The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue could be used to enhance the efficacy of cancer therapy without increasing its toxicity. Specific targeting of cancer cells can be... more
The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue could be used to enhance the efficacy of cancer therapy without increasing its toxicity. Specific targeting of cancer cells can be achieved through the use of pH-low insertion peptides (pHLIP), which take advantage of the acidity of the tumor microenvironment to deliver cargoes selectively to tumor cells. We developed a pHLIP–peptide nucleic acid (PNA) conjugate as an antisense reagent to reduce expression of the otherwise undruggable DNA double-strand break repair factor, KU80, and thereby radiosensitize tumor cells. Increased antisense activity of the pHLIP–PNA conjugate was achieved by partial mini-PEG sidechain substitution of the PNA at the gamma position, designated pHLIP-αKu80(γ). We evaluated selective effects of pHLIP-αKu80(γ) in cancer cells in acidic culture conditions as well as in two subcutaneous mouse tumor models. Fluorescently labeled pHLIP-αKu80(γ) delivers spec...
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The delivery of cancer therapeutics can be limited by pharmacological issues such as poor bioavailability and high toxicity to healthy tissue. pH-low insertion peptides (pHLIPs) represent a promising tool to overcome these limitations.... more
The delivery of cancer therapeutics can be limited by pharmacological issues such as poor bioavailability and high toxicity to healthy tissue. pH-low insertion peptides (pHLIPs) represent a promising tool to overcome these limitations. pHLIPs allow for the selective delivery of agents to tumors on the basis of pH, taking advantage of the acidity of the hypoxic tumor microenvironment. This review article highlights the various applications in which pHLIPs have been utilized for targeting and treating diseases in hypoxic environments, including delivery of small molecule inhibitors, toxins, nucleic acid analogs, fluorescent dyes, and nanoparticles.
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DNA double-strand breaks (DSB) are the most cytotoxic DNA lesions, and up to 90% of DSBs require repair by nonhomologous end joining (NHEJ). Functional and genomic analyses of patient-derived melanomas revealed that PTEN loss is... more
DNA double-strand breaks (DSB) are the most cytotoxic DNA lesions, and up to 90% of DSBs require repair by nonhomologous end joining (NHEJ). Functional and genomic analyses of patient-derived melanomas revealed that PTEN loss is associated with NHEJ deficiency. In PTEN-null melanomas, PTEN complementation rescued the NHEJ defect; conversely, suppression of PTEN compromised NHEJ. Mechanistic studies revealed that PTEN promotes NHEJ through direct induction of expression of XRCC4-like factor (/XLF), which functions in DNA end bridging and ligation. PTEN was found to occupy the gene promoter and to recruit the histone acetyltransferases, PCAF and CBP, inducing XLF expression. This recruitment activity was found to be independent of its phosphatase activity, but dependent on K128, a site of regulatory acetylation on PTEN. These findings define a novel function for PTEN in regulating NHEJ DSB repair, and therefore may assist in the design of individualized strategies for cancer therapy. ...
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The development of small-molecule tyrosine kinase inhibitors (TKI) specific for epidermal growth factor receptors (EGFR) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients.... more
The development of small-molecule tyrosine kinase inhibitors (TKI) specific for epidermal growth factor receptors (EGFR) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key microenvironmental stress in solid tumors that is associated with poor prognosis due, in part, to acquired resistance to conventional therapy. This study documents that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line HCC827, which harbors an activating EGFR mutation. Following hypoxic growth conditions, HCC827 cells treated with gefitinib upregulated N-cadherin, fibronectin, and vimentin expression and downregulated E-cadherin, characteristic of an epithelial-mesenchymal transition (EMT), which prior studies have linked to EGFR TKI resistance. Mechanistically, knockdown of the histone demethylases, LSD1 and PLU-1, prevented and r...
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Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells. Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links. Recent work... more
Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells. Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links. Recent work has suggested that site-specific mutagenesis and recombination might be achieved through the use of targeted psoralen adducts. The fate of plasmids containing psoralen adducts was evaluated in Xenopus oocytes, an experimental system that has well-characterized recombination capabilities and advantages in the analysis of intermediates in DNA metabolism. Psoralen adducts were delivered to a specific site by a triplex-forming oligonucleotide. These lesions are clearly recognized and processed in oocytes, since mutagenesis was observed at the target site. The spectrum of induced mutations was compared with that found in similar studies in mammalian cells. Plasmids carrying multiple random adducts were preferentially degraded, perhaps due to the introduct...
