RAD51 Foci Formation In Response Research Articles

Targeting the DNA Damage Response Pathways and Replication Stress in Colorectal Cancer.

PurposeGenomic instability is a hallmark of cancer and targeting DNA damage response (DDR) is emerging as a promising therapeutic strategy in different solid tumors. The effectiveness of targeting DDR in colorectal cancer has not been extensively explored.Experimental DesignWe challenged 112 cell models recapitulating the genomic landscape of metastatic colorectal cancer with ATM, ATR, CHK1, WEE1, and DNA-PK inhibitors, in parallel with chemotherapeutic agents. We focused then on ATR inhibitors (ATRi) and, to identify putative biomarkers of response and resistance, we analyzed at multiple levels colorectal cancer models highly sensitive or resistant to these drugs.ResultsWe found that around 30% of colorectal cancers, including those carrying KRAS and BRAF mutations and unresponsive to targeted agents, are sensitive to at least one DDR inhibitor. By investigating potential biomarkers of response to ATRi, we found that ATRi-sensitive cells displayed reduced phospho-RPA32 foci at basal level, while ATRi-resistant cells showed increased RAD51 foci formation in response to replication stress. Lack of ATM and RAD51C expression was associated with ATRi sensitivity. Analysis of mutational signatures and HRDetect score identified a subgroup of ATRi-sensitive models. Organoids derived from patients with metastatic colorectal cancer recapitulated findings obtained in cell lines.ConclusionsIn conclusion, a subset of colorectal cancers refractory to current therapies could benefit from inhibitors of DDR pathways and replication stress. A composite biomarker involving phospho-RPA32 and RAD51 foci, lack of ATM and RAD51C expression, as well as analysis of mutational signatures could be used to identify colorectal cancers likely to respond to ATRi.

Functional assessment of missense variants of uncertain significance in the cancer susceptibility gene PALB2

Germline PALB2 pathogenic variants are associated with an increased lifetime risk for breast, pancreatic, and ovarian cancer. However, the interpretation of the pathogenicity of numerous PALB2 missense variants of uncertain significance (VUSs) identified in germline genetic testing remains a challenge. Here we selected ten potentially pathogenic PALB2 VUSs identified in 2279 Chinese patients with breast cancer and evaluated their impacts on PALB2 function by systematic functional assays. We showed that three PALB2 VUSs p.K16M [c.47 A > T], p.L24F [c.72 G > C], and p.L35F [c.103 C > T] in the coiled-coil domain impaired PALB2-mediated homologous recombination. The p.L24F and p.L35F variants partially disrupted BRCA1-PALB2 interactions, reduced RAD51 foci formation in response to DNA damage, abrogated ionizing radiation-induced G2/M checkpoint maintenance, and conferred increased sensitivity to olaparib and cisplatin. The p.K16M variant presented mild effects on BRCA1-PALB2 interactions and RAD51 foci formation. Altogether, we identify two novel PALB2 VUSs, p.L24F and p.L35F, that compromise PALB2 function and may increase cancer risk. These two variants display marked olaparib and cisplatin sensitivity and may help predict response to targeted therapy in the clinical treatment of patients with these variants.

DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.

Functional analysis of patient-derived PALB2 missense variants of uncertain significance.

