Segui questo link per vedere altri tipi di pubblicazioni sul tema: Mcl-1/Bcl-XL.
Articoli di riviste sul tema "Mcl-1/Bcl-XL"
Cita una fonte nei formati APA, MLA, Chicago, Harvard e in molti altri stili
Vedi i top-50 articoli di riviste per l'attività di ricerca sul tema "Mcl-1/Bcl-XL".
Accanto a ogni fonte nell'elenco di riferimenti c'è un pulsante "Aggiungi alla bibliografia". Premilo e genereremo automaticamente la citazione bibliografica dell'opera scelta nello stile citazionale di cui hai bisogno: APA, MLA, Harvard, Chicago, Vancouver ecc.
Puoi anche scaricare il testo completo della pubblicazione scientifica nel formato .pdf e leggere online l'abstract (il sommario) dell'opera se è presente nei metadati.
Vedi gli articoli di riviste di molte aree scientifiche e compila una bibliografia corretta.
1
Oh, Doyeun, Seongmin Yoon, Seonyang Park e Jeehyeon Bae. "Differential Regulations and Roles of Mcl-1 and Bcl-Xl during Megakaryopoiesis". Blood 112, n. 11 (16 novembre 2008): 4727. http://dx.doi.org/10.1182/blood.v112.11.4727.4727.
Testo completoAbstract (sommario):
Abstract Backgrounds: Platelet plays an essential role in thrombosis and hemostasis and is produced from hematopoietic stem cells through a serious of differentiation and maturation processes called megakaryopoiesis. The major factor known to control platelet formation is thrombopoietin (TPO), but recently more proteins including apoptosis regulators have been reported to involve in megakaryopoiesis. Evolutionally conserved Bcl-2 family proteins are central regulators of apoptosis. Antiapoptotic Bcl-2 subfamily comprised of Bcl-xL, Mcl-1, Bcl-2, Bcl-w, and Bfl-1 plays a pivotal role in controlling cell death and survival under various conditions. According a recent study, Bcl-xL is a key molecular clock that determines the life span of platelets, but the role and regulation of Bcl-2 members in megakaryopoiesis are largely unknown. Methods and Results: We have established an in vitro system of megakaryopoiesis and performed the profiling of Bcl-2 family genes during megakaryocytosis. We found that Bcl-xL and Mcl-1 were predominant molecules among other members of the pro-survival proteins as determined by quantitative RT-PCR. TPO differentially regulates Bcl-xL and Mcl-1 in Meg-01 cells, in which Bcl-xL protein was significantly up-regulated while the level of Mcl-1 was attenuated. Furthermore, the roles of Bcl-xL and Mcl-1 during megakaryopoiesis were determined by modulating the expression levels of two proteins. Overexpression of Mcl-1 prominently enhanced the viability of the cells, whereas the knockdown of Mcl-1 promoted apoptosis of the cells. In contrast, forced expression of Bcl-xL did not affect the cell survival but rather significantly stimulated differentiation of megakarycytes. Conclusion: Bcl-xL and Mcl-1 are likely essential molecules during megakaryopoiesis. Moreover, the present study implies that although both Bcl-xL and Mcl-1 are members of antiapoptotic Bcl-2 family that promote the survival of different cells, these two proteins have distinctive and non-redundant functions in megakaryocytes.
2
Seyfried, Felix, Felix Uli Stirnweiß, Alexandra Niedermayer, Stefanie Enzenmüller, Rebecca Louise Hörl, Vera Münch, Stefan Köhrer, Klaus-Michael Debatin e Lüder Hinrich Meyer. "Synergistic activity of combined inhibition of anti-apoptotic molecules in B-cell precursor ALL". Leukemia 36, n. 4 (14 gennaio 2022): 901–12. http://dx.doi.org/10.1038/s41375-021-01502-z.
Testo completoAbstract (sommario):
AbstractTargeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.
3
Debrincat, Marlyse A., Emma C. Josefsson, Chloé James, Katya J. Henley, Sarah Ellis, Marion Lebois, Kelly L. Betterman et al. "Mcl-1 and Bcl-xL coordinately regulate megakaryocyte survival". Blood 119, n. 24 (14 giugno 2012): 5850–58. http://dx.doi.org/10.1182/blood-2011-12-398834.
Testo completoAbstract (sommario):
Abstract Mature megakaryocytes depend on the function of Bcl-xL, a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-xL does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1Pf4Δ/Pf4Δ animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-xL, and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-xL in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-xPf4Δ/Pf4ΔMcl-1Pf4Δ/Pf4Δ embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-xL and Mcl-1 is essential for the viability of the megakaryocyte lineage.
4
Senichkin, Viacheslav V., Nikolay V. Pervushin, Alexey V. Zamaraev, Elena V. Sazonova, Anton P. Zuev, Alena Y. Streletskaia, Tatiana A. Prikazchikova et al. "Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors". Cancers 14, n. 1 (30 dicembre 2021): 181. http://dx.doi.org/10.3390/cancers14010181.
Testo completoAbstract (sommario):
BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.
5
Bernardo, Paul H., Thirunavukkarasu Sivaraman, Kah-Fei Wan, Jin Xu, Janarthanan Krishnamoorthy, Chun Meng Song, Liming Tian et al. "Synthesis of a rhodanine-based compound library targeting Bcl-XL and Mcl-1". Pure and Applied Chemistry 83, n. 3 (5 febbraio 2011): 723–31. http://dx.doi.org/10.1351/pac-con-10-10-29.
Testo completoAbstract (sommario):
A small library of pyridine-based rhodanine analogues of BH3I-1 were synthesized and screened against B-cell lymphoma-extra large (Bcl-XL) and myeloid cell leukemia sequence 1 (Mcl-1) for the ability to displace 5-carboxyfluorescein-labeled Bak peptide (Flu-Bak). Differences in selectivity toward Bcl-XL and Mcl-1 were observed, and the binding modes of selected compounds were studied further. The results may be useful in designing potent small-molecule inhibitors of Bcl-XL and Mcl-1 as well as selective Mcl-1 inhibitors.
6
Haselager, Marco V., Karoline Kielbassa, Johanna ter Burg, Danique J. C. Bax, Stacey M. Fernandes, Jannie Borst, Constantine Tam et al. "Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL". Blood 136, n. 25 (17 dicembre 2020): 2918–26. http://dx.doi.org/10.1182/blood.2019004326.
Testo completoAbstract (sommario):
Abstract Chronic lymphocytic leukemia (CLL) cells cycle between lymph node (LN) and peripheral blood (PB) and display major shifts in Bcl-2 family members between those compartments. Specifically, Bcl-XL and Mcl-1, which are not targeted by the Bcl-2 inhibitor venetoclax, are increased in the LN. Because ibrutinib forces CLL cells out of the LN, we hypothesized that ibrutinib may thereby affect expression of Bcl-XL and Mcl-1 and sensitize CLL cells to venetoclax. We investigated expression of Bcl-2 family members in patients under ibrutinib or venetoclax treatment, combined with dissecting functional interactions of Bcl-2 family members, in an in vitro model of venetoclax resistance. In the PB, recent LN emigrants had higher Bcl-XL and Mcl-1 expression than did cells immigrating back to the LN. Under ibrutinib treatment, this distinction collapsed; significantly, the pretreatment profile reappeared in patients who relapsed on ibrutinib. However, in response to venetoclax, Bcl-2 members displayed an early increase, underlining the different modes of action of these 2 drugs. Profiling by BH3 mimetics was performed in CLL cells fully resistant to venetoclax due to CD40-mediated induction of Bcl-XL, Mcl-1, and Bfl-1. Several dual or triple combinations of BH3 mimetics were highly synergistic in restoring killing of CLL cells. Lastly, we demonstrated that proapoptotic Bim interacts with antiapoptotic Bcl-2 members in a sequential manner: Bcl-2 > Bcl-XL > Mcl-1 > Bfl-1. Combined, the data indicate that Bcl-XL is more important in venetoclax resistance than is Mcl-1 and provide biological rationale for potential synergy between ibrutinib and venetoclax.
7
Geng, Xiangrong, Chenguang Wang, Ashley Wolfe, Ira Maine, Suhaib Abdelrahman, Carlos A. Murga-Zamalloa e Ryan A. Wilcox. "Pre-Clinical Activity of Navitoclax in TCR-Driven and Non-ALCL Mature T-Cell Lymphomas". Blood 142, Supplement 1 (28 novembre 2023): 4146. http://dx.doi.org/10.1182/blood-2023-174520.
Testo completoAbstract (sommario):
Background: In contrast to many B-cell lymphomas that highly express BCL-2, BCL-2 expression is observed in <50% of most mature T-cell lymphoma (MTCL) subtypes, although a clinical trial investigating venetoclax in BCL-2 positive relapsed/refractory PTCL is ongoing (NCT03552692). In contrast, BCL-xL and/or MCL-1 are expressed in the majority of MTCL. Consistent with these findings, MCL-1 expressing anaplastic large cell lymphomas (ALCL) and typical (non-ETP) T-ALL, which highly express BCL-xL, have been shown by BH3 profiling to be dependent upon MCL-1 or BCL-xL, respectively. Among the more common MTCL subtypes, including cutaneous T-cell lymphomas (CTCL), the extent to which BCL-2 family members are a therapeutic vulnerability is uncertain. However, we have previously demonstrated that antigen- and costimulatory receptor signaling promote the growth and survival of these lymphomas and confers resistance to chemotherapy, and in conventional (non-malignant) T cells, these same signals upregulate BCL-xL, which is required for their clonal expansion and escape from cell death following T-cell activation. Therefore, given the increasing availability of selective BH3 mimetics and the need for improved therapeutic strategies, we were motivated to examine BCL-2 family member dependencies in these lymphomas. Methods: Peptides generated from BH3-only proteins and selective BH3 mimetics were utilized to perform “BH3 profilng” in MTCL. Cell viability upon exposure to BH3 mimetics was determined using standard approaches. Results: To pre-clinically assess BCL-2 family member dependencies and the mitochondrial apoptotic pathway in T-cell lymphoproliferative neoplasms, we performed BH3 profiling in 17 cell lines. Peptides derived from BIM and PUMA, which are promiscuous, were utilized to assess the extent to which cells are “primed” for apoptosis. Differences in apoptotic priming were not observed across subtypes, and most cell lines examined were highly primed. We next examined the dependence on specific antiapoptotic proteins by BH3 profiling using BAD (antagonizes BCL-2, BCL-xL, and BCL-w), HRK (antagonizes BCL-xL), MS1 (antagonizes MCL-1), and FS1 (antagonizes A1/BFL-1) peptides. BCL-2 dependence was not observed in the T-cell lymphoma cell lines we examined. However, and in stark contrast, most non-ALCL cell lines were BCL-xL dependent, whereas ALCL cell lines were largely MCL-1 dependent. In an effort to further confirm these results, and examine the extent to which peptide-based BH3 profiling predicts sensitivity to selective BH3 mimetics in MTCL, cell lines were treated with venetoclax, A1155463 (a BCL-xL selective antagonist), navitoclax (a BCL-2/BCL-xL antagonist), or S63845 (MCL-1 antagonist), and cell viability determined. Consistent with peptide-based BH3 profiling, all cell lines examined were resistant to venetoclax. ALCL cell lines were largely MCL-1 dependent, whereas the non-ALCL cell lines examined were largely BCL-xL dependent. Therefore, we examined BCL-xL and MCL-1 expression in these cells and observed a strong association between BCL-xL expression and both cytochrome c release in response to HRK peptide and cell viability in the presence of A1155463. A similar association was observed between MCL-1 expression and sensitivity to MS1 and S63845. Transcriptionally profiled MTCL specimens were stratified by MTCL classification (ALCL vs. non-ALCL) and in keeping with the data obtained using cell lines increased BCL-xL expression was observed in non-ALCL MTCL subtypes. Given the observed association between BCL-xL expression and vulnerability to BCL-xL antagonists, we sought to examined mechanisms that promote its expression. Both TCR signaling and CD28 costimulation are exploited by non-ALCL subtypes. We examined the extent to which TCR/CD28 stimulation regulates BCL-xL transcription. In MTCL specimens, a significant increase in BCL-xL transcription and sensitization to Bcl-xL antagonism were observed upon TCR/CD28 stimulation. A TCR-dependent GEM model was utilized, demonstrating that these MTCL are Bcl-xL dependent and sensitive to navitoclax. Conclusions: These findings suggest that BCL-xL is a therapeutic vulnerability for MTCL subsets, particularly non-ALCL subtypes that are TCR dependent, and provide a robust pre-clinical rationale for future studies investigating navitoclax in these lymphomas.
8
Saffari_Chaleshtori, Javad, Ehsan Heidari-Sureshjani, Fahimeh Moradi e Esfandiar Heidarian. "The Effects of Thymoquinone on Viability, and Anti-apoptotic Factors (BCL-XL, BCL-2, MCL-1) in Prostate Cancer (PC3) Cells: An In Vitro and Computer-Simulated Environment Study". Advanced Pharmaceutical Bulletin 9, n. 3 (1 agosto 2019): 490–96. http://dx.doi.org/10.15171/apb.2019.058.
Testo completoAbstract (sommario):
Purpose: Since active plant ingredients can induce apoptosis in many tumors, in this study we evaluate the apoptotic effects of thymoquinone (TQ) on PC3 cells. Also, we predicted the interaction of TQ with BCL-XL, BCL-2, and MCL-1anti-apoptotic factors by computer-simulated environment. Methods: PC3 cells were treated with different concentrations of TQ (0- 80 µM) and IC50 was determined using 3-(4, 5-dimethylthiaztol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptotic and cytotoxicity effects of TQ were analyzed using flowcytometry and comet assay, respectively. Changes in energy and the molecular interactions of TQ with BCL-XL, BCL-2 and MCL-1 anti-apoptotic factors were investigated using simulation. Results: IC50 value was 40 µM. TQ led to the destruction of the genome of PC3 cells and inducing apoptosis. Molecular dynamics (MD) revealed that the root mean-square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and the number of hydrogen and hydrophobic bonds between TQ and residues of BCL-2, BCL-XL and MCL-1were significantly (P<0.001) changed. TQ makes a more stable and stronger connection with BCL-XL compared to BCL-2 and MCL-1 and inhibits BCL-XL non-competitively. Conclusion: Our results demonstrated that TQ not only led to apoptosis, at least partly, due to reduction in the Coil, Turn, and Bend structure of BCL-XL but also caused a decrease in the Rg and RMSD value of BCL-XL, MCL-1, and BCL-2.
