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1
Wang, XiaoZhe, Richard D. Kennedy, Kallol Ray, Patricia Stuckert, Tom Ellenberger, and Alan D. D'Andrea. "Chk1-Mediated Phosphorylation of FANCE Is Required for the Fanconi Anemia/BRCA Pathway." Molecular and Cellular Biology 27, no. 8 (February 12, 2007): 3098–108. http://dx.doi.org/10.1128/mcb.02357-06.
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ABSTRACT The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.
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Draga, Margarethe, Elizabeth B. Madgett, Cassandra J. Vandenberg, David du Plessis, Aisling Kaufmann, Petra Werler, Prasun Chakraborty, Noel F. Lowndes, and Kevin Hiom. "BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells." Molecular and Cellular Biology 35, no. 22 (August 31, 2015): 3829–40. http://dx.doi.org/10.1128/mcb.01497-14.
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The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc−) DT40 cells arrest in G2phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced inFancc−cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G2checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G2arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G2phase.
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Smogorzewska, Agata. "Fanconi Anemia: A Paradigm for Understanding DNA Repair During Replication." Blood 134, Supplement_1 (November 13, 2019): SCI—32—SCI—32. http://dx.doi.org/10.1182/blood-2019-121229.
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Fanconi anemia, the most common hereditary bone marrow failure disorder, results from defective repair of DNA interstrand crosslinks (ICLs), which covalently link complementary DNA strands causing replication stalling. Mutations in 22 different genes (FANCA-FANCW) have been shown to result in Fanconi anemia. Their protein products work at different stages of DNA repair leading to considerable heterogeneity in human phenotypes. The majority of the FANC gene mutations are recessively inherited with the exceptions of FANCB and FANCR/RAD51. FANCB is X-linked, and all FANCR/RAD51 mutations arise de novo, affect only one allele, and the mutant protein acts as a dominant negative against the wild type protein. Despite advances in the molecular diagnosis of Fanconi anemia, if Fanconi anemia is suspected, chromosome breakage (DEB or MMC) testing on patient cells is essential. We have seen a number of patients referred to the International Fanconi Anemia Registry (http://lab.rockefeller.edu/smogorzewska/ifar/) who are misdiagnosed with Fanconi anemia based solely on the presence of a FANC gene variant in gene panel or whole exome sequencing. Conversely, blood mosaicism may lead to a negative blood chromosome breakage test. If there is a high suspicion of Fanconi anemia, but blood breakage results are negative, breakage test on patient fibroblasts should be performed. Diagnosis of Fanconi anemia should also be entertained in young adults presenting with squamous cell carcinoma of the aerodigestive tract, since this may be their initial presentation of Fanconi anemia and conventional chemotherapy dose would precipitate bone marrow failure in these patients. In my talk, I will discuss the mechanism of the Fanconi anemia repair pathway during DNA replication. Then, I will concentrate on the mechanism of bone marrow failure and tumorigenesis in Fanconi anemia. I will explore the hypothesis that the endogenously produced aldehydes including some that are still unknown, contribute to disease development. Fanconi anemia-deficient hematopoietic stem cells have an autonomous DNA repair defect. Accumulation of DNA damage leads to apoptosis due to the activation of p53. If cells escape death, mutagenesis may lead to the development of leukemia. The sources of endogenous DNA damage are poorly understood. Cell cycle induction of Fanconi anemia pathway-deficientmouse hematopoietic stem cells results in DNA damage and bone marrow failure, which implies that the DNA lesions encountered during replication are the culprit. There is mounting evidence that the endogenous aldehydes, including acetaldehyde and formaldehyde,may cause those DNA lesions. To identify other metabolites that may induce bone marrow failure in Fanconi anemia, we used a library of CRISPR guides to target Cas9 to metabolic genes to screen for and identify synthetic lethality with Fanconi anemia deficiency. We have identifiedALDH9A1as the most significantly depleted gene in FANCD2-/- cells. The synthetically lethal interaction was validated using single gene editing in human umbilical cord-derived hematopoietic stem progenitor cells. We propose a model in which aldehydes that are metabolized by ALDH9A1 accumulate in the absence of this enzyme and cause DNA damage that requires the Fanconi anemia pathway proteins for repair, survival, and suppression of tumorigenesis. We are testing this model using Fanca-/-Aldh9a1-/-mice. Disclosures No relevant conflicts of interest to declare.
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Munkhjargal, Anudari, Myung-Jin Kim, Da-Yeon Kim, Young-Jun Jeon, Young-Hoon Kee, Lark-Kyun Kim, and Yong-Hwan Kim. "Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression." International Journal of Molecular Sciences 22, no. 15 (July 21, 2021): 7782. http://dx.doi.org/10.3390/ijms22157782.
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Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.
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Repczyńska, Anna, and Olga Haus. "Genetic background and diagnosis of Fanconi anemia." Postępy Higieny i Medycyny Doświadczalnej 74 (December 31, 2020): 589–600. http://dx.doi.org/10.5604/01.3001.0014.6332.
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Fanconi anemia (FA) is a rare genetic disease caused by mutations in genes whose protein products are involved in important cell processes such as replication, cell cycle control and repair of DNA damage. FA is characterized by congenital malformations, bone marrow failure and high risk of cancer. Phenotypic symptoms, present in about 75% of patients, most often include such abnormalities as short stature, microcephaly, thumb and radial side of the limb defects, abnormal skin pigmentation, gastrointestinal and genitourinary defects. Progressive bone marrow failure occurs in the first decade of life, often initially with leukopenia or thrombocytopenia. The most common cancers occurring in patients with FA are myelodysplastic syndromes and acute myeloid leukemia, as well as solid tumors of the head and neck, skin, gastrointestinal system and genitourinary system. So far, 22 genes of Fanconi anemia (FANC) have been identified, which are located on the autosomal chromosomes, except for FANCB, which is located on the X chromosome. Protein products of FANC genes are the elements of Fanconi anemia pathway, which regulates DNA damage repair systems. Genetic diagnostics of Fanconi anemia should start by testing crosslinking agents: mitomycin C (MMC) or diepoxybutane (DEB) assuring differential diagnosis of chromosome instability syndromes. In patients with Fanconi anemia, an increased number of chromosomal gaps and breaks as well as specific radial structures are observed. In order to detect a mutation underlying Fanconi anemia, molecular techniques should be used, preferentially next generation sequencing (NGS).
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Dan, Chenchen, Hongjing Pei, Buzhe Zhang, Xuan Zheng, Dongmei Ran, and Changzheng Du. "Fanconi anemia pathway and its relationship with cancer." Genome Instability & Disease 2, no. 3 (June 2021): 175–83. http://dx.doi.org/10.1007/s42764-021-00043-0.
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AbstractFanconi Anemia (FA) is a rare inherited hematological disease, caused by mutations in genes involved in the DNA interstrand crosslink (ICL) repair. Up to date, 22 genes have been identified that encode a series of functionally associated proteins that recognize ICL lesion and mediate the activation of the downstream DNA repair pathway including nucleotide excision repair, translesion synthesis, and homologous recombination. The FA pathway is strictly regulated by complex mechanisms such as ubiquitination, phosphorylation, and degradation signals that are essential for the maintenance of genome stability. Here, we summarize the discovery history and recent advances of the FA genes, and further discuss the role of FA pathway in carcinogenesis and cancer therapies.
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Collins, Natalie B., Andrei Tomashevski, and Gary M. Kupfer. "Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function." Blood 108, no. 11 (November 16, 2006): 186. http://dx.doi.org/10.1182/blood.v108.11.186.186.
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Abstract Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.
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Bagby, Grover. "Recent advances in understanding hematopoiesis in Fanconi Anemia." F1000Research 7 (January 24, 2018): 105. http://dx.doi.org/10.12688/f1000research.13213.1.
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Fanconi anemia is an inherited disease characterized by genomic instability, hypersensitivity to DNA cross-linking agents, bone marrow failure, short stature, skeletal abnormalities, and a high relative risk of myeloid leukemia and epithelial malignancies. The 21 Fanconi anemia genes encode proteins involved in multiple nuclear biochemical pathways that effect DNA interstrand crosslink repair. In the past, bone marrow failure was attributed solely to the failure of stem cells to repair DNA. Recently, non-canonical functions of many of the Fanconi anemia proteins have been described, including modulating responses to oxidative stress, viral infection, and inflammation as well as facilitating mitophagic responses and enhancing signals that promote stem cell function and survival. Some of these functions take place in non-nuclear sites and do not depend on the DNA damage response functions of the proteins. Dysfunctions of the canonical and non-canonical pathways that drive stem cell exhaustion and neoplastic clonal selection are reviewed, and the potential therapeutic importance of fully investigating the scope and interdependences of the canonical and non-canonical pathways is emphasized.