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Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected... more
Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy.
Research Interests: Cellular Biology, Biology, DNA damage, Medicine, Mammals, and 15 moreBiological Sciences, Cell line, Mutation, Triple Helix, Animals, Gene, Gene Conversion, Gene Targeting, Genetic Recombination, Homologous Recombination, Nucleic Acid Conformation, Oligonucleotides, Oligonucleotide, Reporter gene, and Medical and Health Sciences
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2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is... more
2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically releva...
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Radiation therapy and DNA-damaging chemotherapy are frequently utilized in the treatment of solid tumors. Innate or acquired resistance to these therapies remains a major clinical challenge in oncology. The development of small molecules... more
Radiation therapy and DNA-damaging chemotherapy are frequently utilized in the treatment of solid tumors. Innate or acquired resistance to these therapies remains a major clinical challenge in oncology. The development of small molecules that sensitize cancers to established therapies represents an attractive approach to extending survival and quality of life in patients. Here, we demonstrate that YU238259, a member of a novel class of DNA double-strand break repair inhibitors, exhibits potent synthetic lethality in the setting of DNA damage response and DNA repair defects. YU238259 specifically inhibits homology-dependent DNA repair (HDR), but not non-homologous end-joining (NHEJ), in cell-based GFP reporter assays. Treatment with YU238259 is not only synergistic with ionizing radiation (IR), etoposide, and PARP inhibition, but this synergism is heightened by BRCA2-deficiency. Further, growth of BRCA2-deficient human tumor xenografts in nude mice is significantly delayed by YU23825...
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Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind... more
Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind with high affinity to single-stranded DNA via Watson–Crick base pairing and can form triple helices via Hoogsteen binding to DNA/PNA duplexes. Dimeric bis-PNAs capable of both strand invasion and triplex formation can form clamp structures on target DNAs. As a strategy to promote site-directed recombination, a bis-PNA was coupled to a 40-nt donor DNA fragment homologous to an adjacent region in the target gene. The PNA–DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency at least 60-fold above background. Induced site-specific recombination was also seen when the bis-PNA and the donor DNA were co-mixed without covalent...
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Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the... more
Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induc...
Research Interests: Biology, DNA damage, DNA repair, Medicine, Multidisciplinary, and 15 moreDNA, Humans, Triple Helix, Nucleotide Excision Repair, DNA Structure, Introns, Genetic Recombination, Base Sequence, Protein Binding, DNA binding proteins, Enzyme Linked Immunosorbent Assay, Xeroderma Pigmentosum, Molecular Sequence Data, binding sites, and Ultraviolet Rays
Research Interests: Molecular Biology, Biology, Confocal Microscopy, Medicine, Gene expression, and 15 moreBiological Sciences, DNA, Cell line, Environmental Sciences, Mutagenesis, Mutation, Mice, Animals, Nucleic Acids, DNA binding, Gene Targeting, Biological activity, Chromosomes, Oligonucleotides, and Oligonucleotide
Research Interests: Molecular Biology, Biology, Medicine, Biological Sciences, DNA, and 15 moreCercopithecus aethiops, Cell line, Environmental Sciences, Mutagenesis, Mice, Animals, Peptide Nucleic Acids, Nucleic Acids, Plasmids, DNA binding, Gene Targeting, Base Sequence, Binding Site, Oligonucleotide, and DNA sequence
TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of... more
TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-c...
