Selective radiosensitization of p53 mutant pancreatic cancer cells by combined inhibition of Chk1 and PARP1

We have recently shown that inhibition of HRR (homologous recombination repair) by Chk1 (checkpoint kinase 1) inhibition radiosensitizes pancreatic cancer cells and others have demonstrated that Chk1 inhibition selectively sensitizes p53 mutant tumor cells. Furthermore, PARP1 [poly (ADP-ribose) polymerase-1] inhibitors dramatically radiosensitize cells with DNA double strand break repair defects. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 [via olaparib (AZD2281)] would selectively sensitize p53 mutant pancreatic cancer cells to radiation. We also used 2 isogenic p53 cell models to assess the role of p53 status in cancer cells and intestinal epithelial cells to assess overall cancer specificity. DNA damage response and repair were assessed by flow cytometry, γH2AX, and an HRR reporter assay. We found that the combination of AZD7762 and olaparib produced significant radiosensitization in p53 mutant pancreatic cancer cells and in all of the isogenic cancer cell lines. The magnitude of radiosensitization by AZD7762 and olaparib was greater in p53 mutant cells compared with p53 wild type cells. Importantly, normal intestinal epithelial cells were not radiosensitized. The combination of AZD7762 and olaparib caused G₂ checkpoint abrogation, inhibition of HRR, and persistent DNA damage responses. These findings demonstrate that the combination of Chk1 and PARP1 inhibition selectively radiosensitizes p53 mutant pancreatic cancer cells. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRCA1/2 ‘deficient-like’ phenotype in p53 mutant tumor cells, while sparing normal tissue.     

Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by P53

PARP-1 interacts with and poly(ADP-ribosyl)ates p53 and topoisomerase I, which both participate in DNA recombination. Previously, we showed that PARP-1 downregulates homology-directed double-strand break (DSB) repair. We also discovered that, despite the well-established role of p53 as a global suppressor of error-prone recombination, p53 enhances homologous recombination (HR) at the RARα breakpoint cluster region (bcr) comprising topoisomerase I recognition sites. Using an SV40-based assay and isogenic cell lines differing in the p53 and PARP-1 status we demonstrate that PARP-1 counteracts HR enhancement by p53, although DNA replication was largely unaffected. When the same DNA element was integrated in an episomal recombination plasmid, both p53 and PARP-1 exerted anti-recombinogenic rather than stimulatory activities. Strikingly, with DNA substrates integrated into cellular chromosomes, enhancement of HR by p53 and antagonistic PARP-1 action was seen, very similar to the HR of viral minichromosomes. siRNA-mediated knockdown revealed the essential role of topoisomerase I in this regulatory mechanism. However, after I-SceI-meganuclease-mediated cleavage of the chromosomally integrated substrate, no topoisomerase I-dependent effects by p53 and PARP-1 were observed. Our data further indicate that PARP-1, probably through topoisomerase I interactions rather than poly(ADP-ribosyl)ation, prevents p53 from stimulating spontaneous HR on chromosomes via topoisomerase I activity.     

Selective radiosensitization of p53 mutant pancreatic cancer cells by combined inhibition of Chk1 and PARP1

We have recently shown that inhibition of HRR (homologous recombination repair) by Chk1 (checkpoint kinase 1) inhibition radiosensitizes pancreatic cancer cells, and others have demonstrated that Chk1 inhibition selectively sensitizes p53 mutant tumor cells. Furthermore, PARP1 [poly (ADP-ribose) polymerase-1] inhibitors dramatically radiosensitize cells with DNA double-strand break repair defects. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 [via olaparib (AZD2281)] would selectively sensitize p53 mutant pancreatic cancer cells to radiation. We also used two isogenic p53 cell models to assess the role of p53 status in cancer cells and intestinal epithelial cells to assess overall cancer specificity. DNA damage response and repair were assessed by flow cytometry, γH2AX and an HRR reporter assay. We found that the combination of AZD7762 and olaparib produced significant radiosensitization in p53 mutant pancreatic cancer cells and in all of the isogenic cancer cell lines. The magnitude of radiosensitization by AZD7762 and olaparib was greater in p53 mutant cells compared with p53 wild-type cells. Importantly, normal intestinal epithelial cells were not radiosensitized. The combination of AZD7762 and olaparib caused G₂ checkpoint abrogation, inhibition of HRR and persistent DNA damage responses. These findings demonstrate that the combination of Chk1 and PARP1 inhibition selectively radiosensitizes p53 mutant pancreatic cancer cells. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRCA1/2 “deficient-like” phenotype in p53 mutant tumor cells, while sparing normal tissue.Key words: pancreatic cancer, Chk1, PARP1, radiosensitization, p53     