9
Eldering, Eric, Victor Peperzak, Hanneke ter Burg, Stacey M. Fernandes, Jennifer R. Brown e Arnon P. Kater. "Bcl-2 Members As Drug Target and Biomarkers for Response to Ibrutinib and Venetoclax in CLL". Blood 128, n. 22 (2 dicembre 2016): 2043. http://dx.doi.org/10.1182/blood.v128.22.2043.2043.
Testo completoAbstract (sommario):
Abstract Introduction. BCL-2 family members are crucial determinants for survival in normal and malignant B cells. Venetoclax, the BCL-2 targeting drug, was recently clinically approved for CLL. CLL cells cycle between the lymph node (LN) and peripheral blood, and between those compartments display large changes in BCL-XL and MCL-1, which are not targeted by Venetoclax. However, novel BH3 mimetics specific for BCL-XL and MCL-1 are under preclinical development. Ibrutinib is a clinically successful BTK inhibitor that forces Chronic Lymphocytic Leukemia (CLL) cells out of the lymph node. After prolonged application resistance develops in a fraction of patients, who then show fast disease progression. Thus, both Venetoclax and Ibrutinib have potential drawbacks when applied as single agents. For long-term successful application of new drug combinations, it is crucial to understand BCL-2 member functionality of malignant B cells in relation to their normal counterparts. In parallel, we studied clinical samples of CLL patients under Ibrutinib or Venetoclax treatment for changes in BCL-2 family members. Experimental procedures. FACS sorting of tonsil B subsets, mRNA and protein profiling, co-culture of CLL cells, co-IP of BCL-2 members, cell death assays with BH3 mimetics specific for BCL-2, BCL-XL or MCL-1 (ABT-199, WEHI-539, A-1210477), intracellular FACS for BCL-2, BCL-XL and MCL-1 in CLL (CD19/CD5/CXCR4). Results.We mapped the clearly distinct expression profiles for BCL-2 members in tonsillar naïve, germinal center (GC), plasma (PC) and memory B cells, which translate into different BH3 mimetic sensitivity profiles. In brief, naïve and memory cells rely on BCL-2, GC cells on MCL-1, and PC on BCL-XL. These approaches were extended to primary CLL cells. In a LN model where cells are fully resistant to single agent ABT-199 due to induction of MCL-1, BCL-XL and BFL-1, we find that dual or triple BH3 mimetic combinations restore killing. Moreover, there is differential sensitivity to (combinations of) BH3 mimetics between healthy and malignant B cells, suggesting there is a therapeutic window. Furthermore, intracellular FACS staining for BCL-2, MCL-1, and BCL-XL was performed on patients treated with Ibrutinib or Venetoclax. In untreated patients, recent LN emigrants (CD5hi/CXCR4lo) have higher BCL-XL and MCL-1 expression than 'old' returning cells (CD5lo/CXCR4hi). This distinction collapses under Ibrutinib treatment, demonstrating that Ibrutinib affects pro-survival BCL-2 members in vivo. Interestingly, in patients that develop Ibrutinib resistance the MCL-1 or BCL-XL pre-treatment profile reappears. In contrast, under Venetoclax treatment BCL-2 member levels do not change and can even increase in some patients, underlining the different mode of action of the two drugs. The pattern differed among patients (n=5) and increases were observed for BCL-2, BCL-XL, or MCL-1. Conclusions. BH3 mimetics have not only found their way to the clinic, but can also be used as tools for BCL-2 functionality profiling. Secondly, our ex vivo data underline that combination treatment between (paired) BH3 mimetics and/or Ibrutinib may afford better long-term efficacy in CLL than single agents. Thirdly, BCL-2 members may be useful as biomarkers for response and progression. Disclosures Eldering: Roche: Research Funding; Gilead: Research Funding. Brown:Pharmacyclics: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Genetech: Consultancy. Kater:Celgene: Research Funding.
10
Eno, Colins O., Guoping Zhao, Kristen E. Olberding e Chi Li. "The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis". Biochemical Journal 444, n. 1 (26 aprile 2012): 69–78. http://dx.doi.org/10.1042/bj20112023.
Testo completoAbstract (sommario):
Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H2O2. The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H2O2-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H2O2 treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H2O2-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H2O2-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H2O2 activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.
11
Touzeau, Cyrille, Jeremy Ryan, Philippe Moreau, Triona Ni Chonghaile, Steven Le Gouill, Paul G. Richardson, Kenneth C. Anderson, Martine Amiot e Anthony G. Letai. "BH3 Profiling Identifies Bcl-2 Dependency in Multiple Myeloma and Predicts Sensitivity to BH3 Mimetics". Blood 124, n. 21 (6 dicembre 2014): 417. http://dx.doi.org/10.1182/blood.v124.21.417.417.
Testo completoAbstract (sommario):
Abstract Despite significant improvement in the treatment of Multiple Myeloma (MM), patients still experience relapse and the disease remains incurable. Cellular fate decision in response to drug therapy is mainly determined by the interactions among BCL-2 family members. Thus anti-apoptotic BCL-2 family proteins represent an attractive target for therapy. BH3 profiling is a functional assay that identifies the tumor cell’s addiction to anti-apoptotic members, such as BCL-2, BCL-XL or MCL-1. In the present study, we determined the BCL-2, BCL-XL and MCL-1 dependency of both MM cell lines and primary myeloma samples, and then tested the ability of BH3 profiling to predict the in vitrosensitivity to BH3 mimetics. First, the BCL-2, BCL-XL or MCL-1 dependence of MM cell lines (n=11) was measured using BH3 profiling. After cell permeabilization, mitochondria were exposed to standardized amounts of peptides derived from the BH3 domains of BH3-only proteins. For instance, the BAD BH3-only peptide binds with high affinity to BCL-2 and BCL-XL, whereas HRK and MS1 peptides bind with high affinity only to BCL-XL and MCL-1, respectively. The cytochrome c release induced by each peptide was quantified by FACS analysis. Dependence on individual anti-apoptotic proteins was found to be very heterogeneous from one cell line to another. Most cell lines (7/11) were found to be MCL-1 dependent. Five cell lines were found to be Bcl-XL dependent and 2 cell lines BCL-2 dependent. Notably, 3 cell lines showed co-dependence to both MCL-1 and BCL-XL anti-apoptotic proteins. We then determined the in vitro sensitivity of MM cell lines to the BH3 mimetics ABT-199 (BC-2 specific) and ABT-263 (BCL-2, BCL-XL and BCL-w specific). Only 3 cell lines out of 11 were found sensitive to ABT-199 and 263 (LD50<100 nM). Interestingly, as we have previously found in other cancers, BH3 profiling predicted sensitivity to ABT-199 and 263 treatment. We then determined the mitochondrial priming of primary plasma cells obtained from 11 MM patients. BH3 profiling was performed on CD138-purified plasma cells. As observed with MM cell lines, patient samples showed heterogeneous dependency to anti-apoptotic proteins: MCL-1 (n=3), BCL-2 (n=2), Bcl-XL (n=2). Of note, mitochondria of 5 patient samples were found to be independent of BCL-2, BCL-XL or MCL-1. Cells were cultured with/without ABT-199 and 263 during 16 hours and then cell death was assessed by flow cytometry after annexin V staining. Two and 4 samples were found sensitive (LD50 < 100 nM) to ABT-199 and ABT-263, respectively. Once again, BH3 profiling with the BAD peptide correlated with in vitro sensitivity to ABT-199 and 263. To conclude, Multiple Myeloma is a heterogeneous disease regarding its dependence to anti-apoptotic proteins, and cannot be considered as monolithically MCL-1 or BCL-2 dependent. Rather, this determination must be made individually. BH3 profiling allows the identification of a BCL-2 dependent subset of MM patients and predicts ABT-199 in vitro sensitivity. It is of clinical interest to identify BCL-2 dependency in MM due to the current development of the oral BH3 mimetic ABT-199 that showed very promising results in BCL-2 dependent malignancies. Disclosures Off Label Use: The presentation discuss the sensitivity of myeloma cells to the Bcl-2 specific BH3 mimetic ABT-199. Le Gouill:Roche: Consultancy; Janssen: Consultancy. Letai:Abbvie: Consultancy.
12
Derenne, Sophie, Brett Monia, Nicholas M. Dean, Jennifer K. Taylor, Marie-Josée Rapp, Jean-Luc Harousseau, Régis Bataille e Martine Amiot. "Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-xL is an essential survival protein of human myeloma cells". Blood 100, n. 1 (1 luglio 2002): 194–99. http://dx.doi.org/10.1182/blood.v100.1.194.
Testo completoAbstract (sommario):
Abstract Multiple myeloma (MM) is a plasma cell malignancy that occurs mainly in bone marrow. As MM cells proliferate slowly, it would seem essential to find means of preventing their growth and accumulation inside bone marrow. The present study used an antisense strategy to elucidate the respective roles of Bcl-2, Bcl-xL, and Mcl-1 proteins in myeloma cell survival. Each antisense oligonucleotide (ASO; Bcl-2, Bcl-xL, or Mcl-1 ASO) introduced into human myeloma cell lines by electroporation induced a marked reduction in the level of the corresponding protein. Mcl-1 ASO triggers an important decrease of viability in all myeloma cell lines tested and in 2 primary myeloma cells, whereas neither Bcl-2 nor Bcl-xL ASO affected the viability of myeloma cells. The decrease of cell viability induced by Mcl-1 ASO treatment was associated with an induction of apoptosis that occurred through the disruption of mitochondrial membrane potential ΔΨm and the activation of executioner caspase-3. Furthermore, we have shown that interleukin 6 cannot prevent the Mcl-1 ASO-induced apoptosis. Finally, although Bcl-2 ASO treatment alone has no effect, it can sensitize myeloma cell lines to dexamethasone (Dex), whereas Bcl-xL ASO in combination with Dex still had no effect. As MM remains an incurable disease despite intensive chemotherapy, these results suggest that Mcl-1 antisense strategy rather than Bcl-2 antisense strategy could be of considerable importance in the treatment of MM.
13
Sobol, Benjamin, Osama Azzam Nieto, Emily Lara Eberlein, Anna-Lena Scherr, Lars Ismail, Annika Kessler, Luisa Nader et al. "Specific Targeting of Antiapoptotic Bcl-2 Proteins as a Radiosensitizing Approach in Solid Tumors". International Journal of Molecular Sciences 23, n. 14 (16 luglio 2022): 7850. http://dx.doi.org/10.3390/ijms23147850.
Testo completoAbstract (sommario):
Avoidance of therapy-induced apoptosis is a hallmark of acquired resistance towards radiotherapy. Thus, breaking resistance still challenges modern cancer therapy. The Bcl-2 protein family is known for its regulatory role in apoptosis signaling, making Bcl-2, Mcl-1 and Bcl-xL promising targets. This study evaluates the effects of highly specific inhibitors for Bcl-xL (WEHI-539), Bcl-2 (ABT-199) and Mcl-1 (S63845) as radiosensitizers. Covering a broad spectrum of solid tumors, Non-Small-Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC) and synovial sarcoma cell lines were exposed to fractionated radiation as standard therapy with or without Bcl-2 protein inhibition. Protein expression was detected by Western blot and cell death was assessed by flow cytometry measuring apoptosis. In contrast to NSCLC, a high level of Bcl-xL and its upregulation during radiotherapy indicated radioresistance in HNSCC and synovial sarcoma. Radioresistant cell lines across all entities benefited synergistically from combined therapy with Bcl-xL inhibition and fractionated radiation. In NSCLC cell lines, Mcl-1 inhibition significantly augmented radiotherapy independent of the expression level. Our data suggest that among antiapoptotic Bcl-2 proteins, targeting Bcl-xL may break resistance to radiation in HNSCC, synovial sarcoma and NSCLC in vitro. In NSCLC, Mcl-1 might be a promising target that needs further investigation.
14
Fortner, Colin, Maren Wichert, Alexandra Niedermayer, Klaus-Michael Debatin, Lüder Hinrich Meyer e Felix Seyfried. "Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia". Blood 142, Supplement 1 (28 novembre 2023): 1608. http://dx.doi.org/10.1182/blood-2023-177613.
Testo completoAbstract (sommario):
T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.
15
Cucarull, Blanca, Anna Tutusaus, Miguel Subías, Milica Stefanovic, Tania Hernáez-Alsina, Loreto Boix, María Reig et al. "Regorafenib Alteration of the BCL-xL/MCL-1 Ratio Provides a Therapeutic Opportunity for BH3-Mimetics in Hepatocellular Carcinoma Models". Cancers 12, n. 2 (1 febbraio 2020): 332. http://dx.doi.org/10.3390/cancers12020332.
Testo completoAbstract (sommario):
Background: The multikinase inhibitor regorafenib, approved as second-line treatment for hepatocellular carcinoma (HCC) after sorafenib failure, may induce mitochondrial damage. BH3-mimetics, inhibitors of specific BCL-2 proteins, are valuable drugs in cancer therapy to amplify mitochondrial-dependent cell death. Methods: In in vitro and in vivo HCC models, we tested regorafenib’s effect on the BCL-2 network and the efficacy of BH3-mimetics on HCC treatment. Results: In hepatoma cell lines and Hep3B liver spheroids, regorafenib cytotoxicity was potentiated by BCL-xL siRNA transfection or pharmacological inhibition (A-1331852), while BCL-2 antagonism had no effect. Mitochondrial outer membrane permeabilization, cytochrome c release, and caspase-3 activation mediated A-1331852/regorafenib-induced cell death. In a patient-derived xenograft (PDX) HCC model, BCL-xL inhibition stimulated regorafenib activity, drastically decreasing tumor growth. Moreover, regorafenib-resistant HepG2 cells displayed increased BCL-xL and reduced MCL-1 expression, while A-1331852 reinstated regorafenib efficacy in vitro and in a xenograft mouse model. Interestingly, BCL-xL levels, associated with poor prognosis in liver and colorectal cancer, and the BCL-xL/MCL-1 ratio were detected as being increased in HCC patients. Conclusion: Regorafenib primes tumor cells to BH3-mimetic-induced cell death, allowing BCL-xL inhibition with A-1331852 or other strategies based on BCL-xL degradation to enhance regorafenib efficacy, offering a novel approach for HCC treatment, particularly for tumors with an elevated BCL-xL/MCL-1 ratio.
