ABSTRACT
Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic gene across human cancers. Mutant IDH1 (mIDH1) generates the oncometabolite (R)-2-hydroxyglutarate, disrupting enzymes involved in epigenetics and other processes. A hallmark of IDH1-mutant solid tumors is T cell exclusion, whereas mIDH1 inhibition in preclinical models restores antitumor immunity. Here, we define a cell-autonomous mechanism of mIDH1-driven immune evasion. IDH1-mutant solid tumors show selective hypermethylation and silencing of the cytoplasmic double-stranded DNA (dsDNA) sensor CGAS, compromising innate immune signaling. mIDH1 inhibition restores DNA demethylation, derepressing CGAS and transposable element (TE) subclasses. dsDNA produced by TE-reverse transcriptase (TE-RT) activates cGAS, triggering viral mimicry and stimulating antitumor immunity. In summary, we demonstrate that mIDH1 epigenetically suppresses innate immunity and link endogenous RT activity to the mechanism of action of a US Food and Drug Administration-approved oncology drug.
Subject(s)
Immune Evasion , Immunity, Innate , Isocitrate Dehydrogenase , Neoplasms , Animals , Humans , Mice , Cell Line, Tumor , DNA/metabolism , DNA Demethylation , DNA Methylation , DNA Transposable Elements , Epigenesis, Genetic , Glutarates/metabolism , Immunity, Innate/genetics , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Mutation , Neoplasms/immunology , Neoplasms/genetics , Nucleotidyltransferases/genetics , Tumor Escape , Immune Evasion/geneticsABSTRACT
Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.
Subject(s)
Isocitrate Dehydrogenase , Leukemia, Myeloid, Acute , Animals , Azacitidine/pharmacology , Enzyme Inhibitors/pharmacology , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice , Mutation/genetics , Stem Cells/metabolismABSTRACT
BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.
Subject(s)
Cancer Vaccines , Glioma , Animals , Glioma/drug therapy , Glioma/genetics , Glioma/pathology , Glutarates , HLA-A2 Antigen/genetics , HLA-DR1 Antigen/genetics , Humans , Immune Checkpoint Inhibitors , Isocitrate Dehydrogenase/genetics , Mice , Mice, Transgenic , Tumor Microenvironment , Up-Regulation , Vaccines, SubunitABSTRACT
Targeting the menin-MLL protein-protein interaction is being pursued as a new therapeutic strategy for the treatment of acute leukemia carrying MLL-rearrangements (MLLr leukemia). Herein, we report M-1121, a covalent and orally active inhibitor of the menin-MLL interaction capable of achieving complete and persistent tumor regression. M-1121 establishes covalent interactions with Cysteine 329 located in the MLL binding pocket of menin and potently inhibits growth of acute leukemia cell lines carrying MLL translocations with no activity in cell lines with wild-type MLL. Consistent with the mechanism of action, M-1121 drives dose-dependent down-regulation of HOXA9 and MEIS1 gene expression in the MLL-rearranged MV4;11 leukemia cell line. M-1121 is orally bioavailable and shows potent antitumor activity in vivo with tumor regressions observed at tolerated doses in the MV4;11 subcutaneous and disseminated models of MLL-rearranged leukemia. Together, our findings support development of an orally active covalent menin inhibitor as a new therapy for MLLr leukemia.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Leukemia, Myeloid, Acute/drug therapy , Proto-Oncogene Proteins/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Models, Molecular , Molecular Structure , Proto-Oncogene Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Anticancer efficacy is driven not only by dose but also by frequency and duration of treatment. We describe a multiscale model combining cell cycle, cellular heterogeneity of B-cell lymphoma 2 family proteins, and pharmacology of AZD5991, a potent small-molecule inhibitor of myeloid cell leukemia 1 (Mcl-1). The model was calibrated using in vitro viability data for the MV-4-11 acute myeloid leukemia cell line under continuous incubation for 72 hours at concentrations of 0.03-30 µM. Using a virtual screen, we identified two schedules as having significantly different predicted efficacy and showed experimentally that a "short" schedule (treating cells for 6 of 24 hours) is significantly better able to maintain the rate of cell kill during treatment than a "long" schedule (18 of 24 hours). This work suggests that resistance can be driven by heterogeneity in protein expression of Mcl-1 alone without requiring mutation or resistant subclones and demonstrates the utility of mathematical models in efficiently identifying regimens for experimental exploration.
