ABSTRACT
Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel ß-strands, offset by an increase in the helical component.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase , DNA Methylation , Humans , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Amino Acids , DNA , Ferrous CompoundsABSTRACT
Salivary gland tumours (SGTs) are a heterogeneous group of benign tumours of various origins and pathologies, showing a number of DNA modifications. Previously, in malignant head and neck cancer (HNSCC), we found overexpression of ALKBH proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate and Fe(II) dependent dioxygenase. Moreover, we proved the connection of some of these dioxygenases with cancer development. Here, we studied the expression of five of these ALKBH dioxygenases: 1, 3, 4, 5, and FTO in benign SGTs. Using Western blot analysis, we found overexpression of three proteins: ALKBH1, 4, and FTO in SGT as compared to the surrounding, unaffected tissue. ALKBH4 was overexpressed in 76% of patient samples, whereas ALKBH1 and FTO in 65% of the samples. These results differ from those obtained in HNSCC, where FTO overexpression has been observed in 90% of patient samples. We also investigated the relationships between ALKBHs' expression levels in normal and SGT tissues and identified two correlated pairs: ALKBH1-ALKBH3 and ALKBH1-ALKBH5. Additionally, in tumour tissue ALKBHs: ALKBH1, ALKBH3, ALKBH4, and ALKBH5 levels were correlated with each other. Together, these findings show that the ALKBH proteins exhibit pro cancerogenic action in SGT, even though the levels ALKBHs are generally lower in benign SGT than in malignant HNSCC. We suggest that the overexpression of the ALKBHs, especially FTO, may be used as a cancer marker and for its grading.
Subject(s)
Dioxygenases , Head and Neck Neoplasms , Salivary Gland Neoplasms , Humans , Dioxygenases/genetics , Dioxygenases/metabolism , Squamous Cell Carcinoma of Head and Neck , Salivary Gland Neoplasms/genetics , AlkB Homolog 1, Histone H2a Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolismABSTRACT
Long noncoding RNAs (lncRNAs) are confirmed as the key regulators of hepatocellular carcinoma (HCC) occurrence and progression, but the role of AlkB homologue 3 antisense RNA 1 (ALKBH3-AS1) in HCC is unclear. We revealed the overexpression of ALKBH3-AS1 in HCC tissues. The upregulated levels of ALKBH3-AS1 were observed in HCC cells. ALKBH3-AS1 was expressed in the nucleus and cytoplasm of HCC cells. The high ALKBH3-AS1 expression was markedly associated with a decreased survival rate of HCC patients. ALKBH3-AS1 knockdown repressed and ALKBH3-AS1 overexpression enhanced HCC cell invasion and proliferation. ALKBH3-AS1 silencing restricted HCC growth in vivo. A significant positive correlation between ALKBH3-AS1 and ALKBH3 mRNA levels was confirmed in HCC specimens. ALKBH3-AS1 silencing reduced ALKBH3 expression by stabilizing its mRNA stability in HCC cells. Notably, the impact of ALKBH3 silencing on HCC cells was similar to that of ALKBH3-AS1 knockdown. ALKBH3 restoration prominently attenuated the suppressive effects resulting from ALKBH3-AS1 silencing in HCCLM3 cells. Hypoxia-inducible factor-1α (HIF-1α) transcriptionally activated ALKBH3-AS1 expression in hypoxic HCC cells. ALKBH3-AS1 knockdown markedly attenuated cell proliferation and invasion in hypoxic Huh7 cells. Collectively, HIF-1α-activated ALKBH3-AS1 exerted an oncogenic role by enhancing ALKBH3 mRNA stability in HCC cells.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , MicroRNAs , RNA Stability , RNA, Long Noncoding , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Neoplasms/pathology , MicroRNAs/genetics , RNA, Antisense , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/geneticsABSTRACT
Studies on biological functions of RNA modifications such as N6-methyladenosine (m6A) in mRNA have sprung up in recent years, while the roles of N1-methyladenosine (m1A) in cancer progression remain largely unknown. We find m1A demethylase ALKBH3 can regulate the glycolysis of cancer cells via a demethylation activity dependent manner. Specifically, sequencing and functional studies confirm that ATP5D, one of the most important subunit of adenosine 5'-triphosphate synthase, is involved in m1A demethylase ALKBH3-regulated glycolysis of cancer cells. The m1A modified A71 at the exon 1 of ATP5D negatively regulates its translation elongation via increasing the binding with YTHDF1/eRF1 complex, which facilitates the release of message RNA (mRNA) from ribosome complex. m1A also regulates mRNA stability of E2F1, which directly binds with ATP5D promoter to initiate its transcription. Targeted specific demethylation of ATP5D m1A by dm1ACRISPR system can significantly increase the expression of ATP5D and glycolysis of cancer cells. In vivo data confirm the roles of m1A/ATP5D in tumor growth and cancer progression. Our study reveals a crosstalk of mRNA m1A modification and cell metabolism, which expands the understanding of such interplays that are essential for cancer therapeutic application.
