Repair Of DNA Interstrand Crosslinks Research Articles

RA09.05: MDM2 AND ERCC1: NOVEL BIOMARKERS TO PREDICT THE RESPONSE TO NEOADJUVANT CHEMOTHERAPY FOR ESOPHAGEAL CANCER PATIENTS

Abstract Background Neoadjuvant chemotherapy (NAC) by 5-fluorouracil (5-FU) and cisplatin followed by esophagectomy has been established as a standard approach for the patients with clinical stage II/III esophageal squamous cell carcinoma (ESCC) in Japan. However, NAC is not always effective in all these patients, and it is important to identify biomarkers for predicting the response. Therefore, the present study aimed to identify markers for predicting the response to chemotherapy and the clinical outcomes. Methods This study included 62 ESCC patients who underwent esophagectomy following NAC. We performed immunohistochemical analyses for murine double minute 2 (MDM2), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and excision repair cross-complementing rodent repair deficiency-1 (ERCC1) in surgical specimens following NAC and assessed the associations of the findings with clinicopathological features in individual patients. Results The MDM2 status and ERCC1 status demonstrated significant inverse associations with the response to NAC (MDM2: P = 0.025, ERCC1: P = 0.021). In addition, a high TP status in the tumor was significantly associated with tumor differentiation (P = 0.008), depth of invasion (P < 0.001), lymphatic invasion (P = 0.003), venous invasion (P < 0.001) and tumor stage (P = 0.006). Moreover, a high DPD status was associated with tumor differentiation (P = 0.008), depth of invasion (P < 0.001), venous invasion (P = 0.003) and tumor stage (P = 0.007). The TP status also significantly predicted the eventual clinical outcome of patients following surgery. Conclusion Recently, overexpression of MDM2 has been reported for poor prognosis in patients with ESCC treated by chemoradiotherapy. 5-FU metabolic enzymes, such as TP andDPD, have been investigated for sensitivity to 5-FU. ERCC1, a key component of homologous recombination-based repair of interstrand DNA cross-links, has been reported to predict sensitivity to cisplatin. In our study, MDM2 and ERCC1 immunoreactivity predict chemosensitivity of NAC based on 5-FU and cisplatin. High intratumoral TP status and DPD status are associated with tumor progression and prognosis in ESCC patients receiving NAC. Disclosure All authors have declared no conflicts of interest.

RUNX Poly(ADP-Ribosyl)ation and BLM Interaction Facilitate the Fanconi Anemia Pathway of DNA Repair

The Fanconi anemia (FA) pathway is a pivotal genome maintenance network that orchestrates the repair of DNA interstrand crosslinks (ICLs). The tumor suppressors RUNX1 and RUNX3 were shown to regulate the FA pathway independent of their canonical transcription activities, by controlling the DNA damage-dependent chromatin association of FANCD2. Here, in further biochemical characterization, we demonstrate that RUNX3 is modified by PARP-dependent poly(ADP-ribosyl)ation (PARylation), which in turn allows RUNX binding to DNA repair structures lackingtranscription-related RUNX consensus motifs. SILAC-based mass spectrometric analysis revealed significant association of RUNX3 with core DNA repair complexes, including PARP1, even in unstressed cells. After DNA damage, the increased interaction between RUNX3 and BLM facilitates efficient FANCD2 chromatin localization. RUNX-Walker motif mutations from breast cancers are impaired for DNA damage-inducible PARylation, unveiling a potential mechanism for FA pathway inactivation in cancers. Our results reinforce the emerging paradigm that RUNX proteins are tumor suppressors with genome gatekeeper function.

