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1.
NAR Cancer ; 5(1): zcad003, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36755959

ABSTRACT

The DNA-dependent protein kinase (DNA-PK) plays a critical role in the DNA damage response (DDR) and non-homologous end joining (NHEJ) double-strand break (DSB) repair pathways. Consequently, DNA-PK is a validated therapeutic target for cancer treatment in certain DNA repair-deficient cancers and in combination with ionizing radiation (IR). We have previously reported the discovery and development of a novel class of DNA-PK inhibitors with a unique mechanism of action, blocking the Ku 70/80 heterodimer interaction with DNA. These Ku-DNA binding inhibitors (Ku-DBi's) display nanomolar activity in vitro, inhibit cellular DNA-PK, NHEJ-catalyzed DSB repair and sensitize non-small cell lung cancer (NSCLC) cells to DSB-inducing agents. In this study, we demonstrate that chemical inhibition of the Ku-DNA interaction potentiates the cellular effects of bleomycin and IR via p53 phosphorylation through the activation of the ATM pathway. This response is concomitant with a reduction of DNA-PK catalytic subunit (DNA-PKcs) autophosphorylation at S2056 and a time-dependent increase in H2AX phosphorylation at S139. These results are consistent with Ku-DBi's abrogating DNA-PKcs autophosphorylation to impact DSB repair and DDR signaling through a novel mechanism of action, and thus represent a promising anticancer therapeutic strategy in combination with DNA DSB-inducing agents.

2.
Front Oncol ; 12: 850883, 2022.
Article in English | MEDLINE | ID: mdl-35463312

ABSTRACT

The vast majority of cancer patients receive DNA-damaging drugs or ionizing radiation (IR) during their course of treatment, yet the efficacy of these therapies is tempered by DNA repair and DNA damage response (DDR) pathways. Aberrations in DNA repair and the DDR are observed in many cancer subtypes and can promote de novo carcinogenesis, genomic instability, and ensuing resistance to current cancer therapy. Additionally, stalled or collapsed DNA replication forks present a unique challenge to the double-strand DNA break (DSB) repair system. Of the various inducible DNA lesions, DSBs are the most lethal and thus desirable in the setting of cancer treatment. In mammalian cells, DSBs are typically repaired by the error prone non-homologous end joining pathway (NHEJ) or the high-fidelity homology directed repair (HDR) pathway. Targeting DSB repair pathways using small molecular inhibitors offers a promising mechanism to synergize DNA-damaging drugs and IR while selective inhibition of the NHEJ pathway can induce synthetic lethality in HDR-deficient cancer subtypes. Selective inhibitors of the NHEJ pathway and alternative DSB-repair pathways may also see future use in precision genome editing to direct repair of resulting DSBs created by the HDR pathway. In this review, we highlight the recent advances in the development of inhibitors of the non-phosphatidylinositol 3-kinase-related kinases (non-PIKKs) members of the NHEJ, HDR and minor backup SSA and alt-NHEJ DSB-repair pathways. The inhibitors described within this review target the non-PIKKs mediators of DSB repair including Ku70/80, Artemis, DNA Ligase IV, XRCC4, MRN complex, RPA, RAD51, RAD52, ERCC1-XPF, helicases, and DNA polymerase θ. While the DDR PIKKs remain intensely pursued as therapeutic targets, small molecule inhibition of non-PIKKs represents an emerging opportunity in drug discovery that offers considerable potential to impact cancer treatment.

3.
Front Oncol ; 12: 826655, 2022.
Article in English | MEDLINE | ID: mdl-35251993

ABSTRACT

Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination, and the DNA damage response (DDR). Retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. Similarly, relative expression of RPA is a predictive marker for response to chemotherapy. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure. We have developed second-generation RPA inhibitors (RPAis) that block the RPA-DNA interaction and optimized formulation for in vivo analyses. Data demonstrate that unlike first-generation RPAis, second-generation molecules show increased cellular permeability and induce cell death via apoptosis. Second-generation RPAis elicit single-agent in vitro anticancer activity across a broad spectrum of cancers, and the cellular response suggests existence of a threshold before chemical RPA exhaustion induces cell death. Chemical RPA inhibition potentiates the anticancer activity of a series of DDR inhibitors and traditional DNA-damaging cancer therapeutics. Consistent with chemical RPA exhaustion, we demonstrate that the effects of RPAi on replication fork dynamics are similar to other known DDR inhibitors. An optimized formulation of RPAi NERx 329 was developed that resulted in single-agent anticancer activity in two non-small cell lung cancer models. These data demonstrate a unique mechanism of action of RPAis eliciting a state of chemical RPA exhaustion and suggest they will provide an effective therapeutic option for difficult-to-treat lung cancers.

