Homology-dependent DNA Repair Research Articles

BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1

Defects in BRCA1, BRCA2 and other genes of the homology-dependent DNA repair (HR) pathway cause an elevated rate of mutagenesis, eliciting specific mutation patterns including COSMIC signature SBS3. Using genome sequencing of knock-out cell lines we show that Y family translesion synthesis (TLS) polymerases contribute to the spontaneous generation of base substitution and short insertion/deletion mutations in BRCA1 deficient cells, and that TLS on DNA adducts is increased in BRCA1 and BRCA2 mutants. The inactivation of 53BP1 in BRCA1 mutant cells markedly reduces TLS-specific mutagenesis, and rescues the deficiency of template switch–mediated gene conversions in the immunoglobulin V locus of BRCA1 mutant chicken DT40 cells. 53BP1 also promotes TLS in human cellular extracts in vitro. Our results show that HR deficiency–specific mutagenesis is largely caused by TLS, and suggest a function for 53BP1 in regulating the choice between TLS and error-free template switching in replicative DNA damage bypass.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering for Cell Therapy Development

Gene-editing technology has revolutionized molecular therapeutics, enabling DNA-engineering-based approaches to treat disease. Despite this, development of medicines using gene editing has been hampered by technological, immunological, and legal limitations. We described previously the discovery of novel type II and type V gene-editing systems from metagenomic data, the characterization of these systems in vitro, and a demonstration of their activity in immortalized cell lines. Here, we substantially advance this work with three separate, novel gene-editing systems, demonstrating their utility for cell therapy development. We express and purify the nuclease components of both type II and type V effectors and show that all three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain constant region occurred in up to 95% of cells, both copies of the TCR beta-chain constant region in up to ~90% of cells, and beta-2 microglobulin in up to 95% of cells. Simultaneous double knock-out of TRAC and TRBC was obtained at an equal frequency. Gene editing with our systems had no effect on T cell viability. Further, we use our novel gene-editing systems to exploit homology-dependent DNA repair to integrate a CAR construct into the TCR alpha-chain locus (in up to ~60% of T cells), and demonstrate high-level CAR expression and antigen-dependent CAR-T cytotoxicity. Such robust editing activity at the TCR loci will permit efficient engineering and manufacture of allogeneic chimeric antigen receptor- (CAR) and TCR-based cell therapies.We next applied our novel gene-editing tools to NK cells and B cells. We achieved almost 100% gene disruption at the CD38 locus in NK cells and integrated a chimeric antigen receptor into the NK cell genome at a frequency of ~40%. Such CAR-NK cells displayed robust CAR-directed cellular cytotoxicity. B cell editing occurred in approximately 80% of target cells with successful transgene integration.Cas9 is notorious for tolerating mismatches and bulges in its target, resulting in high levels of unwanted DNA double-strand break formation. In contrast, interrogation of our gene editing systems using GUIDE-seq reveals no or very few off-target sites even at doses greatly in excess of those required for high-frequency gene editing. Finally, as our systems are taken from microbes found in environmental samples rather than from human pathogens, we expect pre-existing immunity to our nucleases will be quite rare. In all, we show that three new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development. DisclosuresNo relevant conflicts of interest to declare.

Generation of a homozygous ZBTB7A knockout human induced pluripotent stem line by CRISPR/Cas9 editing

ZBTB7A plays important roles in several biological processes, including silencing of the fetal γ-globin genes, hematopoiesis, primed-to-naive transition, etc. Meanwhile, it is also associated with Oncogenic transformation and tumor progression. However, the mechanism of ZBTB7A function is not fully understood yet. Here, we generated a homozygous ZBTB7A knockout human induced pluripotent stem cell (iPSC) line, GZHMCi007-A by the CRISPR/Cas9-mediated homology-dependent DNA repair method. The iPSCs of ZBTB7A-/- established by us is a powerful tool for related research.

An in vivo Interaction Network of DNA-Repair Proteins: A Snapshot at Double Strand Break Repair in Deinococcus radiodurans.