Case study: patient-derived clear cell adenocarcinoma xenograft model longitudinally predicts treatment response

There has been little progress in the use of patient-derived xenografts (PDX) to guide individual therapeutic strategies. In part, this can be attributed to the operational challenges of effecting successful engraftment and testing multiple candidate drugs in a clinically workable timeframe. It also remains unclear whether the ancestral tumor will evolve along similar evolutionary trajectories in its human and rodent hosts in response to similar selective pressures (i.e., drugs). Herein, we combine a metastatic clear cell adenocarcinoma PDX with a timely 3 mouse x 1 drug experimental design, followed by a co-clinical trial to longitudinally guide a patient’s care. Using this approach, we accurately predict response to first- and second-line therapies in so far as tumor response in mice correlated with the patient’s clinical response to first-line therapy (gemcitabine/nivolumab), development of resistance and response to second-line therapy (paclitaxel/neratinib) before these events were observed in the patient. Treatment resistance to first-line therapy in the PDX is coincident with biologically relevant changes in gene and gene set expression, including upregulation of phase I/II drug metabolism (CYP2C18, UGT2A, and ATP2A1) and DNA interstrand cross-link repair (i.e., XPA, FANCE, FANCG, and FANCL) genes. A total of 5.3% of our engrafted PDX collection is established within 2 weeks of implantation, suggesting our experimental designs can be broadened to other cancers. These findings could have significant implications for PDX-based avatars of aggressive human cancers.

Abstract 340: CTDP1 regulates FANCI activation and DNA repair

Abstract Objective: The modular BRCA1 C-terminal (BRCT) domain plays a central role in the regulation of DNA damage repair (DDR). RNA Pol II C-terminal domain phosphatase 1 (CTDP1), the only phosphatase encoding a BRCT domain, is one of the few BRCT domain-containing proteins without a formal role ascribed to DDR. Thus, the goal of this study is to characterize the DDR-specific role of CTDP1 through interrogation of the BRCT domain-protein interaction with FANCI and the repair of DNA interstrand cross-links (ICL) through the Fanconi anemia pathway. Methods: Novel CTDP1 BRCT domain-protein interactions were identified with mass spectrometry and analyzed with gene ontology enrichment. Molecular biology experiments were conducted to examine the effects of CTDP1 knockdown or overexpression on cell growth, survival, apoptosis, regulation of FANCI activation and localization, FANCD2 foci formation and ubiquitination, and homologous recombination repair. Mass spectrometry was used to determine both CTDP1-regulated FANCI phosphorylation sites and FANCI protein interactions. Results: The BRCT domain of CTDP1 was found to interact with three Fanconi anemia proteins-FANCA, FANCD2, and FANCI. This interaction remains unchanged upon mitomycin C treatment. Loss of CTDP1 induces a four-fold decrease in homologous recombination repair efficiency. CTDP1 prevents ICL-induced apoptosis. Knockdown of CTDP1 negatively affects plating efficiency and decreases the rate of cellular growth in breast cancer cell lines, but not the untransformed MCF-10A breast cell line. CTDP1 prevents sensitivity to DNA-damaging agents repaired by homologous recombination, but not UV damage or treatment with paclitaxel. CTDP1 knockdown negatively impacts FANCD2 activation measured by DNA damage-induced foci formation and monoubiquitination, which is critical for the onset of ICL repair by the ID2 complex and downstream effector proteins of the Fanconi anemia pathway. CTDP1 regulates 15 phosphorylation sites in FANCI determined by mass spectrometry profiling of cells overexpressing wild-type CTDP1 compared to phosphatase-dead D302K mutant. Paradoxically, CTDP1 phosphatase significantly increases SQ motif phosphorylation of S559 and S556, which is important for dormant origin firing and replication fork restart. This suggests that phosphorylation regulation of FANCI at non-SQ motif residues could regulate FANCI activation potential, resulting in increased phosphorylation at the SQ motif. Significance: This is the first demonstration of a formal role of CTDP1 in DDR. CTDP1 is implicated in the regulation of the specific ICL-repair Fanconi anemia pathway, and regulates breast cancer cell survival in vitro and in vivo. Delineating the mechanism by which CTDP1 regulates FANCI activation can provide the basis for preclinical studies targeting CTDP1 in breast cancer to promote sensitivity to DNA-damaging therapies. Citation Format: Kimiko L. Krieger, Wen-Feng Hu, Dragana Lagundzin, Nicholas T. Woods. CTDP1 regulates FANCI activation and DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 340.

