Activation Of Fanconi Anemia Pathway Research Articles

The RPA inhibitor HAMNO sensitizes Fanconi anemia pathway-deficient cells

ABSTRACT The Fanconi anemia (FA) DNA repair pathway is required for DNA inter-strand crosslink (ICL) repair. Besides its role in ICL repair, FA proteins play a central role in stabilizing stalled replication forks, thereby ensuring genome integrity. We previously demonstrated that depletion of replication protein A (RPA) induces the activation of FA pathway leading to FANCD2 monoubiquitination and FANCD2 foci formation. Thus, we speculated that FA-deficient cells would be more sensitive to RPA inhibition compared to FA-proficient cells. Following treatment with RPA inhibitor HAMNO, we observed significant induction in FANCD2 monoubiquitination and foci formation as observed in RPA depletion. In addition, HAMNO treatment caused increased levels of γ-H2AX and S-phase accumulation in FA-deficient cells. Importantly, FA-deficient cells showed more increased sensitivity to HAMNO than FA-proficient cells. Moreover, in combination with cisplatin, HAMNO further enhanced the cytotoxicity of cisplatin in FA-deficient cells, while being less toxic against FA-proficient cells. This result suggests that RPA inhibition might be a potential therapeutic candidate for the treatment of FA pathway-deficient tumors.

SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway.

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI‐FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)‐mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error‐free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error‐free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI‐deficient cells are exquisitely sensitive to ICL‐inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR‐mediated ICL repair is defective, and breaks are instead re‐ligated by polymerase θ‐dependent microhomology‐mediated end‐joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error‐free ICL repair.

Regulation of the Fanconi Anemia DNA Repair Pathway by Phosphorylation and Monoubiquitination.

The Fanconi anemia (FA) DNA repair pathway coordinates a faithful repair mechanism for stalled DNA replication forks caused by factors such as DNA interstrand crosslinks (ICLs) or replication stress. An important role of FA pathway activation is initiated by monoubiquitination of FANCD2 and its binding partner of FANCI, which is regulated by the ATM-related kinase, ATR. Therefore, regulation of the FA pathway is a good example of the contribution of ATR to genome stability. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via phosphorylation and monoubiquitination.

Centipeda minima extract sensitizes lung cancer cells to DNA-crosslinking agents via targeting Fanconi anemia pathway

BackgroundIntrinsic and acquired chemoresistance remains a critical challenge in lung cancer chemotherapy. Fanconi anemia (FA) pathway plays an important role in antagonizing the cytotoxic effects of chemotherapeutics by repairing DNA damage. We recently demonstrated that the traditional Chinese medicinal herb, Centipeda minima (C. minima), possessed anti-inflammatory and antioxidant properties. However, the potential anticancer application of C. minima and the underlying mechanisms remain unclear. PurposeWe aimed to investigate the combined anticancer effects of the ethanol extract of C. minima (ECM) and DNA-crosslinking agents on non-small cell lung cancer (NSCLC) and elucidate the underlying mechanisms. MethodsCell viability and flow cytometry assay were performed to determine the synergistic cytotoxicity of ECM and DNA-crosslinking agents, cisplatin (CDDP) or mitomycin C (MMC), in NSCLC cells. Western blotting and immunofluorescence were conducted to examine the effects of ECM on protein expression in DNA damage repair pathway. Comet assay was applied to evaluate DNA damage levels. Subcutaneous xenografts of NSCLC were established to evaluate the combined anticancer effects of ECM and CDDP. ResultsCombined treatments with ECM and DNA-crosslinking agents exhibited synergistic cytotoxic effects against A549 and H1299 cells. FANCD2 was highly expressed in NSCLC that correlates with poor prognosis of NSCLC patients, based on the online database analysis. ECM significantly inhibited DNA damage-induced monoubiquitination and nuclear foci formation of FANCD2, thereby sensitizing NSCLC to CDDP- or MMC-induced DNA damage and apoptosis, as evidenced by increased expression of γ-H2AX, increased cleavage of caspases-3 and PARP, and enhanced Annexin V-FITC/PI staining. Further, ECM can also decrease the protein level of FANCD2 that contributes to the chemosensitizing effects. Moreover, ECM significantly attenuated CDDP-mediated S-phase arrest by antagonizing the activation of ATR/Chk1 pathway in NSCLC cells. Animal experiments further demonstrated that ECM and CDDP combination treatment synergistically inhibited tumor growth by decreasing FANCD2 protein level in tumor tissues. ConclusionOur results demonstrated that ECM can inhibit DNA-crosslinking agents-induced activation of FA pathway by attenuating both the expression and monoubiquitination of FANCD2. ECM and CDDP combination therapy exhibited synergistic anticancer effects both in vitro and in vivo, indicating that ECM and its active components might serve as novel anticancer drugs in the combination chemotherapy.

