FANCD2 counteracts O6-methylguanine-induced mismatch repair-dependent apoptosis.

BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. METHODS AND RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p = .01) and hemoglobin levels (p < .05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p = .001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Identification of the ferroptosis-related prognostic gene signature in mesothelioma.

Mesothelioma, an uncommon yet highly aggressive malignant neoplasm, presents challenges in the effectiveness of current therapeutic approaches. Ferroptosis, a non-apoptotic mechanism of cellular demise, exhibits a substantial association with the progression of diverse cancer forms. It is important to acknowledge that there exists a significant association between ferroptosis and the advancement of various forms of cancer. Nevertheless, the precise role of ferroptosis regulatory factors within the context of mesothelioma remains enigmatic. In our investigation, we initially scrutinized the prognostic significance of 24 ferroptosis regulatory factors in the realm of mesothelioma. Our observations unveiled that heightened expression levels of CARS1, CDKN1A, TFRC, FANCD2, FDFT1, HSPB1, SLC1A5, SLC7A11, coupled with reduced DPP4 expression, were indicative of an unfavorable prognosis. Built upon the nine previously discussed prognostic genes, the ferroptosis prognostic model offers a reliable means to forecast mesothelioma patients' survival with a substantial degree of precision. Furthermore, a notable correlation emerged between these prognostic ferroptosis regulators and parameters such as immune cell infiltration, tumor mutation burden, microsatellite instability, and PD-L1 expression in the context of mesothelioma. Within this cadre of nine ferroptosis regulatory factors with prognostic relevance, FANCD2 exhibited the most pronounced prognostic influence, as elucidated by our analyses. Subsequently, we executed a validation process employing clinical specimens sourced from our institution, thus confirming that heightened FANCD2 expression is a discernible harbinger of an adverse prognosis in the context of mesothelioma. In vitro experiments revealed that knocking down FANCD2 markedly suppressed the proliferation, migration, and ability of mesothelioma cells to attract immune cells. Furthermore, our findings also showed that reducing FANCD2 levels heightened the vulnerability of mesothelioma cells to inducers of ferroptosis. Furthermore, an extensive pan-cancer analysis uncovered a robust association between FANCD2 and the gene expression linked to immune checkpoints, thereby signifying an adverse prognosis across a broad spectrum of cancer types. Additional research is warranted to validate these findings.

Pancancer analysis of the prognostic and immunological role of FANCD2: a potential target for carcinogenesis and survival.

Recent evidence has shed light on the significant role of FANCD2 in cancer initiation, development, and progression. However, a comprehensive pan-cancer analysis of FANCD2 has been lacking. In this study, we have conducted a thorough investigation into the expression profiles and prognostic significance of FANCD2, as well as its correlation with clinicopathological parameters and immune cell infiltration, using advanced bioinformatic techniques. The results demonstrate that FANCD2 is significantly upregulated in various common cancers and is associated with prognosis. Notably, higher expression levels of FANCD2 are linked to poor overall survival, as indicated by Cox regression and Kaplan-Meier analyses. Additionally, we have observed a decrease in the methylation of FANCD2 DNA in some cancers, and this decrease is inversely correlated with FANCD2 expression. Genetic alterations in FANCD2 predominantly manifest as mutations, which are associated with overall survival, disease-specific survival, disease-free survival, and progression-free survival in certain tumor types. Moreover, FANCD2 exhibits a strong correlation with infiltrating cell levels, immune checkpoint genes, tumor mutation burden (TMB), and microsatellite instability (MSI). Enrichment analysis further highlights the potential impact of FANCD2 on Fanconi anemia (FA) pathway and cell cycle regulation. Through this comprehensive pan-cancer analysis, we have gained a deeper understanding of the functions of FANCD2 in oncogenesis and metastasis across different types of cancer.

APLF facilitates interstrand DNA crosslink repair and replication fork protection to confer cisplatin resistance.