An extremophile Deinococcus radiodurans survives massive DNA damage by efficiently mending hundreds of double strand breaks through homology-dependent DNA repair pathways. Although DNA repair proteins that contribute to its impressive DNA repair capacity are fairly known, interactions among them or with proteins related to other relevant pathways remain unexplored. Here, we report in vivo cross-linking of the interactomes of key DNA repair proteins DdrA, DdrB, RecA, and Ssb (baits) in D. radiodurans cells recovering from gamma irradiation. The protein-protein interactions were systematically investigated through co-immunoprecipitation experiments coupled to mass spectrometry. From a total of 399 proteins co-eluted with the baits, we recovered interactions among diverse biological pathways such as DNA repair, transcription, translation, chromosome partitioning, cell division, antioxidation, protein folding/turnover, metabolism, cell wall architecture, membrane transporters, and uncharacterized proteins. Among these, about 80 proteins were relevant to the DNA damage resistance of the organism based on integration of data on inducible expression following DNA damage, radiation sensitive phenotype of deletion mutant, etc. Further, we cloned ORFs of 23 interactors in heterologous E. coli and expressed corresponding proteins with N-terminal His-tag, which were used for pull-down assays. A total of 95 interactions were assayed, in which we confirmed 25 previously unknown binary interactions between the proteins associated with radiation resistance, and 2 known interactions between DdrB and Ssb or DR_1245. Among these, five interactions were positive even under non-stress conditions. The confirmed interactions cover a wide range of biological processes such as DNA repair, negative regulation of cell division, chromosome partitioning, membrane anchorage, etc., and their functional relevance is discussed from the perspective of DNA repair. Overall, the study substantially advances our understanding on the cross-talk between different homology-dependent DNA repair pathways and other relevant biological processes that essentially contribute to the extraordinary DNA damage repair capability of D. radiodurans. The data sets generated and analyzed in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD021822.

Premalignant Oligodendrocyte Precursor Cells Stall in a Heterogeneous State of Replication Stress Prior to Gliomagenesis.

Cancer evolves from premalignant clones that adopt unusual cell states to achieve transformation. We previously pinpointed the oligodendrocyte precursor cell (OPC) as a cell of origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection, determined global gene-expression changes by bulk RNA sequencing, and compared with cell-state fluctuations at the single-cell level by stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1-Trp53 deletion, bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, and stochastic profiling revealed a spectrum of stem-progenitor (Axl, Aldh1a1), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, mutant OPCs that strongly expressed key effectors of nonsense-mediated decay (Upf3b) and homology-dependent DNA repair (Rad51c, Slx1b, Ercc4) were identified along with DNA-damage markers, suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease (Mus81) and Rin1, a Ras-Raf antagonist and possible counterbalance to Nf1 loss, which was microdeleted or downregulated in gliomas at 150 days. These hidden cell-state variations uncover replication stress as a potential bottleneck that must be resolved for glioma initiation. SIGNIFICANCE: Profiling premalignant cell states in a mouse model of glioma uncovers regulatory heterogeneity in glioma cells-of-origin and defines a state of replication stress that precedes tumor initiation.See related articles by Singh and colleagues, p. 1840 and Schaff and colleagues, p. 1853.

Abstract PR04: The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies

Abstract High-grade serous ovarian cancer (HGSOC) is the most frequent and most aggressive histologic subtype of ovarian cancer. The cornerstone of the existing treatment of HGSOC is DNA-damaging chemotherapy; however, practically all patients eventually develop a progressive disease and the 5-year survival is only 40%. Immunotherapy would seem to be an attractive alternative treatment to chemotherapy, yet existing immunotherapies perform poorly in ovarian cancer, with only ~10% of patients responding to checkpoint blockade. Why this is the case remains poorly understood and there is a pressing need to understand the underlying biology of immune evasion in ovarian cancer. One critical area of interest is the role of homology-dependent DNA repair (HR) in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. Hence, we have modeled the biology of ovarian cancer using patient-relevant mutational landscapes in an immune-proficient, syngeneic mouse model in order to help us identify the contribution of common driver mutations to the immune repertoire in the tumor microenvironment, and thus to responses of HGSOC tumors to immunotherapy. We hypothesize that the immune composition and gene expression signatures of the resulting tumors will vary based on the combination of genetic alterations and the DNA repair proficiency of the transformed cells. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in HR-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune /stromal analysis using flow cytometry, scRNAseq transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean Smith, David Pepin, Raghav Mohan, Ferenc Reinhardt, Tian Xia, Tony E. Chavarria, Esmee Hoefsmit, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Benjamin G. Neel, Robert A. Weinberg. The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR04. doi:10.1158/1535-7163.TARG-19-PR04