Abstract 2825: PIN1-SCFFBW7 proteolytic signaling regulates the Fanconi anemia pathway

Abstract Genome instability caused by defective DNA repair mechanisms is a hallmark of cancer and drives tumorigenesis. The Fanconi anemia (FA) DNA repair pathway contributes to the integrity of the genome by resolving DNA interstrand cross-links (ICLs) encountered during DNA replication. Deregulation of the FA pathway is associated with cancer predisposition and affects therapeutic outcomes against DNA-damaging cytotoxic chemotherapy. FANCD2 monoubiquitination by a multi-subunit ubiquitin E3 ligase, the FA core complex, is an essential gateway that connects the DNA damage response to enzymatic steps of DNA ICL repair. We have previously identified FAAP20 as a critical component of the FA pathway; and demonstrated that the phosphorylation of the Cdc4 phospho-degron (CPD) motif in FAAP20 by GSK3β is required for its proteasomal degradation mediated by the E3 ligase, SCFFBW7 complex. However, the upstream signaling that governs the FAAP20 phosphorylation status and its detailed mode of action for FAAP20 degradation remain elusive. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, is a key regulator of the integrity of the FA core complex and, therefore, FA pathway activity as a whole. By catalyzing a proline cis-trans isomerization of the phosphorylated backbone and thereby converting the substrate into a conformation that is favorable or refractory to downstream proteolytic signaling, PIN1 modulates the rate of protein turnover. We demonstrate that PIN1 interacts and catalyzes a phosphorylated Ser48-Pro49 motif of FAAP20 and by inducing a conformational change of FAAP20, PIN1 enhances the affinity of FAAP20 with PP2A (Protein phosphatase 2), the phosphatase of the CPD motif in FAAP20 and thereby prevents its degradation from the GSK3β-FBW7 mediated proteolytic signaling. Collectively, our studies uncover PIN1-dependent isomerization as a new regulatory mechanism for DNA ICL repair. Given that PIN1 overexpression is prevalent in diverse human cancers, identifying a way to regulate PIN1 activity in the FA pathway may help develop a PIN1 inhibitor as a chemosensitizer for cytotoxic chemotherapy to increase its therapeutic efficacy. Citation Format: Jingming Wang, Bryan Chan, Michael Tong, Markus Seeliger, Hyungjin Kim. PIN1-SCFFBW7 proteolytic signaling regulates the Fanconi anemia pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2825.

Map of synthetic rescue interactions for the Fanconi anemia DNA repair pathway identifies USP48

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.

Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading

SUMMARYDNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate.

Co-inhibition of Pol η and ATR sensitizes cisplatin-resistant non-small cell lung cancer cells to cisplatin by impeding DNA damage repair.

For the majority of patients with advanced non-small cell lung cancer (NSCLC), the standard of care remains platinum-based chemotherapy. However, cisplatin resistance is a big obstacle to the treatment, and elucidation of its mechanism is warranted. In this study, we showed that there was no difference in intracellular uptake of cisplatin or the removal of platinum-DNA adducts between a cisplatin-resistant NSCLC cell line (A549/DR) and a cisplatin-sensitive NSCLC cell line (A549). However, the capacity to repair DNA interstrand crosslinks (ICLs) and double-strand breaks (DSBs) was significantly enhanced in the A549/DR cell line compared to 3 cisplatin-sensitive cell lines. We found that the protein and mRNA expression levels of Pol η, a Y-family translesion synthesis (TLS) polymerase, were markedly increased upon cisplatin exposure in A549/DR cells compared with A549 cells. Furthermore, intracellular co-localization of Pol η and proliferation cell nuclear antigen (PCNA) induced by cisplatin or cisplatin plus gemcitabine treatment was inhibited by depleting ataxia telangiectasia mutated and Rad-3-related (ATR). Pol η depletion by siRNA sensitized A549/DR cells to cisplatin; co-depletion of Pol η and ATR further increased A549/DR cell death induced by cisplatin or cisplatin plus gemcitabine compared to depletion of Pol η or ATR alone, concomitant with inhibition of DNA ICL and DSB repair and accumulation of DNA damage. No additional sensitization effect of co-depleting Pol η and ATR was observed in A549 cells. These results demonstrate that co-inhibition of Pol η and ATR reverses the drug resistance of cisplatin-resistant NSCLC cells by blocking the repair of DNA ICLs and DSBs induced by cisplatin or cisplatin plus gemcitabine.

