TSN Disrupts Fanconi Anemia Pathway Activation Through JAK/STAT1-Mediated Transcriptional Repression of FA Core Subunits in Bladder Cancer

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options

Mechanistic Insight into Site-Restricted Monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.

Upregulation of Fanconi Anemia DNA Repair Genes in Melanoma Compared with Non-Melanoma Skin Cancer

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein “core complex” containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.

The Fanconi anemia (FA) gene family comprises at least 12 genes interacting in a common pathway involved in DNA repair. To gain insight into the role of FA gene inactivation occurring in tumors among the general population, we endogenously targeted in cancer cells four FA genes that act at different stages of the FA pathway. After successful mono-allelic deletion of all genes, the sequential homozygous deletion was achieved only for FANCC and FANCG, acting upstream, but not for BRCA2 or FANCD2, acting downstream in the FA path¬way. Targeting of the second allele in BRCA2 and FANCD2 heterozygote clones resulted in re-deletion exclusively of the already defective allele in multiple instances (13x concerning BRCA2, 25x concerning FANCD2), strongly suggesting a detrimental phenotype. Unlike com¬plete FANCD2 disruption, the mere reduction of FANCD2 protein levels had no discernible effect. In addition, we confirmed that human cancer cells harboring the Seckel ATR mutation display impaired FANCD2 monoubiquitination and FANCD2 nuclear focus formation, as well as an increased sensitivity to DNA interstrand-crosslinking agents. Nevertheless, these cells were viable, indicating an ATR-independent function of FANCD2, distinct from its major known functions, to be responsible for the detrimental effects of FANCD2 loss. In conclusion, we established the downstream FA genes FANCD2 and BRCA2 to represent particularly vulnerable parts of the FA pathway, providing direct evidence for the paradoxical assumption that their inactivation could be predominantly selected against in cancer cells. This would explain why certain FA gene defects, despite an apparent selection for FA pathway inactivation in cancer, are rarely observed in tumors among the general population.

Gene promoter methylation is an epigenetic mechanism used by cells to control gene expression. Over recent decades, scientists have made various discoveries linking DNA methylation to several adverse outcomes, including human cancers. The Fanconi Anemia (FA) pathway is involved in homologous recombination, one of the major mechanisms of DNA repair. This pathway is essential for human cells to maintain integrity following DNA damage. Cancers with defective FA pathways are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. The FA pathway contains at least 19 genes, and some of the members have been implicated in susceptibility to a number of cancers by genetic or epigenetic alterations. Promoter methylation in FA genes is thought to play a role in the occurrence of cancer. Recently we screened 139 non-small cell lung cancer (NSCLC) formalin-fixed, paraffin-embedded (FFPE) tumors for FANCD2 foci formation by FA triple stain immunofluorescence (FATSI) analysis. Among the 104 evaluable tumors, 23 (22%) were FANCD2 foci negative. Since epigenetic inactivation can be one of the mechanisms for FA functional deficiency in these tumors, we evaluated 39 NSCLC samples (21 foci positive and 18 foci negative; 21 adenocarcinomas, 17 squamous cell carcinomas, 1 large cell carcinoma) for FANCF, FANCL and FANCS (BRCA1) promoter methylation. Human lung tumor tissue samples were obtained from The Tissue Procurement Shared Resources of the Ohio State University after IRB approval. Genomic DNA and total RNA samples were isolated from frozen lung tumor and matching non-tumor tissues. The promoter methylation status of FANCF, FANCL and FANCS was evaluated using methylation-specific PCR (MS-PCR). Among the 18 FATSI negative tumors, promoter methylation was found in FANCF (1 adenocarcinoma), FANCL (1 adenocarcinoma) and FANCS (1 adenocarcinoma). Among the 21 FATSI positive tumors, no promoter methylation was detected in FANCF or FANCL. Promoter methylation in FANCS was found in 1 (squamous cell carcinoma) of 21 FATSI positive tumors. The above observations suggest that epigenetic alterations, specifically methylation, can be one of the factors that contribute to FA functional deficiency in NSCLC patients. These findings may have clinical implications, since these tumors may be more sensitive to cross-link based therapy. However, an important caveat is that these changes may not be stable and could revert during treatment. Further studies in FA gene expression are needed to determine the impact of FA gene promoter methylation on FA repair foci formation. Citation Format: Andrew Fink, Arjun Kalvala, Li Gao, Kathleen Dotts, Brittany Aguila, Shirley Tang, Gregory A. Otterson, Miguel A. Villalona-Calero, Wenrui Duan. Promoter hypermethylation status of Fanconi Anemia (FA) pathway genes FANCF, FANCL and FANCS in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4438.