BRCA1 intronic Alu elements drive gene rearrangements and PARP inhibitor resistance

BRCA1 mutant carcinomas are sensitive to PARP inhibitor (PARPi) therapy; however, resistance arises. BRCA1 BRCT domain mutant proteins do not fold correctly and are subject to proteasomal degradation, resulting in PARPi sensitivity. In this study, we show that cell lines and patient-derived tumors, with highly disruptive BRCT domain mutations, have readily detectable BRCA1 protein expression, and are able to proliferate in the presence of PARPi. Peptide analyses reveal that chemo-resistant cancers contain residues encoded by BRCA1 intron 15. Mechanistically, cancers with BRCT domain mutations harbor BRCA1 gene breakpoints within or adjacent to Alu elements in intron 15; producing partial gene duplications, inversions and translocations, and terminating transcription prior to the mutation-containing BRCT domain. BRCA1 BRCT domain-deficient protein isoforms avoid mutation-induced proteasomal degradation, support homology-dependent DNA repair, and promote PARPi resistance. Taken together, Alu-mediated BRCA1 gene rearrangements are responsible for generating hypomorphic proteins, and may represent a biomarker of PARPi resistance.

Abstract SY21-02: Oncometabolites suppress homologous recombination DNA repair by inhibition of chromatin remodeling at the DNA double-strand break

Abstract Elevated levels of the metabolites, 2-hydroxyglutarate (2HG), fumarate, and succinate, can occur in human malignancies due to somatic mutations in the isocitrate dehydrogenase 1/2 (IDH1/2) genes or germline mutations in fumarate hydratase (FH) and succinate dehydrogenase (SDH) genes, respectively. Mutations in IDH1 and IDH2 are found in a large number of human malignancies, most notably gliomas and acute myelogenous leukemias, along with cholangiocarcinomas, chondrosarcomas, and melanomas. Inherited mutations in FH and SDH, are linked to the familial cancer predisposition syndromes, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) and Hereditary Paraganglioma and Pheochromocytoma (SDH PGL/PCC), respectively. These structurally related metabolites inhibit α-ketoglutarate-dependent enzymes, including dioxygenases that modify chromatin. We have shown that they consequently suppress the pathway of homology-dependent DNA repair (HDR), conferring an exquisite sensitivity to PARP inhibitors that is currently being tested in clinical trials. In this presentation, we will discuss our recent work elucidating the mechanistic basis for this suppression of DNA repair. Rather than acting indirectly through epigenetic regulation of gene expression, we find that these metabolites act directly by inhibiting HDR factor recruitment to DNA double-strand breaks (DSBs). The metabolites inhibit the lysine demethylases, KDM4A/B, causing aberrant hypermethylation of H3K9 and disrupting signaling at the break, thereby delaying the stepwise recruitment of key HDR factors. These results define a novel mechanism of decreased DNA repair in oncometabolite producing human tumors and may provide the basis for the rational design of novel therapeutic strategies for these malignancies. Citation Format: Parker S. Sulkowski, Sebstian Oeck, Jing Li, Brian Shuch, Megan C. King, Ranjit S. Bindra, Peter M. Glazer. Oncometabolites suppress homologous recombination DNA repair by inhibition of chromatin remodeling at the DNA double-strand break [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY21-02.

Comparison of the effect of three different topoisomerase II inhibitors combined with cisplatin in human glioblastoma cells sensitized with double strand break repair inhibitors

Topoisomerase II (Topo2) inhibitors in combination with cisplatin represent a common treatment modality used for glioma patients. The main mechanism of their action involves induction of DNA double-strand breaks (DSBs). DSBs are repaired via the homology-dependent DNA repair (HRR) and non-homologous end-joining (NHEJ). Inhibition of the NHEJ or HRR pathway sensitizes cancer cells to the treatment. In this work, we investigated the effect of three Topo2 inhibitors—etoposide, NK314, or HU-331 in combination with cisplatin in the U-87 human glioblastoma cell line. Etoposide as well as NK314 inhibited Topo2 activity by stabilizing Topo2-DNA cleavable complexes whereas HU-331 inhibited the ATPase activity of Topo2 using a noncompetitive mechanism. To increase the effectiveness of the treatment, we combined cisplatin and Topo2 inhibitor treatment with DSB repair inhibitors (DRIs). The cells were sensitized with NHEJ inhibitor, NU7441, or the novel HRR inhibitor, YU238259, prior to drug treatment. All of the investigated Topo2 inhibitors in combination with cisplatin efficiently killed the U-87 cells. The most cytotoxic effect was observed for the cisplatin + HU331 treatment scheme and this effect was significantly increased when a DRI pretreatment was used; however, we did not observed DSBs. Therefore, the molecular mechanism of cytotoxicity caused by the cisplatin + HU331 treatment scheme is yet to be evaluated. We observed a concentration-dependent change in DSB levels and accumulation at the G2/M checkpoint and S-phase in glioma cells incubated with NK314/cisplatin and etoposide/cisplatin. In conclusion, in combination with cisplatin, HU331 is the most potent Topo2 inhibitor of human glioblastoma cells.