16
Massardi, Adhie, Sandy Samsul Bahry, Nur Anindya Rahmawati, Carissa Azmi Shabirah e Artini Pangastuti. "Bioactive Compounds from Penicillium sp. Inhibit Antiapoptotic Bcl-2, Bcl-XL and Mcl-1: An in silico Study". Molecular and Cellular Biomedical Sciences 7, n. 2 (18 luglio 2023): 81. http://dx.doi.org/10.21705/mcbs.v7i2.330.
Testo completoAbstract (sommario):
Background: Antiapoptotic Bcl-2 proteins are overexpressed in cancer cells, leading to inhibition of apoptosis and the development of therapeutic resistance. Targeting only one type of antiapoptotic protein may have limited efficacy in cancer therapy. Anticancer drugs capable of inhibiting Bcl-2, Bcl-XL and Mcl-1 simultaneously are necessary to be explored. Penicillium sp. produces various bioactive compounds with anticancer, antibacterial, and antiviral activities. The aim of this research was to determine the best bioactive compound candidates for inhibiting Bcl-2, Bcl-XL, and Mcl-1 proteins.Materials and methods: Molecular docking analysis was conducted to estimate the binding affinity of Penicillium sp. bioactive compounds with Bcl-2, Bcl-XL, and Mcl-1 proteins. Compounds with the lowest binding energies were visualized using PyMol and Ligplot+ and further subjected to drug-likeness testing based on Lipinski's rule of five.Results: Bioactive compounds with the highest binding affinities were verruculogen and wortmannin. Wortmannin complied with Lipinski's rule of five. Meanwhile, verruculogen violated one out of the five rules by having a molecular weight >500 Da. Both compounds could be used as oral drugs.Conclusion: Verruculogen and wortmannin from Penicillium sp. show significant potential as oral anticancer drug candidates.Keywords: Bcl-2, Bcl-XL, Mcl-1, Penicillium sp., in silico
17
Touzeau, Cyrille, Carole Brosseau, Christelle Dousset, Catherine Pellat-Deceunynck, Steven Le Gouill e Martine Amiot. "Mantle-Cell Lymphoma (MCL) Cells Are Highly Sensitive To ABT-199 But Their Sensitivity May Be Altered By The Microenvironment Via The Up-Regulation Of Bcl-Xl and Bcl2A1". Blood 122, n. 21 (15 novembre 2013): 4285. http://dx.doi.org/10.1182/blood.v122.21.4285.4285.
Testo completoAbstract (sommario):
Abstract Despite improvement in the treatment of Mantle-cell lymphoma (MCL), relapse invariably occurs and innovative strategies are needed. Bcl-2 inhibitors such as ABT-737 and ABT-263 (navitoclax), which target both Bcl-2 and Bcl-xL, demonstrated antitumor activity in B-cell malignancies. However, the clinical development of navitoclax is limited by a deep thrombocytopenia, which is induced by inhibition of Bcl-xL in platelets. To overcome this toxicity, ABT-199, the first-in-class orally bioavailable Bcl-2-selective BH3 mimetic, has been developed and showed promising antitumor activity in B-cell lymphoma while sparing platelets. In the present study, the apoptotic efficiency of ABT-199 in comparison with that of ABT-737 was evaluated in seven MCL cell lines. We found two MCL cell lines sensitive to ABT-199 (LD50 of 100 and 200 nM), one intermediate (LD50 of 1000nM) and 4 resistant (LD50 from 5000 to 10000 nM). Surprisingly, LD50 values of the 2 sensitive cell lines (MINO, GRANTA-519) were slightly higher for ABT-199 than for ABT-737. We further demonstrated that the Bcl-2/Mcl-1 ratio determined by RT-PCR is a predictive biomarker for ABT-199 sensitivity. To further determine the role of Mcl-1 in ABT-199 resistance, Mcl-1 siRNA were transfected in Z-138 and JEKO-1 cells. Mcl-1 silencing sensitized these 2 cell lines to low dose ABT-199 confirming the importance of Mcl-1 in ABT-199 resistance as previously shown for ABT-737. Moreover, in Z-138 cells, which highly express Bcl-xL, we showed that Bcl-xL silencing sensitized them to ABT-199. These results show that in addition to Mcl-1, Bcl-xL might also confer resistance to ABT-199-induced apoptosis in MCL. This could explain the slight difference of sensitivity of MCL cells between ABT-199 and ABT-737. In contrast to MCL cell lines, we found so far that ABT-199 efficiency killed all tested circulating primary cells from MCL patients (n=7) with LD50 values inferior to 10 nM. Because MCL cells reside mainly in lymph nodes, we wondered whether mimicking the microenvironment could impact the sensitivity of MCL cells to BH3 mimetics like it was previously demonstrated for chronic lymphoid leukemia cells. Thus, the ABT-199 sensitive MINO and GRANTA-519 cells were cultured on CD40L-expressing fibroblasts L in order to mimic the lymph node microenvironment. Both cell lines and primary cells became resistant to ABT-199 within 24h. Investigation of the underlying mechanism revealed a strong up-regulation of both Bcl-xL and Bcl2A1 protein expression. By contrast, culture of MCL cells with parental CD40L- fibroblasts or in conditioned medium from CD40L+ L fibroblasts culture failed to induce ABT-199 resistance. These results highlight the implication of the CD40L pathway in ABT-199 resistance through the up-regulation of Bcl-xL and Bcl2A1 in MCL. In conclusion, while circulating primary MCL cells are highly sensitive to ABT-199, it would be important to address the impact of microenvironment on long-term survival of MCL cells within lymph nodes under ABT-199 treatment. Disclosures: No relevant conflicts of interest to declare.
18
Morales, Alejo A., David Siefker, Shannon M. Matulis, Delia M. Gutman e Lawrence H. Boise. "Bak Binding to Bcl-xL and Not to Mcl-1 Is Associated with ABT-737 Sensitivity in Multiple Myeloma Cell Lines". Blood 112, n. 11 (16 novembre 2008): 3675. http://dx.doi.org/10.1182/blood.v112.11.3675.3675.
Testo completoAbstract (sommario):
Abstract ABT-737 is a Bad-like BH3 mimetic and an effective inhibitor of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL and Bcl-w, but not Mcl-1. Recent studies have shown this new drug as a promising anti-cancer agent with activity in multiple myeloma cells. The purpose of this study was to evaluate the role of Bcl-2 family members in both determining the sensitivity and the mechanism of action of ABT-737 in multiple myeloma cell lines. ABT-737, as a single agent, induced apoptosis in six myeloma cell lines, although the sensitivity was quite different among cell lines. Three cell lines 8226/S, MM.1s and KMS18, were highly sensitive to ABT-737 with EC50 values of 0.30, 0.39 and 0.58 μM, respectively. In contrast, three cell lines, KMS11, U266 and OPM2 displayed lower sensitivity to the drug with EC50 values of 1.60, 2.58 and 2.57 μM ABT-737. No correlation between the sensitivity to ABT-737 and the expression pattern of the Bcl-2 family members was found. Interestingly, Mcl-1, a critical anti-apoptotic protein involved in myeloma cell survival that has also been shown to confer resistance to ABT-737, did not correlate with sensitivity to the drug. Bfl-1, an anti-apoptotic Bcl-2 family member with similar functions to Mcl-1, was only expressed in two sensitive cell lines, MM.1s and KMS18. Since the expression pattern did not reveal any strong correlation, we determined the effects of ABT-737 on association of Bcl-2 proteins. Co-immunoprecipitation experiments in MM.1s and KMS11, demonstrated that ABT-737 released Bak and Bim from Bcl-xL and Bim from Bcl-2 while no change was observed for Bak and Bim bound to Mcl-1. A closer look at the interaction of Bcl-2 family members revealed that Bak is equally bound to Mcl-1 and Bcl-xL in the less sensitive cell lines while it is primarily bound to Bcl-xL in the more sensitive cell lines 8226/S and KMS18. Interestingly, Bak in equally bound to Mcl-1 and Bcl-xL in MM.1s, the third sensitive cell line; however, Bim is also highly bound to Bcl-xL, suggesting an easier release of Bak and Bim by ABT-737 from a Bim-primed-Bcl-xL. Consistent with this idea, Bcl-xL overexpression significantly protected 8226/S but not KMS11 from ABT-737-induced death. Additionally, while silencing of Bim significantly protected MM.1s and KMS11 from ABT-737-induced apoptosis, release of Bak from Bcl-xL was not observed after Bim silencing in the MM.1s cells. Together these data suggest that the interaction pattern not the expression pattern of Bcl-2 proteins is a more accurate measure of ABT-737 function in cells. This is important in diseases like multiple myeloma where Mcl-1 in addition to other anti-apoptotic Bcl-2 proteins are typically expressed.
19
Slomp, Anne, Laura M. Moesbergen, Constantijn J. M. Halkes, Pieter Sonneveld, Monique C. Minnema e Victor Peperzak. "MCL-1 Inhibition Is Highly Effective Against Multiple Myeloma Cells from Poor Prognosis Patients". Blood 132, Supplement 1 (29 novembre 2018): 1916. http://dx.doi.org/10.1182/blood-2018-99-117990.
Testo completoAbstract (sommario):
Abstract Introduction: Despite recent advances in treatment, multiple myeloma (MM) is still considered incurable. Several novel therapies with different mechanisms of action are currently being studied for use in MM. These include targeted therapies such as pathway inhibitors and BCL-2 homology domain 3 (BH3) mimetics. BH3-mimetics overcome apoptosis resistance by binding and inhibiting select pro-survival BCL-2 family proteins. In MM, overexpression of MCL-1 leads to apoptosis resistance and is associated with shorter patient survival. In addition to MCL-1, overexpression of BCL-2 and/or BCL-XL has been observed in MM, suggesting that these three pro-survival proteins are promising targets for therapy. Finding tumor characteristics that can predict responses to MCL-1, BCL-2 or BCL-XL inhibitors is essential for finding optimal therapy combinations for each patient. Besides MM patient age, fitness and disease characteristics, several genetic aberrations are associated with treatment response and patient survival. This study aims at finding predictors of BH3-mimetic inhibitors in MM. Methods: Using primary MM (n=42) and monoclonal gammopathy of undetermined significance (MGUS) (n=10) bone marrow aspirates and a collection of human myeloma cell lines (HMCL), we determine dependence on MCL-1, BCL-2, and BCL-XL, and investigate whether this correlates to protein expression, disease stage, tumor cytogenetics, and patient characteristics. We use potent and specific BH3-mimetics S63845 (MCL-1), ABT-199/Venetoclax (BCL-2), and A-1155463 (BCL-XL) to assess pro-survival protein dependence. In addition, combinations of these BH3-mimetics are used to determine whether they act in synergy. Results: In HMCL, sensitivity to BCL-2 family inhibitors depends to a large extent on expression of BCL-2 and BCL-XL, as was shown previously. Inhibition of MCL-1 efficiently induces apoptosis in HMCL and acts in synergy with BCL-XL inhibition. For primary MM cells, we observed that MCL-1 and BCL-2 inhibitor sensitivity varies greatly between patients. In contrast, MM plasma cells (PC) are generally insensitive to BCL-XL inhibitors as single treatment. As observed with HMCL, combining BCL-2 family inhibitors further increased the level of apoptosis in these cultures. The t(11;14) translocation was previously linked to increased sensitivity towards Venetoclax in MM patients. Our experimental setup and data confirms this correlation (fold difference = 2.3, P=0.009 at 100 nM). Interestingly, we found an even greater difference in Venetoclax sensitivity when comparing patient samples with high versus low BCL-2 protein expression in MM PC (fold difference = 3.0, P=0.0002 at 100 nM). MGUS PC were generally insensitive to BCL-2 and BCL-XL inhibition, and sensitivity to MCL-1 inhibition was limited. These data reveal that MM PC are more dependent on BCL-2 family proteins for survival than PC from premalignant MGUS patients. Next, we investigated whether known poor prognostic characteristics correlate to inhibitor sensitivity and found that MM cells from patients with advanced disease stage (i.e. ISS 3), renal failure, or poor cytogenetics were markedly more sensitive to MCL-1 inhibition (ISS 3: P=0.029, β2M: P=0.020, renal failure: P=0.045, 1q+/17p+/t(4;14)+/t(14;16)+: P=0.039, and HD-/t(11;14)-: P=0.005). Conclusions: We observed for the first time that cells from MM patients with poor prognosis disease are more sensitive to inhibition of MCL-1. Combining BH3-mimetic inhibitors synergistically increased apoptosis compared to the single inhibitors. In addition, including flow cytometric determination of BCL-2 protein expression in MM cells at diagnosis may aid in predicting patient sensitivity to Venetoclax. Combined, our data indicate that BCL-2 protein expression, in combination with disease characteristics and cytogenetic testing, may predict sensitivity to BCL-2 and MCL-1 inhibitors in subsets of MM patients. Disclosures Sonneveld: Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Minnema:Amgen: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Janssen: Consultancy; Servier: Consultancy.
20
Chauhan, Dharminder, Mohan Brahmandam, Teru Hideshima, Klaus Podar, Nikhil Munshi, Noopur Raje e Kenneth C. Anderson. "Bcl-2, Mcl-1 and p53 Expression Confer Sensitivity to Bcl-2 Inhibitor ABT-737 in Multiple Myeloma." Blood 108, n. 11 (16 novembre 2006): 3474. http://dx.doi.org/10.1182/blood.v108.11.3474.3474.