Replication stress converts the stalled forks into reversed forks, which is an important protection mechanism to prevent fork degradation and collapse into poisonous DNA double-strand breaks (DSBs). Paradoxically, the mechanism also acts in cancer cells to contribute to chemoresistance against various DNA-damaging agents. PARP1 binds to and is activated by stalled forks to facilitate fork reversal. Aprataxin and polynucleotide kinase/phosphatase-like factor (APLF) binds to PARP1 through the poly(ADP-ribose) zinc finger (PBZ) domain and is known to be involved in non-homologous end joining (NHEJ). Here, we identify a novel function of APLF involved in interstrand DNA crosslink (ICL) repair and fork protection. We demonstrate that PARP1 activity facilitates the APLF recruitment to stalled forks, enabling the FANCD2 recruitment to stalled forks. The depletion of APLF sensitizes cells to cisplatin, impairs ICL repair, reduces the FANCD2 recruitment to stalled forks, and results in nascent DNA degradation by MRE11 nucleases. Additionally, cisplatin-resistant cancer cells show high levels of APLF and homologous recombination-related gene expression. The depletion of APLF sensitizes cells to cisplatin and results in fork instability. Our results reveal the novel function of APLF to facilitate ICL repair and fork protection, thereby contributing to cisplatin-resistant phenotypes of cancer cells.

Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.

Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2-/- fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2-/- fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2-/- long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.

FANCD2 deficiency sensitizes SHH medulloblastoma to radiotherapy via ferroptosis.

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.

The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export.

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway lead to R-loop accumulation, which contributes to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export.

A Fanca knockout mouse model reveals novel Fancd2 function.

Fanconi anemia (FA) is a genetically and clinically heterogenous inherited disorder. Clinically, Fanca subtype patients exhibited milder phenotypes compared to Fancd2 subtypes. Increasing evidence suggests that Fancd2 perform independent functions, but the detailed mechanisms are not well characterized. In this study, we developed a Fanca KO mice model in C57BL/6 background with ATG region deletion, then performed a detailed FA phenotypes characterization and analysis with Fanca KO mice and Fancd2 KO mice in the same congenic background. We found that both the Fanca KO and Fancd2 KO cause severe FA phenotypes in mice. However, Fanca KO mice exhibited milder FA phenotypes comparing to Fancd2 KO mice. Fanca KO mice showed higher embryonic and postnatal survival rate, less congenital eye defects in early development. At adult stage, Fanca KO mice showed increased HSC number and reconstitution function. Furthermore, we did RNA-seq study and identified differential expression of Dlk1 and Dlk1 pathway genes in Fanca KO and Fancd2 KO embryonic cells and adult HSCs. Finally, we revealed that Fancd2 was expressed and physically interact with Dlk1 in Fanca KO cells. Collectively, our findings suggested that Fancd2 has distinct functions in the absence of Fanca.

STIM1 translocation to the nucleus protects cells from DNA damage.

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.

Identification of arnicolide C as a novel chemosensitizer to suppress mTOR/E2F1/FANCD2 axis in non-small cell lung cancer.

BACKGROUND AND PURPOSE: The mammalian target of rapamycin (mTOR) pathway plays critical roles in intrinsic chemoresistance by regulating Fanconi anaemia complementation group D2 (FANCD2) expression. However, the mechanisms by which mTOR regulates FANCD2 expression and related inhibitors are not clearly elucidated. Extracts of Centipeda minima (C. minima) showed promising chemosensitizing effects by inhibiting FANCD2 activity. Here, we have aimed to identify the bioactive chemosensitizer in C. minima extracts and elucidate its underlying mechanism. EXPERIMENTAL APPROACH: The chemosensitizing effects of arnicolide C (ArC), a bioactive compound in C. minima, on non-small cell lung cancer (NSCLC) were investigated using immunoblotting, immunofluorescence, flow cytometry, the comet assay, small interfering RNA (siRNA) transfection and animal models. The online SynergyFinder software was used to determine the synergistic effects of ArC and chemotherapeutic drugs on NSCLC cells. KEY RESULTS: ArC had synergistic cytotoxic effects with DNA cross-linking drugs such as cisplatin and mitomycin C in NSCLC cells. ArC treatment markedly decreased FANCD2 expression in NSCLC cells, thus attenuating cisplatin-induced FANCD2 nuclear foci formation, leading to DNA damage and apoptosis. ArC inhibited the mTOR pathway and attenuated mTOR-mediated expression of E2F1, a critical transcription factor of FANCD2. Co-administration of ArC and cisplatin exerted synergistic anticancer effects in the A549 xenograft mouse model by suppressing mTOR/FANCD2 signalling in tumour tissues. CONCLUSION AND IMPLICATIONS: ArC suppressed DNA cross-linking drug-induced DNA damage response by inhibiting the mTOR/E2F1/FANCD2 signalling axis, serving as a chemosensitizing agent. This provides insight into the anticancer mechanisms of ArC and offers a potential combinatorial anticancer therapeutic strategy.

Pan-cancer analysis of the tumorigenic role of Fanconi anemia complementation group D2 (FANCD2) in human tumors.