Poly(ADP-ribose) polymerase (PARP-1) and p53 independently function in regulating double-strand break repair in primate cells

PARP-1 is rapidly activated by DNA strand breaks, which finally leads to the modulation of multiple protein activities in DNA replication, DNA repair and checkpoint control. PARP-1 may be involved in homologous recombination, and poly(ADP-ribosyl)ation of p53 represents one possible mechanism that activates p53 as a recombination surveillance factor. Here, we examined the influence of PARP-1 on homology-directed double-strand break (DSB) repair by use of a fluorescence- and I-SceI- meganuclease-based assay with either episomal or chromosomally integrated DNA substrates. Surprisingly, the transient expression of both full-length PARP-1 and of a dominant negative mutant, retaining the DNA-binding but lacking the catalytic domain, down-regulated DSB repair in a dose-dependent manner. This effect was seen regardless of p53 status, however, with enhanced inhibition in the presence of wild-type p53. Taken together, our data reveal that PARP-1 overexpression counteracts DSB repair independently of its enzymatic activity and of poly(ADP-ribosyl)ation of p53 in particular, but synergizes with p53 in suppressing chromosomal rearrangements.     

p21 promotes error-free replication-coupled DNA double-strand break repair

p21 is a well-established regulator of cell cycle progression. The role of p21 in DNA repair, however, remains poorly characterized. Here, we describe a critical role of p21 in a replication-coupled DNA double-strand break (DSB) repair that is mechanistically distinct from its cell cycle checkpoint function. We demonstrate that p21-deficient cells exhibit elevated chromatid-type aberrations, including gaps and breaks, dicentrics and radial formations, following exposure to several DSB-inducing agents. p21(-/-) cells also exhibit an increased DNA damage-inducible DNA-PK(CS) S2056 phosphorylation, indicative of elevated non-homologous DNA end joining. Concomitantly, p21(-/-) cells are defective in replication-coupled homologous recombination (HR), exhibiting decreased sister chromatid exchanges and HR-dependent repair as determined using a crosslinked GFP reporter assay. Importantly, we establish that the DSB hypersensitivity of p21(-/-) cells is associated with increased cyclin-dependent kinase (CDK)-dependent BRCA2 S3291 phosphorylation and MRE11 nuclear foci formation and can be rescued by inhibition of CDK or MRE11 nuclease activity. Collectively, our results uncover a novel mechanism by which p21 regulates the fidelity of replication-coupled DSB repair and the maintenance of chromosome stability distinct from its role in the G1-S phase checkpoint.     

DNA Oligonucleotides and Plasmids Perform Equally as Donors for Targeted Gene Conversion

Site-specific gene modifications in cells are initiated by the introduction of exogenous DNA. We used a recently established cell assay to compare the ability of DNA donors to induce a single point mutation that converts a target gene encoding blue fluorescent protein (BFP) into expressing green fluorescent protein (GFP). In a chromosomal assay with cells stably expressing BFP, we showed that fluorescently labeled single-stranded oligonucleotides and a donor plasmid cotranscribing a red fluorescent protein provide similar efficiencies in triggering BFP–GFP conversions. In transient cotransfections, an isogenic donor plasmid comprising a nonfunctional GFP gene yielded a greater efficiency for the conversion of the BFP target gene than a nonisogenic donor, and all plasmid donors were superior to oligonucleotides.     

MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways

Purpose: Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK inhibitors have shown promising anti-tumor responses in recent preclinical and clinical studies, and are currently being tested in combination with radiation in clinical trials. Here, we have evaluated the radiosensitizing potential of a novel MEK1/2 inhibitor GSK1120212 (GSK212,or trametinib) and evaluated whether MEK1/2 inhibition alters DNA repair mechanisms in multiple PC cell lines.Methods: Radiosensitization and DNA double-strand break (DSB) repair were evaluated by clonogenic assays, comet assay, nuclear foci formation (γH2AX, DNA-PK, 53BP1, BRCA1, and RAD51), and by functional GFP-reporter assays for homologous recombination (HR) and non-homologous end-joining (NHEJ). Expression and activation of DNA repair proteins were measured by immunoblotting.Results: GSK212 blocked ERK1/2 activity and radiosensitized multiple KRAS mutant PC cell lines. Prolonged pre-treatment with GSK212 for 24-48 hours was required to observe significant radiosensitization. GSK212 treatment resulted in delayed resolution of DNA damage by comet assays and persistent γH2AX nuclear foci. GSK212 treatment also resulted in altered BRCA1, RAD51, DNA-PK, and 53BP1 nuclear foci appearance and resolution after radiation. Using functional reporters, GSK212 caused repression of both HR and NHEJ repair activity. Moreover, GSK212 suppressed the expression and activation of a number of DSB repair pathway intermediates including BRCA1, DNA-PK, RAD51, RRM2, and Chk-1.Conclusion: GSK212 confers radiosensitization to KRAS-driven PC cells by suppressing major DNA-DSB repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of PC.     

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.     

The role of the BRCA1 tumor suppressor in DNA double-strand break repair

The tumor suppressor gene BRCA1 was cloned in 1994 based on its linkage to early-onset breast and ovarian cancer. Although the BRCA1 protein has been implicated in multiple cellular functions, the precise mechanism that determines its tumor suppressor activity is not defined. Currently, the emerging picture is that BRCA1 plays an important role in maintaining genomic integrity by protecting cells from double-strand breaks (DSB) that arise during DNA replication or after DNA damage. The DSB repair pathways available in mammalian cells are homologous recombination and nonhomologous end-joining. BRCA1 function seems to be regulated by specific phosphorylations in response to DNA damage and we will focus this review on the roles played by BRCA1 in DNA repair and cell cycle checkpoints. Finally, we will explore the idea that tumor suppression by BRCA1 depends on its control of DNA DSB repair, resulting in the promotion of error-free and the inhibition of error-prone recombinational repair.     

RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner

Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.     

DNA双链断裂修复的选择性调控机制

在各种DNA损伤中,DNA双链断裂(double-strand break,DSB)是最为严重的一种,快速准确地修复DSB对维持基因组稳定性起着至关重要的作用。真核生物细胞通过一系列复杂的信号转导途径激活对DSB的修复,其中最为重要的是同源重组和非同源末端连接机制。最近的研究表明,这两种方式在DSB修复的早期是相互竞争的关系,其选择在很大程度上受到53BP1及同源蛋白质的调控。将讨论53BP1作为DSB修复途径的核心因子,在染色质水平整合BRCA1、Ct IP等修复因子和多种组蛋白修饰构成的信号途径,介导同源重组和非同源末端连接通路选择的分子机制。     

Double strand break repair by homologous recombination is regulated by cell cycle-independent signaling via ATM in human glioma cells

To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM(+), p53(+)) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G(0)/G(1), suggesting that ATM is also important for HRR outside of the S and G(2) cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.     

Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences

Mutation of BRCA2 causes familial early onset breast and ovarian cancer. BRCA2 has been suggested to be important for the maintenance of genome integrity and to have a role in DNA repair by homology- directed double-strand break (DSB) repair. By studying the repair of a specific induced chromosomal DSB we show that loss of Brca2 leads to a substantial increase in error-prone repair by homology-directed single-strand annealing and a reduction in DSB repair by conservative gene conversion. These data demonstrate that loss of Brca2 causes misrepair of chromosomal DSBs occurring between repeated sequences by stimulating use of an error-prone homologous recombination pathway. Furthermore, loss of Brca2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C-induced DNA cross-links at the expense of error-free repair by sister chromatid recombination. This provides insight into the mechanisms that induce genome instability in tumour cells lacking BRCA2.     

