ABSTRACT
The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, we report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10â¯000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme's active site. We demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. These data supported progressing AG-270 into current clinical studies (ClinicalTrials.gov NCT03435250).
Subject(s)
Enzyme Inhibitors/chemistry , Methionine Adenosyltransferase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/genetics , Binding Sites , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/therapeutic use , Homozygote , Humans , Methionine Adenosyltransferase/metabolism , Molecular Dynamics Simulation , Neoplasms/drug therapy , Purine-Nucleoside Phosphorylase/metabolism , S-Adenosylmethionine/metabolism , Structure-Activity RelationshipABSTRACT
The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.
Subject(s)
DNA Damage/drug effects , Enzyme Inhibitors/pharmacology , Methionine Adenosyltransferase/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/genetics , Purine-Nucleoside Phosphorylase/genetics , RNA Splicing/drug effects , RNA, Messenger/genetics , Animals , Cell Line , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p16 , DNA Damage/genetics , Gene Deletion , HCT116 Cells , HEK293 Cells , Humans , Methionine Adenosyltransferase/genetics , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasms/drug therapy , Neoplasms/genetics , RNA Splicing/genetics , S-Adenosylmethionine/metabolismABSTRACT
Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity.
Subject(s)
Cyclin-Dependent Kinases/drug effects , Phenylenediamines/pharmacology , Proto-Oncogene Protein c-fli-1/genetics , Pyrimidines/pharmacology , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Oncogene Proteins, Fusion/drug effects , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/drug effects , RNA-Binding Protein EWS/drug effects , Synthetic Lethal Mutations/drug effects , Synthetic Lethal Mutations/geneticsABSTRACT
In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.
Subject(s)
Carcinogenesis/genetics , Gene Expression Regulation, Neoplastic/genetics , RNA, Long Noncoding/genetics , Tumor Suppressor Protein p53/genetics , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Line, Tumor , DNA Damage , Flow Cytometry , Humans , Immunoblotting , Immunohistochemistry , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , MCF-7 Cells , Mice , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Precancerous Conditions/genetics , Proportional Hazards Models , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Skin Neoplasms/genetics , Survival AnalysisABSTRACT
Phosphorylation signaling networks have primarily been studied from an activation perspective, with protein phosphatases viewed as simple counter-balances that functioned passively in the wake of kinase activity. Indeed, there have been only sporadic efforts to investigate the independent role of phosphatases in DNA damage response (DDR). However, global phosphoproteomic analysis of the DDR revealed that over one-third of observed phosphorylation sites were down-regulated within minutes of DNA damage, suggesting a more robust role for phosphatases in DNA repair. Consistent with these observations, recent studies reveal that dephosphorylation of DNA repair factors during specific phases of the cell cycle may be a pre-requisite for their participation in the DDR. Here, we summarize recent literature and speculate on the emerging role of phosphatases in the DDR.
Subject(s)
DNA Repair , DNA/metabolism , Phosphoprotein Phosphatases/genetics , Signal Transduction , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA/chemistry , DNA Breaks, Double-Stranded , Gene Expression Regulation , Humans , Phosphoprotein Phosphatases/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , PhosphorylationABSTRACT
Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion are unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1-T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting nonhomologous end-joining. We also reveal that protein phosphatase complex PP4C/R3ß dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.
Subject(s)
DNA Breaks, Double-Stranded , Intracellular Signaling Peptides and Proteins/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , G1 Phase , HeLa Cells , Humans , Mitosis , Molecular Sequence Data , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Binding , Protein Transport , Tumor Suppressor p53-Binding Protein 1ABSTRACT
The p16INK4a protein is a principal cyclin-dependent kinase inhibitor that decelerates the cell cycle. Abnormally high levels of p16INK4a are commonly observed in human papillomavirus (HPV)-positive head and neck squamous cell carcinomas (HNSCC). We and others found that p16INK4a overexpression is associated with improved therapy response and survival of patients with HNSCC treated with radiotherapy. However, the functional role of p16INK4a in HNSCC remains unexplored. Our results implicate p16INK4a in regulation of homologous recombination-mediated DNA damage response independently from its role in control of the cell cycle. We found that expression of p16INK4a dramatically affects radiation sensitivity of HNSCC cells. p16INK4a overexpression impairs the recruitment of RAD51 to the site of DNA damage in HPV-positive cells by downregulating of cyclin D1 protein expression. Consistent with the in vitro findings, immunostaining of HNSCC patient samples revealed that high levels p16INK4a expression significantly correlated with decreased cyclin D1 expression. In summary, these findings reveal an unexpected function of p16INK4a in homologous recombination-mediated DNA repair response and imply p16INK4a status as an independent marker to predict response of patients with HNSCC to radiotherapy.