9
Müller, Lars U. W., Michael D. Milsom, Chad E. Harris, Rutesh Vyas, Kristina M. Brumme, Kalindi Parmar, Lisa A. Moreau, et al. "Overcoming reprogramming resistance of Fanconi anemia cells." Blood 119, no. 23 (June 7, 2012): 5449–57. http://dx.doi.org/10.1182/blood-2012-02-408674.
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Abstract Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs.
10
Trottier, Magan, and Stephen Meyn. "Fanconi Anemia and the Response of Human Hematopoietic Cells to DNA Damage." Blood 112, no. 11 (November 16, 2008): 441. http://dx.doi.org/10.1182/blood.v112.11.441.441.
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Abstract Given their critical role in the generation and renewal of the hematopoietic system, we hypothesize that hematopoietic stem and primitive cells employ unusually stringent DNA damage responses to maintain genome stability. We also propose that Fanconi anemia proteins prevent bone marrow failure through their participation in these DNA repair and damage response pathways. We are testing both hypotheses by characterizing cellular responses to DNA double-strand breaks (DSBs) in 3 lineage-negative populations of hematopoietic cells isolated from human umbilical cord blood: hematopoietic stem cell-containing primitive CD34+ CD38− CD45RA−, early progenitor CD34+ CD38+ and late progenitor CD34− CD38+. Two key events in the initial cellular response to DSBs are phosphorylation of H2AX histones in chromatin at or near DSBs, and association of 53BP1 with gamma-H2AX at DSB sites. Once DSBs are detected, they then are repaired by two major pathways: error-prone non-homologous end-joining (NHEJ), utilized throughout the cell cycle, and error-free homologous recombination (HR), used when sister chromatids are present during S/G2 phases of the cell cycle. To study the response to and repair of DSBs in our sorted hematopoietic populations, we exposed quiescent and cytokine-stimulated haematopoietic cells and human fibroblasts to ionizing radiation (IR). Irradiation with 3 Gy rapidly induced formation of 53BP1 foci in all cell types, both cycling and non-cycling. In contrast, X-irradiation only induced foci of the Fanconi anemia protein FANCD2 in cycling cells. The majority of the induced FANCD2 foci colocalized with foci of gamma-H2AX. The subsequent persistence of 53BP1 foci at X-ray induced damage sites differed significantly between the lin− hematopoietic cells and the fibroblasts. During the first 6 hours following IR, cycling primitive and progenitor hematopoietic cells resolved 53BP1 foci more slowly than cycling human primary fibroblasts. In addition, X-ray induced 53BP1 foci persisted even longer in quiescent primitive CD34+ CD38− and early progenitor CD34+ CD38+ cells than in their cycling counterparts, while foci resolution kinetics were similar in quiescent and cycling late progenitor CD34− CD38+ cells and in fibroblasts. We also used a neutral comet assay to determine the kinetics of DSB rejoining following exposure to 15 Gy. We found that resolution of 53BP1 foci is not directly linked to rejoining of DSBs in lin− hematopoietic cells, as they rejoin DSBs more rapidly than primary fibroblasts, yet resolve 53BP1 foci more slowly. The effect is most prominent in primitive CD34− CD38− cells. Both cycling and non-cycling fibroblasts displayed similar kinetics of DSB rejoining. However, quiescent lin− hematopoietic cells displayed persistent breaks compared to cycling counterparts or to fibroblasts. Of particular note, quiescent lin− hematopoietic cells still had not rejoined a significant fraction of induced DSBs 24 hours post irradiation. Our results indicate that human primitive and progenitor hematopoetic cells differ from fibroblasts in their DNA damage responses and DSB repair kinetics. Our findings also suggest that, unlike fibroblasts, progression through the cell cycle plays a key role in the rapid repair of DSBs in hematopoietic cells. Because Lin− hematopoietic cells are more efficient at rejoining DSBs when cycling, we suggest that error-free HR repair plays an important role in DSB repair in these cells. Our detection of DNA damage-induced FANCD2 foci only in cycling lin− hematopoietic cells provides indirect support for this conclusion, as FA proteins are thought to be involved in HR repair. We are currently testing this hypothesis by determining the effect of knocking down FA protein expression on DSB repair and DNA damage responses in our three hematopoietic populations.
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Ninou, Anna Huguet, Jemina Lehto, Dimitrios Chioureas, Hannah Stigsdotter, Korbinian Schelzig, Emma Åkerlund, Greta Gudoityte, et al. "PFKFB3 Inhibition Sensitizes DNA Crosslinking Chemotherapies by Suppressing Fanconi Anemia Repair." Cancers 13, no. 14 (July 18, 2021): 3604. http://dx.doi.org/10.3390/cancers13143604.
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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.
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García-de-Teresa, Benilde, Alfredo Rodríguez, and Sara Frias. "Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences." Genes 11, no. 12 (December 21, 2020): 1528. http://dx.doi.org/10.3390/genes11121528.
Full textAbstract:
Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.
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Singh, Thiyam R., Abdullah M. Ali, Chang-hu Du, and Ruhikanta A. Meetei. "ATR Dependent Phosphorylation of FANCM Is Required for the Fanconi Anemia Pathway." Blood 110, no. 11 (November 16, 2007): 837. http://dx.doi.org/10.1182/blood.v110.11.837.837.
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Abstract Fanconi anemia (FA) is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. We have shown that FANCM which is one of the FA core complex proteins is hyperphosphorylated in response to DNA damage suggesting that it may serve as a signal transducer through which the activity of the FA-core complex is regulated. The cell cycle checkpoint kinase, ATR has been shown to act upstream of the FA pathway, however, its substrate within the FA-core complex has not been identified yet. FANCM contains multiple predicted ATR phosphorylation sites suggesting that FANCM could be a direct ATR target. In this study, we examined the roles of ATR in regulating FANCM phosphorylation in response to DNA damage: by kinetics study we found that phosphorylation of FANCM is concurrent with FANCD2 monoubiquitination; siRNA mediated suppression of ATR activity abrogates both phosphorylation of FANCM and monoubiquitination of FANCD2; and ATR knock out HCT116 cells display defective phosphorylation of FANCM as well as defective monoubiquitination of FANCD2 indicating that DNA damage induced phosphorylation of FANCM is ATR dependant. Furthermore, we used mass spectrometry to identify the in vivo phosphorylation sites of FANCM and found a novel DNA damage-inducible phosphorylation site (S-1045; one of the potential ATR phosphorylation sites) within FANCM protein. Using ATR knock out HCT116 cells and the anti-p-S1045 antibody, we show that phosphorylation of FANCM at S-1045 is ATR dependant. The biological relevance of phosphorylation of FANCM at S1045 in FA pathway will be investigated by functional complementation analysis with non phosphorylatable FANCM mutants in FANCM deficient cells.
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Jenkins, Chelsea, Jenny Kan, and Maureen E. Hoatlin. "Targeting the Fanconi Anemia Pathway to Identify Tailored Anticancer Therapeutics." Anemia 2012 (2012): 1–7. http://dx.doi.org/10.1155/2012/481583.
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The Fanconi Anemia (FA) pathway consists of proteins involved in repairing DNA damage, including interstrand cross-links (ICLs). The pathway contains an upstream multiprotein core complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and a downstream pathway that converges with a larger network of proteins with roles in homologous recombination and other DNA repair pathways. Selective killing of cancer cells with an intact FA pathway but deficient in certain other DNA repair pathways is an emerging approach to tailored cancer therapy. Inhibiting the FA pathway becomes selectively lethal when certain repair genes are defective, such as the checkpoint kinase ATM. Inhibiting the FA pathway in ATM deficient cells can be achieved with small molecule inhibitors, suggesting that new cancer therapeutics could be developed by identifying FA pathway inhibitors to treat cancers that contain defects that are synthetic lethal with FA.
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Hodson, Charlotte, and Helen Walden. "Towards a Molecular Understanding of the Fanconi Anemia Core Complex." Anemia 2012 (2012): 1–10. http://dx.doi.org/10.1155/2012/926787.
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Fanconi Anemia (FA) is a genetic disorder characterized by the inability of patient cells to repair DNA damage caused by interstrand crosslinking agents. There are currently 14 verified FA genes, where mutation of any single gene prevents repair of DNA interstrand crosslinks (ICLs). The accumulation of ICL damage results in genome instability and patients having a high predisposition to cancers. The key event of the FA pathway is dependent on an eight-protein core complex (CC), required for the monoubiquitination of each member of the FANCD2-FANCI complex. Interestingly, the majority of patient mutations reside in the CC. The molecular mechanisms underlying the requirement for such a large complex to carry out a monoubiquitination event remain a mystery. This paper documents the extensive efforts of researchers so far to understand the molecular roles of the CC proteins with regard to its main function in the FA pathway, the monoubiquitination of FANCD2 and FANCI.
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Mi, Jun, Andrei Tomashevski, and Gary M. Kupfer. "Differential Phosphorylation of FANCA and FANCG Determines Response of Fanconi Anemia Core Complex to DNA Damage and S Phase." Blood 104, no. 11 (November 16, 2004): 726. http://dx.doi.org/10.1182/blood.v104.11.726.726.