1531 Background: Inherited PALB2 pathogenic variants are associated with an increased lifetime risk for breast cancer development. However, the interpretation of numerous PALB2 missense variants of uncertain significance (VUS) identified in germline genetic testing remains a challenge. Here, we assessed the impact of breast cancer patient-derived VUS on PALB2 function and identified pathogenic PALB2 missense variants that may increase cancer risk. Methods: A total of seven potentially pathogenic PALB2 VUS identified in 2,279 breast cancer patients were selected for functional analysis. All these selected VUS were assessed by SIFT, Align-GVGD, and PolyPhen2 in silico and were predicted to be deleterious by at least two in silico algorithms. The p.L35P [c.104T > C] variant was also included, for which pathogenicity has been recently confirmed. The effects of the VUS on the homologous recombination (HR) activity of PALB2 were tested by U2OS/DR-GFP reporting system. Functional characterization was further validated by protein co-immunoprecipitation and RAD51 recruitment assay. Results: PALB2 variants p.L24F [c.72G > C] and p.L35P [c.104T > C] showed the most significant disruption to the HR activity of PALB2 relative to the wild-type condition, retaining only 52.2% ( p = 0.0013) and 8.5% ( p < 0.0001) of HR activity respectively. Moderate but statistically significant HR deficiency was observed for four other variants (p.P405A [c.1213C > G], p.T1012I [c.3035C > T], p.E1018D [c.3054G > C], and p.T1099M [c.3296C > T]). We found no statistical differences for the p.K628N [c.1884G > T] and p.R663C [c.1987C > T] in the HR activity compared to wild-type PALB2. The p.L24F and p.L35P variants compromised the BRCA1-PALB2 interaction and reduced RAD51 foci formation in response to DNA damage. Conclusions: We have identified a novel patient-derived pathogenic PALB2 missense variant, p.L24F [c.72G > C], that compromises PALB2-mediated HR activity. We suggest the integration of the identified pathogenic variants into breast cancer genetic counseling and individualized treatment regimens for better clinical outcomes.

Functional characterization of 84 PALB2 variants of uncertain significance

PurposeInherited pathogenic variants in PALB2 are associated with increased risk of breast and pancreatic cancer. However, the functional and clinical relevance of many missense variants of uncertain significance (VUS) identified through clinical genetic testing is unclear. The ability of patient-derived germline missense VUS to disrupt PALB2 function was assessed to identify variants with potential clinical relevance. MethodsThe influence of 84 VUS on PALB2 function was evaluated using a cellular homology directed DNA repair (HDR) assay and VUS impacting activity were further characterized using secondary functional assays. ResultsFour (~5%) variants (p.L24S,c.71T>C; p.L35P,c.104T>C; pI944N,c.2831T>A; and p.L1070P,c.3209T>C) disrupted PALB2-mediated HDR activity. These variants conferred sensitivity to cisplatin and a poly(ADP-ribose) polymerase (PARP) inhibitor and reduced RAD51 foci formation in response to DNA damage. The p.L24S and p.L35P variants disrupted BRCA1–PALB2 protein complexes, p.I944N was associated with protein instability, and both p.I944N and p.L1070P mislocalized PALB2 to the cytoplasm. ConclusionThese findings show that the HDR assay is an effective method for screening the influence of inherited variants on PALB2 function, that four missense variants impact PALB2 function and may influence cancer risk and response to therapy, and suggest that few inherited PALB2 missense variants disrupt PALB2 function in DNA repair.

RAD51 regulates REV1 recruitment to DNA double-strand break sites

REV1 is one of the major Y-family DNA polymerases. It not only functions as a scaffold protein to mediate other specialized DNA polymerases to sites of lesions, but also inserts deoxycytidine across the lesion strand during translesion DNA synthesis (TLS). Meanwhile, REV1 has been reported to be involved in homologous recombination (HR) repair. Here we further explore the roles of REV1-interacting proteins RAD51 and RAD51C in REV1-mediated DNA double-strand break (DSB) repair. We found that RAD51 but not RAD51C regulates REV1 recruitment to DSB sites via pulsed laser microirradiation. Interestingly, immunofluorescence staining exhibits that REV1 also regulates RAD51 focus formation in response to CPT treatment. These results suggest that REV1 and RAD51 might be mutually dependent on each other in the REV1-related HR pathway.

