Suppression of homology-dependent DNA double-strand break repair induces PARP inhibitor sensitivity in VHL-deficient human renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of VHL 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 VHL-deficient renal cancer cells relative to wild type VHL-complemented cells. Functionally, this gene repression is associated with impaired DNA double-strand break repair in VHL-deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand breaks and reduced repair activity in a homology-dependent plasmid reactivation assay. Furthermore, VHL deficiency conferred increased sensitivity to PARP inhibitors, analogous to the synthetic lethality observed between hypoxia and these agents. Finally, we discovered a correlation between VHL inactivation and reduced HR gene expression in a large panel of human renal carcinoma samples. Together, our data elucidate a novel connection between VHL-deficient renal carcinoma and hypoxia-induced down-regulation of DNA repair, and identify potential opportunities for targeting DNA repair defects in human renal cell carcinoma.

A cell-penetrating antibody inhibits human RAD51 via direct binding.

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.

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.

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

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.

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.

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 a pathway that is independent of reactive oxygen species and iron, and that the potent cytotoxicity of (-)-lomaiviticin A arises from the 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. Labelling studies suggest that 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 that of other members of this family.

Hypoxia and DNA repair.

Hypoxia is a characteristic feature of solid tumors and occurs very early in neoplastic development. Hypoxia transforms cell physiology in multiple ways, with profound changes in cell metabolism, cell growth, susceptibility to apoptosis, induction of angiogenesis, and increased motility. Over the past 20 years, our lab has determined that hypoxia also induces genetic instability. We have conducted a large series of experiments revealing that this instability occurs through the alteration of DNA repair pathways, including nucleotide excision repair, DNA mismatch repair, and homology dependent repair. Our work suggests that hypoxia, as a key component of solid tumors, can drive cancer progression through its impact on genomic integrity. However, the acquired changes in DNA repair that are induced by hypoxia may also render hypoxic cancer cells vulnerable to tailored strategies designed to exploit these changes.

Targeting cancer with a lupus autoantibody.

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.

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.

Triplex-stimulated intermolecular recombination at a single-copy genomic target.

Gene targeting via homologous recombination offers a potential strategy for therapeutic correction of mutations in disease-related human genes. However, there is a need to improve the efficiency of site-specific recombination by transfected donor DNAs. Oligonucleotide-mediated triple helix formation has been shown to constitute a DNA lesion sufficient to provoke DNA repair and thereby stimulate recombination. However, the ability of triplex-forming oligonucleotides (TFOs) to induce recombination between a target locus and a donor DNA has so far been demonstrated only with multicopy episomal targets in mammalian cells. Using cell lines containing the firefly luciferase reporter gene, we have now established the ability of TFOs to induce gene correction by exogenous donor DNAs at a single-copy chromosomal locus. We find that cotransfection of TFOs and short, single-stranded DNA donor molecules into mammalian cells yields gene correction in a dose-dependent manner at frequencies up to 0.1%, which is five- to ninefold above background. We demonstrate both oligonucleotide-specific and target site-specific effects. We also find that recombination can be induced by both parallel and antiparallel triple helix formation. These results provide further support for the development of TFOs as reagents to stimulate site-specific correction of defective human genes.