Subject(s)
Carcinoma, Squamous Cell/genetics , Cyclin-Dependent Kinase Inhibitor p16/physiology , Head and Neck Neoplasms/genetics , Papillomavirus Infections/genetics , Recombinational DNA Repair , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/therapy , Carcinoma, Squamous Cell/virology , Cell Line, Tumor , Chemoradiotherapy , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , DNA Damage , Head and Neck Neoplasms/mortality , Head and Neck Neoplasms/therapy , Head and Neck Neoplasms/virology , Humans , Kaplan-Meier Estimate , Papillomavirus Infections/mortality , Papillomavirus Infections/therapy , Papillomavirus Infections/virology , Rad51 Recombinase/metabolism , Radiation Tolerance , Treatment OutcomeABSTRACT
The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively engaging two components of the exocyst complex, EXO84 and SEC5. RALB employs SEC5 to trigger innate immunity signalling, whereas RALB-EXO84 interaction induces autophagocytosis. How this differential interaction is achieved molecularly by the RAL GTPase remains unknown. We found that whereas GTP binding turns on RALB activity, ubiquitylation of RALB at Lys 47 tunes its activity towards a particular effector. Specifically, ubiquitylation at Lys 47 sterically inhibits RALB binding to EXO84, while facilitating its interaction with SEC5. Double-stranded RNA promotes RALB ubiquitylation and SEC5-TBK1 complex formation. In contrast, nutrient starvation induces RALB deubiquitylation by accumulation and relocalization of the deubiquitylase USP33 to RALB-positive vesicles. Deubiquitylated RALB promotes the assembly of the RALB-EXO84-beclin-1 complexes driving autophagosome formation. Thus, ubiquitylation within the effector-binding domain provides the switch for the dual functions of RALB in autophagy and innate immune responses.
Subject(s)
Autophagy/physiology , Immunity, Innate/physiology , Ubiquitin Thiolesterase/metabolism , ral GTP-Binding Proteins/metabolism , Autophagy/genetics , Cells, Cultured , HEK293 Cells , HeLa Cells , Humans , Immunity, Innate/genetics , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Signal Transduction , Ubiquitin Thiolesterase/genetics , Ubiquitination , ral GTP-Binding Proteins/chemistry , ral GTP-Binding Proteins/geneticsABSTRACT
Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G(1) to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non-small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic.
Subject(s)
Biomarkers, Pharmacological , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Neoplasms/diagnosis , Poly(ADP-ribose) Polymerase Inhibitors , Protein Phosphatase 2/genetics , Recombinational DNA Repair/genetics , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Pharmacological/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/physiology , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Down-Regulation/genetics , Drug Resistance, Neoplasm/drug effects , Enzyme Inhibitors/therapeutic use , Gene Deletion , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Prognosis , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/physiology , Treatment OutcomeABSTRACT
The kinase oncogenes are well-characterized drivers of cancer development, and several targeted therapies focused on both specific and selectively nonselective kinase inhibitors have now been approved for clinical use. In contrast, much less is known about the role of protein phosphatases, although modulation of their activities might form the foundation for an effective anti-cancer approach. The serine-threonine protein phosphatase 2A (PP2A) is implicated in the regulation of numerous signaling pathways and may function as a tumor suppressor. Recently pharmacological modulation of PP2A activity has been showed to have a potent anti-tumor activity in both in vitro and in vivo cancer models. These studies implicate PP2A as a promising therapeutic target for the treatment of cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Neoplasms/drug therapy , Neoplasms/enzymology , Protein Phosphatase 2/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , Neoplasms/metabolism , Protein Phosphatase 2/metabolism , Structure-Activity RelationshipABSTRACT
The repair of double-strand breaks in mammalian cells is carried out by two pathways: homologous recombination and nonhomologous end joining. The factors that regulate the mechanism through which a specific repair pathway is activated are still not clearly defined. To study whether the complexity of the double-strand break ends is a factor that determines the choice of the repair pathway, we examined the involvement of homologous recombination by the formation of Rad51 foci in human HeLa cells treated with bleomycin and ionizing radiation. The quantity of double-strand breaks was determined by gel electrophoresis and the formation of gamma-H2AX foci. Two hours after treatment with low doses of the agents that induced similar quantities of double-strand breaks that could be repaired effectively by the cells, Rad51 foci were observed only in the irradiated cells. Rad51 foci appeared in bleomycin-treated cells after prolonged exposure to the drug when the cells were arrested in the G2 phase of the cell cycle. Since bleomycin produces double-strand breaks that are less complex than the breaks induced by ionizing radiation, these results indicate that the complexity of the break ends is a factor in the choice of repair pathway and that homologous recombination is recruited in the repair of breaks with more complex multiply damaged ends during the late S and G2 phases of the cell cycle.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Bleomycin/pharmacology , Cell Cycle , Chromosomes/ultrastructure , Dose-Response Relationship, Radiation , Flow Cytometry , G2 Phase , HeLa Cells , Histones/metabolism , Humans , Microscopy, Fluorescence/methods , Rad51 Recombinase/metabolism , Recombination, Genetic , Time FactorsABSTRACT
It has been shown that the key homologous recombination protein Rad51 accumulates in DNA damage-induced nuclear foci that are attached to the nuclear matrix. In the present communication we attempted to find whether Rad51 contains a functional domain responsible for nuclear matrix binding. By alignments of the sequences encoding nuclear matrix targeting signals of human nuclear matrix binding proteins with the whole length human Rad51sequence a putative nuclear matrix targeting signal was identified. To prove that it is responsible for the nuclear matrix association of Rad51 18 base pairs encoding a cluster of hydrophobic amino acids in the human Rad51 Flag-tagged gene were deleted. The formation of damage-induced Rad51 foci and their association with the nuclear matrix were monitored in HeLa cells transfected with the wild-type and the mutated Rad51gene after treatment with mitomycin C. The results showed that while the wild-type protein formed Rad51 foci attached to the nuclear matrix, the mutated Rad51 failed to form DNA damage-induced nuclear foci. The loss of foci formation activity of the mutated protein was not due to impaired ability to bind double-stranded DNA in an ATP-dependent way in vitro and to bind chromatin in vivo. These data suggest that the assembly of Rad51 into nuclear foci is assisted by association with the nuclear matrix, which may support the spatial organization of the process of repair by homologous recombination.