Targeted mass spectrometry enables robust quantification of FANCD2 mono-ubiquitination in response to DNA damage

The Fanconi anemia pathway is an important coordinator of DNA repair pathways and is particularly relevant to repair of DNA inter-strand crosslinks. Central to the pathway is monoubiquitination of FANCD2, requiring the function of multiple proteins in an upstream Fanconi core complex. We present development and analytical characterization of a novel assay for quantification of unmodified and monoubiquitinated FANCD2 proteoforms, based on peptide immunoaffinity enrichment and targeted multiple reaction monitoring mass spectrometry (immuno-MRM). The immuno-MRM assay is analytically characterized using fit-for-purpose method validation. The assay linear range is >3 orders of magnitude with total repeatability <16% CV. In proof-of-principle experiments, we demonstrate application of the multiplex assay by quantifying the FANCD2 proteoforms following mitomycin-c treatment in an isogenic pair of FancA-corrected and uncorrected cell lines, as well as primary peripheral blood mononuclear cells from Fanconi Anemia patients. Additionally, we demonstrate detection of endogenous FANCD2 monoubiquitination in human breast cancer tissue. The immuno-MRM assay provides a potential functional diagnostic for patients with Fanconi Anemia with defects in the upstream FA complex or FANCD2, and a potential test for predicting sensitivity to DNA cross-linking agents in human cancers.

Reduced recruitment of 53BP1 during interstrand crosslink repair is associated with genetically inherited attenuation of mitomycin C sensitivity in a family with Fanconi anemia.

The Fanconi anemia (FA) pathway is implicated in the repair of DNA interstrand crosslinks (ICL). In this process, it has been shown that FA factors regulate the choice for DNA double strand break repair towards homologous recombination (HR). As this mechanism is impaired in FA deficient cells exposed to crosslinking agents, an inappropriate usage of non-homologous end joining (NHEJ) leads to the accumulation of toxic chromosomal abnormalities. We studied a family with two FANCG patients and found a genetically inherited attenuation of mitomycin C sensitivity resulting in-vitro in an attenuated phenotype for one patient or in increased resistance for two healthy relatives. A heterozygous mutation in ATM was identified in these 3 subjects but was not directly linked to the observed phenotype. However, the attenuation of ICL sensitivity was associated with a reduced recruitment of 53BP1 during the course of ICL repair, and increased HR levels. These results further demonstrate the importance of favoring HR over NHEJ for the survival of cells challenged with ICLs.

PAK4 Inhibition Impacts Growth and Survival, and Increases Sensitivity to DNA-Damaging Agents in Waldestrostom Macroglobulinemia

