ABSTRACT
In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.
Subject(s)
Apoptosis , Crystallography, X-Ray , bcl-2-Associated X Protein/chemistry , Amino Acid Sequence , Animals , Cytochromes c/metabolism , Dimerization , Embryo, Mammalian/metabolism , Hydrophobic and Hydrophilic Interactions , Liposomes/metabolism , Liver/metabolism , Mice , Mitochondria/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Sequence Alignment , bcl-2-Associated X Protein/metabolismABSTRACT
The BCL-2 protein family determines the commitment of cells to apoptosis, an ancient cell suicide programme that is essential for development, tissue homeostasis and immunity. Too little apoptosis can promote cancer and autoimmune diseases; too much apoptosis can augment ischaemic conditions and drive neurodegeneration. We discuss the biochemical, structural and genetic studies that have clarified how the interplay between members of the BCL-2 family on mitochondria sets the apoptotic threshold. These mechanistic insights into the functions of the BCL-2 family are illuminating the physiological control of apoptosis, the pathological consequences of its dysregulation and the promising search for novel cancer therapies that target the BCL-2 family.
Subject(s)
Apoptosis , Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2/physiology , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/physiology , Humans , Models, Molecular , Molecular Sequence Data , Molecular Targeted Therapy , Protein Multimerization , Protein Structure, Secondary , Proto-Oncogene Proteins c-bcl-2/chemistry , Signal TransductionABSTRACT
Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.
Subject(s)
Leukemia, Myeloid, Acute , Proto-Oncogene Proteins c-bcl-2 , Humans , Aged , bcl-2-Associated X Protein/genetics , Cell Line, Tumor , Proto-Oncogene Proteins c-bcl-2/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Apoptosis , MutationABSTRACT
Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Bridged Bicyclo Compounds, Heterocyclic , Hematologic Neoplasms , Leukemia, Lymphocytic, Chronic, B-Cell , Mutation , Myelopoiesis/drug effects , Myeloproliferative Disorders , Neoplasms, Second Primary , Sulfonamides , bcl-2-Associated X Protein , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Bridged Bicyclo Compounds, Heterocyclic/adverse effects , Female , Hematologic Neoplasms/genetics , Hematologic Neoplasms/metabolism , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Middle Aged , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/metabolism , Neoplasms, Second Primary/genetics , Neoplasms, Second Primary/metabolism , Sulfonamides/administration & dosage , Sulfonamides/adverse effects , Vidarabine/administration & dosage , Vidarabine/adverse effects , Vidarabine/analogs & derivatives , bcl-2-Associated X Protein/antagonists & inhibitors , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.
Subject(s)
Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/immunology , Drug Design , Membrane Proteins/agonists , Animals , Benzimidazoles/administration & dosage , Benzimidazoles/therapeutic use , Humans , Ligands , Membrane Proteins/immunology , Mice , Models, Molecular , Nucleotides, Cyclic/metabolismABSTRACT
Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in gamma-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from gamma-irradiation-induced death, because their glucocorticoid-mediated decimation in Puma-deficient mice activated cycling of stem/progenitor cells and restored thymic lymphomagenesis. Our demonstration that cycles of cell attrition and repopulation by stem/progenitor cells can drive tumorigenesis has parallels in human cancers, such as therapy-induced malignancies.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Apoptosis/radiation effects , Gamma Rays , Lymphoma/physiopathology , Thymus Neoplasms/physiopathology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Cells, Cultured , Dexamethasone/pharmacology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/radiation effects , Leukocytes/drug effects , Leukocytes/pathology , Leukocytes/radiation effects , Lymphoma/genetics , Mice , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Survival Analysis , Thymus Neoplasms/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between alpha6 helices. Cysteine residues placed in alpha6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha6:alpha6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in alpha6 was not required to form the alpha6:alpha6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha6:alpha6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.
Subject(s)
Apoptosis , Protein Multimerization , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Animals , Binding Sites , Cytochromes c/metabolism , Mice , Protein Structure, Secondary , Sequence Deletion , Signal Transduction , Structure-Activity Relationship , Zinc/metabolismABSTRACT
The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.