Subject(s)
Glycolysis , Mitochondrial Proton-Translocating ATPases , Neoplasms , RNA, Messenger , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Glycolysis/genetics , Humans , Methylation , Mitochondrial Proton-Translocating ATPases/metabolism , Neoplasms/enzymology , Neoplasms/genetics , RNA, Messenger/metabolismABSTRACT
Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N2-methyl-dG (N2-MedG) and N2-n-butyl-dG (N2-nBudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N2-MedG and N2-nBudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N2-MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N2-MedG and N2-nBuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N2-alkyl-dG lesions in mammalian cells.
Subject(s)
Deoxyguanosine , Tandem Mass Spectrometry , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Animals , Chromatography, Liquid , DNA , DNA Damage , DNA Repair , DNA-Directed DNA Polymerase/metabolism , Deoxyguanosine/metabolism , Genomics , Humans , Mammals/genetics , Mammals/metabolismABSTRACT
Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase , DNA Repair , Nuclear Proteins , Polyubiquitin , Ubiquitin , Ubiquitins , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , DNA/metabolism , Models, Molecular , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polyubiquitin/genetics , Polyubiquitin/metabolism , Protein Binding , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitins/genetics , Ubiquitins/metabolismABSTRACT
Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Cell Nucleus/enzymology , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Neoplasms/enzymology , Nuclear Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA, Neoplasm/metabolism , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Cell Nucleus/genetics , DNA Helicases/genetics , DNA Methylation , DNA-Binding Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Methylation , Neoplasms/genetics , Nuclear Proteins/genetics , R-Loop Structures , RNA, Neoplasm/genetics , Spliceosomes/genetics , Spliceosomes/metabolism , Transcription, Genetic , UbiquitinationABSTRACT
BACKGROUND: tRNA-derived small RNAs (tDRs), which are widely distributed in human tissues including blood and urine, play an important role in the progression of cancer. However, the expression of tDRs in colorectal cancer (CRC) plasma and their potential diagnostic values have not been systematically explored. METHODS: The expression profiles of tDRs in plasma of CRC and health controls (HCs) are investigated by small RNA sequencing. The level and diagnostic value of 5'-tRF-GlyGCC are evaluated by quantitative PCR in plasma samples from 105 CRC patients and 90 HCs. The mechanisms responsible for biogenesis of 5'-tRF-GlyGCC are checked by in vitro and in vivo models. RESULTS: 5'-tRF-GlyGCC is dramatically increased in plasma of CRC patients compared to that of HCs. The area under curve (AUC) for 5'-tRF-GlyGCC in CRC group is 0.882. The combination of carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) with 5'-tRF-GlyGCC improves the AUC to 0.926. Consistently, the expression levels of 5'-tRF-GlyGCC in CRC cells and xenograft tissues are significantly greater than that in their corresponding controls. Blood cells co-cultured with CRC cells or mice xenografted with CRC tumors show increased levels of 5'-tRF-GlyGCC. In addition, we find that the increased expression of 5'-tRF-GlyGCC is dependent on the upregulation of AlkB homolog 3 (ALKBH3), a tRNA demethylase which can promote tRNA cleaving to generate tDRs. CONCLUSIONS: The level of 5'-tRF-GlyGCC in plasma is a promising diagnostic biomarker for CRC diagnosis.
Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , RNA, Transfer/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/blood , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Animals , Biomarkers, Tumor/blood , Cell Line, Tumor , Colorectal Neoplasms/blood , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Male , Mice, Inbred BALB C , RNA, Transfer/blood , RNA, Transfer/metabolism , Xenograft Model Antitumor AssaysSubject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/deficiency , Collagen Type I/biosynthesis , DNA Methylation , Hodgkin Disease/enzymology , Neoplasm Proteins/metabolism , RNA Interference , RNA Processing, Post-Transcriptional , RNA, Neoplasm/metabolism , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Base Sequence , Cell Line, Tumor , Collagen Type I/genetics , Collagen Type I, alpha 1 Chain , CpG Islands/genetics , DNA Methylation/drug effects , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Datasets as Topic , Decitabine/pharmacology , Hodgkin Disease/genetics , Hodgkin Disease/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Lymphocytes/metabolism , Methylation/drug effects , Neoplasm Proteins/genetics , Promoter Regions, Genetic/genetics , RNA Processing, Post-Transcriptional/drug effects , Sequence Alignment , tRNA Methyltransferases/metabolismABSTRACT
AlkB is a bacterial Fe(II)- and 2-oxoglutarate-dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid-alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N6-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.
Subject(s)
AlkB Enzymes/chemistry , DNA Repair , DNA, Bacterial/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , AlkB Enzymes/genetics , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/chemistry , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/chemistry , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , DNA/chemistry , DNA/genetics , DNA, Bacterial/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , HumansABSTRACT
The nine identified human homologues of E. coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Anthraquinones/pharmacology , Biomarkers, Tumor/metabolism , Head and Neck Neoplasms/metabolism , Squamous Cell Carcinoma of Head and Neck/metabolism , Aged , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/antagonists & inhibitors , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Biomarkers, Tumor/genetics , Female , Follow-Up Studies , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/pathology , Humans , Ketoglutaric Acids/metabolism , Male , Middle Aged , Prognosis , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/pathology , Substrate Specificity , Tumor Cells, CulturedABSTRACT
5-Methylcytosine (5mC) in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It is oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases. In this work, we demonstrate that the epigenetic marker 5mC is modified to 5hmC, 5fC, and 5caC in vitro by another class of α-KG/Fe(II)-dependent proteins-the DNA repair enzymes in the AlkB family, which include ALKBH2, ALKBH3 in huamn and AlkB in Escherichia coli. Theoretical calculations indicate that these enzymes may bind 5mC in the syn-conformation, placing the methyl group comparable to 3-methylcytosine, the prototypic substrate of AlkB. This is the first demonstration of the AlkB proteins to oxidize a methyl group attached to carbon, instead of nitrogen, on a DNA base. These observations suggest a broader role in epigenetics for these DNA repair proteins.
Subject(s)
5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , AlkB Enzymes/metabolism , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Cytosine/analogs & derivatives , AlkB Enzymes/genetics , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Animals , Computational Biology , CpG Islands , Cytosine/metabolism , DNA/genetics , DNA Methylation , Epigenesis, Genetic , Humans , Molecular Structure , Oxidation-ReductionABSTRACT
Transfer RNA is heavily modified and plays a central role in protein synthesis and cellular functions. Here we demonstrate that ALKBH3 is a 1-methyladenosine (m1A) and 3-methylcytidine (m3C) demethylase of tRNA. ALKBH3 can promote cancer cell proliferation, migration and invasion. In vivo study confirms the regulation effects of ALKBH3 on growth of tumor xenograft. The m1A demethylated tRNA is more sensitive to angiogenin (ANG) cleavage, followed by generating tRNA-derived small RNAs (tDRs) around the anticodon regions. tDRs are conserved among species, which strengthen the ribosome assembly and prevent apoptosis triggered by cytochrome c (Cyt c). Our discovery opens a potential and novel paradigm of tRNA demethylase, which regulates biological functions via generation of tDRs.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Cell Proliferation/genetics , Neoplasms/genetics , RNA, Transfer/genetics , Adenosine/analogs & derivatives , Adenosine/genetics , Animals , Apoptosis/genetics , Cell Movement/genetics , Cytidine/analogs & derivatives , Cytidine/genetics , Disease Progression , HeLa Cells , Humans , Mice , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasms/enzymology , Neoplasms/pathology , Ribonuclease, Pancreatic/genetics , Xenograft Model Antitumor AssaysABSTRACT
Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self-assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic "thermal tag" with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes: FTO (7; IC50 =2.6â µm) and ALKBH3 (8; IC50 =3.7â µm). To date, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/antagonists & inhibitors , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/antagonists & inhibitors , Combinatorial Chemistry Techniques/methods , Enzyme Inhibitors/chemistry , Oxidoreductases, O-Demethylating/antagonists & inhibitors , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/chemistry , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 5, RNA Demethylase/antagonists & inhibitors , AlkB Homolog 5, RNA Demethylase/chemistry , AlkB Homolog 5, RNA Demethylase/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/chemistry , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Catalysis , Fluorometry/methods , Humans , Hydrazones/chemistry , Kinetics , Ligands , Molecular Structure , Oxidoreductases, O-Demethylating/chemistry , Oxidoreductases, O-Demethylating/genetics , Peptides/chemistry , Peptides/genetics , Protein Denaturation , Protein Engineering , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Transition TemperatureABSTRACT
Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.