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Abstract Fanconi anemia (FA) is a genetic disease marked by bone marrow failure, congenital defects, and cancer. In spite of the identification of at least 8 genes, the biochemistry of the disease and its normal pathway in the cell remains elusive. The FA core complex is composed of at least 5 proteins, 2 of which, FANCA and FANCG, we have shown to be phosphorylated. In these studies, we show that both FANCA and FANCG are phosphorylated in response to DNA damage. In the case of FANCG, we have mapped the site of this phosphorylation to serine 7, using a phosphoserine 7 FANCG antiserum. Because of the link of FA function and the FA core complex-dependent monoubiquitination that occurs both as a result of DNA damage as well as at S phase, we also examined if phosphorylation occurred at S phase as well. While FANCG serine 7 phosphorylation occurs both at S phase and after DNA damage (similar to FANCD2 monoubiquitination), FANCA phosphorylation occurs only after DNA damage. Recent data have implicated the kinase ATR as important in the pathway. In order to assess whether a downstream target of ATR is differentially phosphorylated in FA cells, we tested the phosphorylation status of chk1 in FA-A mutant and corrected cells. Chk1 kinase is phosphorylated at serine 318 in response to DNA damage only in corrected cells but not mutant FA cells, while signaling through chk2 kinase is unaffected. These data suggest the importance of phosphorylation in the FA pathway in the regulation of both cellular responses to DNA damage as well as engagement of the cell cycle.
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Arkinson, Connor, Viduth K. Chaugule, Rachel Toth, and Helen Walden. "Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1." Life Science Alliance 1, no. 5 (October 2018): e201800162. http://dx.doi.org/10.26508/lsa.201800162.
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The Fanconi anemia pathway for DNA interstrand crosslink repair and the translesion synthesis pathway for DNA damage tolerance both require cycles of monoubiquitination and deubiquitination. The ubiquitin-specific protease-1 (USP1), in complex with USP1-associated factor 1, regulates multiple DNA repair pathways by deubiquitinating monoubiquitinated Fanconi anemia group D2 protein (FANCD2), Fanconi anemia group I protein (FANCI), and proliferating cell nuclear antigen (PCNA). Loss of USP1 activity gives rise to chromosomal instability. Whereas many USPs hydrolyse ubiquitin–ubiquitin linkages, USP1 targets ubiquitin–substrate conjugates at specific sites. The molecular basis of USP1's specificity for multiple substrates is poorly understood. Here, we reconstitute deubiquitination of purified monoubiquitinated FANCD2, FANCI, and PCNA and show that molecular determinants for substrate deubiquitination by USP1 reside within the highly conserved and extended N-terminus. We found that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for direct PCNA or FANCI deubiquitination. Furthermore, we show that the N-terminus of USP1 is sufficient to engineer specificity in a more promiscuous USP.
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Kim, Yonghwan, Gabriella S. Spitz, Uma Veturi, Francis P. Lach, Arleen D. Auerbach, and Agata Smogorzewska. "Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4." Blood 121, no. 1 (January 3, 2013): 54–63. http://dx.doi.org/10.1182/blood-2012-07-441212.
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Abstract SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases.
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Larin, Meghan, David Gallo, Laura Tamblyn, Jay Yang, Hudson Liao, Nestor Sabat, Grant W. Brown, and J. Peter McPherson. "Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair." Nucleic Acids Research 42, no. 15 (July 23, 2014): 9807–20. http://dx.doi.org/10.1093/nar/gku676.
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AbstractIndividuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.
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Patel, Ketan J. "Links Between DNA Damage and Metabolism, Pathways Causing Bone Marrow Failure in Fanconi Anemia, and Therapeutic Implications." Blood 120, no. 21 (November 16, 2012): SCI—3—SCI—3. http://dx.doi.org/10.1182/blood.v120.21.sci-3.sci-3.
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Abstract Abstract SCI-3 Recent work from my lab has discovered that metabolism generates reactive aldehydes. These reactive molecules are potent damagers of DNA. The consequences of this are revealed by the inactivation of enzymes that detoxify these aldehydes and the Fanconi anemia DNA repair pathway in mice and vertebrate cell lines. The scientific session presentation will discuss this work and recent unpublished research on how natural aldehydes damage blood stem cells. This work has consequences for understanding how metabolism and ethanol exposure can be genotoxic, particularly in the vast population of Southeast Asians carrying a genetic defect in aldehyde catabolism (“pink flushers”). It is also relevant to the emergence of bone marrow failure and leukemia in Fanconi anemia. Disclosures: No relevant conflicts of interest to declare.
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Geng, Liyi, Catherine J. Huntoon, and Larry M. Karnitz. "RAD18-mediated ubiquitination of PCNA activates the Fanconi anemia DNA repair network." Journal of Cell Biology 191, no. 2 (October 11, 2010): 249–57. http://dx.doi.org/10.1083/jcb.201005101.
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The Fanconi anemia (FA) network is important for the repair of interstrand DNA cross-links. A key event in FA pathway activation is the monoubiquitylation of the FA complementation group I (FANCI)–FANCD2 (ID) complex by FA complementation group L (FANCL), an E3 ubiquitin ligase. In this study, we show that RAD18, another DNA damage–activated E3 ubiquitin ligase, also participates in ID complex activation by ubiquitylating proliferating cell nuclear antigen (PCNA) on Lys164, an event required for the recruitment of FANCL to chromatin. We also found that monoubiquitylated PCNA stimulates FANCL-catalyzed FANCD2 and FANCI monoubiquitylation. Collectively, these experiments identify RAD18-mediated PCNA monoubiquitination as a central hub for the mobilization of the FA pathway by promoting FANCL-mediated FANCD2 monoubiquitylation.
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Liang, Fengshan, Adam S. Miller, Caroline Tang, David Maranon, Elizabeth A. Williamson, Robert Hromas, Claudia Wiese, Weixing Zhao, Patrick Sung, and Gary M. Kupfer. "The DNA-binding activity of USP1-associated factor 1 is required for efficient RAD51-mediated homologous DNA pairing and homology-directed DNA repair." Journal of Biological Chemistry 295, no. 24 (April 29, 2020): 8186–94. http://dx.doi.org/10.1074/jbc.ra120.013714.
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USP1-associated factor 1 (UAF1) is an integral component of the RAD51-associated protein 1 (RAD51AP1)–UAF1-ubiquitin-specific peptidase 1 (USP1) trimeric deubiquitinase complex. This complex acts on DNA-bound, monoubiquitinated Fanconi anemia complementation group D2 (FANCD2) protein in the Fanconi anemia pathway of the DNA damage response. Moreover, RAD51AP1 and UAF1 cooperate to enhance homologous DNA pairing mediated by the recombinase RAD51 in DNA repair via the homologous recombination (HR) pathway. However, whereas the DNA-binding activity of RAD51AP1 has been shown to be important for RAD51-mediated homologous DNA pairing and HR-mediated DNA repair, the role of DNA binding by UAF1 in these processes is unclear. We have isolated mutant UAF1 variants that are impaired in DNA binding and tested them together with RAD51AP1 in RAD51-mediated HR. This biochemical analysis revealed that the DNA-binding activity of UAF1 is indispensable for enhanced RAD51 recombinase activity within the context of the UAF1–RAD51AP1 complex. In cells, DNA-binding deficiency of UAF1 increased DNA damage sensitivity and impaired HR efficiency, suggesting that UAF1 and RAD51AP1 have coordinated roles in DNA binding during HR and DNA damage repair. Our findings show that even though UAF1's DNA-binding activity is redundant with that of RAD51AP1 in FANCD2 deubiquitination, it is required for efficient HR-mediated chromosome damage repair.
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Koptyra, Mateusz, Scott Houghtaling, Marcus Grompe, and Tomasz Skorski. "Fanconi Anemia D2 Protein Contributes to BCR/ABL-Mediated Transformation of Hematopoietic Cells." Blood 106, no. 11 (November 16, 2005): 2878. http://dx.doi.org/10.1182/blood.v106.11.2878.2878.
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Abstract Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) or genotoxic treatment. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts. In addition, although the expression of FANCD2 was stimulated by BCR/ABL and growth factors, higher levels of FANCD2 monoubiquitination was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, monoubiquitination of FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. BCR/ABL kinase displayed an impaired transformation potential in FANCD2-/- murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Growth ability of BCR/ABL-positive FANCD2-/- cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DNA damage than +/+ counterparts as indicated by enzymatic assays converting oxidative DNA lesions into gaps detectable by comet assay. In addition, enhanced oxidative DNA damage in BCR/ABL-positive FANCD2-/- cells produced a variety of DNA lesions including abasic sites, and single- and double-strand breaks assessed by neutral comet assay. Moreover, BCR/ABL-positive FANCD2-/- cells accumulated higher numbers of DSBs detected by γ-H2AX immunostaining and displayed discrete apoptosis. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in the initial steps of BCR/ABL dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.