Testo completoAbstract (sommario):
Abstract Bcl-2 family of proteins confers resistance to chemotherapy in multiple myeloma (MM). Identification of BH3-mimetic drugs that inactivate pro-survival targets by interfering with the interaction of Bcl-2 proteins may therefore be therapeutically useful. A recent study identify ABT-737, a potent small-molecule inhibitor of anti-apoptotic proteins Bcl-2, Bcl-xL, and Bcl-w with an affinity 2–3 orders of magnitude more potent than any previously reported compounds (Nature2005, 435:672–681). Here we show that ABT-737 triggers apoptosis in MM cells resistant to conventional therapies. The functional specificity of ABT-737 binding to Bcl-2 or Bcl-xL was confirmed by using the less active enantiomer of ABT-737, which had no significant effect on viability of cells, even at higher concentration. Washout experiments show that exposure of MM cells to ABT-737 for 3h is sufficient to cause an early and irreversible commitment to cell death. Given that ABT-737 binds Bcl-2, Bcl-xL, and Bcl-w with high affinity but has far lower affinity for Mcl-1, we asked whether the anti-MM activity of ABT-737 is regulated by the relative expression of Bcl-2, Bcl-xL, or Mcl-1. For these studies, we selected MM.1S (sensitive to ABT-737: IC50-2 μM) and OPM-1 (least sensitive to ABT-737; IC50: 10–15 μM) and examined the basal expression levels of Bcl-2, Bcl-xL, and Mcl-1. MM.1S cells, in contrast to OPM-1 cells, express high basal levels of both Bcl-2 and Bcl-xL, but low Mcl-1 protein; conversely, OPM-1 cells express high Mcl-1 and low Bcl-2 levels. Since ABT-737 cannot neutralize Mcl-1, it is likely that the high Mcl-1 and low Bcl-2/Bcl-xL expression profile in OPM-1 renders these cells less sensitive to ABT-737; whereas low Mcl-1 and high Bcl-2/Bcl-xL expression in MM.1S cells allows for efficient killing by ABT-737. Importantly, treatment of MM.1S MM cells with ABT-737 and proteasome inhibitor Bortezomib induces additive cytotoxicity (combination index = 1.0). The mechanism underlying additive anti-MM activity of ABT-737 with Bortezomib includes downregulation of Mcl-1 along with targeting Bcl-2. Our findings have clinical implications: Bortezomib is FDA approved for the treatment of MM, but prolonged treatment can be associated with toxicity (Chauhan et al. Cancer Cell2005, 8:407–419); importantly, combining ABT-737 with Bortezomib would allow for the use of lower doses of Bortezomib. Another mechanism whereby MM cells evades the cytotoxic effects of chemotherapy is via p53 mutations. Bcl-2 is linked to p53-mediated signaling, and we therefore examined whether ABT-737 is able to overcome the tumorigenic effects conferred via p53 mutations. Co-precipitations experiments show that MM.1S cells (ABT-737-sensitive) predominantly carry wild type p53, whereas OPM-1 cell line (less sensitive to ABT-737) has mutant p53. Taken together, these finding suggest that 1) sensitivity to ABT-737 correlates with Bcl-2 expression in MM cells, 2) higher expression of Mcl-1 reduces sensitivity to ABT-737 whereas other anti-MM agents that block Mcl-1 may synergize with ABT-737, and 3) ABT-737 induces apoptosis in MM with p53 (wt) or p53 (mt), albeit with differential sensitivity. A report that ABT-737 enhances the apoptotic activity of chemotherapeutic agents (Olterdorf et al. Nature2005, 435:672–681), together with our present findings, provides the framework for clinical trials of ABT-737, either alone or in combination with other anti-MM agents, to enhance efficacy, reduce toxicity, and overcome drug resistance in MM patients.
21
Ge, Yubin, Chengzhi Xie, Holly Edwards, J. Timothy Caldwell e Jeffrey Taub. "At Clinically Achievable Concentrations Obatoclax (GX15-070) Potentiates The Cytotoxic Effect Of Cytarabine On Acute Myeloid Leukemia Cells By Enhancing DNA Damage". Blood 122, n. 21 (15 novembre 2013): 3829. http://dx.doi.org/10.1182/blood.v122.21.3829.3829.
Testo completoAbstract (sommario):
Abstract AML accounts for one-fourth of acute leukemias in children, but is responsible for more than half of the pediatric acute leukemia deaths. Most adult AML patients treated with chemotherapeutic regimens relapse and die of their disease or associated complications. One major cause of treatment failure in this disease is resistance to anthracycline [e.g. daunorubicin (DNR)] and cytarabine (ara-C)-based chemotherapy. Therefore more effective therapies are urgently needed. One common cancer-causing defect arises from the overexpression of the anti-apoptotic Bcl-2 family members, including Bcl-2, Bcl-xL, and Mcl-1, which inactivate the intrinsic apoptotic pathway in many types of cancer cells. Overexpression of Bcl-2, Bcl-xL, and/or Mcl-1 has been associated with chemoresistance in leukemia cell lines and with a poor clinical outcome in studies of adult patients with leukemia, including AML. Therefore, small molecules that inhibit the anti-apoptotic Bcl-2 family proteins could be novel therapeutic agents for the treatment of leukemia. In this study, we investigated the effects of clinically achievable doses (175 nM and lower) of obatoclax (a hydrophobic small molecule pan-Bcl-2 inhibitor that binds to the BH3-binding site of Bcl-2, Bcl-xL and Mcl-1) on cytarabine sensitivity in AML cell lines (n=5) and diagnostic AML blasts (n=6). The majority of the AML cell lines and diagnostic AML blast samples expressed Bcl-2, Bcl-xL, and/or Mcl-1 proteins as determined by Western blots, though the levels were variable and did not correlate with sensitivities to cytarabine or obatoclax. Simultaneous combination of obatoclax and cytarabine resulted in synergistic inhibition of proliferation of 4 AML cell lines and 4 diagnostic AML blast samples. Cytarabine IC50s were reduced by 3.4-26.6 fold in the presence of 175 nM obatoclax. The two drugs also synergistically induced apoptosis in THP-1 and OCI-AML3 cells (combination index values ranged from 0.23 to 0.55). Bax activation was detected in the combined drug treatments but not in the individual drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential (MMP). Interestingly, co-treatment of THP-1 and OCI-AML3 cells with cytarabine and obatoclax resulted in decreased levels of Mcl-1 and increased nuclear accumulation of Bcl-2 and Bcl-xL that can occur as early as 24h after combined treatment with cytarabine and obatoclax. Apoptosis and MMP loss induced by the combination of cytarabine and obatoclax could be completely attenuated by ectopic overexpression of Bcl-xL. Emerging evidence suggests that Bcl-2, Bcl-xL, and Mcl-1 also possess nuclear functions and can influence the repair of DNA double-strand breaks (DSBs) and/or the regulation of cell cycle checkpoints. It is conceivable that obatoclax may target the nuclear functions of these anti-apoptotic Bcl-2 family proteins to enhance ara-C-induced apoptosis in AML cells. Interestingly, combined drug treatment showed an increase in DNA DSBs, as indicated by increased levels of phosphorylated H2AX (γH2AX) and comet assay, in both THP-1 and OCI-AML3 cells. Further, γH2AX foci coincided with increased nuclear localization of Bcl-2 and Bcl-xL. Furthermore, a time course experiment revealed that γH2AX levels in AML cells treated with combined cytarabine and obatoclax positively correlated with cleavage of caspase-3 and PARP and inversely correlated with Mcl-1 levels. Additional experiments suggest that induction of a decrease of Mcl-1 and nuclear accumulation of Bcl-2 and Bcl-xL may represent a common mechanism underlying apoptosis induced by DNA DSBs. Our study provides convincing evidence that obatoclax enhances cytarabine-induced DNA DSBs which may trigger a decrease of Mcl-1 and nuclear accumulation of Bcl-2 and Bcl-xL, tipping the balance toward apoptosis. Our new findings strongly support the clinical use of obatoclax in combination with DNA damaging agents for treating AML and possibly a broader range of malignancies. Disclosures: No relevant conflicts of interest to declare.
22
Erlacher, Miriam, e Verena Labi. "MCL-1 and BCL-XL: blood brothers". Blood 137, n. 14 (8 aprile 2021): 1850–51. http://dx.doi.org/10.1182/blood.2020010569.
Testo completo
23
Matulis, Shannon M., Cathy Sharp, Ajay K. Nooka, Jonathan L. Kaufman, Sagar Lonial e Lawrence H. Boise. "Efficacy Of ABT-199 In Multiple Myeloma". Blood 122, n. 21 (15 novembre 2013): 4453. http://dx.doi.org/10.1182/blood.v122.21.4453.4453.
Testo completoAbstract (sommario):
Great strides have been made in the last 15 years in the treatment of multiple myeloma, with FDA approval of both IMIDs and proteasome inhibitors, 10-year survival rates are now achievable in around 25% of patients. However, for the remaining 75%, disease relapse due to drug resistance remains a significant clinical concern. Therefore novel therapeutic approaches to be used in combination with the current active agents are required. The Bcl-2 family of proteins regulates apoptosis and presents an exciting target for therapy. We have demonstrated that myeloma cell lines can be dependent on the anti-apoptotic protein Mcl-1 or can be co-dependent on Mcl-1 and Bcl-2/xL for survival. The distinction between Mcl-1 dependence and co-dependence is the distribution of the pro-apoptotic BH3-only protein Bim. In Mcl-1-dependent lines, Bim is primarily associated with Mcl-1. In contrast in the co-dependent lines, Bim is either predominately associated with Bcl-2/xL or when it is released from Bcl-2/xL it cannot bind to Mcl-1 because of the presence of the Mcl-1 inhibitor, Noxa. This renders these cells sensitive to the Bcl-2/xL inhibitor ABT-737. We have confirmed these findings in freshly isolated patient samples and demonstrated that among the 5 patient samples tested, Bim was associated with both Mcl-1 and Bcl-xL and the cells were sensitive to ABT-737. This suggests that co-dependence on Mcl-1 and Bcl-2/xL may be a common phenomena in myeloma. However, given the adverse effects seen with Bcl-xL inhibition, ABT-737 or the related compound Navitoclax may be difficult to use for the treatment of multiple myeloma. However, ABT-199, a Bcl-2-specific inhibitor, has been developed and we report on preclinical testing in multiple myeloma. Freshly isolated plasma cells from 3 myeloma patients were treated with either ABT-737 or ABT-199 for 24 h and IC50s for each drug were compared. In each patient sample, the plasma cells were less sensitive to ABT-199 than to ABT-737 (MM49: 199 IC50 1.4 μM vs. 737 IC50 0.9 μM; MM51: 199 IC50 0.34 μM vs. 737 IC50 0.07 μM; MM52: 199 IC50 1.3 μM vs. 737 IC50 0.25 μM), and only MM51 was truly sensitive to ABT-199. CoIP studies in MM51 revealed very little Bim bound to Mcl-1 and none bound to Bcl-xL, suggesting the majority of the Bim present in the cell must be bound to Bcl-2. These data suggest that myeloma cells are more likely to be co-dependent on Mcl-1 and Bcl-xL for survival than Mcl-1 and Bcl-2. However Bcl-2 dependence can exist in myeloma. We also performed dose curves in 4 multiple myeloma cells lines, representing 4 different Bim binding patterns and sensitivity to ABT-737. 8226, MM.1s, and KMS18 are all co-dependent cell lines (sensitive to ABT-737), while KMS11 is Mcl-1 dependent. Of these cell lines, KMS18 is the only one with the majority of Bim bound to Bcl-2, therefore should be sensitive to ABT-199. Surprisingly this was not the case. 8226 was the only cell line that was sensitive to ABT-199 (IC50 2.2 μM), while MM1.s, KMS18, and KMS11 were insensitive with IC50s of 6.2 μM, 6.8 μM, and 24.7 μM respectively. In order to gain a mechanistic understanding of these data, we performed CoIP studies to determine the pattern of Bim binding to Mcl-1, Bcl-xL, and Bcl-2 following treatment with ABT-199. We found in KMS18 cells, upon treatment, Bim is released from Bcl-2 and bound by Mcl-1 thereby preventing apoptosis. We have already demonstrated that very little Bim is bound to Bcl-2 in KMS11 and MM.1s, which is consistent with the lack of sensitivity in these cell lines. However in 8226 cells, the high expression of Noxa prevents the Bim released from Bcl-2 from binding to Mcl-1, thereby promoting apoptosis. Silencing of Noxa in this cell line raises the IC50 of ABT-199 2-fold. Next we investigated the effectiveness of combining ABT-199 with the proteasome inhibitor carfilzomib, which has been shown to be a potent inducer of Noxa. A synergistic response to this drug combination was seen in KMS18 cells. CoIP studies revealed that in the presence of Cz, the Bim released from Bcl-2 by ABT-199 could no longer bound to Mcl-1, and was free to activate Bak and Bax. Only additive responses were seen in 8226, MM.1s and KMS11 cell lines. Taken together these data suggest ABT-199 alone may only be an effective treatment for multiple myeloma in a small subset of patients. However, combining it with either Noxa inducers or Mcl-1 inhibitors could be a promising approach for the treatment of this disease. Disclosures: Kaufman: Jansenn: Consultancy; Millennium Pharmaceuticals: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Onyx: Consultancy; Merck: Research Funding. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise:Onyx Pharmaceuticals: Consultancy.
24
Seyfried, Felix, Felix Stirnweiß, Alexandra Niedermayer, Stefanie Enzenmüller, Rebecca Hörl, Stefan Köhrer, Klaus-Michael Debatin e Lüder Hinrich Meyer. "Synergistic Activity of BH3-Mimetics By Combined Targeting of Anti-Apoptotic Regulators in B-Cell Precursor ALL". Blood 138, Supplement 1 (5 novembre 2021): 706. http://dx.doi.org/10.1182/blood-2021-150162.