Subject(s)
AlkB Enzymes/antagonists & inhibitors , Antineoplastic Agents, Alkylating/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Enzyme Inhibitors/therapeutic use , Glutaminase/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/drug therapy , AlkB Enzymes/genetics , AlkB Enzymes/metabolism , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/antagonists & inhibitors , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Alkylation/drug effects , Animals , Antineoplastic Agents, Alkylating/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Damage , Enzyme Inhibitors/pharmacology , Glutaminase/metabolism , Humans , Male , Mice , Mice, Nude , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Neoplasms/pathology , RNA Interference , Random Allocation , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: DNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer. METHODS: Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation. RESULTS: The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20% CpG methylation was found to be statistically significantly associated with reduced survival (HR = 2.3; P = 0.012). By thresholding at the clinically relevant CpG methylation level (>20%), we find the incidence of ALKBH3 promoter methylation to be 5% (13 out of 265). CONCLUSIONS: ALKBH3 is a novel addition to the catalogue of DNA repair genes found inactivated in breast cancer. Our results underscore a link between defective alkylation repair and breast cancer which, additionally, is found in association with poor disease outcome.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Breast Neoplasms/genetics , CpG Islands , DNA Methylation , DNA Repair , Promoter Regions, Genetic , Adult , Aged , Aged, 80 and over , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Alkylation , Biomarkers, Tumor , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cohort Studies , Computational Biology/methods , DNA Damage , Epigenesis, Genetic , Epithelial Cells/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Middle Aged , Neoplasm Grading , Neoplasm Staging , Prognosis , Tissue Array AnalysisABSTRACT
Human AlkB homolog 3 (ALKBH3) is overexpressed in non-small cell lung cancers (NSCLC) and its high expression is significantly correlated with poor prognosis. While ALKBH3 knockdown induces apoptosis in NSCLC cells, the underlying anti-apoptotic mechanisms of ALKBH3 in NSCLC cells remain unclear. Here we show that ALKBH3 knockdown induces cell cycle arrest or apoptosis depending on the TP53 gene status in NSCLC cells. In comparison to parental cells, TP53-knockout A549 cells showed DNA damage-responsive signal induced by ALKBH3 knockdown. TP53 knockout shifted the phenotypes of A549 cells induced by ALKBH3 knockdown from cell cycle arrest to apoptosis induction, suggesting that the TP53 gene status is a critical determinant of the phenotypes induced by ALKBH3 knockdown in NSCLC cells.
Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/deficiency , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Tumor Suppressor Protein p53/genetics , Apoptosis/genetics , Cell Cycle Checkpoints/genetics , Cell Proliferation/genetics , DNA Damage , Humans , Phenotype , Tumor Cells, CulturedABSTRACT
Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (d- and l-2HG) have been found at abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC50 values of d-2HG on the enzymes, and the results have not been correlated to physiologically relevant concentrations of 2HG and αKG in cancer cells. In this work, we performed detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3, and the bacterial AlkB in the presence of d- and l-2HG in both double- and single-stranded DNA contexts. We determined the kinetic parameters of inhibition, including kcat, KM, and Ki. We also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both d- and l-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 at pathologically relevant concentrations (73-88% for d-2HG and 31-58% for l-2HG inhibition). This work provides a new perspective that the elevation of the d- or l-2HG concentration in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair performed by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.
Subject(s)
AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Glutarates/metabolism , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/antagonists & inhibitors , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/antagonists & inhibitors , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , Base Sequence , Chromatography, High Pressure Liquid , DNA Repair , Enzyme Assays , Glutarates/analysis , Glutarates/chemistry , Humans , Inhibitory Concentration 50 , Ketoglutaric Acids/analysis , Ketoglutaric Acids/chemistry , Ketoglutaric Acids/metabolism , Kinetics , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , StereoisomerismABSTRACT
The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.