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Lambert, W. Clark, Monique M. Brown, and Santiago A. Centurion. "The Co-Recessive Inheritance Model: A Paradigm for Fanconi Anemia and Other Bone Marrow Failure Syndromes." Blood 106, no. 11 (November 16, 2005): 3760. http://dx.doi.org/10.1182/blood.v106.11.3760.3760.
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Abstract One of us (WCL) has previously proposed a mathematical model, Co-Recessive Inheritance, for inherited diseases associated with DNA repair deficiencies (Lambert WC, Lambert MW: Mutat. Res., 1985;145:227–234; Lambert WC: Keynote Address, 21st Anniversary Celebration, MRC Cell Mutation Unit, University of Sussex, UK. Mutat. Res., 1992;273:179–102). The model is also applicable to diseases associated with defective cell cycle modulation following specific types of DNA damage, such as Fanconi Anemia, with or without additional defects in DNA repair. The model proposes that in some complementation groups of these diseases defective alleles at more than one locus are required for the disease phenotype to be expressed. It follows from the model (A readily understandable derivation will be presented.) that the carrier frequencies of the genes involved are very much higher than would be predicted based on classical population genetics. This may impact on recent observations of higher than expected co-inheritance of defective alleles of Fanconi Anemia and Bloom Syndrome genes along with BRCA genes in certain populations (e.g., Koren-Michowitz, M, et al.: Am. J. Hematol., 2005;78:203–206), and provides an explanation for the lower than expected incidence of cancer in these individuals. It also provides an explanation for finding biallelic defects in the same DNA repair genes in more than one complementation group of Fanconi Anemia (Howlett NG, et al.: Science, 2002;297:606–609). The Co-Recessive Model predicts that other findings of this nature are to be expected, and provides some guidelines that may be helpful in the process of gene discovery in Fanconi Anemia. Among the more important of these are 1) that the search for defective genes in each complementation group should not cease when one such gene is found, even if one or more patients in the group is homozygous or compound heterozygous for defective alleles of that gene, and 2) that carrier frequencies for some Fanconi Anemia genes may be much higher than would otherwise be anticipated, with a significant proportion of the normal population being carriers. If the latter hypothesis is correct, it follows that the relevance of these rare diseases and their associated genes to disease, including bone marrow failure, in the general population is dramatically greater than has been generally believed.
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Wang, Rui, Walter F. Lenoir, Chao Wang, Dan Su, Megan McLaughlin, Qianghua Hu, Xi Shen, et al. "DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress." Proceedings of the National Academy of Sciences 117, no. 52 (December 21, 2020): 33436–45. http://dx.doi.org/10.1073/pnas.2008821117.
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Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4. As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.
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Collins, Natalie B., James B. Wilson, Thomas Bush, Andrei Thomashevski, Kate J. Roberts, Nigel J. Jones, and Gary M. Kupfer. "ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function." Blood 113, no. 10 (March 5, 2009): 2181–90. http://dx.doi.org/10.1182/blood-2008-05-154294.
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Abstract Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage–specific event that is downstream of ATR and is functionally important in the FA pathway.
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Gueiderikh, Anna, Frédérique Maczkowiak-Chartois, Guillaume Rouvet, Sylvie Souquère-Besse, Sébastien Apcher, Jean-Jacques Diaz, and Filippo Rosselli. "Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis." Science Advances 7, no. 1 (January 2021): eabb5414. http://dx.doi.org/10.1126/sciadv.abb5414.
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Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex–independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.
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Guo, Yingying, Wanjuan Feng, Shirley M. H. Sy, and Michael S. Y. Huen. "ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response." Journal of Biological Chemistry 290, no. 46 (September 29, 2015): 27545–56. http://dx.doi.org/10.1074/jbc.m115.672626.
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Stoklosa, Tomasz, Mateusz Koptyra, Grazyna Hoser, Ilona Seferynska, Eliza Glodkowska, and Tomasz Skorski. "BCR/ABL Requires Fanconi Anemia D2 (FANCD2) Protein to Transform Hematopoetic Stem Cells." Blood 114, no. 22 (November 20, 2009): 3249. http://dx.doi.org/10.1182/blood.v114.22.3249.3249.
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Abstract Abstract 3249 Poster Board III-1 Fanconi D2 protein (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) and phosphorylated on S222 (FANCD2-phosphoS222) in response to DNA double-strand breaks (DSBs). FANCD2-Ub interacts with RAD51 to facilitate homologous recombination repair (HRR), and FANCD2-phosphoS222 activates the S phase checkpoint. We detected an increased amount of FANCD2-Ub in CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) patients and in BCR/ABL-positive cell lines in comparison to normal counterparts. This effect was not associated with up-regulation of FANCD2 ubiquitinase FANCL or down-regulation of FANCD2 deubiquitinase USP1, but was reversed after inhibition of BCR/ABL kinase with imatinib and reduction of reactive oxygen species (ROS) with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). In addition mitomycin C routinely used for diagnostic testing in Fanconi anemia, strongly elevated FANCD2-Ub in CD34+ CML cells. Therefore we postulate that FANCD2-Ub may play a role in BCR/ABL transformation. In support for this hypothesis, we observed that clonogenic potential of BCR/ABL-positive murine leukemia stem cells (LSCs)-enriched FANCD2-/- Sca1+Kit+lin- bone marrow cells was reduced by approximately 10-fold in comparison to BCR/ABL-positive FANCD2+/+ counterparts; non-transformed -/- and +/+ cells displayed similar clonogenic potential stimulated by SCF and GM-CSF. Restoration of FANCD2 expression “rescued” the impaired clonogenic activity of BCR/ABL-positive FANCD2-/- cells. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Negative effect of BCR/ABL kinase on FANCD2-/- cell growth was reversed by expression of exogenous FANCD2. The in vitro growth defect of BCR/ABL-positive FANCD2-/- cells was accompanied by delayed leukemogenesis in SCID mice. These results suggest that FANCD2, a key regulator of DNA damage response, may play an important role in the initiation and/or maintenance of BCR/ABL-positive leukemias. We showed before that CD34+ CML-CP and CML-BC cells contain higher number of ROS-induced DSBs in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that LSC-enriched (CD34+CD38-) CML-CP and CML-BC cells display more DSBs than normal counterparts. Thus, BCR/ABL-mediated leukemogenesis is associated with accumulation of an excess of ROS-induced DSBs, which if not repaired, may induce apoptosis. We hypothesize that FANCD2 is necessary to “protect” leukemia cells from potentially lethal effect of BCR/ABL-induced oxidative DNA damage (including DSBs) at early stages of transformation and possibly also during the progression to CML-BC. This suggestion is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DSBs in comparison to +/+ counterparts. This effect did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by VE and NAC reduced the number of DSBs and eliminated the growth defect of BCR/ABL-positive FANCD2-/- cells. Accumulation of excessive DNA damage (DSBs) and impairment of growth potential of BCR/ABL-positive FANCD2-/- cells could be prevented by expression of FANCD2 wild-type (proficient in DNA repair and S phase checkpoint) and S222A phosphorylation-deficient mutant (proficient in DNA repair, but deficient in S phase checkpoint), but not the K561R monoubiquitination-deficient mutant (deficient in DNA damage, but proficient in S phase checkpoint). Since FANCD2-Ub interacts with RAD51 to promote HRR and BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, it is plausible that elevated expression of FANCD2-Ub may facilitate DSB repair to protect leukemia cells from lethal effects of DSBs. In concordance, co-localization of FANCD2-Ub and RAD51 was readily detected in the nuclei of BCR/ABL-positive leukemia cells. In conclusion our results indicate that FANCD2 plays an important role during the induction and perhaps also progression of Philadelphia-chromosome positive leukemias due to its ability to facilitate the repair of numerous, potentially lethal DSBs. Disclosures: No relevant conflicts of interest to declare.
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Yagasaki, Hiroshi, Daiki Adachi, Tsukasa Oda, Irene Garcia-Higuera, Nii Tetteh, Alan D. D'Andrea, Makoto Futaki, Shigetaka Asano, and Takayuki Yamashita. "A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA." Blood 98, no. 13 (December 15, 2001): 3650–57. http://dx.doi.org/10.1182/blood.v98.13.3650.
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Abstract Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FAgenes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA− cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.
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Zhang, Haojian, David Kozono, Kevin O'Connor, Sofia Vidal-Cardenas, Abigail Hamilton, Emily Gaudiano, Joel S. Greenberger, Markus Grompe, Kalindi Parmar, and Alan D. D'Andrea. "Bone Marrow Failure in Fanconi Anemia from Hyperactive TGF-β Signaling." Blood 124, no. 21 (December 6, 2014): 356. http://dx.doi.org/10.1182/blood.v124.21.356.356.