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR)1–3. However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ)4. We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells4, 5 and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants6 for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

YU238259 Is a Novel Inhibitor of Homology-Dependent DNA Repair That Exhibits Synthetic Lethality and Radiosensitization in Repair-Deficient Tumors.

Radiotherapy and DNA-damaging chemotherapy are frequently utilized in the treatment of solid tumors. Innate or acquired resistance to these therapies remains a major clinical challenge in oncology. The development of small molecules that sensitize cancers to established therapies represents an attractive approach to extending survival and quality of life in patients. Here, we demonstrate that YU238259, a member of a novel class of DNA double-strand break repair inhibitors, exhibits potent synthetic lethality in the setting of DNA damage response and DNA repair defects. YU238259 specifically inhibits homology-dependent DNA repair, but not non-homologous end-joining, in cell-based GFP reporter assays. Treatment with YU238259 is not only synergistic with ionizing radiation, etoposide, and PARP inhibition, but this synergism is heightened by BRCA2 deficiency. Further, growth of BRCA2-deficient human tumor xenografts in nude mice is significantly delayed by YU238259 treatment even in the absence of concomitant DNA-damaging therapy. The cytotoxicity of these small molecules in repair-deficient cells results from an accumulation of unresolved DNA double-strand breaks. These findings suggest that YU238259 or related small molecules may have clinical benefit to patients with advanced BRCA2-negative tumors, either as a monotherapy or as an adjuvant to radiotherapy and certain chemotherapies. We have identified a novel series of compounds that demonstrate synthetic lethality in DNA repair-deficient cell and animal models and have strong potential for clinical translation.

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria.

Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis of recF and recR in mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCE This study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284-2295, 2013, http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

Abstract SY42-03: Novel DNA repair inhibitors for radiosensitization and cancer therapy

Abstract There is growing interest in the development of molecules that can inhibit DNA repair or disrupt DNA damage response pathways for application to cancer therapy. Such inhibitors offer the potential to achieve synthetic lethality in the context of DNA repair deficient cancers and/or to sensitize tumors to chemotherapy and radiation. We have identified a series of structurally related small molecules that demonstrate increased toxicity to human cancer cells deficient in BRCA2, PTEN, ATM, and the Fanconi pathway. These compounds are not PARP inhibitors but rather impact DNA damage response pathways at upstream steps, causing impairment of both homology-dependent DNA repair (HDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks. The molecules also reduce the formation of DNA repair foci by several damage response factors, including 53BP1, BRCA1, and DNA-PK, following ionizing radiation treatment of cancer cells. Efforts to identify the molecular target of these compounds are underway. In addition, we have found that a highly unusual lupus-derived, cell-penetrating autoantibody, 3E10, preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we find that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low dose doxorubicin. These findings identify a new antibody that may have clinical application in cancer, particularly in BRCA-related malignancies. Because lupus is associated with circulating autoantibodies reactive against host DNA, the work also suggests that lupus autoantibodies may be a valuable source of other potential therapeutic agents. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the unexpectedly low rates of breast and ovarian cancers observed in lupus patients. Citation Format: Peter M. Glazer. Novel DNA repair inhibitors for radiosensitization and cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY42-03. doi:10.1158/1538-7445.AM2014-SY42-03

Abstract 2648: Pharmacological modulation of TH-302-mediated in vitro cytotoxicity