10509 Loss of the fanconi anemia (FA) pathway function has been described in a number of sporadic tumor types including breast, ovarian, pancreatic, head and neck and hematological malignancies. Functionally, the FA pathway responds to stalled DNA replication following DNA damage. Given the importance of the FA pathway in the response to DNA damage, we hypothesized that cells deficient in this pathway may become hyper-dependent on alternative DNA damage response pathways in order to respond to endogenous genotoxic stress such as occurs during metabolism. Therefore, targeting these alternative pathways could offer therapeutic strategies in FA pathway deficient tumors. To identify new therapeutic targets we treated FA pathway competent and deficient cells with a DNA damage response siRNA library, that individually knocked out 230 genes. We identified a number of gene targets that were specifically toxic to FA pathway deficient cells, amongst which was the DNA damage response kinase Ataxia Telangiectasia Mutated (ATM). To test the requirement for ATM in FA pathway deficient cells, we interbred Fancg ± Atm± mice. Consistent with the siRNA screen result, Fancg-/- Atm-/- mice were non viable and Fancg± Atm-/- and Fancg-/- Atm ± progeny were less frequent that would have been expected. Several human cell lines with FA gene mutations were observed to have constitutive activation of ATM which was markedly reduced on correction with the appropriate wild-type FA gene. Interestingly, FA pathway deficient cells, including the FANCC mutant and FANCG mutant pancreatic cancer cell lines, were selectively sensitive to monotherapy with the ATM inhibitor KU55933, as measured by dose inhibition and colony count assays. FA pathway deficient cells also demonstrated an increased level of chromosomal breakage, cell cycle arrest and apoptosis following KU55933 treatment when compared to FA pathway corrected cells. We conclude that FA pathway deficient cells have an increased requirement for ATM activation in order to respond to sporadic DNA damage. This offers the possibility that monotherapy with ATM inhibitors could be a therapeutic strategy for tumors that are deficient for the FA pathway. No significant financial relationships to disclose.

PLK1 Acts in Homologous Recombinatorial Repair and in Mitosis As Synthetically Lethal with the Fanconi Anemia/BRCA Pathway

The BACH1 helicase was initially identified by its direct binding to BRCA1 and, thus, was linked to hereditary breast cancer. More recently, BACH1 was identified as the gene defective in the J complementation group of Fanconi anemia (FA). FA is a multigenetic disorder characterized by cellular sensitivity to crosslinkers and chromosome instability. Because FANCD2 monoubiquitination is intact in BACH1 deficient cells, BACH1 appears to act downstream in the FA pathway akin to BRCA2/FANCD1. Interestingly, while BRCA1 has various interactions with FA proteins it has not been identified as an FA gene. As the race to uncover the last few unknown FA complementation groups comes to an end, future work will be required to uncover how these gene products function to combat the effects of DNA damage and maintain genomic stability. In particular, it remains elusive whether BRCA1 is functionally linked to the FA pathway through its interaction with BACH1/FANCJ. This review focuses on a model for the connection of BRCA1 to BACH1 in the FA pathway. We predict that BRCA1 regulates the BACH1 helicase activity to coordinate the timely displacement of Rad51 from nucleofilaments, promoting error free repair and ultimately maintaining chromosomal integrity.

Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

Every cell in our body other than red blood cells has a genome, the stability of which is crucial for life. The DNA is precisely replicated during cell proliferation, and is stably maintained, even after terminal differentiation, as a repository of information that orchestrates the cell’s metabolism. Unfortunately, our world is full of potential threats to genomic stability. DNA may degrade spontaneously, errors may occur during its replication, or metabolic byproducts, such as oxygen radicals or aldehydes, may chemically modify its nucleotide bases (i.e. DNA adducts). Ionizing radiation, ultraviolet light, and chemotherapeutic drugs are all well-known exogenous sources of DNA damage [1, 2]. To ensure cellular fitness, organisms have developed an elaborate molecular network to detect and repair DNA damage [3]. If deleterious effects of DNA damage exceed the cell’s repair capacity, it accumulates in the genome, leading to activation of cell cycle checkpoints (buying time for repair), cell death (apoptosis or necrosis), or, in the failure of checkpoints or cell death, conversion of DNA damage to mutations. This may result in poor cell proliferation, emergence of malignancy, impaired stem cell maintenance, or early-onset aging [1, 2]. Since all cellular activity in a way relies on the genome, the mechanisms that govern genomic stability are fundamentally important in biomedical research in general. Blood cells are no exception. As our knowledge of genomic stability has expanded massively in recent years, a rare hematological disorder, Fanconi anemia (FA), has become a prototypical example among hereditary conditions involving a defect in the DNA damage response (DDR). FA is one of several disorders discovered by the prominent Swiss pediatrician Guido Fanconi [4]. His first report in 1927 described brothers presenting with a pernicious anemia-like condition, congenital anomalies, and hyper-pigmentation of the skin. In the 1960s and 70s, it was established that FA cells display chromosomal instability, which is particularly pronounced following treatment with mitomycin C [5, 6]. The first molecular cloning of an FA gene was achieved by functional complementation using a cDNA expression library in the early 1990s [7]. However, until quite recently, the true molecular defect in FA, which is now considered to affect the response to replication stress, had not been defined. There seem to be a number of reasons why FA attracts widespread attention today. First of these is that novel FA genes continue to be identified year after year. This trend began at the turn of the 21st century, and has continued into this year, with the current number of the FA genes now totaling 15 [8–10]. This is surprising. Second, FA patients with disparate mutations display essentially similar phenotypes, strongly suggesting the presence of a common signal transduction pathway consisting of the FA gene products (i.e. FA pathway) [11, 12]. This prediction was well confirmed by the identification of the FA core complex (i.e. interactions between the FA core components FANCA/B/C/E/F/G/M/L) as an E3 ubiquitin ligase, and DNA damage-induced monoubiquitination of FANCD2 and FANCI, which is absent in cells lacking the core complex members [11, 12]. Of note, posttranslational protein modifications, such as ubiquitination, are currently the focus of intensive research in this field [13, 14]. Third, the discovery that the FANCD1 gene is the breast cancer M. Takata (&) Laboratory of DNA Damage Signaling, Department of Late Effect Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan e-mail: mtakata@house.rbc.kyoto-u.ac.jp

ABSTRACTThe Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

Inducible Loss of the Fanconi Anemia Pathway in iPSC Causes Rapid Cell Cycle Arrest and Apoptosis through ATM/ATR and p53 Signaling