Testo completoAbstract (sommario):
Abstract In acute lymphoblastic leukemia (ALL), the most frequent malignancy in children and adolescents, deregulated cell death pathways contribute to leukemia development and therapy failure. Apoptosis (programmed cell death) is controlled at the mitochondrial level by different pro- and anti-apoptotic regulators. Molecules of the BCL-2 family are key in regulating intrinsic apoptosis signaling. Pro-apoptotic BH3-only proteins such as BIM and BID activate pro-death proteins like BAX and BAK leading to cell death, while anti-apoptotic BCL-2 family members including BCL-2, MCL-1 and BCL-XL sequester pro-apoptotic molecules, thereby preventing pro-death protein activation and apoptosis induction. Small molecule inhibitors have been developed, which bind to the anti-apoptotic molecules BCL-2, MCL-1 and BCL-XL leading to cell death induction. In particular inhibition of BCL-2, a key regulator of survival in B-cell malignancies including BCP-ALL, by the specific inhibitor venetoclax (VEN) has shown substantial, clinical anti-tumor activity. However, in BCP-ALL heterogeneous sensitivities for VEN have been described suggesting that other BCL-2 family members like MCL-1 and BCL-XL interfere with BCL-2 inhibition thereby counteracting VEN activity leading to poor treatment response. In this study, we compared the effects of inhibition of the anti-apoptotic BCL-2 family members BCL-2 (venetoclax, VEN), MCL-1 (S63845) and BCL-XL (A-1331852), investigated molecular mechanisms and determinants of inhibitor sensitivity, and addressed potential synergistic activity upon simultaneous blockage of BCL-2 together with MCL-1 or BCL-XL in BCP-ALL. First, we investigated the activity of the single inhibitors to induce cell death (positive staining for propidium iodide) in 7 BCP-ALL cell lines and a series of 27 BCP-ALL patient-derived xenograft samples (PDX). Titrating increasing concentrations, we estimated half-maximal effective concentrations (EC 50) for each inhibitor showing heterogenous responses of individual samples to the different inhibitors. Importantly, sensitivities to either inhibitor were not associated with leukemia characteristics including recurrent genetic alterations described in BCP-ALL. We also did not observe similar sensitivities to the inhibitors of individual samples, however some VEN insensitive samples showed sensitivity to MCL-1 and/or BCL-XL-inhibition, suggesting functional substitution of the anti-apoptotic regulators. Next, we investigated anti-apoptotic addictions of BCP-ALL cells to BCL-2 family proteins upon VEN treatment. Analyzing apoptosis signaling after exposure to VEN and BH3-peptides, we identified that ALL cells adapt their anti-apoptotic addiction to MCL-1 and BCL-XL as an escape strategy from VEN-induced cell death. Analyzing protein complexes by co-immunoprecipitation, we found that exposure of ALL cells to VEN rapidly led to reduced BIM/BCL-2 and compensatory increased BIM/MCL-1 complexes. Conversely, S63845 reduced BIM/MCL-1 complexes and led to increased BIM/BCL-2 binding. Importantly, both protein complexes could be effectively disrupted by combination treatment with VEN and S63845, which resulted in release of BIM and promotion of apoptosis signaling. Based on our mechanistic findings, we evaluated BH3-mimetic combinations for cell death induction using multi-dose matrix assays to calculate synergy metrics based on the Bliss independence model. Analyzing combination effects in seven BCP-ALL cell lines and four PDX samples, we identified positive mean synergy scores of VEN with S63845 and A-1331852 in all cases. However, the most synergistic area of the multi-dose-response matrices differed among different leukemias, indicating different extents of addictions and thereby synergies at different drug concentrations. Finally, combined BCL-2 and MCL-1 inhibition was evaluated in vivo in a PDX model of KMT2A-ENL positive pro-B ALL. Combination treatment of VEN with S63845 led to reduced leukemia loads in spleen, bone marrow and CNS as compared to single agent treatment. Taken together, we found heterogeneous responses of BCP-ALL samples to BH3-mimetics antagonizing BCL-2, MCL-1 and BCL-XL. The ability of leukemia cells to adapt their anti-apoptotic dependency from BCL-2 to MCL-1 or BCL-XL can be used as target for combination therapy, demonstrating synergistic activity in PDX samples ex vivo and in vivo. Disclosures No relevant conflicts of interest to declare.
25
Jain, Reema, Julie M. Sheridan, Antonia Policheni, Melanie Heinlein, Luke C. Gandolfo, Grant Dewson, Gordon K. Smyth et al. "A critical epithelial survival axis regulated by MCL-1 maintains thymic function in mice". Blood 130, n. 23 (7 dicembre 2017): 2504–15. http://dx.doi.org/10.1182/blood-2017-03-771576.
Testo completo
26
Chen, Min, Alan D. Guerrero, Li Huang, Zainuer Shabier, Michael Pan, Tse-Hua Tan e Jin Wang. "Caspase-9-induced Mitochondrial Disruption through Cleavage of Anti-apoptotic BCL-2 Family Members". Journal of Biological Chemistry 282, n. 46 (24 settembre 2007): 33888–95. http://dx.doi.org/10.1074/jbc.m702969200.
Testo completoAbstract (sommario):
Mitochondrial disruption during apoptosis results in the release of cytochrome c that forms apoptosomes with Apaf-1 and caspase-9. Activation of caspase-9 by dimerization in apoptosomes then triggers a caspase signaling cascade. In addition, other apoptosis signaling molecules released from the mitochondrion, such as apoptosis-inducing factor and endonuclease G, may induce caspase-9-independent apoptosis. To determine the signaling events induced by caspase-9, we used chemically induced dimerization for specific activation of caspase-9. We observed that caspase-9 dimerization resulted in the loss of mitochondrial membrane potential and the cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1. Moreover, cleavage-resistant Bcl-2, Bcl-xL, or Mcl-1 potently inhibited caspase-9-dependent loss of mitochondrial membrane potential and the release of cytochrome c. Our data suggest that a caspase-9 signaling cascade induces feedback disruption of the mitochondrion through cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1.
27
Matulis, Shannon M., Jonathan L. Kaufman, Sagar Lonial e Lawrence H. Boise. "Bone Marrow Stromal Cells Enforce MCL-1 Dependence In Multiple Myeloma Through the Secretion of A Soluble Factor",. Blood 118, n. 21 (18 novembre 2011): 3950. http://dx.doi.org/10.1182/blood.v118.21.3950.3950.
Testo completoAbstract (sommario):
Abstract Abstract 3950 It is well established that the bone marrow microenvironment contributes to the growth and survival of multiple myeloma plasma cells, however the molecular mechanisms that control these changes within the myeloma cell have not been completely elucidated. We have recently demonstrated that myeloma cell lines can be dependent on the anti-apoptotic protein MCL-1 or can be co-dependent on MCL-1 and Bcl-2/xL. The primary distinction between MCL-1 dependence and co-dependence is the distribution of the pro-apoptotic BH3-only protein Bim. In MCL-1-dependent lines, Bim is primarily associated with MCL-1. In contrast in co-dependent lines Bim is either predominantly associated with Bcl-2/xL or when it is released from Bcl-2/xL it can not bind to MCL-1 because of the presence of the MCL-1 inhibitor, Noxa. This renders these cells sensitive to the Bcl-2/xL inhibitor ABT-737. We have confirmed these findings in freshly isolated myeloma cells and demonstrated that among the 4 patient samples tested, Bim was associated with both MCL-1 and Bcl-xL and the cells were sensitive to ABT-737. This suggests that co-dependence on MCL-1 and Bcl-xL may be a common phenomena in myeloma. We now demonstrate that in about 50% of patient samples tested, CD138 column-purified MM cells are more sensitive to ABT-737 than cells that were not purified following ficoll separation of the bone marrow aspirate. This suggests that stromal components in the marrow alter the dependence on Bcl-2 proteins resulting in a cell that is more MCL-1 dependent when in the presence of stroma. To directly test this possibility we determined the effect of co-culturing the co-dependent line, MM.1s with the human stromal line Hs-5 on ABT-737 (0.6 mM) sensitivity and Bim binding. MM1.s cells were incubated with Hs-5 cells for 30 min prior to addition of drug or were cultured in medium containing 50% conditioned medium (48 h growth) from Hs-5 cells to determine if any effects observed were due to a soluble factor. Following a 24 h culture, cells were subjected to CD138 column purification and apoptosis analyzed by Annexin V-FITC/PI flow cytometry. While mono-cultured MM1.s were very sensitive to ABT-737 (88.5% +/− 3.6% control apoptosis, mean +/− SE), the addition of Hs-5 cells or conditioned medium had a profound effect on cell survival resulting in significant resistance to Bcl-2/xL inhibition (23% +/− 4.4% and 24.5% +/− 2.3% control apoptosis respectively). To determine the molecular basis in this shift from co-dependence to MCL-1 dependence, western blot and co-immunoprecipitation analyses were performed. While neither co-culture nor conditioned medium had any significant effect on the expression of levels of Bim, MCL-1 or Bcl-xL, both conditions resulted in a shift of Bim binding from being equally split amongst MCL-1 and Bcl-xL to a binding pattern where Bim was preferentially bound to MCL-1. This is consistent with loss of sensitivity to ABT-737 and a shift to MCL-1 dependence. We next tested the effects of Hs5 co-culture and conditioned medium on other inducers of cell death. We focused on bortezomib (5 nM) and arsenic trioxide (2 mM) as both can induce the MCL-1 inhibitor Noxa, therefore sensitivity would not be expected to be significantly influenced by a change from co-dependence to MCL-1 dependence. Consistent with this possibility we found that bortezomib induced cell death was only modestly affected by co-culture (79% +/− 2.3% vs. 66.5% +/− 1.9% control apoptosis) and no protection was observed with Hs5 conditioned medium. Similar results were observed with arsenic trioxide (monoculture – 31% +/− 2.3%, Hs5 co-culture −15% +/− 1.0%, conditioned medium – 30% +/− 4.2% control apoptosis). Taken together these data demonstrate that a soluble factor produced by stromal support can alter Bim binding to anti-apoptotic Bcl-2 family members, rendering myeloma cells primarily dependent on MCL-1 and protecting them from cell death signals that function through inhibition of Bcl-2/xL. Moreover these data in part explain why bortezomib can overcome survival signaling provided by stromal support, as proteasome inhibition is capable of inducing the MCL-1 inhibitor Noxa. Disclosures: Kaufman: Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Boise:University of Chicago: Patents & Royalties.
28
Napoli, Giulia C., Andres F. Leon, Erica L. Beatson, Keith T. Schmidt, Emily N. Risdon, Cindy H. Chau, Douglas K. Price e William D. Figg. "Abstract B068: Treatment combinations targeting MCL-1 and BCL-xL exert synergistic anti-tumor effects in AR-V7 expressing CRPC cell lines". Cancer Research 83, n. 11_Supplement (2 giugno 2023): B068. http://dx.doi.org/10.1158/1538-7445.prca2023-b068.
Testo completoAbstract (sommario):
Abstract Therapeutic agents that overcome acquired resistance to current androgen receptor (AR) pathway targeted therapies in patients with castration-resistant prostate cancer (CRPC) remain an unmet clinical need. A multitude of pathways are implicated in acquired treatment resistance, including the PI3K/mTOR/AKT, WNT/β-catenin, and DNA damage repair pathways. Notably, the androgen receptor splice variant 7 (AR-V7) activates the AR pathway in the absence of androgens, playing a crucial role in CRPC progression. An unbiased high throughput drug screen evaluating an array of inhibitors targeting diverse signaling pathways revealed highly synergistic activity of BH3 mimetics in AR-V7 expressing CRPC cell lines (e.g. LNCaP95, 22Rv1, VCaP-CR). Combinations targeting MCL-1 and BCL-xL (S63845 with A-1331852 or Navitoclax) displayed increased antitumor activity compared to those targeting MCL-1 and BCL-2 (S63845 with Venetoclax). Synergy among BCL-xL and MCL-1 targeted combinations was demonstrated using 2D and 3D CellTiter-Glo viability assays. Combination treatments in 3D spheroid cultures of 22RV1 and LNCaP95 cells confirmed the synergy observed in 2D assays. Furthermore, combined BCL-xL and MCL-1 inhibition altered AR-V7 expression and activated c-PARP cleavage. Drug combinations synergistically targeting BCL-xL and MCL-1 should be further explored for the treatment of therapy-resistant CRPC. Citation Format: Giulia C. Napoli, Andres F. Leon, Erica L. Beatson, Keith T. Schmidt, Emily N. Risdon, Cindy H. Chau, Douglas K. Price, William D. Figg. Treatment combinations targeting MCL-1 and BCL-xL exert synergistic anti-tumor effects in AR-V7 expressing CRPC cell lines [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B068.
29
Cakar, Burcu, Pinar Gursoy, Harika Atmaca, Asli Kisim, Emir Bozkurt, Selim Uzunoglu, Canfeza Sezgin et al. "Paclitaxel in combination with AT-101 induces apoptosis via supressing Bcl-2, bcl-XL, mcl-1 proteins in human breast cancer cells." Journal of Clinical Oncology 31, n. 15_suppl (20 maggio 2013): e13578-e13578. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e13578.
Testo completoAbstract (sommario):
e13578 Background: Paclitaxel, a microtubule stabilizing agent, has been a standard of care for breast cancer. AT-101, is an -/- enantiomer of gossypol, inhibits the Bcl-2 family proteins which contain BH3 domain. We reported previously that Paclitaxel in combination with AT-101 showed strong synergistic cytotoxic and apoptotic effects in human breast cancer cells. In this study, to elucidate the molecular mechanisms involved in the apoptotic effect of AT-101/Paclitaxel combination treatment in breast cancer cells, we investigated the possible roles of anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins which contain BH3 domain. Involvement of caspase 3 and 7 activation was also investigated. Methods: Human breast cancer cells were treated with increasing concentrations of drugs alone or with the synergistic combination doses of AT-101 and Paclitaxel. Cell Death Detection Elisa Plus Kit (Roche) was used to detect apoptosis. Caspase 3/7 activity was evaluated by Caspase-Glo 3/7 (Promega, Madison, WI) kit. Changes in the mRNA levels of Bcl-2, Bcl-XL and Mcl-1 genes were evaluated by qRT-PCR. Expression levels of these proteins were also investigated by Western blot analysis. Results: Combined treatment was shown to have strong synergistic apoptotic effects in MCF-7 and MDA- MB-231 human breast cancer cells. mRNA levels of Bcl-2, Bcl-XL and Mcl-1 molecules were reduced by the combination treatment in both cell lines. In parallel with mRNA levels, Bcl-2, Bcl-XL and Mcl-1 protein levels were significantly reduced after this novel drug combination. Combined treatment also induced caspase 3/7 activation in breast cancer cells. Conclusions: These preliminary data suggest that anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 may play important role in the underlying mechanistic rationale of apoptotic effect of AT-101/paclitaxel combination, while pro-apoptotic Bcl-2 related genes (Caspase-3 and Caspase-7) also seem to regulate this synergistic interaction.
30
Fenton, Robert G., Michele L. Cramer, Richard B. Thompson e Bin Zhang. "Affinities of Mcl-1 and Bcl-XL for BH3-Only Proteins Define Distinct Survival Functions at the Outer Mitochondrial Membrane of Multiple Myeloma Cells." Blood 104, n. 11 (16 novembre 2004): 788. http://dx.doi.org/10.1182/blood.v104.11.788.788.