Abstract IA21: Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer

Abstract Pancreas cancer is projected to cause approximately 42,000 deaths in 2016, making it the third greatest cause of cancer mortality (behind lung and colorectal, but greater even than breast). Systemic failure remains the dominant cause of death, although local failure is responsible for up to 1/3 of the pancreatic cancer related mortality. We have focused on improving the treatment of locally advanced pancreatic cancer, defined as disease which cannot be resected but has not overtly metastasized, where the issue of local control becomes even more important, and have hypothesized that the treatment of locally advanced pancreatic cancer will require control of both the gross primary tumor as well as the microscopic disease that exists in virtually every patient. Gemcitabine has been a mainstay of treatment for pancreatic cancer, and we have standardized combining it with radiation for locally advanced disease. Because our initial studies revealed that activation of the DNA damage response, particularly activation of CHK1 and WEE1, were crucial in the defense pancreatic cancers mount against chemotherapy and radiation, we have studied the effect of CHK1 and WEE1 inhibitors on gemcitabine and gemcitabine-radiation mediated cytotoxicity. These inhibitors abrogate the G2 checkpoint after DNA damage. The initial hypothesis for using CHK1 and WEE1 inhibitors was that cancer cells lacking a p53 checkpoint would be selectively sensitized, compared to normal cells, because normal cells would be protected by an intact G1 checkpoint. After initial studies with several agents, we have focused on the WEE1 inhibitor AZD1775, chiefly because it was available for a clinical trial. We have carried out both mechanistic and clinical studies using AZD1775. Mechanistically, we have evaluated both the contributions of homologous recombination repair (HRR) inhibition and G2 checkpoint abrogation by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistoneH3 flow cytometry, respectively in pancreatic cancer model systems. AZD1775 causes inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. Sensitization by AZD1775 is associated with persistent γH2AX and inhibition of RAD51 focus formation. Using BRCA2 isogenic cells, we have found that AZD1775 sensitizes to gemcitabine-radiation in BRCA2 wild type cells, while in BRCA2 null cells AZD1775 did not sensitize despite significant G2 checkpoint abrogation. In vivo, in patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibits tumor growth and impairs RAD51 focus formation in response to gemcitabine-radiation. We've concluded that, whereas sensitization is accompanied by inhibition of CDK1 phosphorylation and abrogation of the G2 checkpoint, this mechanism is not sufficient for sensitization. Our findings suggest that sensitization to chemoradiation by WEE1 inhibition also requires inhibition of HRR. The importance of HRR has also supported studies combining CHK1 or WEE1 inhibition with a PARP inhibitor, which would be anticipated to potentiate the effects of HRR inhibition. Based on these studies, we have launched a clinical trial combining gemcitabine and gemcitabine-radiation, at standard doses, with dose escalating AZD1775 for patients with locally advanced pancreatic cancer. Patients receive one cycle of gemcitabine with AZD1775, then 2 cycles of gemcitabine-radiation with AZD1775, then another cycle of AZD1775-gemcitabine, followed by assessment for response and toxicity. Optional cycles of AZD1775 and gemcitabine are then given to patients who have not progressed. AZD1775 has been escalated according to a Time-to-Event Continual Reassessment Model, in which the dose for each patient is based on the toxicity profile of all prior patients, and the goal is to establish a tolerable dose of the combination therapy and to estimate the response and survival. With 21 patients entered onto the study, the median survival has not been reached, and the 64% survival at the 18-month landmark point exceeds the 18-month median survival that was the agreed on endpoint for triggering a larger trial. The trial also includes a pharmacodynamic endpoint of a hair follicle biopsy to assess target engagement. In summary, these findings suggest that WEE1 inhibition may act through both G2 checkpoint abrogation but, more importantly, inhibition of HRR, and is a promising potentiator of gemcitabine-radiation in the treatment of locally advanced pancreatic cancer. Supported by P50 CA130810, R01 CA163895, P30 CA46592, and the Taubman Institute Citation Format: Theodore S. Lawrence, Meredith A. Morgan. Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer [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 IA21.

Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair

To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.

