploits Myeloid Cell Leukemia 1 (MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis

Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.

Both IGF1R and INSR Knockdown Exert Antitumorigenic Effects in Prostate Cancer In Vitro and In Vivo.

The IGF network with its main receptors IGF receptor 1 (IGF1R) and insulin receptor (INSR) is of major importance for cancer initiation and progression. To date, clinical studies targeting this network were disappointing and call for thorough analysis of the IGF network in cancer models. We highlight the oncogenic effects controlled by IGF1R and INSR in prostate cancer cells and show similarities as well as differences after receptor knockdown (KD). In PC3 prostate cancer cells stably transduced with inducible short hairpin RNAs, targeting IGF1R or INSR attenuated cell growth and proliferation ultimately driving cells into apoptosis. IGF1R KD triggered rapid and strong antiproliferative and proapoptotic responses, whereas these effects were less pronounced and delayed after INSR KD. Down-regulation of the antiapoptotic proteins myeloid cell leukemia-1 and survivin was observed in both KDs, whereas IGF1R KD also attenuated expression of prosurvival proteins B cell lymphoma-2 and B cell lymphoma-xL. Receptor KD induced cell death involved autophagy in particular upon IGF1R KD; however, no difference in mitochondrial energy metabolism was observed. In a mouse xenograft model, induction of IGF1R or INSR KD after tumor establishment eradicated most of the tumors. After 20 days of receptor KD, tumor cells were found only in 1/14 IGF1R and 3/14 INSR KD tumor remnants. Collectively, our data underline the oncogenic functions of IGF1R and INSR in prostate cancer namely growth, proliferation, and survival in vitro as well as in vivo and identify myeloid cell leukemia-1 and survivin as important mediators of inhibitory and apoptotic effects.