p21-activated kinases (PAKs) are effectors for the small GTPases Cdc42 and Rac that control a wide range of cellular processes including cytoskeleton reorganization, cell proliferation, survival and motility. Based on our initial results showing significant biological and functional role of PAK4 in plasma cell malignancies, we investigated its role in Waldenström macroglobulinemia (WM). We first confirmed high level of PAK4 expression and activity in WM cells. Both genetic depletion via stable lentiviral knock down and pharmacological inhibition of PAK4 via allosteric modulator KPT-9274 decreased tumor cell growth and survival in a dose and time dependent manner in a panel of WM cell lines. The growth inhibitory effect was associated with induction of apoptotic cell death, caspases activation and PARP cleavage as well as modulation of pro-apoptotic proteins such as PUMA. In addition, related-PAK4 biological processes such as migration, invasion and adhesion to bone marrow stromal cells (BMSCs) were significantly impacted by the inhibitor. A significant effect of KPT9274 was also observed in primary tumor cells from both newly diagnosed and relapse WM patients, including primary cells from ibrutinib resistant patients.To gain further insight into the consequences of PAK4 inhibition, we performed gene expression profile in WM cell lines treated with KPT-9274. Intriguingly we detected a significant impact on expression of Fanconi Anemia (FA)/BRCA pathway related genes, a DNA-damage response pathway essential for the repair of DNA interstrand crosslinks (ICLs) and regulation of cellular responses to replication stress. Since FA/BRCA deficient cells are defective in the formation of Rad51 foci, a crucial component of the homologous recombination (HR) repair machinery, we examined the effect of PAK4/NAMPT inhibition on Rad51 as well its impact on HR. We observed that, apart from inducing DNA damage, assessed by γH2AX expression, KPT-9274 specifically inhibited Rad51 and the double strand break repair by the homologous recombination pathway. Protein pull down analysis revealed the existence of a complex between nicotinamide phosphoribosyltransferase (NAMPT) and the conserved PAK binding domain; treatment with KPT-9274 disrupted this interaction and inhibited the enzymatic activity of NAMPT. NAD is necessary for PARP function especially with increasing DNA damage; and its depletion severely hamper DNA repair. Consistent with this, we observed that nicotinic acid supplementation rescued the effect of KPT-9274 on NAMPT inhibition as well as DNA damage and repair.As FA/BRCA pathway has been shown to affects sensitivity to alkylating agents, we next combined KPT-9274 with either melphalan or bendamustine in MM and WM cells. The combination treatment resulted in a synergistic time- and dose-dependent inhibition of cell survival and growth in cell lines and primary patient tumor cells. We observed a synergistic induction of apoptosis and activation of caspase-3/7, -8 and -9 following treatment with combined versus single-agent therapy. Similarly, in PAK4 knock-down WM cells bendamustine significantly increased the rate of early and late apoptotic cells (60%) compared to scrambled knockdown cells (37%).In summary, we report a significant anti-proliferative activity of dual PAK4-NAMPT inhibition in WM cells, with induction of apoptosis, DNA-damage response and FA/BRCA pathway disruption and a strong synergistic activity in combination with DNA damaging agents such as Bendamustine. Altogether, our data suggests dual PAK4-NAMPT inhibition represents a novel therapeutic strategies in WM. DisclosuresSenapedis:Karyopharm Therapeutics Inc: Employment, Equity Ownership. Baloglu:Karyopharm Therapeutics Inc: Employment, Equity Ownership. Oliva:Takeda: Honoraria; Celgene: Honoraria. Anderson:Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees. Treon:Pharmacyclics: Consultancy, Research Funding.

Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors.

Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes.

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells.

Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear. We hypothesized that metformin is cytotoxic in a tumor-specific environment such as glucose deprivation and/or low oxygen (O2) tension. We herein demonstrated that metformin was highly cytotoxic under glucose-depleted, but not hypoxic (2% O2) conditions. In order to elucidate the underlying mechanisms of this selective cytotoxicity, we treated exposed DNA repair-deficient chicken DT40 cells with metformin under glucose-depleted conditions and measured cellular sensitivity. Under glucose-depleted conditions, metformin specifically killed fancc and fancl cells that were deficient in FANCC and FANCL proteins, respectively, which are involved in DNA interstrand cross-link repair. An analysis of chromosomal aberrations in mitotic chromosome spreads revealed that a clinically relevant concentration of metformin induced DNA double-strand breaks (DSBs) in fancc and fancl cells under glucose-depleted conditions. In summary, metformin induced DNA damage under glucose-depleted conditions and selectively killed cells. This metformin-mediated selective toxicity may suppress the growth of malignant tumors that are intrinsically deprived of glucose.

