Denise C. Hegan

Inhibition of poly(ADP-ribose) polymerase down-regulates BRCA1 and RAD51 in a pathway mediated by E2F4 and p130

Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy, on the basis of the role of PARP in recruitment of base excision repair (BER) factors to sites of DNA damage. Here we show that PARP inhibition to block BER is toxic to hypoxic cancer cells, in which homology-dependent repair (HDR) is known to be down-regulated. However, we also report the unexpected finding that disruption of PARP, itself, either via chemical PARP inhibitors or siRNAs targeted to PARP-1, can inhibit HDR by suppressing expression of BRCA1 and RAD51, key factors in HDR of DNA breaks. Mechanistically, PARP inhibition was found to cause increased occupancy of the BRCA1 and RAD51 promoters by repressive E2F4/p130 complexes, a pathway prevented by expression of HPV E7, which disrupts p130 activity, or by siRNAs to knock down p130 expression. Functionally, disruption of p130 by E7 expression or by siRNA knockdown also reverses the cytotoxicity and radiosensitivity associated with PARP inhibition, suggesting that the down-regulation of BRCA1 and RAD51 is central to these effects. Direct measurement of HDR using a GFP-based assay demonstrates reduced HDR in cells treated with PARP inhibitors. This work identifies a mechanism by which PARP regulates DNA repair and suggests new strategies for combination cancer therapies.

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

The oncometabolite 2-hydroxyglutarate renders IDH1/2 mutant cancer cells deficient in homologous recombination and confers vulnerability to synthetic lethal targeting with PARP inhibitors. Target 2HG or not 2HG, that is the question Mutations in isocitrate dehydrogenase 1 and 2, which result in overproduction of 2-hydroxyglutarate (2HG), are observed in multiple tumor types, including gliomas and acute myelogenous leukemia. An additional form of 2HG is produced under hypoxia, which is also frequent in tumors. 2HG is considered to be an oncometabolite, or a metabolite that promotes carcinogenesis, and inhibitors of mutant isocitrate dehydrogenase are in development to target this process. However, Sulkowski et al. found that it may be possible to take advantage of 2HG overproduction instead. The authors discovered that 2HG overproduction impairs homologous recombination used in DNA repair and sensitizes cancer cells to treatment with PARP inhibitors, another class of cancer drugs that are already in clinical use. 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 relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Targeting Cancer with a Lupus Autoantibody

A cell-penetrating lupus anti-DNA antibody inhibits DNA repair, sensitizes cancer cells to DNA-damaging therapy in vitro and in vivo, and is synthetically lethal to BRCA2-deficient human cancer cells. Taming the Big Bad Wolf Just like the wolves for which lupus is named, the antibodies involved in its pathogenesis can attack almost any part of a patient, causing widespread damage. Now, Hansen et al. show that these biological wolves can sometimes be tamed and their ferociousness put to use in treating another deadly disease. Lupus is an autoimmune disease associated with antibodies that target host DNA, wreaking havoc on patients’ cells throughout the body. Recently, cancer researchers have tried to co-opt some of these antibodies, particularly those that can penetrate human cells, for use as vehicles for therapeutic agents. While using lupus antibodies to deliver proteins to protect normal cells from therapeutic ionizing radiation delivered to a tumor, researchers discovered that one antibody, 3E10, could itself sensitize cancer cells to radiation treatment. The authors then characterized this observed effect in malignant cells and determined its mechanism. They found that 3E10 bound single-stranded DNA and interfered with its repair, making the cells more susceptible to DNA-damaging agents such as doxorubicin and radiation. In addition, 3E10 alone was toxic to cancer cells with deficient DNA repair pathways, such as those that harbor BRCA2 mutations. Further research is necessary to identify other pathways that make tumor cells susceptible to 3E10 and to analyze the pharmacokinetics and other characteristics of this treatment. However, 3E10 was already shown to be safe in a previous phase 1 trial in lupus patients and should now be able to transition into clinical trials for cancer patients as well. Although researchers have not yet discovered a cure for lupus, the big bad wolf’s offspring may potentially tame another life-threatening illness. Systemic lupus erythematosus (SLE) is distinct among autoimmune diseases because of its association with circulating autoantibodies reactive against host DNA. The precise role that anti-DNA antibodies play in SLE pathophysiology remains to be elucidated, and potential applications of lupus autoantibodies in cancer therapy have not previously been explored. We report the unexpected finding that a cell-penetrating lupus autoantibody, 3E10, has potential as a targeted therapy for DNA repair–deficient malignancies. We find that 3E10 preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes cultured tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we demonstrate that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low-dose doxorubicin. Our results establish an approach to cancer therapy that we expect will be particularly applicable to BRCA2-related malignancies such as breast, ovarian, and prostate cancers. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the intrinsic deficiencies in DNA repair and the unexpectedly low rates of breast, ovarian, and prostate cancers observed in SLE patients. In summary, this study provides the basis for the potential use of a lupus anti-DNA antibody in cancer therapy and identifies lupus autoantibodies as a potentially rich source of therapeutic agents.