Subject(s)
Apoptosis/physiology , Mitochondrial Membranes/metabolism , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Cysteine/chemistry , Humans , Mice , Mitochondrial Membranes/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Stilbenes , Sulfhydryl Reagents , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/geneticsABSTRACT
The B-cell CLL/lymphoma 2 (Bcl2) relative Myeloid cell leukemia sequence 1 (Mcl1) is essential for cell survival during development and for tissue homeostasis throughout life. Unlike Bcl2, Mcl1 turns over rapidly, but the physiological significance of its turnover has been unclear. We have gained insight into the roles of Mcl1 turnover in vivo by analyzing mice harboring a modified allele of Mcl1 that serendipitously proved to encode an abnormally stabilized form of Mcl1 due to a 13-aa N-terminal extension. Although the mice developed normally and appeared unremarkable, the homozygous males unexpectedly proved infertile due to defective spermatogenesis, which was evoked by enhanced Mcl1 prosurvival activity. Under unstressed conditions, the modified Mcl1 is present at levels comparable to the native protein, but it is markedly stabilized in cells subjected to stresses, such as protein synthesis inhibition or UV irradiation. Strikingly, the modified Mcl1 allele could genetically complement the loss of Bcl2, because introduction of even a single allele significantly ameliorated the severe polycystic kidney disease and consequent runting caused by Bcl2 loss. Significantly, the development of c-MYC-induced acute myeloid leukemia was also accelerated in mice harboring that Mcl1 allele. Our collective findings reveal that, under certain circumstances, the N terminus of Mcl1 regulates its degradation; that some cell types require degradation of Mcl1 to induce apoptosis; and, most importantly, that rapid turnover of Mcl1 can serve as a tumor-suppressive mechanism.
Subject(s)
Apoptosis , Cell Transformation, Neoplastic/genetics , Infertility, Male/genetics , Myeloid Cell Leukemia Sequence 1 Protein/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Alleles , Animals , Cell Death , Cell Survival , Female , Fibroblasts/metabolism , Flow Cytometry , Gene Expression Regulation , HEK293 Cells , Humans , Infertility, Male/metabolism , Leukemia, Myeloid, Acute/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Polycystic Kidney Diseases/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-myc/metabolism , Spermatogenesis , Testis/pathology , Time Factors , Ultraviolet RaysABSTRACT
The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.
Subject(s)
Apoptosis/physiology , Protein Conformation , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , BH3 Interacting Domain Death Agonist Protein/genetics , BH3 Interacting Domain Death Agonist Protein/metabolism , Bcl-2-Like Protein 11 , Cells, Cultured , Dimerization , Disulfides/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Models, Molecular , Molecular Sequence Data , Mutation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Signal Transduction/physiology , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Apoptosis, the major form of programmed cell death in metazoan organisms, plays critical roles in normal development, tissue homeostasis and immunity, and its disturbed regulation contributes to many pathological states, including cancer, autoimmunity, infection and degenerative disorders. In vertebrates, it can be triggered either by engagement of 'death receptors' of the tumour necrosis factor receptor family on the cell surface or by diverse intracellular signals that act upon the Bcl-2 protein family, which controls the integrity of the mitochondrial outer membrane through the complex interactions of family members. Both pathways lead to cellular demolition by dedicated proteases termed caspases. This review discusses the groundbreaking experiments from many laboratories that have clarified cell death regulation and galvanised efforts to translate this knowledge into novel therapeutic strategies for the treatment of malignant and perhaps certain autoimmune and infectious diseases.
Subject(s)
Apoptosis , Neoplasms/therapy , Gene Expression RegulationABSTRACT
The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/chemistry , Animals , Apoptosis , Benzothiazoles/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Humans , Hydrazones/chemistry , Kinetics , Mice , Models, Chemical , Myeloid Cell Leukemia Sequence 1 Protein , Protein Binding , Proto-Oncogene Proteins c-bcl-2/geneticsABSTRACT
Activation and oligomerisation of Bax, a key pro-apoptotic Bcl-2 family protein, are key steps in the mitochondrial pathway to apoptosis. The signals for apoptosis are conveyed by the distantly related BH3-only proteins, which use their short BH3 domain, an amphipathic α-helix, to interact with other Bcl-2 family members. Here we report an NMR study of interactions between BaxΔC and BH3 domain-containing peptides in the absence and presence of CHAPS, a zwitterionic detergent. We find for the first time that CHAPS interacts weakly with BaxΔC (fast exchange on the NMR chemical shift timescale), at concentrations below micelle formation and with an estimated Kd in the tens of mM. Direct and relatively strong-interactions (slow exchange on the NMR chemical shift timescale) were also observed for BaxΔC with BaxBH3 (estimated Kd of circa 150µM) or BimBH3 in the absence of CHAPS. The interaction with either peptide alone induced widespread chemical shift perturbations to BaxΔC in solution which implies that BaxΔC might have undergone significant conformation change upon binding the BH3 peptide. However, BaxΔC remained monomeric upon binding either CHAPS or a BH3 peptide alone, but the presence of both provoked it to form a dimer.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Cholic Acids/metabolism , Detergents/metabolism , Membrane Proteins/metabolism , Peptide Fragments/metabolism , Proto-Oncogene Proteins/metabolism , bcl-2-Associated X Protein/metabolism , Apoptosis , Apoptosis Regulatory Proteins/chemistry , Bcl-2-Like Protein 11 , Humans , Membrane Proteins/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Protein Conformation , Protein Interaction Maps , Protein Multimerization , Proto-Oncogene Proteins/chemistry , bcl-2-Associated X Protein/chemistryABSTRACT
Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.