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Abstract Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life due to attrition of hematopoietic stem and progenitor cells (HSPCs). FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutants in one of sixteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA may result, directly or indirectly, from hyperactivation of cell autonomous or microenvironmental growth suppressive pathways induced due to genotoxic stress. Recent studies suggest that one suppressive pathway may be the hyperactive p53 response observed in HSPCs from FA patients. In order to further identify suppressive mechanisms accounting for bone marrow failure in FA, we performed a whole genome-wide shRNA screen in FA cells. Specifically, we screened for candidate genes whose knockdown would rescue cellular growth inhibition and genotoxic stress induced by a DNA cross-linking agent mitomycin C (MMC). We transduced a FA-deficient human fibroblast line with pools of shRNAs and screened for rescue of MMC-inhibited growth. Selected shRNA inserts were identified by next generation sequencing. The top hits in the screen were shRNAs directed against multiple components of the TGF-β signaling pathway. Consistent with this, disruption of the TGF-β signaling pathway by shRNA/sgRNA-mediated knockdown of SMAD3 or TGFR1 (downstream components of the TGF- β pathway) rescued growth of multiple cell lines from several FA complementation groups in presence of genotoxic agents (e.g. MMC or acetaldehyde). Pharmacologic inhibition of the TGF- β pathway using small molecule inhibitors resulted in improved survival of FA-deficient lymphoblast cells in presence of MMC or acetaldehyde, suggesting that a hyperactive, TGF-β-mediated, suppression pathway may account, at least in part, for reduced FA cell growth. Interestingly, genes encoding TGF-β pathway signaling components were highly expressed in the bone marrow from FA patients and FA mice. Moreover, disruption of the TGF- β pathway by shRNA-mediated knockdown of SMAD3 rescued the growth defects of primary HSPCs from FA-deficient murine bone marrow. To further implicate the TGF-β pathway, we established primary stromal cell lines from the bone marrow of FA-deficient mice as well as human FA patients. We confirmed that TGF-β signaling was hyperactive in these stroma cells resulting in growth suppression and elevated phospho-ERK levels due to non-canonical signaling of the pathway. Inhibitors of TGF-β signaling partially rescued the growth defects and reduced phospho-ERK levels in these FA stroma cells. The deficiency of FA DNA repair pathway leads to cellular defects in homologous recombination (HR) repair and hyperactivation of toxic non-homologous end joining (NHEJ)-mediated repair. We therefore tested whether inhibition of the TGF-β pathway in FA cells could rescue HR defects and account for the improvement of FA cellular growth. Interestingly, disruption of the TGF-β signaling pathway caused a decrease in NHEJ activity. Disruption of the TGF-β pathway also resulted in reduced MMC-mediated DNA damage and increased HR. Taken together, our results demonstrate that primary FA hematopoietic and bone marrow stromal cells exhibit hyperactive TGF-β signaling accounting at least in part for the bone marrow failure in FA. Inhibitors of the TGF-β signaling pathway may therefore be useful in the clinical treatment of patients with bone marrow failure and Fanconi anemia. Disclosures No relevant conflicts of interest to declare.
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Vinciguerra, Patrizia, Susana Godinho, Kalindi Parmar, David Pellman, and Alan D'Andrea. "Cytokinesis Failure in Fanconi Anemia Pathway Deficient Murine Hematopoietic Stem Cells." Blood 114, no. 22 (November 20, 2009): 495. http://dx.doi.org/10.1182/blood.v114.22.495.495.
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Abstract Abstract 495 Fanconi Anemia (FA) is a rare recessive chromosomal-instability disorder characterized by congenital malformations, a high predisposition to cancer, and progressive bone marrow failure. FA is genetically heterogeneous and, to date, thirteen FA genes have been identified (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N). The thirteen encoded FA proteins cooperate in a common DNA repair pathway active during the Synthesis (S) phase of the cell cycle. DNA damage detected during replication results in the monoubiquitination of two FA proteins, FANCD2 and FANCI, that translocate into chromatin-associated DNA repair foci where they colocalize with downstream components of the pathway. Partial colocalization with BLM, the RecQ helicase mutated in Bloom's syndrome, has also been described. How disruption of this pathway leads to bone marrow failure is a critical unanswered question. Interestingly, FA cells also have abnormalities that suggest a defect in mitosis, including micronuclei and multinucleation. The objectives of this study were to 1) investigate the role of the FA pathway in normal mitosis and 2) determine whether defects in this function underlie the bone marrow failure of FA patients. For this study, we used HeLa cells transiently or stably knocked down for FA genes, FA patient derived cell lines and hematopoietic stem cells from Fanconi mice models generated in our laboratory (Fancd2-/- and Fancg-/-). First, a polyclonal antibody was raised against FANCI and, together with an anti-FANCD2 antibody, used to investigate the localization of the FANCD2-I complex throughout the cell cycle by immunostaining. FANCI and FANCD2 colocalized to discrete foci on condensed chromosomes in a population of cells in Mitosis (M) phase, consistent with results of Chan et al. (Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol. 2009;11:753-760), Naim and Rosselli (The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat Cell Biol. 2009;11:761-768). These foci were dependent on an intact FA pathway, but did not localize at centromeres and did not increase when the spindle assembly checkpoint was challenged. By immunofluorescence, we showed an increase in the presence of Hoechst positive DNA bridges and PICH positive / BLM positive DNA bridges (Hoechst positive and negative) in anaphase and telophase of FA deficient cells compared to FA proficient cells. This increase of DNA bridges between separating sister chromatids in FA deficient cells correlated with an increase of multinucleated cells. Multinuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Furthermore, inhibition of apoptosis increased the number of binucleated cells, suggesting that cytokinesis failure led to apoptosis. Importantly, an increase in binucleated cells was also observed in the hematopoietic stem cells population from Fancd2-/- and Fancg-/- mice, compared to wild-type sibling mice, and this increase correlated with elevated apoptosis in those cells. Based on these new findings, we conclude that the Fanconi pathway is required for normal mitosis and hypothesize that apoptosis induced by cytokinesis failure of hematopoietic stem cells may cause the bone marrow failure commonly found in FA patients. Disclosures: No relevant conflicts of interest to declare.
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Rego, Meghan A., Frederick W. Kolling, Elizabeth A. Vuono, Maurizio Mauro, and Niall G. Howlett. "Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein." Blood 120, no. 10 (September 6, 2012): 2109–17. http://dx.doi.org/10.1182/blood-2012-02-410472.
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Abstract The Fanconi anemia (FA)–BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that although mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2, and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation.
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Liang, Fengshan, Simonne Longerich, Caroline Tang, Olga Buzovestsky, Yong Xiong, David Maranon, Claudia Wiese, Adam Miller, Patrick Sung, and Gary M. Kupfer. "The Role of UAF1 in the Fanconi Anemia Pathway Regulation of Homologous Recombination-Mediated Genome Maintenance." Blood 128, no. 22 (December 2, 2016): 1041. http://dx.doi.org/10.1182/blood.v128.22.1041.1041.
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Abstract Background: Fanconi anemia (FA), a cancer-prone genetic disease, is caused by defects in the FA-DNA repair pathway. In response to DNA interstrand crosslink (ICL)-induced DNA damage, FANCI-FANCD2 mono-ubiquitination licenses the execution of downstream DNA damage signaling and repair steps, including repair by homologous recombination (HR) that utilizes the recombinase RAD51 and its cohort of accessory factors. Timely deubiquitination of FANCD2 by the UAF1-USP1 deubiquitinating enzyme complex is also critically important for the FA pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1 deficient cells are also impaired for DNA repair by homologous recombination. UAF1 physically associates with RAD51AP1, a protein that enhances the activity of the RAD51 recombinase. It remains to be defined how UAF1 regulates homologous recombination and genome stability. Methods: Highly purified proteins were used to define the DNA binding activity and protein interaction of UAF1. In vitroD-loop formation reaction and synaptic complex assembly assay were used to discover the function of UAF1 in RAD51 recombinase enhancement. HeLa and U2OS-DR-GFP cell lines with impaired UAF1-RAD51AP1 interaction or UAF1 DNA binding were generated to examine DNA-damage agent sensitivity and HR efficiency. Results: (1) UAF1 possesses a DNA binding activity capable of engaging ssDNA, dsDNA and has a preference for the D-loop DNA substrate. We further identified that the N-terminus but not C-terminal SLD domain of UAF1 binds DNA. (2) UAF1 forms a dimeric complex with RAD51AP1. Our results also revealed a trimeric complex of RAD51-RAD51AP1-UAF1, with RAD51AP1 providing a tethering function between the other two proteins. (3) The RAD51AP1-UAF1 interaction interface was defined showing a novel SIM motif in the middle portion of RAD51AP1and the SLD1-SLD2 domain of UAF1 mediate protein complex formation. Based on the domain mapping results, point mutants of RAD51AP1 and UAF1 that are specifically compromised for the formation of the RAD51AP1-UAF1 complex were generated. (4) UAF1 synergizes with RAD51AP1 in the RAD51-mediated D-loop reaction and that this functional synergy requires the RAD51AP1-UAF1 complex and also the DNA and RAD51 binding attributes of RAD51AP1. (5) RAD51AP1-UAF1 works in conjunction with the RAD51 presynaptic filament in the capture of the duplex DNA partner and in the assembly of the synaptic complex. (6) Human cell lines impaired for RAD51AP1-UAF1 complex formation are compromised for the ability to repair DNA damage and to execute HR. (7) DNA repair function of the RAD51AP1-UAF1 complex is likely USP1-independent. Conclusions: The physical interaction between UAF1 and RAD51AP1 is indispensable for functional synergy in vitro and, accordingly, for the biological function of UAF1 in HR and DNA damage repair. Our findings provide insights into a novel USP1-independent regulatory mechanism of UAF1 on homologous recombination-mediated genome maintenance. Disclosures No relevant conflicts of interest to declare.