The cytotoxicity of (−)-lomaiviticin A arises from induction of double-strand breaks in DNA

The metabolite (–)-lomaiviticin A, which contains two diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups, inhibits the growth of cultured human cancer cells at nanomolar–picomolar concentrations; however, the mechanism responsible for the potent cytotoxicity of this natural product is not known. Here we report that (–)-lomaiviticin A nicks and cleaves plasmid DNA by an ROS- and iron-independent pathway and that the potent cytotoxicity of (–)-lomaiviticin A arises from induction of DNA double-strand breaks (dsbs). In a plasmid cleavage assay, the ratio of single-strand breaks (ssbs) to dsbs is 5.3±0.6:1. Labeling studies suggest this cleavage occurs via a radical pathway. The structurally related isolates (–)-lomaiviticin C and (–)-kinamycin C, which contain one diazofluorene, are demonstrated to be much less effective DNA cleavage agents, thereby providing an explanation for the enhanced cytotoxicity of (–)-lomaiviticin A compared to other members of this family.

Targeted gene modification of hematopoietic progenitor cells in mice following systemic administration of a PNA-peptide conjugate.

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

Mechanism of Action Studies of Lomaiviticin A and the Monomeric Lomaiviticin Aglycon. Selective and Potent Activity Toward DNA Double-Strand Break Repair-Deficient Cell Lines

(-)-Lomaiviticin A (1) and the monomeric lomaiviticin aglycon [aka: (-)-MK7-206, (3)] are cytotoxic agents that induce double-strand breaks (DSBs) in DNA. Here we elucidate the cellular responses to these agents and identify synthetic lethal interactions with specific DNA repair factors. Toward this end, we first characterized the kinetics of DNA damage by 1 and 3 in human chronic myelogenous leukemia (K562) cells. DSBs are rapidly induced by 3, reaching a maximum at 15 min post addition and are resolved within 4 h. By comparison, DSB production by 1 requires 2-4 h to achieve maximal values and >8 h to achieve resolution. As evidenced by an alkaline comet unwinding assay, 3 induces extensive DNA damage, suggesting that the observed DSBs arise from closely spaced single-strand breaks (SSBs). Both 1 and 3 induce ataxia telangiectasia mutated- (ATM-) and DNA-dependent protein kinase- (DNA-PK-) dependent production of phospho-SER139-histone H2AX (γH2AX) and generation of p53 binding protein 1 (53BP1) foci in K562 cells within 1 h of exposure, which is indicative of activation of nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. Both compounds also lead to ataxia telangiectasia and Rad3-related- (ATR-) dependent production of γH2AX at later time points (6 h post addition), which is indicative of replication stress. 3 is also shown to induce apoptosis. In accord with these data, 1 and 3 were found to be synthetic lethal with certain mutations in DNA DSB repair. 1 potently inhibits the growth of breast cancer type 2, early onset- (BRCA2-) deficient V79 Chinese hamster lung fibroblast cell line derivative (VC8), and phosphatase and tensin homologue deleted on chromosome ten- (PTEN-) deficient human glioblastoma (U251) cell lines, with LC50 values of 1.5 ± 0.5 and 2.0 ± 0.6 pM, respectively, and selectivities of >11.6 versus the isogenic cell lines transfected with and expressing functional BRCA2 and PTEN genes. 3 inhibits the growth of the same cell lines with LC50 values of 6.0 ± 0.5 and 11 ± 4 nM and selectivities of 84 and 5.1, for the BRCA2 and PTEN mutants, respectively. These data argue for the evaluation of these agents as treatments for tumors that are deficient in BRCA2 and PTEN, among other DSB repair factors.