5.
Mol Cell ; 81(15): 3128-3144.e7, 2021 08 05.
Article in English | MEDLINE | ID: mdl-34216544

ABSTRACT

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.


Subject(s)
BRCA1 Protein/genetics , DNA Replication/drug effects , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Cell Line , Cisplatin/pharmacology , DNA/genetics , DNA/metabolism , DNA, Single-Stranded/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Fanconi Anemia Complementation Group Proteins/genetics , Homologous Recombination/drug effects , Humans , Mice, Inbred NOD , RNA Helicases/genetics , Rad51 Recombinase/genetics , Replication Protein A/genetics , Tumor Suppressor p53-Binding Protein 1/genetics
6.
Cancers (Basel) ; 13(13)2021 Jul 03.
Article in English | MEDLINE | ID: mdl-34283091

ABSTRACT

Genome stability and maintenance pathways along with their requisite proteins are critical for the accurate duplication of genetic material, mutation avoidance, and suppression of human diseases including cancer. Many of these proteins participate in these pathways by binding directly to DNA, and a subset employ oligonucleotide/oligosaccharide binding folds (OB-fold) to facilitate the protein-DNA interactions. OB-fold motifs allow for sequence independent binding to single-stranded DNA (ssDNA) and can serve to position specific proteins at specific DNA structures and then, via protein-protein interaction motifs, assemble the machinery to catalyze the replication, repair, or recombination of DNA. This review provides an overview of the OB-fold structural organization of some of the most relevant OB-fold containing proteins for oncology and drug discovery. We discuss their individual roles in DNA metabolism, progress toward drugging these motifs and their utility as potential cancer therapeutics. While protein-DNA interactions were initially thought to be undruggable, recent reports of success with molecules targeting OB-fold containing proteins suggest otherwise. The potential for the development of agents targeting OB-folds is in its infancy, but if successful, would expand the opportunities to impinge on genome stability and maintenance pathways for more effective cancer treatment.

7.
Nucleic Acids Res ; 48(20): 11536-11550, 2020 11 18.
Article in English | MEDLINE | ID: mdl-33119767

ABSTRACT

DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi's) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi's directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi's cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi's. The utility of Ku-DBi's was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi's, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.


Subject(s)
DNA End-Joining Repair/drug effects , DNA-Activated Protein Kinase/antagonists & inhibitors , Ku Autoantigen/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Cells, Cultured , DNA/chemistry , DNA Breaks, Double-Stranded , Gene Editing , Humans , Ku Autoantigen/chemistry , Mice , Protein Kinase Inhibitors/chemistry
8.
Mol Cancer Res ; 18(11): 1699-1710, 2020 11.
Article in English | MEDLINE | ID: mdl-32801161

ABSTRACT

Platinum resistance is a common occurrence in high-grade serous ovarian cancer and a major cause of ovarian cancer deaths. Platinum agents form DNA cross-links, which activate nucleotide excision repair (NER), Fanconi anemia, and homologous recombination repair (HRR) pathways. Chromatin modifications occur in the vicinity of DNA damage and play an integral role in the DNA damage response (DDR). Chromatin modifiers, including polycomb repressive complex 1 (PRC1) members, and chromatin structure are frequently dysregulated in ovarian cancer and can potentially contribute to platinum resistance. However, the role of chromatin modifiers in the repair of platinum DNA damage in ovarian cancer is not well understood. We demonstrate that the PRC1 complex member RING1A mediates monoubiquitination of lysine 119 of phosphorylated H2AX (γH2AXub1) at sites of platinum DNA damage in ovarian cancer cells. After platinum treatment, our results reveal that NER and HRR both contribute to RING1A localization and γH2AX monoubiquitination. Importantly, replication protein A, involved in both NER and HRR, mediates RING1A localization to sites of damage. Furthermore, RING1A deficiency impairs the activation of the G2-M DNA damage checkpoint, reduces the ability of ovarian cancer cells to repair platinum DNA damage, and increases sensitivity to platinum. IMPLICATIONS: Elucidating the role of RING1A in the DDR to platinum agents will allow for the identification of therapeutic targets to improve the response of ovarian cancer to standard chemotherapy regimens.