Testo completoAbstract (sommario):
Abstract The Bcl-2 family member Mcl-1 is a critical survival factor for MM cells. However, the functions of Mcl-1 that distinguish it from other anti-apoptotic factors remain unclear. We used fluorescence polarization (FP) to measure the affinity of purified Mcl-1 for amphipathic peptides derived from the BH3-domains of Bad, Bid, Bim, Bik and PUMA, and compared these results with data obtained with Bcl-XL. Functional assays were then performed with mitochondria isolated from 8226 MM cells or clones engineered to overexpress Mcl-1 or Bcl-XL. In contrast to Bcl-XL, Mcl-1 binds to BadBH3 and BikBH3 with very low affinity (>1 uM), confirmed by its inability to bind full-length Bad or Bik proteins. Mcl-1 exhibited high affinity for BimBH3 (90 ± 27 nM), and intermediate affinities for Bid and PUMA. Bcl-XL had intermediate affinities for each of the BH3-peptides tested (175–400 nM). Mitochondria from parental 8226 cells were very sensitive to BidBH3 and BimBH3 as assayed by cytochrome c (cyt c) release; BadBH3 and BikBH3 did not directly induce cyt c release. Mitochondria from 8226-Mcl-1 (which overexpress Mcl-1 5-fold) were very resistant to BidBH3 (even at 500 uM), suggesting that sequestration of BH3-peptide by Mcl-1 was not the sole mechanism of apoptosis inhibition. Paradoxically, Mcl-1-overexpressing mitochondria were more sensitive to BimBH3 (despite the higher affinity of Mcl-1 for Bim), and neither BadBH3 nor BikBH3 could potentiate BidBH3- or BimBH3-induced cyt c release from these mitochondria. In contrast, 8226-Bcl-XL mitochondria were more sensitive to BidBH3, were similarly resistant to BimBH3, and were sensitized by BadBH3 and BikBH3. Protein crosslinking of Mcl-1-overexpressing mitochondria after culture with BidBH3 indicated that peptide sequestration could account for resistance at low peptide concentrations (5–30 uM), but not at higher doses (≥100 uM) where a Bid-induced Bak conformational change occurred as confirmed by exposure of a trypsin-sensitive site. Despite this, formation of Bak multimers was inhibited. (Bax was not detected in 8226 mitochondria). BimBH3 (30 uM) induced detectable cyt c release and Bak dimerization in Mcl-1-overexpressing mitochondria, but 500 uM BimBH3 failed to enhance either effect, and most of the cyt c remained in the mitochondrial pellet. Taken together, these data demonstrate that Mcl-1 and Bcl-XL are not equivalent in their anti-apoptotic functions at the OMM of MM cells. Bad and Bik do not bind Mcl-1, and cannot displace other BH3-only peptides from Mcl-1, suggesting that Mcl-1 has evolved to specifically bind apoptosis-inducing BH3-only proteins in a resistant complex. Mcl-1 confers exceptional protection against BidBH3, and experiments are underway to confirm this with tBid protein. We propose a model in which low levels of BH3-peptide can be sequestered by Mcl-1, while at higher concentrations Mcl-1 binding capacity becomes saturated, allowing peptides to bind Bak and induce a conformational change. However pore formation is inhibited, perhaps by direct binding of Mcl-1 to Bak. To induce pore formation, BH3-peptides must dissociate the Mcl-1/Bak complex: BimBH3, which has a high affinity for Mcl-1 can perform this function, while BidBH3 cannot. This model is consistent with our measured affinity of Mcl-1 for BakBH3 as compared with its affinities for BimBH3 or BidBH3. We suggest that Mcl-1 prevents full cyt c release by preventing activation of the permeability transition pore complex.
31
Pearce, Alicia F., e Douglas S. Lyles. "Vesicular Stomatitis Virus Induces Apoptosis Primarily through Bak Rather than Bax by Inactivating Mcl-1 and Bcl-XL". Journal of Virology 83, n. 18 (8 luglio 2009): 9102–12. http://dx.doi.org/10.1128/jvi.00436-09.
Testo completoAbstract (sommario):
ABSTRACT Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The mitochondrial pathway is regulated by the Bcl-2 family of proteins, which consists of both pro- and antiapoptotic members. To determine the relative importance of the multidomain proapoptotic Bcl-2 family members Bak and Bax, HeLa cells were transfected with Bak and/or Bax small interfering RNA (siRNA) and subsequently infected with recombinant wild-type VSV. Our results showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is regulated by two antiapoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-XL, which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in the degradation of Mcl-1 but not Bcl-XL. However, inactivation of both Mcl-1 and Bcl-XL was required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its expression, inactivation of Bcl-XL indicates a role for one or more BH3-only Bcl-2 family members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, the initiator caspase associated with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.
32
Boise, Lawrence H., Alejo A. Morales, Metin Kurtoglu, Shannon M. Matulis, Rebecca Markovitz, Kelvin P. Lee e Sagar Lonial. "Distribution of Bim Determines MCL-1 Dependence or Co-Dependence on BCL-xL/BCL-2 In Multiple Myeloma". Blood 116, n. 21 (19 novembre 2010): 4054. http://dx.doi.org/10.1182/blood.v116.21.4054.4054.
Testo completoAbstract (sommario):
Abstract Abstract 4054 The inappropriate activation of oncogenes can result in the up-regulation of pro-apoptotic signals often in the form of BH3-only proteins such as Bim, Noxa or Puma. This renders cells dependent on anti-apoptotic proteins including BCL-2, BCL-xL and MCL-1. Thus cancer cells would be predicted to be more susceptible to inhibition of BCL-2 family proteins, prompting the development and testing of several small molecule inhibitors of this class of proteins. Multiple myeloma is a plasma cell malignancy of the bone marrow and like normal plasma cells, myeloma plasma cells express MCL-1. A search of gene expression profile data from normal plasma cells (n=22), MGUS plasma cells (n=12), plasma cells from patients with asymptomatic (smoldering) myeloma (n=44) or newly diagnosed multiple myeloma (n=538) revealed no significant difference in MCL-1 mRNA expression associated with progression of disease. We then determined MCL-1 dependence through the introduction of siRNA in 4 MM cell lines and consistent with previous findings using anti-sense oligonucleotides in additional lines, we demonstrated that all cell lines tested were MCL-1 dependent. However using the BCL-2/BCL-xL/BCL-w-selective inhibitor ABT-737 we found that 3 of the 6 MCL1-dependent cell lines tested were sensitive (IC50 for Annexin V-FITC positive at 24 hrs of 300 – 600 nM) and therefore also dependent on BCL-xL/BCL-2. Taken together this is the first formal demonstration that cells can be co-dependent on multiple Bcl-2 family members. We have previously reported that ABT-737 sensitivity, and what we now refer to as co-dependence on MCL-1 and BCL-xL/BCL-2, is determined by the distribution of BIM on the anti-apoptotic BCL-2 proteins in these cells. We have now expanded these findings to patient samples that displayed sensitivity to ABT-737 that is similar to what we have observed in the co-dependent cell line MM.1s. Consistent with these findings co-immunoprecipation revealed BIM binding predominantly to BCL-xL. Additionally we have now demonstrated that BIM binding is not simply controlled by the expression levels of BCL-xL or MCL-1 as enforced over-expression of each protein could alter the sensitivity of co-dependent cell lines to ABT-737 but did not alter the initial distribution of BIM amongst these proteins. These data suggest that additional factors regulate the association of BIM with anti-apoptotic BCL-2 proteins. These factors could include differences in cellular localization of these proteins as well as differences in post-translational modifications of either pro- or anti-apoptotic BCL-2 family proteins. Disclosures: Boise: University of Chicago: Patents & Royalties.
33
Conage-Pough, Jason E., Vikas A. Gupta, Shannon M. Matulis e Lawrence H. Boise. "Phosphorylation Influences The Binding Of Bim To Anti-Apoptotic Proteins In Multiple Myeloma". Blood 122, n. 21 (15 novembre 2013): 4446. http://dx.doi.org/10.1182/blood.v122.21.4446.4446.
Testo completoAbstract (sommario):
Multiple myeloma cells are strongly dependent on the anti-apoptotic protein Mcl-1 for survival. However, several studies have shown that some myeloma cells are sensitive to treatment with ABT-737 and related compounds, which target Bcl-2 and Bcl-xL, but not Mcl-1. These studies suggest that sensitive cells have more of the direct-activating, pro-apoptotic protein Bim bound to Bcl-2 and Bcl-xL than to Mcl-1. We have previously shown that the expression levels of pro- and anti-apoptotic proteins have little influence on which anti-apoptotic protein binds Bim, leading us to further explore what factors influence the preferential binding of Bim. We have found that IL-6 stimulation of myeloma cells results in both the phosphorylation of Bim at serine 69, and a shift in Bim from Bcl-2 and Bcl-xL to Mcl-1. These observations suggest a role for phosphorylation in determining the anti-apoptotic protein that Bim binds to. To determine if constitutive phosphorylation of Bim regulates the steady state binding of Bim to anti-apoptotic Bcl-2 family members, we utilized phospho-affinity gel electrophoresis (Phos-Tag). We determined that Bim is constitutively phosphorylated in unstimulated myeloma cells. However, unlike what is observed following IL-6 stimulation, phosphorylation does not occur at serine 69. Through use of co-immunopreciptation studies, we found that phosphorylation status of Bim affects its binding to anti-apoptotic proteins. The phosphorylated species are preferentially bound to Bcl-2 and Bcl-xL, and not to Mcl-1. Additionally, we observed that phosphorylated Bim species bound to Bcl-2 and Bcl-xL were present at a significantly lower level in myeloma cells that were more dependent on Mcl-1, such as OPM2, than in KMS18 cells, which exhibited greater Bcl-2/Bcl-xL dependency. This pattern also held true for myeloma cells that had been selected for resistance to ABT-737. ABT-737 resistant KMS18 cells had no detectable phosphorylated species of Bim, and all of the Bim was bound to Mcl-1. We have begun to identify the phosphorylated amino acids in Bim through the use of phospho-Bim-specific antibodies. In addition to serine 69, we have determined that the constitutive phosphorylation does not occur at serine 59 or serine 87. However, preliminary data suggests that Bim phosphorylated at threonine 116 preferentially binds to Bcl-xL in KMS18 cells. We have also taken initial steps to identify signaling pathways and kinases that are phosphorylating Bim, and altering its affinity. We treated KMS18 myeloma cells for 12 hours with 10 μM of the MEK inhibitor U0126, the JNK inhibitor SP600125, or the p38 inhibitor SB203580, and ran lysates from each of these conditions on PhosTag gels. While U0126 and SB203580 treatment did not result in the loss of any phosphorylated Bim species, treatment with SP600125 resulted in a loss of phosphorylated Bim species comparable to treatment of myeloma cell lysates with lambda protein phosphatase. These results suggest that JNK mediates phosphorylation of Bim in myeloma cells. Interestingly, threonine 116 is contained within a JNK consensus sequence. Therefore, JNK could directly influence the preferential binding of Bim. Together, these results suggest that combining Bim kinase inhibitors with agents that result in decreased Mcl-1 (e.g. CDK9 inhibitors) or inducers of the Mcl-1 inhibitor Noxa (proteasome inhibitors) represents a novel approach in cancer cells that demonstrate Mcl-1 dependence. Disclosures: Boise: Onyx Pharmaceuticals: Consultancy.
34
Gaudette, Brian, Neal N. Iwakoshi e Lawrence H. Boise. "Bcl-xL Protects From UPR-Associated Apoptosis During Plasma Cell Differentiation". Blood 120, n. 21 (16 novembre 2012): 3288. http://dx.doi.org/10.1182/blood.v120.21.3288.3288.
Testo completoAbstract (sommario):
Abstract Abstract 3288 Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. As part of the program that allows for B cell differentiation to a plasma cell, a required signal includes the activation of an unfolded protein response (UPR). However unlike stress-induced activation of the UPR, induction of apoptosis does not occur, suggesting that compensatory survival signals are also activated during plasma cell differentiation. The compensatory survival pathways are less defined and require further research. Therefore we employed a model of plasma cell differentiation to better define the survival signaling during this process. The murine B cell lymphoma cell line, Bcl1 can be stimulated to secrete immunoglobulin using IL-5 and LPS. To determine the effects of exogenous ER stress on plasma cell differentiation, we treated the cells with the inhibitor of N-linked glycosylation, tunicamycin, for 5 hours prior to the differentiation signal. The 5 hour pulse of tunicamycin was sufficient to induce significant apoptosis in undifferentiated cells or cells treated with IL-5, resulting in 78% and 74% cell death respectively by 24 hours post treatment. However, if LPS was included in the differentiation stimulus the cells were able to differentiate into IgM-secreting plasma cells with similar kinetics as cells differentiated in the absence of tunicamycin pretreatment. Thus LPS-induced differentiation is sufficient to block ER stress-induced cell death. Since these cells also activate a UPR during differentiation, we hypothesized that part of the differentiation program included protection from UPR-associated cell death. To investigate this effect, we first examined the levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1 during plasma cell differentiation. We found that differentiation induced Bcl-xL and caused the loss of Mcl-1. From this data we hypothesized that the differentiation of these cells resulted in Bcl-xL dependence during plasma cell differentiation. To test this we used ABT-737, which selectively blocks the binding pocket of Bcl-xL and Bcl-2 but not Mcl-1 and kills cells that are dependent on Bcl-2 or Bcl-xL. Undifferentiated Bcl1 cells were insensitive to ABT-737 with an IC50 > 2μM. However ABT-737 sensitized LPS-treated Bcl1 cells to tunicamycin pretreatment resulting in 89% death in 24 h compared to 23% in untreated cells. These data suggest that the induction of Bcl-xL is responsible for the survival of cells undergoing ER stress. Most importantly, cells treated with LPS and IL-5 for differentiation became sensitive to ABT-737 with 59% cell death versus 26% in untreated cells, thus demonstrating that during plasma cell differentiation, cells switch to a Bcl-xL-dependent state. To determine the molecular basis for these findings we investigated the effects of ABT-737 on the expression levels of Bcl-2 proteins as well as the effects of differentiation on their interactions. ABT-737 did not induce changes in the expression of Bcl-2 family proteins. However, co-immunoprecipitation demonstrated a shift in Bim binding from Mcl-1 in untreated cells to Bcl-xL in differentiating cells. This latter finding is consistent with a shift from Mcl-1 dependence to Bcl-xL during plasma cell differentiation. To validate these data, primary C57BL/6 splenocytes were isolated, depleted of non-B cells and subsequently stimulated with IL-4 and LPS to differentiate into plasmablasts. Realtime qPCR showed an increase in Bcl-xL mRNA and loss of Mcl-1 and Bcl-2 mRNA in both the primary B cells and the Bcl1 cell line. Western blotting of primary B cell lysates also showed an increase in Bcl-xL protein and loss of Bcl-2 and Mcl-1 protein. Together these data indicate that during plasma cell differentiation the cell enters a Bcl-xL-dependent state that protects against differentiation-induced apoptosis. Disclosures: No relevant conflicts of interest to declare.