Abstract TH-302 is a hypoxia-activated prodrug currently in clinical trials for the treatment of cancer. TH-302 releases the DNA cross-linking bromo-isophosphoramidate mustard (Br-IPM) under hypoxic conditions and exhibits 400-fold hypoxia-enhanced cytotoxicity in multiple human cancer cell lines in vitro. In this study, we utilized pharmacological tools to dissect the underlying mechanisms of reductase-dependent prodrug activation and DNA repair processes involved in the cellular response to TH-302. The pharmacological modulators used as tools in this study include the flavin reductase inhibitor diphenyliodonium (DPI) chloride, the NAD(P)H:quinone oxidoreductase (NQO1) inhibitor dicoumarol, the PARP inhibitor ABT-888, and the HDAC inhibitor valproic acid (VPA). An in vitro cytotoxicity assay was used as the primary read-out. The effect of the P450 reductase inhibitor DPI chloride on TH-302 activity was assessed in H460, SiHa, and SiHa cells over-expressing P450 reductase (SiHaPOR). The results demonstrated that TH-302 activity was inhibited by DPI. The HAP Tirapazamine exhibited a profile similar to that of TH-302. We investigated the effect of dicoumarol on TH-302-mediated cytotoxicity in H460 cells. Dicoumarol actually enhanced TH-302 activity in a concentration-dependent manner in this cell line. These results are consistent with an interplay between the POR and NQO1 redox systems. The effect of the PARP inhibitor ABT-888 on TH-302 activity was investigated in three human cancer cell lines: H460, A375 and HCT116. TH-302 activity was not affected by the presence of ABT-888. In contrast, the activity of the monoalkylating agent temozolomide was enhanced by ABT-888 in these cancer cell lines. Similarly, the sensitivity of EM9 cells (deficient in XRCC1) exhibited a heighted sensitivity to temozolomide but not to TH-302. Taken together, these results indicate that SSB (single-strand break) DNA repair mechanisms are not involved in the repair of TH-302 lesions. The effect of HDAC inhibitor VPA on TH-302 was investigated in the human prostate cancer cell line DU-145. Inhibition of HDAC has been shown to downregulate HDR (homology dependent DNA repair). Cisplatin was used as a control. Consistent with the published data, VPA enhanced cisplatin-mediated cytotoxicity. Similarly, TH-302 cytotoxicity was also enhanced by VPA. In summary, TH-302 activity can be modulated by pharmacological modulators of both reductases and DNA damage and repair pathways. TH-302 activity can be inhibited by the flavin reductase inhibitor DPI, while the NQO1 inhibitor dicoumoral can enhance TH-302 activity. TH-302 activity was not affected by the PARP inhibitor ABT-888 but was potentiated by the HDAC inhibitor VPA. Taken together, these results support that HDR and not SSB DNA repair mechanisms is involved in repair of TH-302-induced DNA lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2648. doi:10.1158/1538-7445.AM2011-2648

Potentiation of Temozolomide Cytotoxicity by Inhibition of DNA Polymerase β Is Accentuated by BRCA2 Mutation

Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.

A forward chemical genetic screen reveals an inhibitor of the Mre11–Rad50–Nbs1 complex

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated 6-(4-hydroxyphenyl)-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.

Repression of RAD51 gene expression by E2F4/p130 complexes in hypoxia

We and others have shown that the dysregulation of DNA repair pathways can contribute to the phenomenon of hypoxia-induced genetic instability within the tumor microenvironment. Several studies have revealed that the recombinational repair genes, RAD51 and BRCA1, and the DNA mismatch repair genes, MLH1 and MSH2, are decreased in expression in response to hypoxic stress, prompting interest in elucidating the mechanistic basis for these responses. Here we report that the downregulation of RAD51 by hypoxia is specifically mediated by repressive E2F4/p130 complexes that bind to a single E2F site in the proximal promoter of the gene. Intriguingly, this E2F site is conserved in the promoter of the BRCA1 gene, which is also regulated by a similar mechanism in hypoxia. Mechanistically, we have found that hypoxia induces substantial p130 dephosphorylation and nuclear accumulation, leading to the formation of E2F4/p130 complexes and increased occupancy of E2F4 and p130 at the RAD51 and BRCA1 promoters. These findings reveal a coordinated transcriptional program mediated by the formation of repressive E2F4/p130 complexes that represents an integral response to hypoxic stress. In addition, this co-regulation of key factors within the homology-dependent DNA repair pathway provides a further basis for understanding genetic instability in tumors and may guide the design of new therapeutic strategies for cancer.