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Lambert, Muriel W., Laura W. McMahon, and Deepa M. Sridharan. "α II Spectrin Interacts with Specific Proteins in the Nucleus: Relevance to Fanconi Anemia." Blood 106, no. 11 (November 16, 2005): 184. http://dx.doi.org/10.1182/blood.v106.11.184.184.
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Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, congenital abnormalities and a cellular hypersensitivity to DNA interstrand cross-linking agents. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid spectrin (α SpII∑*) and that this deficiency correlates with a defect in ability to repair DNA interstrand cross-links. In order to get a better understanding of the exact role that α IISp∑* plays in the repair of cross-links and the repair defect in FA, whether it may have additional and potentially critical functions in the nucleus, and the processes that might be most severely affected by a defect in this protein, studies were undertaken to determine precisely what other proteins α IISp∑* interacts with in the nucleus. Co-immunoprecipitation experiments were carried out in which chromatin-associated proteins from normal human lymphoblastoid cells that co-immunoprecipitated (Co-IP) with α II spectrin were examined and identified. These proteins could be grouped into five categories: structural proteins, proteins involved in DNA repair, chromatin remodeling proteins, FA proteins, and transcription and RNA processing proteins. The structural proteins that Co-IP with α II spectrin were: lamin A, actin, protein 4.1B, β IV spectrin, and emerin. This indicates that α II spectrin interacts with proteins in the nucleus that play a role in nuclear cytoskeleton stability, chromatin organization and transcription. A number of proteins that Co-IP with α II spectrin were involved in DNA repair: DNA interstrand cross-link repair (XPF), homologous recombinational repair (HRR) and non-homologous end joining (NHEJ) (MRE11, RAD 50, RAD 51, XRCC2, Ku 70, Ku 80), and nucleotide excision repair (NER) (hHR23B, XPA, RPA, XPB, XPG, XPF, ERCC1). Since both NER and HRR are thought to be involved in repair of DNA interstrand cross-links, association of α II spectrin with XPF and HRR proteins supports our hypothesis that α II spectrin acts as a scaffold for recruitment and alignment of repair proteins at sites of DNA damage. It may act as a scaffolding for proteins involved in more than one repair pathway. α II spectrin also associated with chromatin remodeling proteins: BRG1, hBRM and CSB. This indicates that, like actin, it not only plays a role in nuclear cytoskeletal structure but also in chromatin remodeling as well. In agreement with our previous findings, α II spectrin Co-IP with FANCA and FANCC. The present study showed that it also Co-IP with FANCD2, FANCG and FANCF. There was also a significantly greater association of several FANC proteins, such as FANCA, to α II spectrin after cross-link damage to the cells than in undamaged cells. This further indicates that there is an important interaction between these FANC proteins and α II spectrin during the repair process. Several proteins involved in transcription and RNA processing (p40 and hnRNP A2/B1) also Co-IP with aII spectrin. Again, like actin, aII spectrin in the nucleus may also be involved in these processes. These results indicate that aII spectrin may have multiple roles in the nucleus and, in addition to DNA repair, may be involved in processes such as nuclear cytoskeleton stability, chromatin remodeling, transcription and RNA processing. A deficiency in aII spectrin in FA cells could thus affect multiple pathways where interaction of aII spectrin with functionally important proteins is critical; loss of this interaction in FA cells may explain some of the diverse clinical characteristics of this disorder.
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Jung, Moonjung, Sunandini Sridhar, Audrey Goldfarb, Ryan White, Danielle Keahi, Raymond Noonan, Tom Wiley, Francis P. Lach, and Agata Smogorzewska. "A Novel Source of Endogenous DNA Damage That Requires Repair By the Fanconi Anemia Pathway." Blood 134, Supplement_1 (November 13, 2019): 106. http://dx.doi.org/10.1182/blood-2019-127686.
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Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome. Impaired DNA interstrand crosslink (ICL) repair is the underlying mechanism for BMF in FA. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage are most likely to play an important role in the pathogenesis of FA. Metabolic by-products, such as reactive aldehydes, have been implicated in the acceleration of BMF or leukemia in both humans and mice lacking the ICL repair pathway. However, the potential contribution of other detoxifying metabolic enzymes to genome maintenance has not been systematically investigated. Identification of all sources of endogenous DNA damage will allow us to develop novel strategies to prevent DNA damage from arising, and possibly prevent BMF and leukemia in FA as well as other BMF syndromes. To determine whether detoxifying enzymes other than ALDH2 or ADH5 play a role in the protection of HSPC, we performed a metabolism-focused CRISPR/Cas9 synthetic lethality screen (3000 metabolism genes, 10 sgRNA per gene), using wild-type and FANCD2-/- Jurkat cells. From the screen, we identified ALDH9A1 as the most significantly depleted gene in FANCD2-/- Jurkat cells compared with wild-type. Eight out of ten sgALDH9A1 were significantly depleted in FANCD2-/- Jurkat cells, indicating robust effect of the ALDH9A1 knockout. ADH5, a known synthetic lethal gene with FA, was also depleted in FANCD2-/- Jurkat cells, but to a lesser degree. In vitro fluorescence-based competition assay confirmed synthetic lethal interaction between the two genes, in two independent FANCD2-/- Jurkat clones. To determine whether ALDH9A1 deficiency also caused cell death in FA-deficient human hematopoietic stem progenitor cells (HSPCs), we performed an in vitro validation assay using human umbilical cord blood (UCB). UCB CD34+ cells were edited by ribonucleoprotein delivery of Cas9 and sgRNA, in which either of sgCTRL, sgFANCD2 or sgALDH9A1, or both sgFANCD2 and sgALDH9A1 were used. Edited cells were grown on methylcellulose for 10 to 14 days, after which individual colonies were scored and harvested for sequencing. While CD34+ cells that were targeted by both sgFANCD2 and sgALDH9A1 (double KO) achieved lower editing efficiency for each gene compared with cells targeted by single guides, they produced the fewest hematopoietic colonies and the lowest frequency of GEMM (Granulocyte, Erythrocyte, Macrophage and Megakaryocyte) colonies. We observed fewer colonies targeted for both genes (biallelic double KO; observed to expected ratio 0.33) as compared to either single gene KO. These results suggest that loss of ALDH9A1 is deleterious in FANCD2-deficient HSPC. Lastly, we generated a Fanca-/-Aldh9a1-/- mouse model to examine the in vivo hematopoietic phenotype due to increased endogenous aldehydes. These mice were born at the Mendelian ratio without significant anomalies except rare cases of eye abnormalities. At three months of life, Fanca-/-Aldh9a1-/- mice had lower platelet counts than wild-type, Fanca-/- or Aldh9a1-/- control mice, but total white blood counts and hemoglobin levels were similar between groups. A follow-up result of this mouse model will be presented at the meeting. In conclusion, we identified that cells with ALDH9A1 deficiency require the FA pathway for survival. ALDH9A1 may protect human and mouse HSPC that are deficient in the FA pathway from DNA damage and cell death. Disclosures No relevant conflicts of interest to declare.
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Sobeck, Alexandra, Stacie Stone, Bendert deGraaf, Vincenzo Costanzo, Johan deWinter, Weidong Wang, Hans Joenje, Jean Gautier, and Maureen E. Hoatlin. "Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition." Blood 104, no. 11 (November 16, 2004): 723. http://dx.doi.org/10.1182/blood.v104.11.723.723.
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Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.
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Carreau, Madeleine, Olga I. Gan, Lili Liu, Monica Doedens, Colin McKerlie, John E. Dick, and Manuel Buchwald. "Bone Marrow Failure in the Fanconi Anemia Group C Mouse Model After DNA Damage." Blood 91, no. 8 (April 15, 1998): 2737–44. http://dx.doi.org/10.1182/blood.v91.8.2737.2737_2737_2744.
