Sensitivity of tumor cells deficient in the fanconi anemia pathway to inhibition of ataxia telangiectasia mutated (ATM)

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.

The Fanconi anemia (FA) pathway maintains genomic stability in replicating cells. Some sporadic breast, ovarian, pancreatic, and hematological tumors are deficient in FA pathway function, resulting in sensitivity to DNA-damaging agents. FA pathway dysfunction in these tumors may result in hyperdependence on alternative DNA repair pathways that could be targeted as a treatment strategy. We used a high-throughput siRNA screening approach that identified ataxia telangiectasia mutated (ATM) as a critical kinase for FA pathway-deficient human fibroblasts. Human fibroblasts and murine embryonic fibroblasts deficient for the FA pathway were observed to have constitutive ATM activation and Fancg(-/-)Atm(-/-) mice were found to be nonviable. Abrogation of ATM function in FA pathway-deficient cells resulted in DNA breakage, cell cycle arrest, and apoptotic cell death. Moreover, Fanconi anemia complementation group G- (FANCG-) and FANCC-deficient pancreatic tumor lines were more sensitive to the ATM inhibitor KU-55933 than isogenic corrected lines. These data suggest that ATM and FA genes function in parallel and compensatory roles to maintain genomic integrity and cell viability. Pharmaceutical inhibition of ATM may have a role in the treatment of FA pathway-deficient human cancers.

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

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.

The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of 22% of NSCLC to be FA functionally inactive by Fanconi Anemia Triple Staining Immunofluorescence (FATSI) test. Studies have shown involvement of certain micro RNA (miRNA) as regulatory elements in the development of lung cancer. We set out to evaluate potential involvement of miRNAs in the regulation of the Fanconi Anemia (FA) pathway. Using Nanostring counter miRNA array we screened 734 different miRNA expression in two FA defective lung cancer cells and matched control cells along with two FA pathway deficient lung tumors and matched non-tumor lung tissue samples. Selected miRNA expression were validated with real-time PCR analysis. miRNA target gene expression was analyzed through AmpliSeq RNA gene expression analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. Nanostring data showed that miR-200C was increased 7.5 fold on average in the FA defective lung cancer cells as compared to control cell. An average of 22 fold increase in miR-200C was detected in the FA defective lung tumor tissues comparing to matching non-tumor tissues. AmpliSeq analysis showed the ZEB1(zinc finger E-box binding homeobox 1) mRNA expression was down regulated in10 out 10 lung tumors (100%) comparing to non-tumor tissues, and 9 out of 10 samples (90%) showed reduction in ZEB2 expression. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibiting cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. Our findings indicate that the FA pathway regulates downstream genes through regulation of miRNAs in lung cancer. MiR-200C appears to be one of the most important FA downstream regulators in lung cancer. Validation with a larger sample size will be needed to confirm our findings. Citation Format: Wenrui Duan, Shirley Tang, Li Gao, Kathleen Dotts, Andrew Fink, Arjun Kalvala, Brittany Aguila, Miguel A. Villalona-Calero. Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer [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 1361.

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

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

Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

Background: Fanconi Anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer susceptibility. FA gene mutations are also widespread somatically in non-FA cancer patients. While the FA pathway participates in DNA repair, it is also linked to R-loop metabolism and regulation. R-loops are co-transcriptional RNA:DNA hybrids that form as a result of replication-transcription collisions that are enhanced due to DNA damage, but their aberrant formation drives genomic instability and is an endogenous source of genotoxicity. However, the interplay between FA proteins, DNA repair, and R-loops remains mechanistically ill-defined. Disturbance of mRNA processing, export, and splicing has also been linked to R-loop-mediated genomic instability. The splicing factor SRSF1 participates in mRNA export through the Nuclear Export Factor 1 (NXF1). Aberrant splicing factors expression has been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), which clinically resembles FA. In the present study, we study the coupling of the DNA damage response to the prevention of pathogenic R-loops via mRNA export regulation through the FA pathway and SRSF1. Methods: Cells: FA-D2 mutant, corrected, and ubiquitin-dead mutant; HeLa. siRNA transfections to reduce SRSF1 and FANCD2 protein levels. Cell survival assays against DNA damaging agents. In vitro immunoprecipitation and ubiquitination assays using purified SRSF1 and ID2. DART assay, S9.6 immunofluorescence, and S9.6 slot-blot to measure R loops in cells. Immunoprecipitation of mRNPs used to detect mature mRNA-bound ribonucleoproteins. RNA FISH to measure mRNA export. Results: In this study, we provide strong evidence that the FA pathway coordinates the prevention of R-loop formation and subsequent genomic instability through its interaction with splicing factors via regulation of mRNA export. First, we demonstrate that knockdown of SRSF1, which is known to result in increased R-loops, also is associated with diminished activation of the FA pathway. Second, the central FA complex ID2 binds to SRSF1 in cells and in recombinant protein assays. Further, we demonstrate that SRSF1 activates the FA pathway by binding ID2 and stimulates its monoubiquitination in an RNA-dependent fashion. This activity is associated with co-localization in cells at transcriptional-DNA damage inducible R loops. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, cancer-associated SRSF1 mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export and R-loops accumulation. Conclusion: Our findings uncover a novel mechanism of FANCD2 and SRSF1 in the prevention of R-loop formation via coupling of mRNA export to the DNA damage response, thus preventing genomic instability. Citation Format: Anne Olazabal-Herrero, Fengshan Liang, Arijit Dutta, Yuxin Huang, Zhuobin Liang, Abhishek Gupta, Yaqun Teng, Li Lan, Xiaoyong Chen, Huadong Pei, Manoj Pillai, Patrick Sung, Gary Kupfer. The Fanconi Anemia pathway protein complex FANCI/FANCD2 couples the DNA damage response to R-loop regulation through SRSF1-mediated mRNA export [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6100.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

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

Human MutS and FANCM complexes function as redundant DNA damage sensors in the Fanconi Anemia pathway

A Novel Cell-Free Assay Identifies the Curcumin Analog EF24 as a Potent Inhibitor of the Fanconi Anemia Pathway

Fanconi Anemia Proteins Function in Mitophagy and Immunity

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.