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Oncotarget 2022GZ17-6.02 is undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in renal carcinoma cells and to...
GZ17-6.02 is undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in renal carcinoma cells and to determine whether it interacted with axitinib to enhance tumor cell killing. GZ17-6.02 interacted in an arithmetically greater than additive fashion with axitinib to kill kidney cancer cells. GZ17-6.02 and axitinib cooperated to inactivate ERBB2, c-MET, c-KIT, c-SRC, the AMPK, STAT3, STAT5 and eIF2α and to activate PERK, ULK1 and ATG13. The drugs interacted to increase the expression of FAS-L and to decrease the levels of MCL1, BCL-XL, and HDACs 1-3. The drugs as single agents inactivated the Hippo pathway. GZ17-6.02 and axitinib interacted to enhance autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5, eIF2α, toxic BH3 domain proteins or CD95/FADD significantly reduced drug combination lethality. GZ17-6.02 and axitinib increased the expression of BAK, BIM, Beclin1 and ATG5, effects blocked by knock down of eIF2α. The drugs increased phosphorylation of ULK1 S757 and ATG13 S318 and decreased the phosphorylation of mTORC1 and mTORC2, effects blocked by knock down of AMPKα. Knock down of Beclin1 or ATG5 prevented the drug combination reducing expression of HDACs 1-3 and from enhancing the expression of MHCA. Knock down of HDACs 1-3 enhanced MHCA expression. We conclude that GZ17-6.02 and axitinib interact to kill requiring ER stress signaling, autophagy and death receptor signaling. Autophagic degradation of HDACs played a key role in enhancing MHCA expression and of a potential improved response to checkpoint inhibitory immunotherapy.
Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Autophagy; Axitinib; Beclin-1; Carcinoma, Renal Cell; Cell Line, Tumor; Drug Synergism; Eukaryotic Initiation Factor-2; Humans; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Myeloid Cell Leukemia Sequence 1 Protein; Receptors, Death Domain; STAT5 Transcription Factor
PubMed: 35136485
DOI: 10.18632/oncotarget.28189 -
Journal of Medicinal Chemistry May 2021Small-molecule mediated modulation of protein interactions of Bcl-2 (B-cell lymphoma-2) family proteins was clinically validated in 2015 when Venetoclax, a selective... (Review)
Review
Small-molecule mediated modulation of protein interactions of Bcl-2 (B-cell lymphoma-2) family proteins was clinically validated in 2015 when Venetoclax, a selective inhibitor of the antiapoptotic protein BCL-2, achieved breakthrough status designation by the FDA for treatment of lymphoid malignancies. Since then, substantial progress has been made in identifying inhibitors of other interactions of antiapoptosis proteins. However, targeting their pro-apoptotic counterparts, the "executioners" BAX, BAK, and BOK that both initiate and commit the cell to dying, has lagged behind. However, recent publications demonstrate that these proteins can be positively or negatively regulated using small molecule tool compounds. The results obtained with these molecules suggest that pharmaceutical regulation of apoptosis will have broad implications that extend beyond activating cell death in cancer. We review recent advances in identifying compounds and their utility in the exogenous control of life and death by regulating executioner proteins, with emphasis on the prototype BAX.
Topics: Apoptosis; Cell Membrane Permeability; Humans; Mitochondria; Phenyl Ethers; Proto-Oncogene Proteins c-bcl-2; Small Molecule Libraries; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 33939407
DOI: 10.1021/acs.jmedchem.0c02200 -
Cell Death & Disease Jul 2023BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor,...
BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment.
Topics: Humans; Female; Apoptosis; Proto-Oncogene Proteins c-bcl-2; Myeloid Cell Leukemia Sequence 1 Protein; Antineoplastic Agents; bcl-X Protein; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Melanoma; Ovarian Neoplasms; Cell Line, Tumor
PubMed: 37460459
DOI: 10.1038/s41419-023-05963-1 -
Biological Research Jun 2022Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the...
BACKGROUND
Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo.
METHODS
Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light- or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING pathway and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function.
RESULTS
Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-β mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-β were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection.
CONCLUSIONS
Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.
Topics: Animals; DNA, Mitochondrial; Intravitreal Injections; Lipopolysaccharides; Membrane Proteins; Mitochondria; Nucleotidyltransferases; Rats
PubMed: 35659309
DOI: 10.1186/s40659-022-00390-6 -
Cell Death & Disease Feb 2021Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists... (Review)
Review
Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists of prosurvival (e.g., BCL-2, MCL-1) and proapoptotic (e.g., BIM, BAD, HRK) members, the functional balance of which dictates the activation of BAX and BAK. Once activated, BAX/BAK form pores in the MOM, resulting in cytochrome c release from the mitochondrial intermembrane space, leading to apoptosome formation, caspase activation, and cleavage of intracellular targets. This pathway is induced by cellular stress including DNA damage, cytokine and growth factor withdrawal, and chemotherapy/drug treatment. A well-documented defense of leukemia cells is to shift the balance of the BCL-2 family in favor of the prosurvival proteins to protect against such intra- and extracellular stimuli. Small molecule inhibitors targeting the prosurvival proteins, named 'BH3 mimetics', have come to the fore in recent years to treat hematological malignancies, both as single agents and in combination with standard-of-care therapies. The most significant example of these is the BCL-2-specific inhibitor venetoclax, given in combination with standard-of-care therapies with great success in AML in clinical trials. As the number and variety of available BH3 mimetics increases, and investigations into applying these novel inhibitors to treat myeloid leukemias continue apace the need to evaluate where we currently stand in this rapidly expanding field is clear.
Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Drug Design; Humans; Leukemia, Myeloid; Molecular Mimicry; Molecular Targeted Therapy; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides
PubMed: 33637708
DOI: 10.1038/s41419-021-03500-6 -
Frontiers in Cell and Developmental... 2020BCL-2-related ovarian killer (BOK) is-despite its identification over 20 years ago-an incompletely understood member of the BCL-2 family. BCL-2 family proteins are best... (Review)
Review
BCL-2-related ovarian killer (BOK) is-despite its identification over 20 years ago-an incompletely understood member of the BCL-2 family. BCL-2 family proteins are best known for their critical role in the regulation of mitochondrial outer membrane permeabilization during the intrinsic apoptotic pathway. Based on sequence and structural similarities to BAX and BAK, BOK is grouped with these "killers" within the effector subgroup of the family. However, the mechanism of how exactly BOK exerts apoptosis is not clear and controversially discussed. Furthermore, and in accordance with reports on several other BCL-2 family members, BOK seems to be involved in the regulation of a variety of other, "apoptosis-independent" cellular functions, including the unfolded protein response, cellular proliferation, metabolism, and autophagy. Of note, compared with other proapoptotic BCL-2 family members, BOK levels are often reduced in cancer by various means, and there is increasing evidence for BOK modulating tumorigenesis. In this review, we summarize and discuss apoptotic- and non-apoptotic-related functions of BOK, its regulation as well as its physiological and pathophysiological roles.
PubMed: 33043006
DOI: 10.3389/fcell.2020.574338 -
Biophysical Journal Feb 2022Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and...
Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.
Topics: Apoptosis; Lipids; Liposomes; Mitochondrial Membranes; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 34973947
DOI: 10.1016/j.bpj.2021.12.043 -
The EMBO Journal Dec 2022During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we...
During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.
Topics: Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; HCT116 Cells; HeLa Cells; Humans; Mitochondria; Mitochondrial Proteins; Protein Domains; Proteolysis; Proto-Oncogene Proteins c-bcl-2; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 34931711
DOI: 10.15252/embj.2021108690 -
The EMBO Journal Oct 2021BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause...
BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.
Topics: Animals; Binding Sites; Cloning, Molecular; Deuterium Exchange Measurement; Escherichia coli; Gene Expression; Genetic Vectors; Humans; Kinetics; Liposomes; Membrane Lipids; Mice; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Interaction Domains and Motifs; Protein Multimerization; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Thermodynamics; bcl-2 Homologous Antagonist-Killer Protein
PubMed: 34523147
DOI: 10.15252/embj.2020107237 -
Clinical Cancer Research : An Official... Jan 2023Several MCL-1 inhibitors (MCL-1i), including AMG-176 and AZD5991, have shown promise in preclinical studies and are being tested for the treatment of hematologic...
PURPOSE
Several MCL-1 inhibitors (MCL-1i), including AMG-176 and AZD5991, have shown promise in preclinical studies and are being tested for the treatment of hematologic malignancies. A unique feature of these agents is induction and stability of Mcl-1 protein; however, the precise mechanism is unknown. We aim to study the mechanism of MCL-1i-induced Mcl-1 protein stability.
EXPERIMENTAL DESIGN
Using several B-cell leukemia and lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) lymphocytes, we evaluated molecular events associated with Mcl-1 protein stability including protein half-life, reverse-phase protein array, protein-protein interaction, phosphorylation, ubiquitination, and de-ubiquitination, followed by molecular simulation and modeling.
RESULTS
Using both in vivo and in vitro analysis, we demonstrate that MCL-1i-induced Mcl-1 protein stability is predominantly associated with defective Mcl-1 ubiquitination and concurrent apoptosis induction in both cell lines and primary CLL subjects. These MCL1i also induced ERK-mediated Mcl-1Thr163 phosphorylation, which partially contributed to Mcl-1 stability. Disruption of Mcl-1:Noxa interaction followed by Noxa degradation, enhanced Mcl-1 de-ubiquitination by USP9x, and Mule destabilization are the major effects of these inhibitors. However, unlike other BH3 proteins, Mule:Mcl-1 interaction was unaffected by MCL-1i. WP1130, a global deubiquitinase (DUB) inhibitor, abrogated Mcl-1 induction reaffirming a critical role of DUBs in the observed Mcl-1 protein stability. Further, in vitro ubiquitination studies of Mcl-1 showed distinct difference among these inhibitors.
CONCLUSIONS
We conclude that MCL-1i blocked Mcl-1 ubiquitination via enhanced de-ubiquitination and dissociation of Mcl-1 from Noxa, Bak and Bax, and Mule de-stabilization. These are critical events associated with increased Mcl-1 protein stability with AMG-176 and AZD5991.
Topics: Humans; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Leukemia, Lymphocytic, Chronic, B-Cell; Myeloid Cell Leukemia Sequence 1 Protein; Protein Stability; Proto-Oncogene Proteins c-bcl-2; Ubiquitin Thiolesterase
PubMed: 36346691
DOI: 10.1158/1078-0432.CCR-22-2088