Subject(s)
Histones/metabolism , Ovarian Neoplasms/genetics , Platinum/therapeutic use , Polycomb Repressive Complex 1/metabolism , Animals , Female , Humans , Ovarian Neoplasms/pathology , Phosphorylation , Ubiquitination
9.
ACS Med Chem Lett ; 11(6): 1118-1124, 2020 Jun 11.
Article in English | MEDLINE | ID: mdl-32550990

ABSTRACT

Replication protein A (RPA) is the major human single stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and DNA-damage response (DDR). Inhibition of RPA-DNA interactions represents a therapeutic strategy for cancer drug discovery and has great potential to provide single agent anticancer activity and to synergize with both common DNA damaging chemotherapeutics and newer targeted anticancer agents. In this letter, a new series of analogues based on our previously reported TDRL-551 (4) compound were designed to improve potency and physicochemical properties. Molecular docking studies guided molecular insights, and further SAR exploration led to the identification of a series of novel compounds with low micromolar RPA inhibitory activity, increased solubility, and excellent cellular up-take. Among a series of analogues, compounds 43, 44, 45, and 46 hold promise for further development of novel anticancer agents.

10.
Methods Mol Biol ; 1999: 217-221, 2019.
Article in English | MEDLINE | ID: mdl-31127579

ABSTRACT

With the recent interest in targeting the DNA damage response (DDR) and DNA repair, new screening methodologies are needed to broaden the scope of targetable proteins beyond kinases and traditional enzymes. Many of the proteins involved in the DDR and repair impart their activity by making specific contacts with DNA. These protein-nucleic acid interactions represent a tractable target for perturbation with small molecules. We describe a high throughput, solution-based equilibrium binding fluorescence polarization assay that can be applied to a wide array of protein-nucleic acid interactions. The assay is sensitive, stable, and able to identify small molecules capable of blocking DNA-protein interactions.


Subject(s)
DNA Repair/drug effects , High-Throughput Screening Assays/methods , Replication Protein A/antagonists & inhibitors , Xeroderma Pigmentosum Group A Protein/antagonists & inhibitors , DNA/genetics , DNA/metabolism , DNA Damage , Fluorescence Polarization/methods , Protein Binding/drug effects , Protein Binding/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Replication Protein A/genetics , Replication Protein A/metabolism , Xeroderma Pigmentosum Group A Protein/genetics , Xeroderma Pigmentosum Group A Protein/metabolism
11.
ACS Chem Biol ; 13(2): 389-396, 2018 02 16.
Article in English | MEDLINE | ID: mdl-29210569

ABSTRACT

Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either nonhomologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site. However, genetic recombination in mammalian systems through the HDR pathway is an inefficient process and requires cumbersome laboratory methods to identify the desired accurate insertion events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repairs the majority of nuclease induced DNA DSBs and also is responsible for mutagenic insertion and deletion events at off-target locations throughout the genome. Various methodologies have been developed to increase the efficiency of designer nuclease-based HDR mediated gene editing. Here, we review these advances toward modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering.


Subject(s)
CRISPR-Cas Systems/genetics , DNA End-Joining Repair/genetics , Genome/genetics , Recombinational DNA Repair/drug effects , Recombinational DNA Repair/genetics , Animals , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , DNA/genetics , DNA Breaks, Double-Stranded , DNA End-Joining Repair/drug effects , Endonucleases/metabolism , Gene Editing , Humans
12.
J Med Chem ; 60(19): 8055-8070, 2017 10 12.
Article in English | MEDLINE | ID: mdl-28933851

ABSTRACT

XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA-DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC50 of 0.82 ± 0.18 µM and 1.3 ± 0.22 µM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure-activity relationships and drug development efforts of this novel target.