Research Interests: Molecular Biology, Biology, Cytotoxicity, DNA damage, Medicine, and 15 moreProdrugs, Humans, Animals, Phosphorylation, Comet Assay, Histones, Oxygen, Neoplasms, Molecular Cancer Therapeutics, Cell Survival, Minimum inhibitory concentration, Molecular Structure, CHO cells, Cricetinae, and Pharmacology and pharmaceutical sciences
Research Interests: Biology, Biological Chemistry, Medicine, Biological Sciences, DNA, and 15 moreMutagenesis, Magnesium, Mice, Animals, Cationic polymerization, Biological, Genome, CHEMICAL SCIENCES, Base Sequence, Cations, Chromosomes, Nucleic Acid Conformation, Molecular Sequence Data, DNA mutational analysis, and Medical and Health Sciences
Research Interests: Biology, DNA replication, Biological Chemistry, DNA repair, Medicine, and 15 moreBiological Sciences, Cercopithecus aethiops, Cell line, Humans, Mutagenesis, Animals, Light, CHEMICAL SCIENCES, HeLa cells, Base Sequence, Nucleic Acid Conformation, Oligonucleotide, Molecular Sequence Data, Medical and Health Sciences, and In Vitro Techniques
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Research Interests: Molecular Biology, Biology, Gene Therapy, Nanoparticles, Medicine, and 15 moreBiological Sciences, DNA, Hematopoietic Stem Cells, Cell line, Humans, Genetic Enhancement, Mice, Animals, Gene transfer techniques, Peptide Nucleic Acids, Gene Delivery, In Vivo, Gene Targeting, Genetic Therapy, and Medical and Health Sciences
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<b>Copyright information:</b>Taken from "Triplex-induced recombination and repair in the pyrimidine motif"Nucleic Acids Research 2005;33(11):3492-3502.Published online 16 Jun 2005PMCID:PMC1151591.© The Author 2005.... more
<b>Copyright information:</b>Taken from "Triplex-induced recombination and repair in the pyrimidine motif"Nucleic Acids Research 2005;33(11):3492-3502.Published online 16 Jun 2005PMCID:PMC1151591.© The Author 2005. Published by Oxford University Press. All rights reserved The first lane in each panel contains duplex alone (0 concentration of TFO), the second lane contains 10 M oligonucleotide, and each subsequent column contains a 10-fold dilution. Third-strand binding conditions: () pH 5.4, 10 mM MgCl and 0 mM KCl; () pH 7.2, 10 mM MgCl and 0 mM KCl; () pH 7.2, 0.1 mM MgCl and 140 mM KCl. The bands of reduced mobility relative to the duplex alone represent triplex formation.
Hypoxia induces genomic instability through replication stress and dysregulation of vital DNA repair pathways. The Fanconi anemia (FA) proteins, FANCD2 and FANCI, are key members of a DNA repair pathway that responds to replicative... more
Hypoxia induces genomic instability through replication stress and dysregulation of vital DNA repair pathways. The Fanconi anemia (FA) proteins, FANCD2 and FANCI, are key members of a DNA repair pathway that responds to replicative stress, suggesting that they undergo regulation by hypoxic conditions. Here acute hypoxic stress activates the FA pathway via ubiquitination of FANCD2 and FANCI in an ATR-dependent manner. In addition, the presence of an intact FA pathway is required for preventing hypoxia-induced DNA damage measurable by the comet assay, limiting the accumulation of gH2AX (a marker of DNA damage or stalled replication), and protecting cells from hypoxia-induced apoptosis. Furthermore, prolonged hypoxia induces transcriptional repression of FANCD2 in a manner analogous to the hypoxic downregulation of BRCA1 and RAD51. Thus, hypoxia-induced FA pathway activation plays a key role in maintaining genome integrity and cell survival, while FA protein downregulation with prolong...
Conflict of Interest Statement: None of the authors have any professional or financial affiliations relevant to this work that could be perceived as biasing the presentation.
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b-globin mutation induced by pseudocomplementary peptide nucleic acids
Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity... more
Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of β-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.
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Hypoxia is a hallmark of the tumour microenvironment with profound effects on tumour biology, influencing cancer progression, the development of metastasis and patient outcome. Hypoxia also contributes to genomic instability and mutation... more
Hypoxia is a hallmark of the tumour microenvironment with profound effects on tumour biology, influencing cancer progression, the development of metastasis and patient outcome. Hypoxia also contributes to genomic instability and mutation frequency by inhibiting DNA repair pathways. This review summarises the diverse mechanisms by which hypoxia affects DNA repair, including suppression of homology-directed repair, mismatch repair and base excision repair. We also discuss the effects of hypoxia mimetics and agents that induce hypoxia on DNA repair, and we highlight areas of potential clinical relevance as well as future directions.
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Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could... more
Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the β-globin gene in a mouse model of human β-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may pro...