Abstract 3019: GIPC high expression rescues BRCA2 deficiency and promotes tumorigenesis

Abstract Breast cancer is the most prevalent cancer in women worldwide and mutation in breast cancer susceptible gene 2 (BRCA2) is one of the genetic factors conferring highest risk (40-70%) of developing breast cancer. Although BRCA2 proteins are known to maintain genomic stability mainly by homologous recombination-mediated DNA repair, a detailed mechanism of how BRCA2 loss induces tumorigenesis remains unclear. Interestingly, BRCA2 null mice display embryonic lethality, while inactivation of both alleles of BRCA2 in tumor cells renders them able to survive and are predisposed to tumorigenesis only when there are mutations in BRCA2 genetic interactors. In the current study, we tried to identify such genes using a mouse embryonic stem cell (mESC)-based insertional mutagenesis screen. In mESCs with one knockout allele of Brca2 and the other allele flanked by loxP sites, cell death can be induced by Cre-mediated loss of the conditional allele. To identify genes whose up-regulation can rescue cell death induced by loss of BRCA2, we transduced these mESC with murine stem cell retrovirus (MSCV) expressing Cre recombinase. We obtained Brca2-null mESCs that were rescued by the viral insertion and used them to clone the viral insertion sites. We identified several candidate genes that were up-regulated in viable mESCs. One of these genes encodes a PDZ domain-containing protein, GIPC3, and expression of Gipc3 mRNA was elevated 3 to 4-fold in the rescued clone. We were able to rescue lethality of Brca2-null mESC by overexpressing GIPC3. The rescued cells lost RAD51 foci formation in response to irradiation and were hypersensitive to DNA damaging agents and exhibited an overall increase in genomic instability. Mechanistically, GIPC3 can bind to APPL1 and APPL2, two adaptor proteins involved in AKT-mediated cell survival pathway. We found an increased level of the active form of AKT in GIPC3-rescued cells, and a knock down of APPL1 and APPL2 blocked the rescue by GIPC3 overexpression. We found that the Gipc3 transgene can partially rescue the lethality of Brca2 null embryos and contribute to tumorigenesis in K14-Cre; Brca2cko/cko mice. Besides GIPC3, another GIPC family member, GIPC1 can also rescue BRCA2 loss-induced lethality in mESCs. Oncomine analysis showed Gipc2 mRNA up-regulation in BRCA1 deficient human breast tumors, suggesting the possibility that high expression of all the GIPC family members (GIPC1, 2 and 3) might facilitate BRCA1 or BRCA2-loss induced tumorigenesis. Citation Format: Xia Ding, Shyam K. Sharan. GIPC high expression rescues BRCA2 deficiency and promotes tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3019. doi:10.1158/1538-7445.AM2015-3019

Hypoxia Enhances the Radioresistance of Mouse Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are radioresistant bone marrow progenitors that support hematopoiesis and its reconstitution following total body irradiation. MSCs reside in hypoxic niches within the bone marrow and tumor microenvironments. The DNA damage response (DDR) represents a network of signaling pathways that enable cells to activate biological responses to DNA damaging agents. Hypoxia-mediated alterations in the DDR contribute to the increased radioresistance of hypoxic cancer cells, limiting therapeutic efficacy. The DDR is important in mediating mouse MSC radioresistance. However, the effects of hypoxia on MSC radioresistance are currently unknown. In this report, hypoxia was found to (a) increase MSC proliferation rate and colony size; (b) increase long-term survival post-irradiation (IR), and (c) improve MSC recovery from IR-induced cell cycle arrest. DNA double-strand break (DSB) repair in MSCs was upregulated in hypoxia, accelerating the resolution of highly genotoxic IR-induced DNA DSBs. In addition, HIF-1α was found to contribute to this enhanced DSB repair by regulating (a) the expression of DNA ligase IV and DNA-PKcs and (b) Rad51 foci formation in response to DNA DSBs in hypoxic MSCs. We have demonstrated, for the first time, that hypoxia enhances mouse MSC radioresistance in vitro. These findings have important implications for our understanding of MSC functions in supporting allogeneic bone marrow transplantation and in tumorigenesis.