Subject(s)
Apoptosis , Biphenyl Compounds/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Nitrophenols/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/pharmacology , Animals , Biphenyl Compounds/metabolism , Biphenyl Compounds/therapeutic use , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/metabolism , Nitrophenols/metabolism , Nitrophenols/therapeutic use , Piperazines/metabolism , Piperazines/pharmacology , Piperazines/therapeutic use , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sulfonamides/metabolism , Sulfonamides/therapeutic use , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
The traditional view of hematopoiesis is that myeloid cells derive from a common myeloid progenitor (CMP), whereas all lymphoid cell populations, including B, T, and natural killer (NK) cells and possibly plasmacytoid dendritic cells (pDCs), arise from a common lymphoid progenitor (CLP). In Max41 transgenic mice, nearly all B cells seem to be diverted into the granulocyte lineage. Here, we show that these mice have an excess of myeloid progenitors, but their CLP compartment is ablated, and they have few pDCs. Nevertheless, T cell and NK cell development proceeds relatively normally. These hematopoietic abnormalities result from aberrant expression of Gata6 due to serendipitous insertion of the transgene enhancer (Eµ) in its proximity. Gata6 mis-expression in Max41 transgenic progenitors promoted the gene-regulatory networks that drive myelopoiesis through increasing expression of key transcription factors, including PU.1 and C/EBPa. Thus, mis-expression of a single key regulator like GATA6 can dramatically re-program multiple aspects of hematopoiesis.
Subject(s)
GATA6 Transcription Factor , Hematopoiesis , Mice, Transgenic , GATA6 Transcription Factor/metabolism , GATA6 Transcription Factor/genetics , Animals , Mice , Cell Lineage , Killer Cells, Natural/metabolism , Killer Cells, Natural/immunology , Mice, Inbred C57BL , Dendritic Cells/metabolism , Cell Differentiation , T-Lymphocytes/metabolism , T-Lymphocytes/cytology , Proto-Oncogene Proteins , Trans-ActivatorsABSTRACT
Impaired apoptosis is a cancer hallmark, and some types of lymphomas and other cancers harbor mutations that directly affect key cell death regulators, such as Bcl-2 family members. However, because the majority of tumors seem to lack such mutations, we are examining the hypothesis that tumorigenesis can be sustained at least initially by the normal expression of specific endogenous pro-survival Bcl-2 family members. We previously demonstrated that the lymphomagenesis in Εµ-myc transgenic mice, which constitutively overexpress the c-Myc oncoprotein in B-lymphoid cells and develop pre-B and B-cell lymphomas, does not require endogenous Bcl-2. In striking contrast, we report here that loss in these mice of its close relative Bcl-x(L) attenuated the pre-neoplastic expansion of pro-B and pre-B cells otherwise driven by c-Myc overexpression, sensitized these cells to apoptosis and ablated lymphoma formation. Remarkably, even loss of a single bcl-x allele delayed the lymphomagenesis. These findings identify Bcl-x(L) as a prerequisite for the emergence of c-Myc-driven pre-B/B lymphoma and suggest that BH3 mimetic drugs may provide a prophylactic strategy for c-Myc-driven tumors.