35
Wu, Jenny, Jeremy Ross, Franklin V. Peale, John D. Shaughnessy, Ryan K. Van Laar, Gareth J. Morgan, Jeffrey M. Venstrom e Elizabeth A. Punnoose. "A Favorable BCL-2 Family Expression Profile May Explain the Increased Susceptibility of the t(11;14) Multiple Myeloma Subgroup to Single Agent Venetoclax". Blood 128, n. 22 (2 dicembre 2016): 5613. http://dx.doi.org/10.1182/blood.v128.22.5613.5613.
Testo completoAbstract (sommario):
Abstract Background: In preclinical studies, multiple myeloma (MM) cells are sensitive to selective inhibition of the pro-survival protein BCL-2 by venetoclax (VEN). Resistance to VEN in these models was mediated through BCL-XL and MCL-1, and MCL-1 mediated resistance was neutralized by co-treatment with the proteasome inhibitor bortezomib (BTZ). MM cells harboring the t(11;14) translocation have a higher ratio of BCL-2 relative to MCL-1 mRNA and increased sensitivity to VEN alone, compared to cells with other cytogenetic abnormalities, suggesting that MM subgroups with a favorable BCL-2 family profile might be particularly sensitive to VEN. Clinically, VEN has shown activity in CLL, a disease typically expressing high BCL-2 and low BCL-XL and MCL-1 expression. In MM, VEN is being evaluated as a single agent (NCT01794520) and in combination with BTZ and dexamethasone (Dex) (NCT01794507, NCT02755597) in relapsed/refractory (R/R) patients. As a single agent, improved objective response rates were observed in patients with R/R t(11;14) MM (40% in t(11;14) vs 6% in non-t(11;14)-). In the BTZ/Dex combination study, patients with high BCL-2 expression had an increased objective response rate. Thus, BCL-2 family profiling may explain increased susceptibility of certain MM subgroups to antitumor activity of VEN both as a single agent and in combination. We analyzed MM subgroups for expression of BCL-2 family members to better understand and identify the patient population that can benefit with VEN. Methods: We used publically available data (GSE4581; GSE9782) to analyze BCL-2 family mRNA expression within molecular/cytogenetic defined subgroups among newly diagnosed (NDMM n=414), and R/R (n=264) MM patients, and in comparison to other hematological malignancies (genealogic database). Results: BCL-2, BCL-XL and MCL-1 expression was assessed in MM and compared with 12 major hematologic malignancies. Based on median expression, BCL-2 in MM ranked 6th of 12 tested, with highest BCL-2 expression observed in small lymphocytic leukemia and mantle cell lymphoma. In MM, BCL-2 expression varied significantly across molecular and cytogenetic subgroups. Highest expression was among patients with t(11;14) molecular subtypes (CD1, CD2), in addition to subtypes that are not enriched with t(11;14) like the hyperdiploid and low bone disease subtypes, and lowest in the high-risk subtypes (proliferation, MMSET, MAF/MAFB) (p=0.0001), both in NDMM and R/R MM. BCL-XL and MCL-1 were highly expressed in MM relative to other hematologic malignancies. Expression of BCL-XL and MCL-1 were lowest in the t(11;14) MM population (both p<0.001). Correspondingly, t(11;14) MM was enriched for the highest ratios of BCL-2/MCL-1 (p<0.0001) and BCL-2/BCL-XL (p<0.0001), further supporting the strong single agent VEN activity observed in these patients in clinical study. The t(11;14) positive CD2 subtype can be further characterized by high expression of CD20, (p<0.0001, CD2 vs other), and by other B-cell phenotypic markers (high for PAX5, CD79A and VPREB, and low for CD56). Thus, the CD2 molecular subtype may identify a CLL-like subset of t(11;14) MM that is uniquely susceptible to VEN single agent activity. Finally, when we assessed BCL-2 family expression in R/R MM across lines of treatment, we found patients with 1-3 prior lines of therapy had a higher BCL-2/MCL-1 ratio (p<0.001) compared to patients with greater lines of therapy, due primarily to lower MCL-1 expression (p<0.001), but similar BCL-2 expression. Conclusions: Our analysis suggests that MM subgroups are associated with distinct BCL-2 family expression profiles, and the t(11;14) subgroup amongst MM subgroups may be particularly suited for single agent VEN activity due to a high BCL-2/MCL-1 and BCL-2/BCL-XL ratio. In the non-t(11;14) MM subgroups, VEN treatment in earlier lines of therapy, as well as combination strategies with agents that will down-modulate BCL-XL and/or MCL-1, such as BTZ, may enable maximum VEN activity, and will be further assessed in clinical studies. Disclosures Wu: Genentech: Employment. Ross:AbbVie: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Shaughnessy:Signal Genetics: Consultancy, Equity Ownership. Van Laar:Signal Genetics, Inc.: Employment. Morgan:Takeda: Consultancy, Honoraria; Janssen: Research Funding; Bristol Meyers: Consultancy, Honoraria; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding. Venstrom:Genentech: Employment. Punnoose:Genetech, Inc.: Employment.
36
Chen, Shuang, Yun Dai, Xin-Yan Pei e Steven Grant. "Bim Upregulation by Histone Deacetylase Inhibitors Mediates Interactions with the Bcl-2 Antagonist ABT-737: Evidence for Distinct Roles for Bcl-2, Bcl-xL, and Mcl-1". Molecular and Cellular Biology 29, n. 23 (5 ottobre 2009): 6149–69. http://dx.doi.org/10.1128/mcb.01481-08.
Testo completoAbstract (sommario):
ABSTRACT The Bcl-2 antagonist ABT-737 kills transformed cells in association with displacement of Bim from Bcl-2. The histone deactetylase (HDAC) inhibitor suberoyl bis-hydroxamic acid (SBHA) was employed to determine whether and by what mechanism ABT-737 might interact with agents that upregulate Bim. Expression profiling of BH3-only proteins indicated that SBHA increased Bim, Puma, and Noxa expression, while SBHA concentrations that upregulated Bim significantly potentiated ABT-737 lethality. Concordance between SBHA-mediated Bim upregulation and interactions with ABT-737 was observed in various human leukemia and myeloma cells. SBHA-induced Bim was largely sequestered by Bcl-2 and Bcl-xL, rather than Mcl-1; ABT-737 attenuated these interactions, thereby triggering Bak/Bax activation and mitochondrial outer membrane permeabilization. Knockdown of Bim (but not Puma or Noxa) by shRNA or ectopic overexpression of Bcl-2, Bcl-xL, or Mcl-1 diminished Bax/Bak activation and apoptosis. Notably, ectopic expression of these antiapoptotic proteins disabled death signaling by sequestering different proapoptotic proteins, i.e., Bim by Bcl-2, both Bim and Bak by Bcl-xL, and Bak by Mcl-1. Together, these findings indicate that HDAC inhibitor-inducible Bim is primarily neutralized by Bcl-2 and Bcl-xL, thus providing a mechanistic framework by which Bcl-2 antagonists potentiate the lethality of agents, such as HDAC inhibitors, which upregulate Bim.
37
Morales, Alejo A., Metin Kurtoglu, Shannon M. Matulis, Jiangxia Liu, David Siefker, Delia M. Gutman, Jonathan L. Kaufman, Kelvin P. Lee, Sagar Lonial e Lawrence H. Boise. "Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1–expressing myeloma cells". Blood 118, n. 5 (4 agosto 2011): 1329–39. http://dx.doi.org/10.1182/blood-2011-01-327197.
Testo completoAbstract (sommario):
Abstract Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-xL/Bcl-2 dependence. Surprisingly, analysis of Mcl-1–dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-xL in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.
38
Sumarni, Uly, Jiaqi Zhu, Tobias Sinnberg e Jürgen Eberle. "Sensitivity of Cutaneous T-Cell Lymphoma Cells to the Mcl-1 Inhibitor S63845 Correlates with the Lack of Bcl-w Expression". International Journal of Molecular Sciences 23, n. 20 (18 ottobre 2022): 12471. http://dx.doi.org/10.3390/ijms232012471.
Testo completoAbstract (sommario):
Long-term, curative treatment of cutaneous T-cell lymphomas (CTCL) remains a major challenge. Therapy resistance is often based on apoptosis deficiency, and may depend on antiapoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, Bcl-w and Mcl-1. For their targeting, several antagonists have been generated, which mimic the Bcl-2 homology domain 3 (BH3 mimetics). As dysregulation and overexpression of Mcl-1 has been reported in CTCL, the use of Mcl-1 inhibitors appears as an attractive strategy. Here, we investigated the effects of the selective Mcl-1 inhibitor S63845 in a series of four CTCL cell lines, in comparison to ABT-263 and ABT-737 (inhibitors of Bcl-2, Bcl-xL and Bcl-w). In two cell lines (HH, HuT-78), S63845 resulted in significant apoptosis induction, decrease in cell viability, loss of mitochondrial membrane potential and caspase activation, while two other cell lines (MyLa, SeAx) remained completely resistant. An inverse correlation was found, as S63845-resistant cells were highly sensitive to ABT-263/-737, and S63845-sensitive cells showed only moderate sensitivity to ABTs. Combinations of S63845 and ABT-263 partially yielded synergistic effects. As concerning Bcl-2 protein expression, weaker Mcl-1 expression was found in S63845-resistant MyLa and SeAx, while for Bcl-2 and Bcl-xL, the lowest expression was found in the highly sensitive cell line HH. The most striking difference between S63845-resistant and -sensitive cells was identified for Bcl-w, which was exclusively expressed in S63845-resistant cells. Thus, CTCL may be efficiently targeted by BH3 mimetics, providing the right target is preselected, and Bcl-w expression may serve as a suitable marker.
39
Uren, Rachel T., Grant Dewson, Lin Chen, Stephanie C. Coyne, David C. S. Huang, Jerry M. Adams e Ruth M. Kluck. "Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak". Journal of Cell Biology 177, n. 2 (23 aprile 2007): 277–87. http://dx.doi.org/10.1083/jcb.200606065.
Testo completoAbstract (sommario):
The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)–only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-xl, Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-xl–like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-xl and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption.
40
Cardone, Michael H., Andrew Kinloch, Mine Canakci e Jingping Ge. "Measurements of Mcl-1 protein dependencies using dimerization-specific antibodies as predictive biomarkers for BH-3 mimetic class therapies in AML." Journal of Clinical Oncology 38, n. 15_suppl (20 maggio 2020): e19505-e19505. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e19505.
Testo completoAbstract (sommario):
e19505 Background: The anti-apoptotic Bcl-2 family proteins facilitate pro-survival and resistance to anti-cancer therapies. Measuring the function of these proteins has shown utility in predicting response to treatment. A biomarker platform that assesses the functionality of these proteins by measuring BH3 mediated signaling potential in individual patients’ cancers, could provide a potential guiding platform for treating AML. In AML venetoclax is becoming widely prescribed and highly effective in combination with other drugs. Recent data indicate that Mcl-1 dependence is a resistance factor to venetoclax and that methods for identifying this could provide guidance for combination treatments. We are developing a technology to directly measure the occurrence of heterodimers of Mcl-1, or Bcl-xL, bound to pro-apoptotic BH3-only protein Bim. These measurements are combined in algorithms developed to indicate the cancer cell apoptotic priming state and offer great potential for identifying best AML treatment options. Methods: Monoclonal antibodies against conformation-specific epitopes induced in Mcl-1/Bim and Bcl-xL/Bim protein complexes were made. Selective bonding of Heterodimer Specific Mcl-1 Bim (HSMCB) and Heterodimer Specific Bcl-xL Bim (HSBXB) mabs were confirmed using ELISA, fluorescence polarization, immunofluorescence microscopy and flow, and by immunohistochemistry in genetically defined cell lines and in AML patient biopsied samples. Results: We show that HSMCB signal depends on both Mcl-1 and Bim protein levels while HSBXB requires Bc-xL and Bim levels. The relative signals of HSMCB to unbound Mcl-1 signal ([HsMcB]/[Mcl-1]) provide a biomarker for Mcl-1 dependence. We carefully established binding metrics that correlate to drug response in cell lines. The pharmacological disruption of these complexes is monitored by the ratiometric readouts that serve as predictive biomarkers for BH-3 mimetic drugs in AML cells. Conclusions: Mcl-1 dependence is a predictive biomarker for venetoclax resistance and for response to Mcl-1 targeted therapies. Flow cytometric and IHC based measurements of a heterodimer complex offer a direct and simpler approach that harbors potential for use in clinical settings. Additional antibodies targeting Mcl-1/Bak and Bcl-2/Bim complexes are being tested.
41
Luciano, Frederic, Maryla Krajewska, Xiaokun Zhang, Arnold Satterthwait e John C. Reed. "Orphan Nuclear Receptor TR3 (Nur77) Binds and Converts the Phenotype of Bcl-B, an Anti-Apoptotic Bcl-2-Family Protein Predominantly Expressed in Human Plasma Cells." Blood 108, n. 11 (16 novembre 2006): 82. http://dx.doi.org/10.1182/blood.v108.11.82.82.