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , DNA/drug effects , Intercalating Agents/chemical synthesis , Intercalating Agents/pharmacology , Xeroderma Pigmentosum Group A Protein/antagonists & inhibitors , Antineoplastic Agents/chemistry , Computer Simulation , DNA Repair/drug effects , Drug Design , Drug Evaluation, Preclinical , Humans , Intercalating Agents/chemistry , Models, Molecular , Molecular Docking Simulation , Solubility , Structure-Activity Relationship
13.
Pharmacol Ther ; 160: 65-83, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26896565

ABSTRACT

The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested that all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy.


Subject(s)
Antineoplastic Agents/therapeutic use , DNA Breaks, Double-Stranded/drug effects , DNA Repair/drug effects , DNA/drug effects , Neoplasms/drug therapy , Animals , DNA Adducts/drug effects
14.
Clin Cancer Res ; 20(24): 6504-16, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25316809

ABSTRACT

PURPOSE: To investigate SGI-110 as a "chemosensitizer" in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer. EXPERIMENTAL DESIGN: Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR. RESULTS: We demonstrate in vitro that SGI-110 resensitized a range of platinum-resistant ovarian cancer cells to cisplatin (CDDP) and induced significant demethylation and reexpression of TSG, differentiation-associated genes, and putative drivers of ovarian cancer cisplatin resistance. In vivo, SGI-110 alone or in combination with CDDP was well tolerated and induced antitumor effects in ovarian cancer xenografts. Pyrosequencing analyses confirmed that SGI-110 caused both global (LINE1) and gene-specific hypomethylation in vivo, including TSGs (RASSF1A), proposed drivers of ovarian cancer cisplatin resistance (MLH1 and ZIC1), differentiation-associated genes (HOXA10 and HOXA11), and transcription factors (STAT5B). Furthermore, DNA damage induced by CDDP in ovarian cancer cells was increased by SGI-110, as measured by inductively coupled plasma-mass spectrometry analysis of DNA adduct formation and repair of cisplatin-induced DNA damage. CONCLUSIONS: These results strongly support further investigation of hypomethylating strategies in platinum-resistant ovarian cancer. Specifically, SGI-110 in combination with conventional and/or targeted therapeutics warrants further development in this setting.


Subject(s)
Azacitidine/analogs & derivatives , DNA Methylation/drug effects , Drug Resistance, Neoplasm/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Azacitidine/administration & dosage , Azacitidine/pharmacology , Cell Line, Tumor , Cisplatin/administration & dosage , Cisplatin/pharmacology , DNA Adducts , Disease Models, Animal , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing , Histones/metabolism , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
15.
Cancer Discov ; 4(10): 1118-9, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25274682

ABSTRACT

DNA repair has been shown to affect the cellular response to platinum-based therapy in a variety of cancers; however, translating this knowledge to the clinic has proven difficult and yielded mixed results. In this issue of Cancer Discovery, Van Allen and colleagues have analyzed responders and nonresponders to neoadjuvant platinum-based therapy with locally advanced urothelial cancer and identified a series of mutations in the nucleotide excision repair (NER) gene ERCC2 that correlate with the response to platinum-based therapy. This work provides evidence that defects in NER can be exploited to maximize the efficacy of conventional platinum-based chemotherapy.


Subject(s)
Cisplatin/therapeutic use , Drug Resistance, Neoplasm/genetics , Mutation , Urologic Neoplasms/drug therapy , Urologic Neoplasms/genetics , Urothelium/pathology , Xeroderma Pigmentosum Group D Protein/genetics , Female , Humans , Male
16.
Mol Cell Biol ; 34(12): 2162-75, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24687855

ABSTRACT

DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a "hinge" region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.


Subject(s)
DNA-Activated Protein Kinase/metabolism , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Cisplatin/pharmacology , Cricetinae , Cricetulus , DNA Adducts/drug effects , DNA Adducts/metabolism , DNA Damage , DNA-Activated Protein Kinase/chemistry , Enzyme Activation/drug effects , Genes, Dominant , Microtubules/drug effects , Microtubules/metabolism , Models, Molecular , Molecular Sequence Data , Phenotype , Phosphorylation/drug effects , VDJ Recombinases/metabolism
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