Analysis of DNA repair and recombination responses in mouse cells depleted for Brca2 by SiRNA

The tumor suppressor BRCA2 is considered to play an important role in the maintenance of genome integrity through the repair of DNA lesions by homologous recombination. A mechanistic understanding of BRCA2 has been complicated by the embryonic lethality of mice bearing allelic knockouts of Brca2, and by variation in the DNA damage response in cells bearing BRCA2 deficiencies. It would be advantageous to develop approaches that avoid the cell lethality associated with complete inactivation of the gene, or the use of established tumor cell lines in which other genes in addition to BRCA2 may be mutant. In this study, SiRNA was used in stable transformation assays to knockdown Brca2 in mouse hybridoma cells by at least 75%. The Brca2-depleted cells were analyzed with respect to cell growth, sensitivity to DNA damaging agents (mitomycin C, methylmethane sulfonate, or ionizing radiation), intrachromosomal homologous recombination and gene targeting. Although the effect of Brca2-depletion on cell growth and sensitivity to DNA damaging agents was modest, the Brca2-depleted cells did show a significant shift in homologous recombination from gene conversion to single-strand annealing and a significant decrease in the efficiency of gene targeting. Both of these phenotypes are consistent with the proposed role of Brca2 in DNA repair and recombination.     

Inhibition of poly(ADP-ribose)polymerase does not affect the recombination events in CHO xrs6 and wild type cells

Activation of poly (ADP-ribose) polymerase -1 (PARP-1) is an early DNA damage response event that, together with phosphorylation of p53, prompts various cellular functions important in the maintenance of the genome stability. In mammalian cells, DSB are repaired by nonhomologous end-joining (NHEJ) and by homologous recombination (HR). To investigate the role of PARP-1 in HR, CHO-K1 wild type and xrs-6 mutant cell line were transfected with pLrec plasmids which carry two nonfunctional copies of the β-galactosidase (lacZ) gene in a tandem array. In result of HR they can give rise to a functional copy of β-galactosidase. To test whether PARP-1 affects the frequency of spontaneous and induced recombination repair, we treated CHO-K1 and xrs6 clones carrying chromosomally integrated pLrec with the PARP-1 inhibitor 3-aminobenzamide (3AB). Our results show that the spontaneous homologous intrachromosomal recombination frequency between the two lacZ copies was almost two orders of magnitude higher in xrs6 cells than in CHO-K1 cells, but that it was not affected by 3AB treatment. Induction of DNA damage by irradiation or electroporation of restriction enzymes did not significantly increase the recombination frequency. Furthermore, in both the cell lines, the effect of PARP-1 inhibition on DSB repair was examined using the neutral comet assay. There was no effect of 3AB treatment on DSB rejoining after 10 Gy irradiation. The results presented support the conclusion that PARP-1 is not directly involved in HR.     

BRCA2 is required for homology-directed repair of chromosomal breaks

The BRCA2 tumor suppressor has been implicated in the maintenance of chromosomal stability through a function in DNA repair. In this report, we examine human and mouse cell lines containing different BRCA2 mutations for their ability to repair chromosomal breaks by homologous recombination. Using the I-SceI endonuclease to introduce a double-strand break at a specific chromosomal locus, we find that BRCA2 mutant cell lines are recombination deficient, such that homology-directed repair is reduced 6- to >100-fold, depending on the cell line. Thus, BRCA2 is essential for efficient homology-directed repair, presumably in conjunction with the Rad51 recombinase. We propose that impaired homology-directed repair caused by BRCA2 deficiency leads to chromosomal instability and, possibly, tumorigenesis, through lack of repair or misrepair of DNA damage.     

Measuring nonhomologous end-joining,homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.