Nickel induces transcriptional down-regulation of DNA repair pathways in tumorigenic and non-tumorigenic lung cells

The heavy metal nickel is a known carcinogen, and occupational exposure to nickel compounds has been implicated in human lung and nasal cancers. Unlike many other environmental carcinogens, however, nickel does not directly induce DNA mutagenesis, and the mechanism of nickel-related carcinogenesis remains incompletely understood. Cellular nickel exposure leads to signaling pathway activation, transcriptional changes and epigenetic remodeling, processes also impacted by hypoxia, which itself promotes tumor growth without causing direct DNA damage. One of the mechanisms by which hypoxia contributes to tumor growth is the generation of genomic instability via down-regulation of high-fidelity DNA repair pathways. Here, we find that nickel exposure similarly leads to down-regulation of DNA repair proteins involved in homology-dependent DNA double-strand break repair (HDR) and mismatch repair (MMR) in tumorigenic and non-tumorigenic human lung cells. Functionally, nickel induces a defect in HDR capacity, as determined by plasmid-based host cell reactivation assays, persistence of ionizing radiation-induced DNA double-strand breaks and cellular hypersensitivity to ionizing radiation. Mechanistically, we find that nickel, in contrast to the metalloid arsenic, acutely induces transcriptional repression of HDR and MMR genes as part of a global transcriptional pattern similar to that seen with hypoxia. Finally, we find that exposure to low-dose nickel reduces the activity of the MLH1 promoter, but only arsenic leads to long-term MLH1 promoter silencing. Together, our data elucidate novel mechanisms of heavy metal carcinogenesis and contribute to our understanding of the influence of the microenvironment on the regulation of DNA repair pathways.

Abstract LB-029: Negative transcriptional and epigenetic regulation of DNA repair pathways by the heavy metals nickel and arsenic

Environmental exposure to certain heavy metals, such as nickel and arsenic, has been implicated in a variety of human cancers, including lung, skin, digestive track, and bladder cancers. Importantly, the mechanism underlying the carcinogenicity of nickel and arsenic remains poorly understood as they do not induce direct DNA mutagenesis. However, they do lead to global changes in chromatin structure and transcription, many similar to the effects of hypoxia. Since hypoxia is known to regulate many different DNA repair pathways, we investigated whether nickel and arsenic may similarly affect cellular DNA repair. We discovered that nickel and arsenic can lead to alterations in DNA repair gene expression, stable gene silencing, and decreased DNA repair capacity. First, we measured protein and mRNA levels of different DNA repair genes after NiCl2 or NaAsO2 treatment. We found that both metals induced down-regulation of BRCA1, FANCD2, and MLH1 over 24 to 48 hours at both the protein and mRNA levels. These results were observed in several different cells lines (HeLa, MCF7, BEAS-2B) with one notable exception that high dose arsenic induced up-regulation of BRCA1, FANCD2, and MLH1 in lung cancer-derived cell lines (A549, HCC827, NCI-H460). Next, to study the impact of long-term heavy metal exposure on DNA repair gene expression, we utilized an MLH1 promoter reporter construct that allows selection of cells harboring a silenced MLH1 promoter with ganciclovir. RKO cells stably expressing this construct were grown in the presence of 100 μM NiCl2, 0.5 μM NaAsO2, 1% oxygen, or control conditions. After 3 weeks, we observed that arsenic treatment, like hypoxia, led to a significant increase in promoter silencing compared to control cells, peaking at about 3.7-fold after 4 weeks. Nickel did not increase silencing, which may indicate a different mechanism of gene regulation. Finally, we used a luciferase assay to measure the effect of nickel and arsenic on the two primary DNA double-strand break (DSB) repair mechanisms, homologous recombination (HR) and non-homologous end joining (NHEJ). BEAS-2B cells pretreated with 250 μM NiCl2 or 5 μM NaAsO2 were transfected with a digested, inactive luciferase plasmid and allowed to conduct DSB repair to reactivate luciferase expression. We found that nickel and arsenic led to a 40-50% reduction in cellular HR capacity with no significant effect on NHEJ. To further pursue these results, we are performing chromatin immunoprecipitation studies to identify transcriptional or epigenetic factors mediating nickel and arsenic-induced down-regulation of DNA repair genes. In addition, we are using chromosomal-based assays to further characterize the impact of nickel and arsenic on DNA repair capacity. In conclusion, we have found that nickel and arsenic negatively regulate cellular DNA repair pathways, identifying a novel way in which heavy metals may contribute to carcinogenesis. Citation Format: Susan E. Scanlon, Christine D. Scanlon, Denise C. Hegan, Parker Sulkowski, Peter M. Glazer. Negative transcriptional and epigenetic regulation of DNA repair pathways by the heavy metals nickel and arsenic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-029.