Subject(s)
Antineoplastic Agents/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Macrocyclic Compounds/pharmacology , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cell Line, Tumor/drug effects , Drug Administration Schedule , Drug Resistance, Neoplasm , Humans , Leukemia, Myeloid, Acute/pathology , Macrocyclic Compounds/administration & dosage , Macrocyclic Compounds/therapeutic use , Mice , Models, Animal , Xenograft Model Antitumor Assays/methodsABSTRACT
Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. These results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses, suggesting that sequencing of MCL-1 inhibitors with targeted therapies could overcome such widespread and clinically important resistance.
Subject(s)
Drug Resistance, Neoplasm/genetics , Molecular Targeted Therapy , Proto-Oncogene Proteins c-bcl-2/genetics , RNA Stability/genetics , Animals , Apoptosis , Base Sequence , Cell Line, Tumor , Humans , Male , Mice, Nude , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Tristetraprolin/metabolismABSTRACT
Dysregulation of the mitochondrial apoptotic pathway controlled by members of the Bcl-2 protein family plays a central role in cancer development and resistance to conventional cytotoxic as well as targeted therapies. Hence, selective inhibition of pro-survival Bcl-2 family of proteins to activate apoptosis in malignant cells represents an exciting anti-cancer strategy. The remarkable clinical performance of the selective Bcl-2 antagonist venetoclax has highlighted the potential for selective inhibitors of the other pro-survival members of the Bcl-2 family, particularly Mcl-1. Here we review the latest progress on the discovery and development of selective inhibitors of Mcl-1 that are undergoing clinical evaluation for cancer therapy.
Subject(s)
Antineoplastic Agents/therapeutic use , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Drug Development , HumansABSTRACT
Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).
Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Leukemia, Myeloid, Acute/drug therapy , Multiple Myeloma/drug therapy , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Animals , Bortezomib/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Humans , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Mice, SCID , Multiple Myeloma/pathology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Rats , Rats, Nude , Sulfonamides/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Purpose:fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition.Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivoResults: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor-induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML.Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234-47. ©2017 AACR.
Subject(s)
Apoptosis/genetics , Gene Duplication , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/genetics , Animals , Benzothiazoles/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , Membrane Potential, Mitochondrial , Mice , Phenylurea Compounds/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational , Proteolysis , Proteome/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD.
Subject(s)
DNA Damage , Leukemia, Myeloid, Acute/drug therapy , Oxidative Stress/drug effects , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Topoisomerase II Inhibitors/pharmacology , fms-Like Tyrosine Kinase 3/metabolism , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Biphenyl Compounds/administration & dosage , Biphenyl Compounds/pharmacology , Cytarabine/pharmacology , Drug Synergism , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/genetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/genetics , Proto-Oncogene Proteins c-pim-1/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Thiazolidines/administration & dosage , Thiazolidines/pharmacology , Topoisomerase II Inhibitors/administration & dosageSubject(s)
Biphenyl Compounds/therapeutic use , ELAV-Like Protein 1/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Oncogene Proteins, Fusion/genetics , TYK2 Kinase/genetics , Thiazolidines/therapeutic use , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Leukemic/drug effects , High-Throughput Nucleotide Sequencing , Humans , Leukemia, Myeloid, Acute/pathology , Protein Kinase Inhibitors/therapeutic useABSTRACT
Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCFß-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or ß-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCFß-TRCP-mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1's interaction with ß-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by ß-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers.
Subject(s)
Cell Movement/physiology , Microfilament Proteins/metabolism , Neoplasm Proteins/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , beta-Transducin Repeat-Containing Proteins/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Growth Processes/physiology , Female , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , Microfilament Proteins/genetics , Neoplasm Proteins/genetics , Phosphorylation , SKP Cullin F-Box Protein Ligases/genetics , Transfection , Ubiquitination , beta-Transducin Repeat-Containing Proteins/geneticsABSTRACT
The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF(FBW7) complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Carrier Proteins/chemistry , Cullin Proteins/chemistry , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/chemistry , Ubiquitination/physiology , Adaptor Proteins, Signal Transducing/metabolism , Anaphase-Promoting Complex-Cyclosome , Binding Sites/physiology , Carrier Proteins/metabolism , Crystallography, X-Ray , Cullin Proteins/metabolism , Glomus Tumor/metabolism , Humans , Models, Chemical , Mutagenesis/physiology , Paraganglioma, Extra-Adrenal/metabolism , Protein Binding/physiology , Protein Folding , Protein Structure, Tertiary/physiology , Structure-Activity Relationship , Substrate Specificity/physiology , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers; however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug target for anticancer therapy.