Monoubiquitination of FANCD2 is a central step in the activation of the Fanconi anemia (FA) pathway after DNA damage. Defects in the FA pathway centered around FANCD2 not only lead to genomic instability but also induce tumorigenesis. At present, few studies have investigated FANCD2 in tumors, and no pan-cancer research on FANCD2 has been conducted. We conducted a comprehensive analysis of the role of FANCD2 in cancer using public databases and other published studies. Moreover, we evaluated the role of FANCD2 in the proliferation, migration and invasion of lung adenocarcinoma cells through in vitro and in vivo experiments, and explored the role of FANCD2 in cisplatin chemoresistance. We investigated the regulatory effect of FANCD2 on the cell cycle of lung adenocarcinoma cells by flow cytometry, and verified this effect by western blotting. FANCD2 expression is elevated in most TCGA tumors and shows a strong positive correlation with poor prognosis in tumor patients. In addition, FANCD2 expression shows strong correlations with immune infiltration, immune checkpoints, the tumor mutation burden (TMB), and microsatellite instability (MSI), which are immune-related features, suggesting that it may be a potential target of tumor immunotherapy. We further found that FANCD2 significantly promotes the proliferation, invasion, and migration abilities of lung adenocarcinoma cells and that its ability to promote cancer cell proliferation may be achieved by modulating the cell cycle. The findings indicate that FANCD2 is a potential biomarker and therapeutic target in cancer treatment by analyzing the oncogenic role of FANCD2 in different tumors.

FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination.

Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive understanding of how PCNA K164 ubiquitination promotes genome stability. To evaluate this, we generated stable cell lines expressing PCNAK164R from the endogenous PCNA locus. Our data reveal that the inability to ubiquitinate K164 causes perturbations in global DNA replication. Persistent replication stress generates under-replicated regions and is exacerbated by the DNA polymerase inhibitor aphidicolin. We show that these phenotypes are due, in part, to impaired Fanconi anemia group D2 protein (FANCD2)-dependent mitotic DNA synthesis (MiDAS) in PCNAK164R cells. FANCD2 mono-ubiquitination is significantly reduced in PCNAK164R mutants, leading to reduced chromatin association and foci formation, both prerequisites for FANCD2-dependent MiDAS. Furthermore, K164 ubiquitination coordinates direct PCNA/FANCD2 colocalization in mitotic nuclei. Here, we show that PCNA K164 ubiquitination maintains human genome stability by promoting FANCD2-dependent MiDAS to prevent the accumulation of under-replicated DNA.

FANCD2 as a novel prognostic biomarker correlated with immune and drug therapy in Hepatitis B-related hepatocellular carcinoma.

BACKGROUND: Ferroptosis is related to the immunosuppression of tumors and plays a critical role in cancer progression. Fanconi anemia complementation group D2 (FANCD2) is a vital gene that regulates ferroptosis. However, the mechanism of action of FANCD2 in Hepatitis B-related hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the prognostic significance and mechanism of action of FANCD2 in Hepatitis B-related HCC. METHODS: The expression of FANCD2 in Hepatitis B-related HCC was explored using The Cancer Genome Atlas (TCGA) and validated using the Gene Expression Omnibus (GEO) database. Univariate and multivariate Cox regression analyses and Kaplan-Meier survival curves were used to analyze the relationship between FANCD2 expression and the overall survival of patients with Hepatitis B-related HCC. Protein-protein interaction networks for FANCD2 were built using the STRING website. In addition, correlations between FANCD2 expression and the dryness index, tumor mutational burden, microsatellite instability (MSI), immune pathways, genes involved in iron metabolism, and sorafenib chemotherapeutic response were analyzed. RESULTS: Our results indicated that FANCD2 was significantly overexpressed in Hepatitis B-related HCC and demonstrated a strong predictive ability for diagnosis (Area Under Curve, 0.903) and prognosis of the disease. High FANCD2 expression was associated with poor prognosis, high-grade tumors, high expression of PDL-1, high MSI scores, and low sorafenib IC50 in Hepatitis B-related HCC. BRCA1, BRCA2, FAN1, and FANCC were vital proteins interacting with FANCD2. The expression level of FANCD2 significantly correlated with the infiltration levels of Treg cells, B cells, CD8 + T cells, CD4 + T cells, neutrophils, macrophages, myeloid dendritic cells, and NK cells in Hepatitis B-related HCC. FANCD2 was positively correlated with the tumor proliferation signature pathway, DNA repair, and cellular response to hypoxia. CONCLUSION: Our study indicated that FANCD2 was a potential novel biomarker and immunotherapeutic target against Hepatitis B-related HCC, which might be related to the chemotherapeutic response to sorafenib.

Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ.

Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS). We previously demonstrated that a MiDAS mechanism also exists in untransformed and primary human cells, which is RAD52 independent but requires FANCD2. However, the properties of this form of MiDAS are not well understood. Here, we report that FANCD2-driven MiDAS in untransformed human cells: 1) requires a prerequisite step of FANCD2 mono-ubiquitination by a subset of Fanconi anemia (FA) proteins, 2) primarily acts to preserve CFS stability but not to prevent chromosome mis-segregation, and 3) depends on HELQ, which potentially functions at an early step. Hence, FANCD2-driven MiDAS in untransformed cells is built to protect CFS stability, whereas RAD52-driven MiDAS in cancer cells is likely adapted to prevent chromosome mis-segregation at the cost of CFS expression. Notably, we also identified a novel form of MiDAS, which surfaces to function when FANCD2 is absent in untransformed cells. Our findings substantiate the complex nature of MiDAS and a link between its deficiencies and the pathogenesis of FA, a human genetic disease.

LncRNA SNHG1 upregulates FANCD2 and G6PD to suppress ferroptosis by sponging miR-199a-5p/3p in hepatocellular carcinoma.

Ferroptosis is a form of regulated cell death (RCD) triggered by iron-dependent lipid peroxidation and is closely associated with the occurrence and progression of hepatocellular carcinoma (HCC). The lncRNA SNHG1 (small nucleolar RNA host gene 1) has been shown to play an oncogenic role in HCC, but its function in RCD other than autophagy and apoptosis is still unknown. Here, we investigated the correlation between SNHG1 and 156 typical markers of five RCD types based on RNA sequencing data from The Cancer Genome Atlas database and showed the negative regulators of ferroptosis FANCD2 (Fanconi anemia complementation group D2) and G6PD (glucose-6-phosphate dehydrogenase) to be the most highly and fifth most highly correlating factors with SNHG1, respectively. A competitive endogenous RNA network of SNHG1 - miR-199a-5p/3p - FANCD2/G6PD was constructed bioinformatically. In vitro experiments showed that overexpression of the miR-199a precursor led to a decrease in expression of SNHG1, FANCD2, and G6PD, whereas knockdown of SNHG1 decreased expression of FANCD2 and G6PD but increased levels of miR-199a-5p and miR-199a-3p in HCC cells (Huh7 and HepG2). In addition, knockdown of SNHG1 increased erastin-mediated ferroptosis, iron accumulation, and lipid peroxidation. These results suggest that SNHG1 upregulates FANCD2 and G6PD by sponging miR-199a, thereby inhibiting ferroptosis in HCC. Moreover, a signature based on expression of SNHG1, FANCD2, and G6PD was identified as being associated with overall survival and the immunological microenvironment in HCC. Collectively, this study identified the SNHG1-miR-199a-FANCD2/G6PD axis in HCC, which is a potential marker for the prognosis and therapy of this tumor.

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability.

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells. (2) Independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11 and EXO1. (3) Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and BRCA2 in the FA/BRCA fork protection pathway.

FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress.

Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.

Fanconi anemia DNA crosslink repair factors protect against LINE-1 retrotransposition during mouse development.

Long interspersed nuclear element 1 (LINE-1) is the only autonomous retrotransposon in humans and new integrations are a major source of genetic variation between individuals. These events can also lead to de novo germline mutations, giving rise to heritable genetic diseases. Recently, a role for DNA repair in regulating these events has been identified. Here we find that Fanconi anemia (FA) DNA crosslink repair factors act in a common pathway to prevent retrotransposition. We purify recombinant SLX4-XPF-ERCC1, the crosslink repair incision complex, and find that it cleaves putative nucleic acid intermediates of retrotransposition. Mice deficient in upstream crosslink repair signaling (FANCA), a downstream component (FANCD2) or the nuclease XPF-ERCC1 show increased LINE-1 retrotransposition in vivo. Organisms limit retrotransposition through transcriptional silencing but this protection is attenuated during early development leaving the zygote vulnerable. We find that during this window of vulnerability, DNA crosslink repair acts as a failsafe to prevent retrotransposition. Together, our results indicate that the FA DNA crosslink repair pathway acts together to protect against mutation by restricting LINE-1 retrotransposition.

Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.