The mechanistic role of cardiac glycosides in DNA damage response and repair signaling.

Cardiac glycosides (CGs) are a class of bioactive organic compounds well-known for their application in treating heart disease despite a narrow therapeutic window. Considerable evidence has demonstrated the potential to repurpose CGs for cancer treatment. Chemical modification of these CGs has been utilized in attempts to increase their anti-cancer properties; however, this has met limited success as their mechanism of action is still speculative. Recent studies have identified the DNA damage response (DDR) pathway as a target of CGs. DDR serves to coordinate numerous cellular pathways to initiate cell cycle arrest, promote DNA repair, regulate replication fork firing and protection, or induce apoptosis to avoid the survival of cells with DNA damage or cells carrying mutations. Understanding the modus operandi of cardiac glycosides will provide critical information to better address improvements in potency, reduced toxicity, and the potential to overcome drug resistance. This review summarizes recent scientific findings of the molecular mechanisms of cardiac glycosides affecting the DDR signaling pathway in cancer therapeutics from 2010 to 2022. We focus on the structural and functional differences of CGs toward identifying the critical features for DDR targeting of these agents.

Ku-DNA binding inhibitors modulate the DNA damage response in response to DNA double-strand breaks.

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.

ATR inhibition overcomes platinum tolerance associated with ERCC1- and p53-deficiency by inducing replication catastrophe.

ERCC1/XPF is a heterodimeric DNA endonuclease critical for repair of certain chemotherapeutic agents. We recently identified that ERCC1- and p53-deficient lung cancer cells are tolerant to platinum-based chemotherapy. ATR inhibition synergistically re-stored platinum sensitivity to platinum tolerant ERCC1-deficient cells. Mechanistically we show this effect is reliant upon several functions of ATR including replication fork protection and altered cell cycle checkpoints. Utilizing an inhibitor of replication protein A (RPA), we further demonstrate that replication fork protection and RPA availability are critical for platinum-based drug tolerance. Dual treatment led to increased formation of DNA double strand breaks and was associated with chromosome pulverization. Combination treatment was also associated with increased micronuclei formation which were capable of being bound by the innate immunomodulatory factor, cGAS, suggesting that combination platinum and ATR inhibition may also enhance response to immunotherapy in ERCC1-deficient tumors. In vivo studies demonstrate a significant effect on tumor growth delay with combination therapy compared with single agent treatment. Results of this study have led to the identification of a feasible therapeutic strategy combining ATR inhibition with platinum and potentially immune checkpoint blockade inhibitors to overcome platinum tolerance in ERCC1-deficient, p53-mutant lung cancers.

Synthesis and Biological Evaluation of Cardiac Glycosides for Cancer Therapy by Targeting the DNA Damage Response.

Cardiac glycosides (CGs) are bioactive compounds originally used to treat heart diseases, but recent studies have demonstrated their anticancer activity. We previously demonstrated that Antiaris toxicaria 2 (AT2) possesses anticancer activity in KRAS mutated lung cancers via impinging on the DNA damage response (DDR) pathway. Toward developing this class of molecules for cancer therapy, herein we report a multistep synthetic route utilizing k-strophanthidin as the initial building block for determination of structure-activity relationships (SARs). A systematic structural design approach was applied that included modifications of the sugar moiety, the glycoside linker, stereochemistry, and lactone ring substitutions to generate a library of O-glycosides and MeON-neoglycosides derivatives. These molecules were screened for their anticancer activities and their impact on DDR signaling in KRAS mutant lung cancer cells. These results demonstrate the ability to chemically synthesize CG derivatives and define the SARs to optimize AT2 as a cancer therapeutic.

Recent Advances in the Development of Non-PIKKs Targeting Small Molecule Inhibitors of DNA Double-Strand Break Repair.

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.

Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent.

BACKGROUND: Enediynes are anti-cancer agents that are highly cytotoxic due to their propensity for low thermal activation of radical generation. The diradical intermediate produced from Bergman cyclization of the enediyne moiety may induce DNA damage and cell lethality. The cytotoxicity of enediynes and difficulties in controlling their thermal cyclization has limited their clinical use. We recently showed that enediyne toxicity at 37 °C can be mitigated by metallation, but cytotoxic effects of 'metalloenediynes' on cultured tumor cells are potentiated by hyperthermia. Reduction of cytotoxicity at normothermia suggests metalloenediynes will have a large therapeutic margin, with cell death occurring primarily in the heated tumor. Based on our previous in vitro findings, FeSO4-PyED, an Fe co-factor complex of (Z)-N,N'-bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine, was prioritized for further in vitro and in vivo testing in normal human melanocytes and melanoma cells. METHODS: Clonogenic survival, apopotosis and DNA binding assays were used to determine mechanisms of enhancement of FeSO4-PyED cytotoxicity by hyperthermia. A murine human melanoma xenograft model was used to assess in vivo efficacy of FeSO4-PyED at 37 or 42.5 °C. RESULTS: FeSO4-PyED is a DNA-binding compound. Enhancement of FeSO4-PyED cytotoxicity by hyperthermia in melanoma cells was due to Bergman cyclization, diradical formation, and increased apoptosis. Thermal enhancement, however, was not observed in melanocytes. FeSO4-PyED inhibited tumor growth when melanomas were heated during drug treatment, without inducing normal tissue damage. CONCLUSION: By leveraging the unique thermal activation properties of metalloenediynes, we propose that localized moderate hyperthermia can be used to confine the cytotoxicity of these compounds to tumors, while sparing normal tissue.

In Vivo Targeting Replication Protein A for Cancer Therapy.

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.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.

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.

OB-Folds and Genome Maintenance: Targeting Protein-DNA Interactions for Cancer Therapy.

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.

Discovery and development of novel DNA-PK inhibitors by targeting the unique Ku-DNA interaction.

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.

Platinum-Induced Ubiquitination of Phosphorylated H2AX by RING1A Is Mediated by Replication Protein A in Ovarian Cancer.

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.

Structure-Guided Optimization of Replication Protein A (RPA)-DNA Interaction Inhibitors.

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.

Small-Molecule Inhibitor Screen for DNA Repair Proteins.

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.

Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells.

Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of PyED, platinated PyED (Pt-PyED). Pt-PyED binds to DNA-like cisplatin, and much like PyED, cytotoxicity is greatly enhanced after treatment with the drug at elevated temperatures. However, in contrast to PyED, Pt-PyED is only minimally cytotoxic at 37°C, at concentrations at which cytotoxicity is enhanced by hyperthermia. Further development of cisplatin-based enediynes may result in compounds which, when activated, will possess multiple DNA binding modalities similar to cisplatin, but produce less side effects in tissues at normothermic temperatures.

Modulating DNA Repair Pathways to Improve Precision Genome Engineering.

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.

Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein-DNA Interaction.

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.

DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer.

Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24h after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism.

DNA repair targeted therapy: The past or future of cancer treatment?

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.