PFKFB3 Inhibition Sensitizes DNA Crosslinking Chemotherapies by Suppressing Fanconi Anemia Repair.

Simple SummaryDNA-damaging chemotherapeutics, such as platinum drugs, are cornerstones in cancer treatment. The efficacy of such treatment is intimately linked to the DNA repair capacity of the cancer cells, as DNA damage above a tolerable threshold culminates in cell death. Cancer cells often have deregulated DNA repair mechanisms, making them initially more sensitive to DNA-damaging chemotherapies. Unfortunately, over time, cancer cells often develop resistance to such treatments by rewiring their DNA damage response pathways. Here, we identify that targeting the recognized anti-cancer target 6-phosphofructo-2-kinase/fructose-2,6,-bisphophatase 3 (PFKFB3), commonly overexpressed in cancer, with the small molecule inhibitor KAN0438757, selectively sensitizes cancer cells to platinum drugs, including treatment-resistant cancer cells, while sparing normal cells. Mechanistically, PFKFB3 promotes tolerance to and the repair of platinum-induced DNA interstrand crosslinks (ICLs) through modulation of the Fanconi anemia (FA) DNA repair pathway. Thus targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.Replicative repair of interstrand crosslinks (ICL) generated by platinum chemotherapeutics is orchestrated by the Fanconi anemia (FA) repair pathway to ensure resolution of stalled replication forks and the maintenance of genomic integrity. Here, we identify novel regulation of FA repair by the cancer-associated glycolytic enzyme PFKFB3 that has functional consequences for replication-associated ICL repair and cancer cell survival. Inhibition of PFKFB3 displays a cancer-specific synergy with platinum compounds in blocking cell viability and restores sensitivity in treatment-resistant models. Notably, the synergies are associated with DNA-damage-induced chromatin association of PFKFB3 upon cancer transformation, which further increases upon platinum resistance. FA pathway activation triggers the PFKFB3 assembly into nuclear foci in an ATR- and FANCM-dependent manner. Blocking PFKFB3 activity disrupts the assembly of key FA repair factors and consequently prevents fork restart. This results in an incapacity to replicate cells to progress through S-phase, an accumulation of DNA damage in replicating cells, and fork collapse. We further validate PFKFB3-dependent regulation of FA repair in ex vivo cultures from cancer patients. Collectively, targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.

Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum.

Simple SummaryChemotherapeutics exerting their antiproliferative actions by introducing DNA crosslinks, such as platinum drugs, are used to treat numerous cancers. Unfortunately, their therapeutic potential is limited due to adverse side effects and acquired resistance, the latter often associated with enhanced DNA repair capacity. Thus, targeting DNA repair is a promising strategy to lower effective doses and associated side effects, and to restore sensitivity to treatment. The C-X3-C motif chemokine receptor 1 (CX3CR1) is an emerging anticancer target which expression correlates with worse overall survival in cancer patients undergoing DNA damaging treatments. Here we show for the first time that the clinical-phase small molecule inhibitor KAND567 targeting CX3CR1 augments the efficacy of DNA crosslinking chemotherapeutics in cancer cell lines, including platinum resistant models, by interference of the Fanconi anemia DNA repair pathway. Hence, the interplay between CX3CR1 and FA repair provides novel potential therapeutic opportunities in cancers treated with DNA crosslinking agents.The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.

Acetylation modulates the Fanconi anemia pathway by protecting FAAP20 from ubiquitin-mediated proteasomal degradation

Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitute a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multisubunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl cis-trans isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely un-known. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue that when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability.

Fanconi Anemia Pathway Activation by FOXM1 Is Critical to Bladder Cancer Recurrence and Anticancer Drug Resistance.