Subject(s)
B-Lymphocytes/metabolism , Cell Transformation, Neoplastic/metabolism , Lymphoma, B-Cell/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Up-Regulation , bcl-X Protein/metabolism , Animals , Apoptosis , B-Lymphocytes/cytology , Cells, Cultured , Crosses, Genetic , Female , Heterozygote , Lymphoma, B-Cell/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Biological , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/metabolism , Proto-Oncogene Proteins c-myc/genetics , Survival Analysis , bcl-X Protein/geneticsABSTRACT
A phase I study was conducted to assess the metabolism and excretion of [(14)C]dabrafenib (GSK2118436; N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzene sulfonamide, methanesulfonate salt), a BRAF inhibitor, in four patients with BRAF V600 mutation-positive tumors after a single oral dose of 95 mg (80 µCi). Assessments included the following: 1) plasma concentrations of dabrafenib and metabolites using validated ultra-high-performance liquid chromatography--tandem mass spectrometry methods, 2) plasma and blood radioactivity, 3) urinary and fecal radioactivity, and 4) metabolite profiling. Results showed the mean total recovery of radioactivity was 93.8%, with the majority recovered in feces (71.1% of administered dose). Urinary excretion accounted for 22.7% of the dose, with no detection of parent drug in urine. Dabrafenib is metabolized primarily via oxidation of the t-butyl group to form hydroxy-dabrafenib. Hydroxy-dabrafenib undergoes further oxidation to carboxy-dabrafenib, which subsequently converts to desmethyl-dabrafenib via a pH-dependent decarboxylation. The half-lives for carboxy- and desmethyl-dabrafenib were longer than for parent and hydroxy-dabrafenib (18-20 vs. 5-6 hours). Based on area under the plasma concentration-time curve, dabrafenib, hydroxy-, carboxy-, and desmethyl-dabrafenib accounted for 11%, 8%, 54%, and 3% of the plasma radioactivity, respectively. These results demonstrate that the major route of elimination of dabrafenib is via oxidative metabolism (48% of the dose) and biliary excretion. Based on our understanding of the decarboxylation of carboxy-dabrafenib, a low pH-driven, nonenzymatic mechanism involving participation of the aryl nitrogen is proposed to allow prediction of metabolic oxidation and decarboxylation of drugs containing an aryl nitrogen positioned α to an alkyl (ethyl or t-butyl) side chain.
Subject(s)
Carbon/metabolism , Decarboxylation/physiology , Imidazoles/metabolism , Neoplasms/metabolism , Nitrogen/metabolism , Oximes/metabolism , Administration, Oral , Adult , Feces/chemistry , Female , Half-Life , Humans , Male , Middle Aged , Oxidation-Reduction , Young AdultABSTRACT
Impairment of apoptosis, the physiologic cell death process, is central to cancer development and renders tumors refractory to cytotoxic therapy. Bcl-2, the oncoprotein activated in follicular lymphoma, inhibits the conserved cell death pathway triggered by diverse cytotoxic agents, as do several close relatives. A small-molecule antagonist of these proteins has now been designed by Oltersdorf et al. Strikingly, ABT-737 sensitizes many tumors to cytotoxic agents and is effective as a single agent against certain lymphomas and solid tumors, provoking stable regression in some tumor xenografts. Hence, this work validates Bcl-2-like proteins as important new targets in cancer therapy.
Subject(s)
Apoptosis , Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Biphenyl Compounds/pharmacology , Humans , Neoplasms/metabolism , Nitrophenols , Piperazines , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides , bcl-2-Associated X ProteinABSTRACT
Defective apoptosis not only promotes tumorigenesis, but also can confound chemotherapeutic response. Here we demonstrate that the proapoptotic BH3-only protein BIM is a tumor suppressor in epithelial solid tumors and also is a determinant in paclitaxel sensitivity in vivo. Furthermore, the H-ras/mitogen-activated protein kinase (MAPK) pathway conferred resistance to paclitaxel that was dependent on functional inactivation of BIM. Whereas paclitaxel induced BIM accumulation and BIM-dependent apoptosis in vitro and in tumors in vivo, the H-ras/MAPK pathway suppressed this BIM induction by phosphorylating BIM and targeting BIM for degradation in proteasomes. The proteasome inhibitor Velcade (P-341, Bortezomib) restored BIM induction, abrogated H-ras-dependent paclitaxel resistance, and promoted BIM-dependent tumor regression, suggesting the potential benefits of combinatorial chemotherapy of Velcade and paclitaxel.