Testo completoAbstract (sommario):
Abstract TR3 (Nur77) is an orphan member of the steroid/retinoid family of Nuclear Receptors (NRs) that translocates from nucleus to cytosol, where it binds Bcl-2 and converts its phenotype from anti-apoptotic to pro-apoptotic (Li, et al. Science289: 1159, 2000; Lin, et al CELL116: 527, 2004). We surveyed TR3 (Nur77) for interactions with all six human anti-apoptotic Bcl-2-family proteins: Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1, and Bcl-B. We observed that Bcl-2, Bfl-1, and Bcl-B interact with TR3 as determined by co-immunoprecipitation assays using lysates from transfected cells and by GST pull down assays using GST-Bcl-2-family fusion proteins. In contrast, Bcl-XL, Bcl-W, and Mcl-1 displayed little or no binding to TR3 (Nur77). Co-localization experiments using fluorescent protein tagging and confocal microscopy corroborated these findings, showing co-localization of a fragment of TR3 (Nur77) that accumulates in cytosol (rather than nucleus) [TR3 lacking the DNA-binding domain [DDBD]) with Bcl-2, Bfl-1, and Bcl-B on mitochondria and other intracellular organelles in intact cells, but not co-localization with Bcl-XL, Mcl-1 or Bcl-W. Co-expression of Bcl-2, Bfl-1, or Bcl-B with TR3DDBD by transfection resulted in robust apoptosis induction, while co-expression of Bcl-XL, Bcl-W, or Mcl-1 with TR3DDBD did not. In contrast to results obtained in TR3DDBD co-expression studies, expressing any of the anti-apoptotic Bcl-2-family proteins (Bcl-2, Bfl-1, Bcl-B, Bcl-XL, Mcl-1, Bcl-W) individually resulted in suppression of apoptosis induced by Staurosporine, illustrating the role of TR3 in converting the phenotypes of Bcl-2, Bfl-1 and Bcl-B from protector to killer. Because binding of TR3 (Nur77) to Bcl-B appeared to be strongest among the Bcl-2-family proteins, we focused on this Bcl-B for additional studies. (Ke, N. et al. J. Biol Chem276: 12481, 2001. Using monospecific antibodies, the endogenous Bcl-B protein was localized in human tissues, revealing predominant expression in plasma cells. Several myeloma cell lines also expressed Bcl-B protein, as determined by immunoblotting. Stimulating myeloma cell line RPMI8226 with ionomycin and phorbol ester TPA (agents that induce TR3 expression and accumulation in cytosol) resulted in association of endogenous TR3 with endogenous Bcl-B, as determined by co-immunoprecipitation experiments. Reducing endogenous Bcl-B levels using small interfering RNA (siRNA) reduced apoptosis induced by transfected TR3DDBD as well as apoptosis induced by a synthetic peptide that mimics TR3. We conclude that cytosolic TR3 (Nur77) interacts selectively with certain anti-apoptotic members of the Bcl-2-family (Bcl-2, Bfl-1, Bcl-B), converting them from protectors to killers. The ability of TR3 (Nur77) to convert Bcl-B suggests a possible novel strategy for triggering apoptosis of Bcl-B-expressing cells, which may be of utility for eradicating long-lived autoantibody-producing plasma cells or for killing malignant myeloma cells. (Supported by NIH GM60554 and SASS Foundation).
42
Phillips, D. C., Y. Xiao, L. T. Lam, E. Litvinovich, L. Roberts-Rapp, A. J. Souers e J. D. Leverson. "Loss in MCL-1 function sensitizes non-Hodgkin’s lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199)". Blood Cancer Journal 5, n. 11 (novembre 2015): e368-e368. http://dx.doi.org/10.1038/bcj.2015.88.
Testo completoAbstract (sommario):
Abstract As a population, non-Hodgkin’s lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2 High ) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2 High cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2 High NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2 Low NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2 Low ) that could benefit from BCL-XL (navitoclax)-driven combination therapy.
43
Gillissen, Bernhard, Jana Wendt, Antje Richter, Anja Richter, Annika Müer, Tim Overkamp, Nina Gebhardt et al. "Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma". Journal of Cell Biology 188, n. 6 (22 marzo 2010): 851–62. http://dx.doi.org/10.1083/jcb.200912070.
Testo completoAbstract (sommario):
Tumor necrosis factor (α)–related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that preferentially kills tumor cells with limited cytotoxicity to nonmalignant cells. However, signaling from death receptors requires amplification via the mitochondrial apoptosis pathway (type II) in the majority of tumor cells. Thus, TRAIL-induced cell death entirely depends on the proapoptotic Bcl-2 family member Bax, which is often lost as a result of epigenetic inactivation or mutations. Consequently, Bax deficiency confers resistance against TRAIL-induced apoptosis. Despite expression of Bak, Bax-deficient cells are resistant to TRAIL-induced apoptosis. In this study, we show that the Bax dependency of TRAIL-induced apoptosis is determined by Mcl-1 but not Bcl-xL. Both are antiapoptotic Bcl-2 family proteins that keep Bak in check. Nevertheless, knockdown of Mcl-1 but not Bcl-xL overcame resistance to TRAIL, CD95/FasL and tumor necrosis factor (α) death receptor ligation in Bax-deficient cells, and enabled TRAIL to activate Bak, indicating that Mcl-1 rather than Bcl-xL is a major target for sensitization of Bax-deficient tumors for death receptor–induced apoptosis via the Bak pathway.
44
Gaudette, Brian, Neal Iwakoshi e Lawrence Boise. "Bcl-xL protects from UPR-associated apoptosis during plasma cell differentiation. (P1457)". Journal of Immunology 190, n. 1_Supplement (1 maggio 2013): 174.11. http://dx.doi.org/10.4049/jimmunol.190.supp.174.11.
Testo completoAbstract (sommario):
Abstract Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. Plasma cell differentiation requires activation of the unfolded protein response, a set of signaling pathways in which prolonged activation leads to apoptosis in other cell types. Plasma cells activate this pathway during differentiation suggesting compensatory survival signals are activated. Consistent with this hypothesis, we found that LPS-driven differentiation protects Bcl1 cells against tunicamycin-activated ER stress-induced death. LPS plus cytokine-induced differentiation was associated with an increase in Bcl-xL expression accompanied by a decrease in Mcl-1 or Bcl-2 in Bcl1 cells and primary B cells respectively. Moreover, differentiation-induced protection against ER stress was shown to be Bcl-xL-dependent as tunicamycin-induced death was restored by the addition of the Bcl-xL/Bcl-2 inhibitor, ABT-737. Importantly, we also showed that during differentiation, Bcl1 cells and primary B cells are Bcl-xL-dependent as ABT-737 resulted in significant apoptosis in the absence of any additional death signal. Finally, we found that differentiation was accompanied by a change in Bim binding from primarily Mcl-1 and/or Bcl-2 to Bcl-xL in differentiating cells. These data demonstrate that Bcl-xL induction protects from death signals activated during plasma cell differentiation.
45
Delbridge, Alex R. D., Joseph T. Opferman, Stephanie Grabow e Andreas Strasser. "Antagonism between MCL-1 and PUMA governs stem/progenitor cell survival during hematopoietic recovery from stress". Blood 125, n. 21 (21 maggio 2015): 3273–80. http://dx.doi.org/10.1182/blood-2015-01-621250.
Testo completoAbstract (sommario):
Abstract Understanding the critical factors that govern recovery of the hematopoietic system from stress, such as during anticancer therapy and bone marrow transplantation, is of clinical significance. We investigated the importance of the prosurvival proteins myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma–extra large (BCL-XL) in stem/progenitor cell survival and fitness during hematopoietic recovery from stress. Loss of a single Mcl-1 allele, which reduced MCL-1 protein levels, severely compromised hematopoietic recovery from myeloablative challenge and following bone marrow transplantation, whereas BCL-XL was dispensable in both contexts. We identified inhibition of proapoptotic p53 upregulated modulator of apoptosis (PUMA) as the key role of MCL-1 in both settings, with Mcl-1+/−;Puma−/− mice completely protected from the deleterious effects of loss of 1 Mcl-1 allele. These results reveal the molecular mechanisms that govern cell survival during hematopoietic recovery from stress.
46
Cleveland, Abigail, Katherine Veleta e Timothy Gershon. "CBIO-23. ANTIAPOPTOTIC Bcl-xL RESTRICTS APOPTOSIS IN SHH MEDULLOBLASTOMA AND PROMOTES PROGRESSION". Neuro-Oncology 23, Supplement_6 (2 novembre 2021): vi31—vi32. http://dx.doi.org/10.1093/neuonc/noab196.122.
Testo completoAbstract (sommario):
Abstract Medulloblastomas in most patients are distinctively sensitive to radiation therapy, but the mechanisms that mediate this sensitivity are unclear. Current treatments still fail 20%-60% of patients with SHH medulloblastoma and can leave survivors with long-term neurocognitive and social deficits. Understanding the mechanisms driving the typical radiation-sensitivity may identify less-toxic therapeutic strategies and provide insight into treatment failure. We previously showed that radiation sensitivity depends on the intrinsic apoptotic pathway, mediated by pro-apoptotic BAX. In cerebellar granule neuron progenitors (CGNPs), the cell of origin for SHH medulloblastoma, BAX activity is directly inhibited by anti-apoptotic BCL-xL; Bcl-xL-deleted CGNPs undergo spontaneous apoptosis. To test the therapeutic potential of disrupting BCL-xL in medulloblastoma, we conditionally deleted Bcl-xL in mice genetically engineered to develop SHH medulloblastoma. Here, I show that Bcl-xL deletion slows SHH medulloblastoma growth and prolongs survival of medulloblastoma-bearing mice. Bcl-xL-deleted tumors initially showed increased rates of spontaneous apoptosis, but this effect waned over time, suggesting the emergence of BCL-xL-independent survival mechanisms. We also noted increased microglial infiltration in Bcl-xL-deleted medulloblastomas. We hypothesize that IGF1 produced by microglia in the tumor microenvironment may be contributing to tumor resistance by upregulating translation of MCL-1, an anti-apoptotic BCL-xL homolog. IGF1 is known to upregulate translation through the mTOR pathway, while anti-apoptotic MCL-1 protein abundance is dependent upon translation regulation. Our on-going studies are testing the efficacy of pharmacologically targeting BCL-xL in mice with medulloblastoma, in combination with targeting IGF1 signaling using mTORC1 inhibitors.
47
Brunelle, Joslyn K., Emelyn H. Shroff, Harris Perlman, Andreas Strasser, Carlos T. Moraes, Richard A. Flavell, Nika N. Danial, Brian Keith, Craig B. Thompson e Navdeep S. Chandel. "Loss of Mcl-1 Protein and Inhibition of Electron Transport Chain Together Induce Anoxic Cell Death". Molecular and Cellular Biology 27, n. 4 (4 dicembre 2006): 1222–35. http://dx.doi.org/10.1128/mcb.01535-06.
Testo completoAbstract (sommario):
ABSTRACT How cells die in the absence of oxygen (anoxia) is not understood. Here we report that cells deficient in Bax and Bak or caspase-9 do not undergo anoxia-induced cell death. However, the caspase-9 null cells do not survive reoxygenation due to the generation of mitochondrial reactive oxygen species. The individual loss of Bim, Bid, Puma, Noxa, Bad, caspase-2, or hypoxia-inducible factor 1β, which are potential upstream regulators of Bax or Bak, did not prevent anoxia-induced cell death. Anoxia triggered the loss of the Mcl-1 protein upstream of Bax/Bak activation. Cells containing a mitochondrial DNA cytochrome b 4-base-pair deletion ([rho −] cells) and cells depleted of their entire mitochondrial DNA ([rho 0] cells) are oxidative phosphorylation incompetent and displayed loss of the Mcl-1 protein under anoxia. [rho 0] cells, in contrast to [rho −] cells, did not die under anoxia. However, [rho 0] cells did undergo cell death in the presence of the Bad BH3 peptide, an inhibitor of Bcl-XL/Bcl-2 proteins. These results indicate that [rho 0] cells survive under anoxia despite the loss of Mcl-1 protein due to residual prosurvival activity of the Bcl-XL/Bcl-2 proteins. Collectively, these results demonstrate that anoxia-induced cell death requires the loss of Mcl-1 protein and inhibition of the electron transport chain to negate Bcl-XL/Bcl-2 proteins.
48
Du, Xing, Richard J. Youle, David J. FitzGerald e Ira Pastan. "Pseudomonas Exotoxin A-Mediated Apoptosis Is Bak Dependent and Preceded by the Degradation of Mcl-1". Molecular and Cellular Biology 30, n. 14 (10 maggio 2010): 3444–52. http://dx.doi.org/10.1128/mcb.00813-09.
Testo completoAbstract (sommario):
ABSTRACT Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax−/−) MEFs but failed in Bak knockout (Bak−/−) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax−/− MEFs, but not in Bak−/− or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-xL, was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-xL in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-xL inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.
49
Haselager, Marco, Rachel Thijssen, Christopher West, Louise Young, Roel Van Kampen, Elaine Willmore, Simon Mackay, Arnon Kater e Eric Eldering. "Regulation of Bcl-XL by non-canonical NF-κB in the context of CD40-induced drug resistance in CLL". Cell Death & Differentiation 28, n. 5 (25 gennaio 2021): 1658–68. http://dx.doi.org/10.1038/s41418-020-00692-w.
Testo completoAbstract (sommario):
AbstractIn chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We determined previously that resistance against various drugs, among which is the clinically applied BH3 mimetic venetoclax, is dominated by upregulation of the anti-apoptotic regulator Bcl-XL. Direct clinical targeting of Bcl-XL by, e.g., Navitoclax is however not desirable due to induction of thrombocytopenia. Since the actual regulation of Bcl-XL in CLL in the context of the LN microenvironment is not well elucidated, we investigated various candidate LN signals to drive Bcl-XL expression. We found a dominance for NF-κB signaling upon CD40 stimulation, which results in activation of both the canonical and non-canonical NF-κB signaling pathways. We demonstrate that expression of Bcl-XL is first induced by the canonical NF-κB pathway, and subsequently boosted and continued via non-canonical NF-κB signaling through stabilization of NIK. NF-κB subunits p65 and p52 can both bind to the Bcl-XL promoter and activate transcription upon CD40 stimulation. Moreover, canonical NF-κB signaling was correlated with Bfl-1 expression, whereas Mcl-1 in contrast, was not transcriptionally regulated by NF-κB. Finally, we applied a novel compound targeting NIK to selectively inhibit the non-canonical NF-κB pathway and showed that venetoclax-resistant CLL cells were sensitized to venetoclax. In conclusion, protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with non-canonical NF-κB signaling.
50
Gupta, Vikas A., Shannon M. Matulis, Jason E. Conage-Pough, Ajay K. Nooka, Jonathan L. Kaufman, Sagar Lonial e Lawrence H. Boise. "Bone marrow microenvironment–derived signals induce Mcl-1 dependence in multiple myeloma". Blood 129, n. 14 (6 aprile 2017): 1969–79. http://dx.doi.org/10.1182/blood-2016-10-745059.
Testo completoAbstract (sommario):
Key Points Bone marrow stromal cell–derived IL-6 induces Mcl-1 dependence through transcriptional and posttranslational changes in the Bcl-2 family. Blocking IL-6 signaling pathways sensitizes myeloma to inhibitors of Bcl-2 and Bcl-2/Bcl-xL.