Although the 5-year survival rate of patients diagnosed with nonmuscle invasive bladder cancer (NMIBC) has reached 85%, more than 50% of patients suffer from frequent recurrences. To identify molecular targets associated with recurrence of NMIBC, we analyzed gene expression data and found that FOXM1 and FANCD2 were involved in recurrence. Therefore, we investigated how these genes were involved in the mechanism of recurrence and confirmed their usefulness as biomarkers. Investigation have shown that FOXM1 directly regulated the transcription of FANCD2, which is the key gene of the Fanconi anemia (FA) pathway. Depletion of FOXM1 resulted in DNA repair defects in the FA pathway and in decreased resistance to chemotherapy. Thus, the FANCD2-associated FA pathway activated by FOXM1 is an important mechanism involved in chemotherapy-related recurrence. In conclusion, FOXM1 and FANCD2 can be used as prognostic factors that are associated with high risk of recurrence and with anticancer drug resistance properties in NMIBC patients.

Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway.

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

New insights into diagnosis and therapeutic options for proliferative hepatoblastoma.

The highly proliferating C2A subtype is characterized by topoisomerase 2-alpha gene up-regulation and FA pathway activation, and the HB therapeutic arsenal could include bortezomib for the treatment of patients with the most aggressive tumors. (Hepatology 2018;68:89-102).

Pharmacological targeting of RAD6 enzyme-mediated translesion synthesis overcomes resistance to platinum-based drugs

Platinum drug-induced cross-link repair requires the concerted activities of translesion synthesis (TLS), Fanconi anemia (FA), and homologous recombination repair pathways. The E2 ubiquitin-conjugating enzyme RAD6 is essential for TLS. Here, we show that RAD6 plays a universal role in platinum-based drug tolerance. Using a novel RAD6-selective small-molecule inhibitor (SMI#9) targeting the RAD6 catalytic site, we demonstrate that SMI#9 potentiates the sensitivities of cancer cells with innate or acquired cisplatin or oxaliplatin resistance. 5-Iododeoxyuridine/5-chlorodeoxyuridine pulse-labeling experiments showed that RAD6 is necessary for overcoming cisplatin-induced replication fork stalling, as replication-restart was impaired in both SMI#9-pretreated and RAD6B-silenced cells. Consistent with the role of RAD6/TLS in late-S phase, SMI#9-induced DNA replication inhibition occurred preferentially in mid/late-S phase. The compromised DNA repair and chemosensitization induced by SMI#9 or RAD6B depletion were associated with decreased platinum drug-induced proliferating cell nuclear antigen (PCNA) and FANCD2 monoubiquitinations (surrogate markers of TLS and FA pathway activation, respectively) and with attenuated FANCD2, RAD6, γH2AX, and POL η foci formation and cisplatin-adduct removal. SMI#9 pretreatment synergistically increased cisplatin inhibition of MDA-MB-231 triple-negative breast cancer cell proliferation and tumor growth. Using an isogenic HCT116 colon cancer model of oxaliplatin resistance, we further show that γH2AX and monoubiquitinated PCNA and FANCD2 are constitutively up-regulated in oxaliplatin-resistant HCT116 (HCT116-OxR) cells and that γH2AX, PCNA, and FANCD2 monoubiquitinations are induced by oxaliplatin in parental HCT116 cells. SMI#9 pretreatment sensitized HCT116-OxR cells to oxaliplatin. These data deepen insights into the vital role of RAD6/TLS in platinum drug tolerance and reveal clinical benefits of targeting RAD6 with SMI#9 for managing chemoresistant cancers.

Rad6 inhibition sensitizes ovarian cancer cells to platinum drugs by attenuating activation of multiple DNA repair networks