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Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in children. However, the specific involvement of FA genes in hematopoiesis and their relation to bone marrow (BM) failure is still unclear. The increased sensitivity of FA cells to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), including the induction of chromosomal aberrations and delay in the G2 phase of the cell cycle, have suggested a role for the FA genes in DNA repair, cell cycle regulation, and apoptosis. We previously reported the cloning of the FA group C gene (FAC) and the generation of a Fac mouse model. Surprisingly, the Fac −/− mice did not show any of the hematologic defects found in FA patients. To better understand the relationship of FA gene functions to BM failure, we have analyzed the in vivo effect of an FA-specific DNA damaging agent in Fac −/− mice. The mice were found to be highly sensitive to DNA cross-linking agents; acute exposure to MMC produced a marked BM hypoplasia and degeneration of proliferative tissues and caused death within a few days of treatment. However, sequential, nonlethal doses of MMC caused a progressive decrease in all peripheral blood parameters of Fac −/− mice. This treatment targeted specifically the BM compartment, with no effect on other proliferative tissues. The progressive pancytopenia resulted from a reduction in the number of early and committed hematopoietic progenitors. These results indicate that the FA genes are involved in the physiologic response of hematopoietic progenitor cells to DNA damage.
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Chesnokova, Vera, and Shlomo Melmed. "Peptide Hormone Regulation of DNA Damage Responses." Endocrine Reviews 41, no. 4 (April 9, 2020): 519–37. http://dx.doi.org/10.1210/endrev/bnaa009.
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Abstract DNA damage response (DDR) and DNA repair pathways determine neoplastic cell transformation and therapeutic responses, as well as the aging process. Altered DDR functioning results in accumulation of unrepaired DNA damage, increased frequency of tumorigenic mutations, and premature aging. Recent evidence suggests that polypeptide hormones play a role in modulating DDR and DNA damage repair, while DNA damage accumulation may also affect hormonal status. We review the available reports elucidating involvement of insulin-like growth factor 1 (IGF1), growth hormone (GH), α-melanocyte stimulating hormone (αMSH), and gonadotropin-releasing hormone (GnRH)/gonadotropins in DDR and DNA repair as well as the current understanding of pathways enabling these actions. We discuss effects of DNA damage pathway mutations, including Fanconi anemia, on endocrine function and consider mechanisms underlying these phenotypes. (Endocrine Reviews 41: 1 – 19, 2020)
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Hamanoue, Satoshi, Miharu Yabe, Hiromasa Yabe, and Takayuki Yamashita. "Hypermethylation of the p15/INK4B Gene in Fanconi Anemia." Blood 104, no. 11 (November 16, 2004): 4296. http://dx.doi.org/10.1182/blood.v104.11.4296.4296.
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Abstract Fanconi anemia (FA) is an inherited bone marrow failure syndrome with multiple complementation groups, characterized by genomic instability and predisposition to MDS and AML. Recent evidence indicates that multiple FA proteins are involved in DNA repair. Thus, increased genetic damage and secondary dysregulation of cell proliferation, differentiation and apoptosis are thought to play important roles in the development of bone marrow failure and subsequent progression to MDS/AML. However, little is known about molecular abnormalities responsible for these hematological disorders. Numerous studies indicated that epigenetic silencing of p15/INK4B, an inhibitor of cyclin-dependent kinases, plays an important role in the pathogenesis of MDS and AML. In the present study, we examined methylation status of 5′ CpG islands of the p15 gene in bone marrow mononuclear cells of FA patients, using methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Bone marrow samples were analyzed in 10 patients and serially studied in 4 of them. Hypermethylation of the p15 promoter region was detected in 5 patients (50%). This group included 3 patients with MDS: FA28-1 with refractory anemia (RA), FA87 with RAEB (RA with excess of blasts), and FA88 with later development of RA and progression to RAEB; whereas myelodysplasia was not observed in 2 patients (FA89, FA90). In two cases (FA88, FA90), p15 hypermethylation became negative during their courses, perhaps because of decreased myeloid cells. On the other hand, none of 5 patients without p15 hypermethylation had MDS. These results suggest that p15 hypermethylation is associated with development of MDS and occurs in the early phase of clonal evolution in the disease. Methylation status of p15 may be a useful prognostic factor of FA. Patient Age at onset (year old) Time from onset (month) Cytopenia MDS Cytogenetic abnormalities p15 methylation MSP b p15 methylation COBRA c a siblings, b MSP: methylation specific PCR, c COBRA: combined bisulfite restriction analysis, d ND: not determined FA28-1a 5 128 severe RA − − + 133 severe RA − + ++ FA87 8 252 severe RAEB + + +++ FA88 5 31 moderate − − + +++ 45 severe RA + − − 58 severe RAEB + + + FA89 5 49 mild − − + + 56 severe − − + + FA90 2 2 mild − − + ++ 31 severe − − − − FA28-2a 5 51 mild − − − NDd FA28-3a 3 12 mild − − − NDd FA47 3 15 mild − − − NDd FA68 5 46 moderate − − − NDd FA91 5 129 mild − − − NDd
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Datta, Arindam, and Robert M. Brosh Jr. "Holding All the Cards—How Fanconi Anemia Proteins Deal with Replication Stress and Preserve Genomic Stability." Genes 10, no. 2 (February 22, 2019): 170. http://dx.doi.org/10.3390/genes10020170.
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Fanconi anemia (FA) is a hereditary chromosomal instability disorder often displaying congenital abnormalities and characterized by a predisposition to progressive bone marrow failure (BMF) and cancer. Over the last 25 years since the discovery of the first linkage of genetic mutations to FA, its molecular genetic landscape has expanded tremendously as it became apparent that FA is a disease characterized by a defect in a specific DNA repair pathway responsible for the correction of covalent cross-links between the two complementary strands of the DNA double helix. This pathway has become increasingly complex, with the discovery of now over 20 FA-linked genes implicated in interstrand cross-link (ICL) repair. Moreover, gene products known to be involved in double-strand break (DSB) repair, mismatch repair (MMR), and nucleotide excision repair (NER) play roles in the ICL response and repair of associated DNA damage. While ICL repair is predominantly coupled with DNA replication, it also can occur in non-replicating cells. DNA damage accumulation and hematopoietic stem cell failure are thought to contribute to the increased inflammation and oxidative stress prevalent in FA. Adding to its confounding nature, certain FA gene products are also engaged in the response to replication stress, caused endogenously or by agents other than ICL-inducing drugs. In this review, we discuss the mechanistic aspects of the FA pathway and the molecular defects leading to elevated replication stress believed to underlie the cellular phenotypes and clinical features of FA.
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Mueller, Lars, Michael D. Milsom, Kristina Brumme, Chad Harris, Kalindi Parmar, Kaya Zhu, London Wendy, et al. "Mechanisms of Resistance to Reprogramming of Cells Defective In the Fanconi Anemia DNA Repair Pathway." Blood 116, no. 21 (November 19, 2010): 196. http://dx.doi.org/10.1182/blood.v116.21.196.196.
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Abstract Abstract 196 Fanconi anemia (FA), the most common inherited bone marrow failure syndrome, is characterized by progressive loss of hematopoietic stem cells, aplastic anemia, genomic instability and cancer predisposition. Induced pluripotent stem (iPS) cells are a promising source of cells for disease-specific investigations and genetic correction. It has been reported that human FA dermal fibroblasts are resistant to direct reprogramming without prior genetic correction (Raya et al., Nature, 2009), but the mechanism remains unclear. In this study we aimed to define the role of the FA pathway during the transition from fibroblasts to iPS cells in murine cells. We transduced Fanca-/- and wild type (wt) tail-tip fibroblasts with four defined factors (Oct3/4, Klf4, Sox2, c-Myc) and enumerated the number of iPS colonies that were derived from 1×105 cells. We noted a >10-fold decrease in the reprogramming efficiency of Fanca-/- cells compared to wt controls [median (range): wt 132 (0 to 1296) colonies, efficiency 0.328%, n=17; Fanca-/- 4 (0 to 80) colonies, efficiency 0.019%, n=10, p<0.0001, Wilcoxon test]. Despite the markedly reduced reprogramming efficiency, Fanca-/- fibroblasts yielded iPS cells that expressed pluripotency markers (Oct3, Nanog, SSEA-1), gave rise to mature teratomas, and were able to generate chimeric mice. Given the defective DNA repair phenotype of FA cells, we compared baseline and reprogramming-induced DNA damage and senescence in wt and Fanca-/- cells. We observed that double strand DNA (dsDNA) breaks (γH2AX foci) and senescence were significantly increased in Fanca-/- cells four days following the transduction with the reprogramming viruses as compared to wt cells (p<0.0001 and p=0.0012, respectively; Table 1). Because reactive oxygen species (ROS) have been implicated as cellular mediators of DNA damage, senescence and genomic instability in FA cells, we measured the ROS levels during reprogramming and observed a 1.7-fold ROS induction in Fanca-/- fibroblasts. Addition of the ROS scavenger N-acetylcysteine (NAC, 100 μ M) reduced the number of dsDNA breaks in the Fanca-/- cells but failed to increase the reprogramming efficiency, possibly due to off-target toxicity. Therefore, to further assess the impact of oxidative DNA damage on the reprogramming of FA iPS cells, we compared the reprogramming efficiency of Fanca-/- and wt fibroblast that were derived concurrently in normoxic (21% O2) or hypoxic (5% O2) conditions. In both Fanca-/- and wt cells, we observed a significant increase of the reprogramming efficiency under hypoxic conditions (p=0.0098 and p=0.0462, respectively). Complementation of Fanca-/- fibroblasts with the FANCA gene in combination with reprogramming under hypoxic conditions led to a significant rescue of the reprogramming efficiency (p=0.0109, Table 2). This correlated with a significant reduction in senescence and a trend towards decreased dsDNA breaks. These data indicate that oxidative DNA damage engages the FA signaling pathway during the reprogramming process, and acts as a negative physiologic regulator of reprogramming in Fanca-/- cells. Our study implicates the FA pathway as essential to the repair of dsDNA breaks that are induced during the reprogramming process, and provides a plausible mechanism for the reduced efficiency of reprogramming in Fanca-/- cells.Table 1:Increased dsDNA breaks (γH2AX foci) and senescence (β-galactosidase) in Fanca-/- fibroblasts four days post infection with reprogramming viruses.Table 2:Comparison of the number of iPS colonies derived from 1×105 input fibroblasts at 5%O2.GenotypeNumber of Experiments (n)Median (range) number of iPS-like colonies per 1×105 input fibroblastsP-valueFA GFP1468 (0 to 488)0.0018WT GFP18494 (22 to 1812)FA GFP1468 (0 to 488)0.0109FA + FANCA15276 (12 to 1196)WT GFP18494 (22 to 1812)0.1514FA +FANCA15276 (12 to 1196) Disclosures: No relevant conflicts of interest to declare.