Subject(s)
Cell Cycle Proteins/genetics , F-Box Proteins/genetics , Gene Expression Regulation , Peptidylprolyl Isomerase/metabolism , Ubiquitin-Protein Ligases/genetics , Amino Acid Sequence , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , F-Box Proteins/antagonists & inhibitors , F-Box Proteins/metabolism , F-Box-WD Repeat-Containing Protein 7 , Genes, Tumor Suppressor , Humans , Molecular Sequence Data , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/genetics , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Cullin Proteins/metabolism , F-Box Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Adaptor Proteins, Signal Transducing/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cullin Proteins/genetics , Cyclin E/genetics , Cyclin E/metabolism , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Glomus Tumor/genetics , Glomus Tumor/metabolism , HEK293 Cells , HeLa Cells , Humans , Paraganglioma, Extra-Adrenal/genetics , Paraganglioma, Extra-Adrenal/metabolism , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s) remain largely unknown. Here, we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone, promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers.
Subject(s)
Carrier Proteins/metabolism , Cullin Proteins/metabolism , Immediate-Early Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Ubiquitination , Amino Acid Motifs , Animals , Carrier Proteins/genetics , Cell Line, Tumor , Cullin Proteins/genetics , Humans , Immediate-Early Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Neoplasms/genetics , Neoplasms/metabolism , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rapamycin-Insensitive Companion of mTOR Protein , TOR Serine-Threonine Kinases , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.
Subject(s)
DNA Methylation , DNA/chemistry , Homeodomain Proteins/chemistry , Hydroxyl Radical/chemistry , Leucine Zippers/genetics , Base Sequence , Binding Sites/genetics , DNA/metabolism , DNA Footprinting , Electrophoretic Mobility Shift Assay , Helianthus/chemistry , Helianthus/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Models, Molecular , Molecular Structure , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Plant homeodomain-leucine zipper (HD-Zip) proteins, unlike many animal homeodomains (HDs), are unable to bind DNA as monomers. To investigate the molecular basis of their different behavior, we have constructed chimeras between the HD of the sunflower HD-Zip protein Hahb-4 and that of Drosophila engrailed (EN). Analysis of the interaction of these proteins with the pseudopalindromic Hahb-4 binding site and the monomeric EN binding site suggests that the loop located between helix I and helix II (amino acids 21-28) of EN is enough to confer efficient DNA binding activity to the Hahb-4 HD. Accordingly, the combined mutation of residues 24 and 25 of Hahb-4 to those present in EN (S24R/R25Y) originated an HD able to interact with the EN binding site, while single mutations were ineffective. We have also determined that a protein with the leucine zipper and helix III of Hahb-4 fused to the rest of the EN HD binds to the Hahb-4 pseudopalindomic binding site with increased affinity and shows extended contacts with DNA respective to Hahb-4. We conclude that the loop located between helix I and helix II of the HD must be regarded as one of the segments that contribute to the present-day diversity in the properties of different HDs.
Subject(s)
Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Binding Sites , DNA/metabolism , Dimerization , Drosophila Proteins , Electrophoretic Mobility Shift Assay , Homeodomain Proteins/chemistry , Leucine Zippers , Molecular Sequence Data , Plant Proteins/chemistry , Point Mutation/genetics , Protein Engineering , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/chemistryABSTRACT
Several families of plant transcription factors contain a conserved DNA binding motif known as the homeodomain. In two of these families, named Hd-Zip and glabra2, the homeodomain is associated with a leucine zipper-like dimerization motif. A group of Hd-Zip proteins, namely Hd-ZipII, contain a set of conserved cysteines within the dimerization motif and adjacent to it. Incubation of one of these proteins, Hahb-10, in the presence of thiol-reducing agents such as dithiothreitol or reduced glutathione produced a significant increase in DNA binding. Under such conditions, the protein migrated as a monomer in non-reducing SDS-polyacrylamide gels. Under oxidizing conditions, a significant proportion of the protein migrated as dimers, suggesting the formation of intermolecular disulfide bonds. A similar behavior was observed for the glabra2 protein HAHR1, which also contains two conserved cysteines within its dimerization domain. Site-directed mutagenesis of the cysteines to serines indicated that each of them has different roles in the activation of the proteins. Purified thioredoxin was able to direct the NADPH-dependent activation of Hahb-10 and HAHR1 in the presence of thioredoxin reductase. The results suggest that redox conditions may operate to regulate the activity of these groups of plant transcription factors within plant cells.