Modulation of the Fanconi anemia pathway via chemically induced changes in chromatin structure.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway.

Abstract 4546: Structural evaluation of mutations identified on Cue domain of FANCD2

Abstract Fanconi anemia complementation group D2 (FANCD2) protein plays pivotal role in DNA interstrand crosslink (ICL) repair and genome stability. FANCD2 comprises conserved CUE domain at N terminus, which is important for DNA ICL repair and protein stability. However, molecular mechanism associated to genetic alteration has not well characterized. Here, we have carried out multimodal approaches to explore folding pattern of FANCD2 Cue domain and functionally relevant mutations. Dynamic Light Scattering and Mass Spectrometry data of purified proteins could revealed that wild- type protein is predominantly monomeric, but mutated counterparts are exhibiting oligomeric properties. Secondary, tertiary structure assessment and their thermal stability of wild- type, mutants were studied by Circular Dichroism (CD) and Fluorescence spectroscopy. Furthermore, binding affinity and kinetics of Cue domain and mutants with monoubiquitin was evaluated by surface plasmon resonance (SPR). Molecular modelling, simulation, normal mode analysis, principal component analysis and docking studies were carried out to understand in-depth conformational dynamics and interactions.

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health

SummaryDefects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health.

The Role of DNA Polymerase Nu and Zeta in the Repair of DNA Interstrand Crosslinks

Interstrand DNA Cross‐links (ICLs) are covalent bonds between the two DNA strands. They can be induced by endogenous aldehydes produced by metabolic pathways or exogenous agents such as chemotherapeutic drugs. ICLs inhibit DNA replication and RNA transcription and are lethal if not repaired. Lesions that are not repaired may cause chromosome breakage and genome instability that could ultimately promote cancer. The Fanconi anaemia (FA) pathway is in charge of removing the ICLs by recruiting specific endonucleases to ‘unhook’ the ICL and DNA translesion synthesis polymerases (TLS) that insert and extend nucleotides opposite the unhooked ICL. Ultimately, the FA pathway directs homologous recombination repair enzymes to complete the repair process and restore the DNA to its original double stranded form. Our major goal is to identify which proteins of the FA pathway regulate TLS polymerase activity. We are currently investigating the roles DNA polymerase Nu (POLN) and DNA polymerase Zeta (REV3) play in TLS during ICL repair. Current models suggest that REV3 is required for TLS but it still unknown if POLN is also involved in the same pathway. Using RNA‐Interference, we depleted HeLa cells of POLN, REV3, or both proteins to determine if they act in the same pathway. First, we determined whether HeLa cells depleted of one or both DNA polymerases induced the similar or enhanced cytotoxicity as measured by colony forming assays following exposure to Cisplatin (CDDP) or Mitomycin‐C (MMC). Second, we examined whether double knockdown cells showed similar or elevated chromosomal aberrations following drug exposure. Third, we performed an immunofluorescence in which we stain for 53BP1 and p‐ATM, markers for DNA double strands breaks. Our preliminary data shows evidence of failure to complete ICL repair when REV3 or POLN are depleted from the cells. Together, the data suggests POLN and REV3 are involved in ICL repair.