Objectives: Ovarian cancer (OC) platinum resistance is often attributable to an increase in DNA repair. Rad6 is an E2 ubiquitin–conjugating enzyme that plays a central role in the activation of several DNA repair pathways. Rad6, in conjunction with its E3 partner Rad18, is vital for the activation of trans-lesion synthesis (TLS) DNA damage tolerance by monoubiquitinating PCNA; and the activation of the Fanconi anemia (FA) DNA repair pathway critical for DNA crosslink repair. We aimed to determine if inhibition of Rad6 could attenuate the DNA damage repair signaling induced by platinum drugs, and thereby overcome OC platinum resistance. Methods: OC cell lines were used to evaluate Rad6-mediated ubiquitin signaling. Both genetic and pharmacologic inhibitors of Rad6 were used and compared with controls in cancer growth and clonogenic assays. Responsiveness to platinum chemotherapy was tested in combination with Rad6 inhibitor. Standard Western blotting techniques were used to measure protein levels with or without Rad6 inhibition. Immunostaining was used to visualize DNA damage foci formation. Results: Rad6B was overexpressed in OC with increasing intensity by immunohistochemistry with increasing stage. Inhibition of Rad6 was effective in blocking DNA repair pathway mediators such as FANCD2, PCNA, yH2AX. Treatment with siRNAs or a small molecule inhibitor led to growth inhibition and attenuated clonogenic potential of OC cells. In addition, OC cells were sensitized to carboplatin by both genetic and pharmacological inhibition of Rad6. Genetic knockdown of Rad6 resulted in a threefold reduction in the IC50 value of carboplatin, and the IC50 value for carboplatin was reduced fivefold after treatment with a small molecule inhibitor. Moreover, Rad6 inhibition potentiated DNA damage induced by platinum drugs and sensitized platinum-resistant OC cells to carboplatin. Immunostaining confirmed that Rad6 inhibition attenuated FANCD2 foci formation, indicating reduced DNA repair. Conclusions: Rad6 inhibition reduced OC cell growth, but also consistently blocked carboplatin-induced PCNA monoubiquitination and FA pathway activation. This resulted in a sustained increase in DNA damage and an increase in cancer cell death. Together, our data indicate that inhibition of Rad6 holds therapeutic potential for the treatment of OC alone or in combination therapy.

Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo.

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18−/− mice. Moreover, primary Rad18−/− mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18−/− HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18−/− mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting.

Advances in the understanding of Fanconi Anemia Complementation Group D2 Protein (FANCD2) in human cancer.

Fanconi anemia (FA) is a rare human genetic disease, resulting from dysfunction in any of 17 known complementation proteins: FANC-A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, Q & S, and other unknowns. Besides the severe bone marrow failure, an extremely high incidence of cancer as well as many other clinic symptoms associated with FA patients, FA cells are known of insufficiency in homologous recombination, DNA mismatch repair, nucleotide excision repair, translesion DNA synthesis, and other molecular defects, leading to genome instability. Those similar molecular and cellular/tissue features show that all FA proteins function in one common signaling pathway, namely, the FA pathway. The monoubiquitination of FANCD2 is the central step of the FA pathway activation upon DNA damage or during DNA replication. The molecular functions of FANCD2 emerge as a very attractive filed of investigation in cancer research. Herein, we review the recent progresses in FANCD2 functions at these rapidly progressed aspects.