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Since their invention in 1991, peptide nucleic acids (PNAs) have been used in a myriad of chemical and biological assays. More recently, peptide nucleic acids have also been demonstrated to hold great potential as therapeutic agents... more
Since their invention in 1991, peptide nucleic acids (PNAs) have been used in a myriad of chemical and biological assays. More recently, peptide nucleic acids have also been demonstrated to hold great potential as therapeutic agents because of their physiological stability, affinity for target nucleic acids, and versatility. While recent modifications in their design have further improved their potency, their preclinical development has reached new heights due to their combination with recent advancements in drug delivery. This review focuses on recent advances in PNA therapeutic applications, in which chemical modifications are made to improve PNA function and nanoparticles are used to enhance PNA delivery.
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The von Hippel-Lindau () tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of loss is currently best understood to occur through stabilization of the hypoxia-inducible... more
The von Hippel-Lindau () tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of loss is currently best understood to occur through stabilization of the hypoxia-inducible factors, activation of hypoxia-induced signaling pathways, and transcriptional reprogramming towards a pro-angiogenic and pro-growth state. However, hypoxia also drives other pro-tumorigenic processes, including the development of genomic instability via down-regulation of DNA repair gene expression. Here, we find that DNA repair genes involved in double-strand break repair by homologous recombination (HR) and in mismatch repair, which are down-regulated by hypoxic stress, are decreased in -deficient renal cancer cells relative to wild type -complemented cells. Functionally, this gene repression is associated with impaired DNA double-strand break repair in -deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand br...
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Peptide nucleic acids (PNAs) can bind duplex DNA in a sequence-targeted manner, forming a triplex structure capable of inducing DNA repair and producing specific genome modifications. Since the first description of PNA-mediated gene... more
Peptide nucleic acids (PNAs) can bind duplex DNA in a sequence-targeted manner, forming a triplex structure capable of inducing DNA repair and producing specific genome modifications. Since the first description of PNA-mediated gene editing in cell free extracts, PNAs have been used to successfully correct human disease-causing mutations in cell culture and in vivo in preclinical mouse models. Gene correction via PNAs has resulted in clinically-relevant functional protein restoration and disease improvement, with low off-target genome effects, indicating a strong therapeutic potential for PNAs in the treatment or cure of genetic disorders. This review discusses the progress that has been made in developing PNAs as an effective, targeted agent for gene editing, with an emphasis on recent in vivo, nanoparticle-based strategies.
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miR-155 is an oncogenic miRNA that is often overexpressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors, including RAD51, MLH1, and MSH6, and its overexpression results in an increased... more
miR-155 is an oncogenic miRNA that is often overexpressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors, including RAD51, MLH1, and MSH6, and its overexpression results in an increased mutation frequency in vitro, although the mechanism has yet to be fully understood. Here, we demonstrate that overexpression of miR-155 drives an increased mutation frequency both in vitro and in vivo, promoting genomic instability by affecting multiple DNA repair pathways. miR-155 overexpression causes a decrease in homologous recombination, but yields a concurrent increase in the error-prone nonhomologous end-joining pathway. Despite repressing established targets MLH1 and MSH6, the identified mutation pattern upon miR-155 overexpression does not resemble that of a mismatch repair–deficient background. Further investigation revealed that all four subunits of polymerase delta, a high-fidelity DNA replication, and repair polymerase are downregulated at ...
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The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming... more
The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% β-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and sim...
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Silencing of MLH1 is frequently seen in sporadic colorectal cancers. We show here that hypoxia causes decreased histone H3 lysine 4 (H3K4) methylation at the MLH1 promoter via the action of the H3K4 demethylases LSD1 and PLU-1 and... more
Silencing of MLH1 is frequently seen in sporadic colorectal cancers. We show here that hypoxia causes decreased histone H3 lysine 4 (H3K4) methylation at the MLH1 promoter via the action of the H3K4 demethylases LSD1 and PLU-1 and promotes durable long-term silencing in a pathway that requires LSD1. Knockdown of LSD1 or its corepressor, CoREST, also prevents the resilencing (and associated cytosine DNA methylation) of the endogenous MLH1 promoter in RKO colon cancer cells following transient reactivation by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The results demonstrate that hypoxia is a driving force for silencing of MLH1 and that the LSD1/CoREST complex is necessary for this process. The results reveal a mechanism by which hypoxia promotes cancer cell evolution to drive malignant progression through epigenetic modulation. Our findings suggest that LSD1/CoREST acts as a colon cancer oncogene by epigenetically silencing MLH1 and also...