Abstract IA06: The Fanconi anemia-BRCA pathway and cancer

Abstract The Fanconi anemia (FA)-BRCA pathway has emerged as an important pathway in cancer biology. FA is a rare genetic disease characterized by chromosomal instability, cancer-susceptibility and cellular sensitivity to interstrand DNA crosslink (ICL)-inducing agents. The FA proteins including breast/ovarian cancer susceptibility proteins, BRCA1(FANCS) and BRCA2(FANCD1), cooperate in a common pathway (the FA-BRCA pathway) required for cellular resistance to ICL-inducing agents. The main function of the pathway is to coordinate multiple DNA repair mechanisms during ICL repair, although many of the components of the pathway also play critical roles in protecting replication forks. Inactivation of this pathway in cancer cells can lead to sensitivity to anti-cancer ICL-inducing agents, such as cisplatin and carboplatin, while reactivation of this pathway is implicated in acquired resistance to ICL-inducing agents. Therefore, inhibition of the pathway is an attractive therapeutic strategy to overcome DNA-crosslinker resistance of tumor cells. In this talk, I will introduce what the FA-BRCA pathway is and how this pathway is inactivated in human cancer. I will also mention some of the mechanisms of acquired resistance to cisplatin and PARP inhibitors in cancer cells, related to the FA-BRCA pathway. I will also discuss our recent findings about the regulation of the FA-BRCA pathway by FANCI protein and a deubiquitinating enzyme, USP28. Citation Format: Toshiyasu Taniguchi. The Fanconi anemia-BRCA pathway and 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 IA06.

Distinct cellular phenotype linked to defective DNA interstrand crosslink repair and homologous recombination.

Repair of DNA interstrand crosslinks (ICLs) predominantly involves the Fanconi anemia (FA) pathway and homologous recombination (HR). The HR repair system eliminates DNA double strand breaks (DSBs) that emerge during ICLs removal. The current study presents a novel cell line, CL-V8B, representing a new complementation group of Chinese hamster cell mutants hypersensitive to DNA crosslinking factors. CL-V8B exhibits increased sensitivity to various DNA-damaging agents, including compounds leading to DSBs formation (bleomycin and 6-thioguanine), and is extremely sensitive to poly (ADP-ribose) polymerase inhibitor (>400-fold), which is typical for HR-defective cells. In addition, this cell line exhibits a reduced number of spontaneous and induced sister chromatid exchanges, which suggests likely impairment of HR in CL-V8B cells. However, in contrast to other known HR mutants, CL-V8B cells do not show defects in Rad51 foci induction, but only slight alterations in the focus formation kinetics. CL-V8B is additionally characterized by a considerable chromosomal instability, as indicated by a high number of spontaneous and MMC-induced chromosomal aberrations, and a twice as large proportion of cells with abnormal centrosomes than that in the wild type cell line. The molecular defect present in CL-V8B does not affect the efficiency and stabilization of replication forks. However, stalling of the forks in response to replication stress is observed relatively rarely, which suggests an impairment of a signaling mechanism. Exposure of CL-V8B to crosslinking agents results in S-phase arrest (as in the wild type cells), but also in larger proportion of G2/M-phase cells and apoptotic cells. CL-V8B exhibits similarities to HR- and/or FA-defective Chinese hamster mutants sensitive to DNA crosslinking agents. However, the unique phenotype of this new mutant implies that it carries a defect of a yet unidentified gene involved in the repair of ICLs.

Preparation of Stable Nitrogen Mustard DNA Interstrand Cross-Link Analogs for Biochemical and Cell Biological Studies.

Nitrogen mustards (NMs) react with two bases on opposite strands of a DNA duplex to form a covalent linkage, yielding adducts called DNA interstrand cross-links (ICLs). This prevents helix unwinding, blocking essential processes such as replication and transcription. Accumulation of ICLs causes cell death in rapidly dividing cells, especially cancer cells, making ICL-forming agents like NMs valuable in chemotherapy. However, the repair of ICLs can contribute to chemoresistance through a number of pathways that remain poorly understood. One of the impediments in studying NM ICL repair mechanisms has been the difficulty of generating site-specific and stable NM ICLs. Here, we describe two methods to synthesize stable NM ICL analogs that make it possible to study DNA ICL repair. As a proof of principle of the suitability of these NM ICLs for biochemical and cell biological studies, we use them in primer extension assays with Klenow polymerase. We show that the NM ICL analogs block the polymerase activity and remain intact under our experimental conditions.