A cell-penetrating antibody inhibits human RAD51 via direct binding

Abstract RAD51, a key factor in homology-directed repair (HDR), has long been considered an attractive target for cancer therapy, but few specific inhibitors have been found. A cell-penetrating, anti-DNA, lupus autoantibody, 3E10, was previously shown to inhibit HDR, sensitize tumors to radiation, and mediate synthetic lethal killing of BRCA2-deficient cancer cells, effects that were initially attributed to its affinity for DNA. However, as the molecular basis for its ability to inhibit DNA repair, we report that 3E10 directly binds to the N-terminus of RAD51, sequesters RAD51 in the cytoplasm, and impedes RAD51 binding to DNA. Further, we generate separation-of-function mutations in the complementarity-determining regions of 3E10 revealing that inhibition of HDR tracks with binding to RAD51 but not to DNA, whereas cell penetration is linked to DNA binding. The consequences of these mutations on putative 3E10 interactions with RAD51 and DNA are correlated with in silico molecular modeling. Taken together, the results identify 3E10 as a novel inhibitor of RAD51 by direct binding, accounting for its ability to suppress HDR and providing the molecular basis to guide pre-clinical development of 3E10 as an anti-cancer agent.

Abstract B25: “TargetDBR”—A DNA repair drug and target discovery collaboration: Exploiting synthetic lethal, high content, and functional cellular reporter assays to accelerate DNA repair targeted drug discovery

Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Inhibition of DSB repair sensitizes cells to these therapies. Mutations have been reported in nearly every DNA repair pathway and these pathways often exhibit redundancy. Inhibition of functional repair factors can induce synthetic lethality in repair-deficient tumors even in the absence of exogenous DNA damage and whilst sparing healthy tissue. We demonstrate a compound and target discovery platform comprising the integration of isogenic cell line screening, high content repair foci assays and relative DSB repair pathway reporting cells, along with chemical biology and medicinal chemistry. Two diverse drug-like compound library screens have been conducted. Screening for synthetic lethality with deficiency in FANCD2 and BRCA2 led to a hit series broadly active against a range of homologous recombination (HR) repair deficiencies, which is now undergoing medicinal chemistry optimization and target deconvolution studies. Outcome of these initial chemoproteomic target pulldown experiments will be presented. A second cellular screen, (50k compounds) for inhibition of BRCA1 and Rad51 foci formation following radiomimetic drug induced DNA damage, has led to identification of compounds that inhibit damage response after ionizing radiation and selectively inhibit HR repair. Focusing on therapeutic targeting of proliferating tumor cells, our research platform has enabled novel hit compound identification, mechanistic profiling, hit-to-lead optimization chemistry and proof of concept efficacy in in vitro tumor models. Effects on tumor cell clonogenic survival and sensitization towards chemotherapeutics and ionizing radiation will be presented, along with comparisons to known DNA repair inhibitors that demonstrate these compounds9 differentiated mechanisms of action. TargetDBR has created a technology platform focused on DNA repair inhibitor discovery and has identified differentiated hits and lead series with potential for development and opportunity to meet the need for new druggable targets in oncology. Citation Format: Jonathan J. Hollick, Laura Abriola, Francoise Bono, Denise Hegan, Pamela Klingbeil, Yanfeng Liu, Ranjini Sundaram, Yulia V. Surovtseva, Mark Whittaker, Ranjit S. Bindra, Peter M. Glazer. “TargetDBR”—A DNA repair drug and target discovery collaboration: Exploiting synthetic lethal, high content, and functional cellular reporter assays to accelerate DNA repair targeted drug discovery [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B25.