Systematic Screen Identifies miRNAs That Target RAD51 and RAD51D to Enhance Chemosensitivity

Homologous recombination mediates error-free repair of DNA double-strand breaks (DSB). RAD51 is an essential protein for catalyzing homologous recombination and its recruitment to DSBs is mediated by many factors including RAD51, its paralogs, and breast/ovarian cancer susceptibility gene products BRCA1/2. Deregulation of these factors leads to impaired DNA repair, genomic instability, and cellular sensitivity to chemotherapeutics such as cisplatin and PARP inhibitors. microRNAs (miRNA) are short, noncoding RNAs that posttranscriptionally regulate gene expression; however, the contribution of miRNAs in the regulation of homologous recombination is not well understood. To address this, a library of human miRNA mimics was systematically screened to pinpoint several miRNAs that significantly reduce RAD51 foci formation in response to ionizing radiation in human osteosarcoma cells. Subsequent study focused on two of the strongest candidates, miR-103 and miR-107, as they are frequently deregulated in cancer. Consistent with the inhibition of RAD51 foci formation, miR-103 and miR-107 reduced homology-directed repair and sensitized cells to various DNA-damaging agents, including cisplatin and a PARP inhibitor. Mechanistic analyses revealed that both miR-103 and miR-107 directly target and regulate RAD51 and RAD51D, which is critical for miR-103/107-mediated chemosensitization. Furthermore, endogenous regulation of RAD51D by miR-103/107 was observed in several tumor subtypes. Taken together, these data show that miR-103 and miR-107 overexpression promotes genomic instability and may be used therapeutically to chemosensitize tumors. These findings demonstrate a role for miR-103 and -107 in regulating DNA damage repair, thereby identifying new players in the progression of cancer and response to chemotherapy.

FANCJ/BACH1 Acetylation at Lysine 1249 Regulates the DNA Damage Response

BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance.

Abstract 1459: Combined inhibition of Wee1 and PARP1 sensitizes pancreatic cancer cells to radiation

Abstract PARP1 [poly (ADP-ribose) polymerase-1] inhibitors are maximally effective in cells with HRR (homologous recombination repair) defects, a concept known as synthetic lethality. We have previously shown that inhibition of HRR and the G2 checkpoint by small molecule inhibitors of Chk1 (checkpoint kinase 1), can induce synthetic lethality by increasing PARP1 inhibitor-mediated radiosensitization selectively in tumor cells and preferentially in tumor cells with p53 mutations. In order to begin to determine whether other inhibitors of the DNA damage response might induce synthetic lethality in combination with PARP1 inhibition and radiation, we evaluated MK-1775, a Wee1 kinase inhibitor. Wee1 regulates the G2 checkpoint by phosphorylation of Cdk1, thus preventing entry into mitosis in the presence of DNA damage, while its influence on HRR is not clear. To determine whether inhibition of Wee1 might synergistically radiosensitize in combination with PARP1 inhibition, we treated pancreatic cancer cells with MK-1775 (100-200nM) and the PARP1 inhibitor, olaparib (1uM) and assessed radiosensitization by clonogenic survival. We found that the combination of MK-1775 with olaparib induced marked radiosensitization in MiaPaCa-2 (RER: 1.9) and AsPC-1 (RER: 1.7) pancreatic cancer cells, which was greater than the radiosensitization produced by either agent alone. In order to determine the mechanism(s) by which Wee1 and PARP1 inhibitors interact to produce radiosensitization, we investigated the G2 checkpoint as well as HRR. In response to radiation, MK-1775 abrogated the G2 checkpoint while olaparib treatment resulted in increased G2 accumulation which was associated with increased γH2AX, suggesting the presence of unrepaired DNA double strand breaks. Abrogation of the olaparib+radiation-mediated G2 checkpoint, by MK-1775 resulted in even greater DNA damage as assessed by γH2AX expression. Given the efficacy of PARP1 inhibitors in HRR defective cells, we also investigated the effect of MK-1775 on HRR. We found that MK-1775 inhibited homology-directed repair of an I-SceI endonuclease-induced DNA double strand break in MiaPaCa-2 cells (Control 5.2% versus MK-1775 3.3%, P<0.05) as well as impaired Rad51 focus formation in response to radiation. Taken together, these results suggest that inhibition of HRR and the G2 checkpoint by inhibition of Wee1 can induce synthetic lethality with PARP1 inhibitors in combination with radiation by increasing unrepaired DNA double strand breaks. These studies support the continued investigation of combined molecularly targeted agents such as Wee1 and PARP1 inhibitors with radiation, to induce synthetic lethality selectively in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1459. doi:1538-7445.AM2012-1459

Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cells

In vertebrate cells, the five RAD51 paralogs (XRCC2/3 and RAD51B/C/D) enhance the efficiency of homologous recombination repair (HRR). Stalling and breakage of DNA replication forks is a common event, especially in the large genomes of higher eukaryotes. When cells are exposed to agents that arrest DNA replication, such as hydroxyurea or aphidicolin, fork breakage can lead to chromosomal aberrations and cell killing. We assessed the contribution of the HRR protein RAD51D in resistance to killing by replication-associated DSBs. In response to hydroxyurea, the isogenic rad51d null CHO mutant fails to show any indication of HRR initiation, as assessed by induction RAD51 foci, as expected. Surprisingly, these cells have normal resistance to killing by replication inhibition from either hydroxyurea or aphidicolin, but show the expected sensitivity to camptothecin, which also generates replication-dependent DSBs. In contrast, we confirm that the V79 xrcc2 mutant does show increased sensitivity to hydroxyurea under some conditions, which was correlated to its attenuated RAD51 focus response. In response to the PARP1 inhibitor KU58684, rad51d cells, like other HRR mutants, show exquisite sensitivity (>1000-fold), which is also associated with defective RAD51 focus formation. Thus, rad51d cells are broadly deficient in RAD51 focus formation in response to various agents, but this defect is not invariably associated with increased sensitivity. Our results indicate that RAD51 paralogs do not contribute equally to cellular resistance of inhibitors of DNAreplication, and that the RAD51 foci associated with replication inhibition may not be a reliable indicator of cellular resistance to such agents.

Mutation of the RAD51C gene in a Fanconi anemia–like disorder

Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers. We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination-mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome.

Selective Anti-Leukemia Targeting of the Interaction Between BCR/ABL and Mammalian RecA Homologs