Abstract 1662: Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells

Abstract Triple negative breast cancers (TNBCs) comprise ∼15-20% of all breast cancers (BrCas), and their treatment is challenging because they are not treatable with ER, PgR or Her2/neu-targeted therapies. Since TNBCs and BRCA-mutated BrCas share many histopathologic features including aberrant DNA repair pathways, the concept of targeting DNA repair defects with agents such as platinum (Pt) compounds is applied for treatment of TNBC patients. Pt drugs induce DNA replication stalling interstrand DNA crosslinks (ICLs), the repair of which require concerted activities of nucleotide excision repair, Fanconi anemia (FA)/BRCA homologous recombination repair (HRR) and translesion synthesis (TLS) pathways. The therapeutic efficacies of these drugs are often limited by the cancer cell's enhanced ability to repair/tolerate these toxic DNA lesions. The TLS pathway a.k.a DNA damage tolerance pathway is critical for cell survival in the face of DNA damage. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the FA network and HR pathway, processes critical for ICL repair. The Rad6 gene is a principal component of the TLS pathway and previous work in our lab has shown its important role in BrCa development/progression and acquisition of cisplatin (CDDP) resistance. To understand the involvements of Rad6/TLS and FA/BRCA in CDDP-induced DNA damage repair, we evaluated CDDP sensitivities, CDDP-induced DNA damage responses and stalled replication restarts in wt-BRCA1 (MDA-MB-231, MDA-MB-468) and mut-BRCA1 (HCC1937, SUM1315) TNBCs. Data from MTT assays showed no correlation between CDDP sensitivity and BRCA1 status; however, pretreatment with our recently developed Rad6 small molecule inhibitor (SMI) enhanced CDDP sensitivity of all TNBC cell lines regardless of their BRCA1 status. Consistent with these data, immunoblot analysis showed that treatment with the Rad6 SMI attenuated the CDDP-induced PCNA ubiquitinations (a hall mark of Rad6/TLS activity), as well as the steady-state levels of FancD2 (a surrogate marker of FA pathway activation), pol eta (TLS polymerase), and Rad51 (critical for HRR) in both wt-BRCA1 and mut-BRCA1 TNBC cells. IdU/CldU labeling assays showed that Rad6 inhibition mitigated the restart of CDDP-induced stalled replication forks. Whereas Rad6 is expressed weakly in normal breast tissues and overexpressed in BrCas, immunohistochemical analysis showed strong nuclear staining for Rad6 in the normal ducts of mut-BRCA1 clinical breast tissues implicating an important role for Rad6 in mut-BRCA1 BrCas. Our data suggest an important role for the Rad6/TLS pathway in processing Pt-induced ICLs in both wt-BRCA1 and mut-BRCA1 TNBCs and the potential therapeutic value of inhibiting this pathway in TNBCs. Supported by NIH CA178117-01. Note: This abstract was not presented at the meeting. Citation Format: Brittany Haynes, Hend Kothayer, Andrew Westwell, Malathy Shekhar. Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells. [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 1662. doi:10.1158/1538-7445.AM2015-1662

Activation of the Fanconi anemia/BRCA pathway at low doses of ionization radiation

Fanconi anemia (FA) is a rare, clinically heterogeneous autosomal recessive or X-linked genetic disease characterized by chromosome fragility, congenital malformations and cancer susceptibility. FA patients are usually radiosensitive when exposed to radiotherapy but the role of the FA in response to ionizing radiation (IR) is controversial. Here we have investigated IR-induced activation of the FA pathway by systematically analyzing monoubiquitination of the central protein FANCD2 and subsequent recruitment to stalled replication forks in primary fibroblasts. We developed an immunolabelling method to simultaneously visualize IR-induced FANCD2 and γH2AX foci in S-phase. We observed FANCD2 foci formation in a subset of IR-induced γH2AX foci in S-phase cells. This was observed at doses of IR ranging from 0.1 to 5.0Gy in a dose dependent non-threshold fashion. Our results indicate that minimum doses of IR can produce replication fork stalling and FA pathway activation during S-phase in primary cells.

Disparate Requirements for the Phosphorylation of Distinct Sites on the Fanconi Anemia Group I Protein