We showed before that cells transformed by BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2 and TEL/PDGFR, inducing chronic myeloproliferative disorders (MPDs), and CD34+ chronic myeloid leukemia (CML) stem/ progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain an excess of DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress [Blood, 2005; Cell Cycle, 2006; DNA Repair, 2006; Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cellenriched populations seem to display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Elevated levels of DSBs were also observed in leukemia cells expressing imatinib-resistant BCR/ABL kinase mutants. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. Numerous ROS- and radiation- induced DSBs are not lethal for BCR/ABL-positive leukemia cells; instead, they induce chromosomal instability implicating enhanced, but unfaithful repair [Leukemia, 2008]. The previous report [Mol. Cell, 2001] and ongoing studies demonstrated that BCR/ABL kinase (non-mutated and imatinib-resistant mutants) modulates expression of the mammalian RecA homologs RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which are responsible for homologous recombination repair (HRR) of DSBs. RAD51 plays a key role in HRR in cells transformed by BCR/ ABL and other FTKs [Mol. Cell, 2001; Mol. Cell. Biol., 2002]. BCR/ABL stimulates the expression of, interacts with and phosphorylates RAD51 on Y315, which is located in a critical fragment of RAD51 essential for its filament formation on DNA. Accordingly, our recent results indicated that BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for nuclear RAD51 foci formation in response to DNA damage. In addition to RAD51, BCR/ABL interacts directly with and phosphorylates RAD51B and XRCC2, but not other RecA homologs. Altogether, it appears that BCR/ABL can deregulate the expression and phosphorylation of some RecA homologs, which may have a significant impact on the efficiency and fidelity of DSB repair resulting in protection from apoptosis and chromosomal instability. Therefore, disassembly of BCR/ABL from RecA homologs should reduce the capability of CML cells to repair numerous ROS-induced DSBs and eventually trigger apoptosis. Based on this hypothesis we investigated the mechanisms of association between BCR/ABL and RecA homologs. Interactions between BCR/ ABL and RAD51 or RAD51B depend on the proline- rich (PP) regions of RAD51 and RAD51B, and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL, which form a pocket binding the PP regions. Disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) abrogated direct interaction with the BCR/ABL SH3-SH2-CD pocket. On the other hand, single amino acid substitutions in the BCR/ABL SH3-SH2-CD pocket, which eliminated its capability of binding the PP regions, prevented complex formation with RAD51 and RAD51B. In addition, RAD51 and RAD51B may interact with members of the BCR/ABL proteome such as Grb2 and Shc (RAD51 and RAD51B), and c-CrkL (only RAD51B), but not Gab2 and c-Cbl. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3-SH2-CD pocket mutant, where single amino acid substitutions disrupted its direct interaction with RAD51, displayed a slower proliferation rate and responded poorly to genotoxic stress despite intact kinase activity in comparison to cells transformed with non-mutated BCR/ABL. Interestingly, expression of RAD51 PP-LL mutant eliminated BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. These results suggest that the interaction between BCR/ABL and RAD51 may be targeted for selective elimination of leukemia cells and/or suppression of genomic instability. To test this hypothesis we are employing the peptide aptamer strategy targeting RAD51 PP regions in CD34+ cells obtained from imatinib-sensitive and imatinib-resistant CML patients and healthy volunteers in vivo and in vitro. In summary, we hypothesize that mechanisms regulating the association of BCR/ABL with RAD51 and other mammalian RecA homologs may be explored for the planning of more effective anti-tumor modalities.

RAD51 foci formation in response to DNA damage is modulated by TIP49

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.

BRCA1 Promotes Induction of ssDNA by Ionizing Radiation

The BRCA1 tumor suppressor contributes to the repair of DNA double-strand breaks (DSB) through homologous recombination, but the mechanism is unknown. The rapid accumulation of BRCA1 into nuclear foci in response to induction of DNA breaks suggests that BRCA1 may function in an early step in the repair pathway. We examined the role of BRCA1 in one such early step, the resection of DSBs to generate ssDNA. The appearance of ssDNA in response to ionizing radiation is similar to that of BRCA1 foci formation, suggesting that the two processes are related. Furthermore, BRCA1 colocalizes to ssDNA sites induced by ionizing radiation. Overexpression of BRCA1 causes an increase in cells exhibiting ssDNA induced by ionizing radiation. Mutant BRCA1 that lacks the COOH-terminal BRCT domain also promotes ssDNA but fails to form nuclear foci. Knockdown of BRCA1 expression reduces ssDNA and Rad51 foci formation in response to ionizing radiation. These results indicate that BRCA1 promotes induction of ssDNA in response to ionizing radiation and accumulates at sites of ssDNA.

P53 modulates homologous recombination by transcriptional regulation of the RAD51 gene

DNA repair by homologous recombination is involved in maintaining genome stability. Previous data report that wild-type p53 suppresses homologous recombination and physically interacts with Rad51. Here, we show the in vivo binding of wild-type p53 to a p53 response element in the promoter of Rad51 and the downregulation of Rad51 messenger RNA and protein by wild-type p53, favoured by DNA damage. Moreover, wild-type p53 inhibits Rad51 foci formation in response to double-strand breaks, whereas p53 contact mutant R280K fails to repress Rad51 mRNA and protein expression and Rad51 foci formation. We propose that transcriptional repression of Rad51 by p53 participates in regulating homologous recombination, and impaired Rad51 repression by p53 mutants may contribute to malignant transformation.

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.