Fanconi Anemia (FA) is a blood disorder characterized by bone marrow failure, predisposition to hematologic malignancy and sensitivity to interstrand crosslinking agents. Patients with FA carry inherited mutations in any one of at least 16 known Fanconi Anemia Group (FANC) proteins that coordinate to function in a DNA repair pathway (the FA pathway). The activation of this pathway centers on two of these, Fanconi Anemia Group D2 protein (FANCD2) and Fanconi Anemia Group I protein (FANCI), which must undergo both phosphorylation and ubiquitination in order for the pathway to function properly. The latter is catalyzed by the FA core complex ubiquitin ligase, which is composed of 8 other FANC proteins.Previous studies suggest that, in response to DNA damage, FANCI is phosphorylated at multiple sites within its evolutionarily conserved SQ cluster domain (SCD). This process is essential for activation of the canonical FA pathway. Failure of FANCI to phosphorylate inhibits FANCD2 ubiquitination, FANCD2 foci formation and cellular resistance to interstrand crosslinkers. However, while FANCI phosphorylation is important for the FA pathway to function, little is known about how this phosphorylation is regulated. Studies on the regulation of FANCI phosphorylation have largely been limited to chicken DT40 cells. Furthermore, the detection of FANCI phosphorylation has been restricted to an electrophoretic mobility-based method, which provides little information on the biology of specific phosphorylation sites. The objective of our work is to better understand the precise regulation of FANCI SCD phosphorylation, in human cells, at sites that have been established to be functionally significant.By performing mass spectrometry on immunoprecipitated human FANCI protein, we established that the human FANCI SCD is indeed phosphorylated on at least two sites. Each of these sites have been found, through mutagenesis studies, to be involved in FA pathway activation. These two sites have also been implicated, through structural studies, in promoting a stable interaction between FANCI and FANCD2. Using this information, we designed immunogenic phospho-peptides to generate antibodies that specifically detect the phosphorylation of each of these two sites. We used these FANCI phospho-antibodies, together with genetically manipulated human cell culture systems, to study factors that modulate FANCI phosphorylation in the context of the human FA pathway.We first established that these antibodies can be used for both immunoblot and immunofluorescence applications. With immunoblot analysis of cells treated with mitomycin C, we made the interesting observation that the phosphorylation of one of the FANCI sites occurred predominantly in the non-ubiquitinated form of the protein, while the other site was phosphorylated predominantly in the ubiquitinated form. This suggested that the phosphorylation of two distinct FANCI sites occurs at different steps of FA pathway activation. By performing siRNA depletion and biochemical experiments in cultured human cells, we found that the phosphorylation of both sites is at least partially dependent on the Ataxia Telangiectasia and Rad 3 related (ATR) kinase. Surprisingly, we found that only one of these sites could be phosphorylated without prior FANCI/D2 ubiquitination. Phosphorylation of the other site was dependent on both FANCI/D2 ubiquitination and the FA core complex. Therefore, contrary to previous models, we found that both ubiquitination-dependent and -independent phosphorylation sites exist within the FANCI SCD. Different FANCI phosphorylation sites that contribute to FA pathway activation therefore have disparate requirements for their phosphorylation.Until now, studies on the regulation of FANCI phosphorylation have been limited by the lack of available phospho-specific FANCI antibodies. By developing antibodies that can specifically detect the phosphorylation of distinct sites within the functionally important SCD of FANCI, we have established new and critical reagents that provide additional insight into how the human FA pathway is activated. Our results suggest a novel model of FA pathway activation that involves a dynamic interplay between FANCI phosphorylation and FANCI/D2 ubiquitination, and reveal that activation of the FA pathway by FANCI phosphorylation is more complex a process than previously thought. DisclosuresNo relevant conflicts of interest to declare.

Hypoxic Stress Facilitates Acute Activation and Chronic Downregulation of Fanconi Anemia Proteins

Hypoxia induces genomic instability through replication stress and dysregulation of vital DNA repair pathways. The Fanconi anemia (FA) proteins, FANCD2 and FANCI, are key members of a DNA repair pathway that responds to replicative stress, suggesting that they undergo regulation by hypoxic conditions. Here acute hypoxic stress activates the FA pathway via ubiquitination of FANCD2 and FANCI in an ATR-dependent manner. In addition, the presence of an intact FA pathway is required for preventing hypoxia-induced DNA damage measurable by the comet assay, limiting the accumulation of γH2AX (a marker of DNA damage or stalled replication), and protecting cells from hypoxia-induced apoptosis. Furthermore, prolonged hypoxia induces transcriptional repression of FANCD2 in a manner analogous to the hypoxic downregulation of BRCA1 and RAD51. Thus, hypoxia-induced FA pathway activation plays a key role in maintaining genome integrity and cell survival, while FA protein downregulation with prolonged hypoxia contributes to genomic instability. This work highlights the critical role of the FA pathway in response to hypoxic stress and identifies the pathway as a therapeutic target under hypoxic conditions.

Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

Modularized Functions of the Fanconi Anemia Core Complex

The Fanconi anemia (FA) core complex provides the essential E3 ligase function for spatially defined FANCD2 ubiquitination and FA pathway activation. Of the seven FA gene products forming the core complex, FANCL possesses a RING domain with demonstrated E3 ligase activity. The other six components do not have clearly defined roles. Through epistasis analyses, we identify three functional modules in the FA core complex: a catalytic module consisting of FANCL, FANCB, and FAAP100 is absolutely required for the E3 ligase function, and the FANCA-FANCG-FAAP20 and the FANCC-FANCE-FANCF modules provide nonredundant and ancillary functions that help the catalytic module bind chromatin or sites of DNA damage. Disruption of the catalytic module causes complete loss of the core complex function, whereas loss of any ancillary module component does not. Our work reveals the roles of several FA gene products with previously undefined functions and a modularized assembly of the FA core complex.