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Yarde, Danielle N., Lori A. Hazlehurst, Vasco A. Oliveira, Qing Chen, and William S. Dalton. "Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function." Blood 108, no. 11 (November 16, 2006): 840. http://dx.doi.org/10.1182/blood.v108.11.840.840.
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Abstract The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.
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Zhang, Shichuan, Hirohiko Yajima, HoangDinh Huynh, Junke Zheng, Elsa Callen, Hua-Tang Chen, Nancy Wong, et al. "Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair." Journal of Cell Biology 193, no. 2 (April 11, 2011): 295–305. http://dx.doi.org/10.1083/jcb.201009074.
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The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)–dependent protein kinase catalytic subunit (DNA-PKcs)–null mice, knockin mice with the DNA-PKcs3A/3A allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs3A/3A HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs3A/3A cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice.
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Chen, Qing, Pieter C. Van der Sluis, Lori Hazlehurst, and William S. Dalton. "Enhanced DNA Repair Via Fanconi Anemia/BRCA Pathway Is Involved in Melphalan-Resistant Myeloma Cells." Blood 104, no. 11 (November 16, 2004): 284. http://dx.doi.org/10.1182/blood.v104.11.284.284.
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Abstract Melphalan, a DNA crosslinker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Interstrand cross-links (ICL) are amongst the most toxic types of DNA damage; therefore, DNA cross-linking agents are important drugs in cancer treatment. Unfortunately, although most patients respond to standard and high dose melphalan therapy, eventually patients acquire drug resistance. Acquired melphalan resistance has been associated with reduced DNA crosslinks, elevated levels of glutathione and increased radiation survival. However, mechanisms associated with resistance are not well understood. Evidence has accumulated to suggest that ICL repair contributes to the melphalan resistance. In this study, we compared the gene expression profile (GEP) of the melphalan-resistant myeloma cell line, 8226/LR5 to the 8226/S drug sensitive cell line, and found genes involved in FANC/BRCA DNA cross-link repair pathway had increased expression in drug resistant cells. The aim of our study was to determine whether FANC-BRCA pathway affects the DNA cross link repair capacity and accounts for acquired melphalan-resistance. Using real time RT-PCR and Western Blotting, we examined the expression levels of FANC/BRCA pathway genes in two different drug sensitive and resistant cell lines: 8226/S 8226/LR5, and U266/S and U266/LR6. The results showed that increased expression of FANC/BRCA pathway genes correlated with the melphalan resistance. The formation of ICL at 5 hours after a 2 hour melphalan exposure was reduced in the LR5 compared to the drug sensitive cell 8226 using single cell comet assay. Using siRNA to knock down FANCL or FANCF in melphalan-resistant cell line LR5 reversed the drug resistance. Conversely, overexpression of FANCL or FANCF in the 8226/S drug sensitive cell line enhanced cell survival. These data show that enhanced DNA repair via Fanconi anemia/BRCA pathway is involved in melphalan-resistant myeloma cells.
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Lambert, Muriel W. "Spectrin and its interacting partners in nuclear structure and function." Experimental Biology and Medicine 243, no. 6 (March 2018): 507–24. http://dx.doi.org/10.1177/1535370218763563.
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Nonerythroid αII-spectrin is a structural protein whose roles in the nucleus have just begun to be explored. αII-spectrin is an important component of the nucleoskelelton and has both structural and non-structural functions. Its best known role is in repair of DNA ICLs both in genomic and telomeric DNA. αII-spectrin aids in the recruitment of repair proteins to sites of damage and a proposed mechanism of action is presented. It interacts with a number of different groups of proteins in the nucleus, indicating it has roles in additional cellular functions. αII-spectrin, in its structural role, associates/co-purifies with proteins important in maintaining the architecture and mechanical properties of the nucleus such as lamin, emerin, actin, protein 4.1, nuclear myosin, and SUN proteins. It is important for the resilience and elasticity of the nucleus. Thus, αII-spectrin’s role in cellular functions is complex due to its structural as well as non-structural roles and understanding the consequences of a loss or deficiency of αII-spectrin in the nucleus is a significant challenge. In the bone marrow failure disorder, Fanconi anemia, there is a deficiency in αII-spectrin and, among other characteristics, there is defective DNA repair, chromosome instability, and congenital abnormalities. One may speculate that a deficiency in αII-spectrin plays an important role not only in the DNA repair defect but also in the congenital anomalies observed in Fanconi anemia , particularly since αII-spectrin has been shown to be important in embryonic development in a mouse model. The dual roles of αII-spectrin in the nucleus in both structural and non-structural functions make this an extremely important protein which needs to be investigated further. Such investigations should help unravel the complexities of αII-spectrin’s interactions with other nuclear proteins and enhance our understanding of the pathogenesis of disorders, such as Fanconi anemia , in which there is a deficiency in αII-spectrin. Impact statement The nucleoskeleton is critical for maintaining the architecture and functional integrity of the nucleus. Nonerythroid α-spectrin (αIISp) is an essential nucleoskeletal protein; however, its interactions with other structural and non-structural nuclear proteins and its functional importance in the nucleus have only begun to be explored. This review addresses these issues. It describes αIISp’s association with DNA repair proteins and at least one proposed mechanism of action for its role in DNA repair. Specific interactions of αIISp with other nucleoskeletal proteins as well as its important role in the biomechanical properties of the nucleus are reviewed. The consequences of loss of αIISp, in disorders such as Fanconi anemia, are examined, providing insights into the profound impact of this loss on critical processes known to be abnormal in FA, such as development, carcinogenesis, cancer progression and cellular functions dependent upon αIISp’s interactions with other nucleoskeletal proteins.
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Al Jabri, Aida, Nusaybah Al Naim, and Abeer Al Dossari. "Homozygous Mutation in the FANCD2 Gene Observed in a Saudi Male Infant with Severe Ambiguous Genitalia." Case Reports in Endocrinology 2021 (July 16, 2021): 1–4. http://dx.doi.org/10.1155/2021/6686312.
Full textAbstract:
Fanconi anemia (FA) is a rare autosomal recessive inherited disease caused by gene mutations that are primarily involved in the response to or repair of DNA damage. FA characterizes by multiple congenital abnormalities and malformations including growth retardation, renal agenesis, absence of radial bones and thumbs as well, progressive bone marrow failure, irregular skin pigmentation patterns, and increased susceptibility to cancer. FANCD2 gene mutation is believed to be one of the causative mutations in Fanconi anemia, and despite many case reports that link the FANC gene mutation to multiple congenital anomalies and disease, there is no case report found to link it with genitalia abnormalities. In our paper, we report a male Saudi infant who presented to the endocrine clinic at the age of 9 months with severe ambiguous genitalia and found that he carries a homozygous variant mutation in the FANCD2 gene and we face a challenge to treat this patient since there was no previous similar case.
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Guenther, Kacey Linnea, Patali Shikhi Cheruku, Richard H. Smith, and Andre Larochelle. "Eltrombopag Promotes DNA Repair in Human Hematopoietic Stem and Progenitor Cells: Implications for the Treatment of Fanconi Anemia." Blood 130, Suppl_1 (December 7, 2017): 776. http://dx.doi.org/10.1182/blood.v130.suppl_1.776.776.
Full textAbstract:
Abstract Fanconi anemia (FA) is an inherited genomic instability syndrome resulting from loss-of-function mutations in any one of at least 21 FANC genes critical for DNA repair. Accumulation of unresolved DNA double strand breaks (DSBs) results in chromosomal rearrangement, accounting in part for the high incidence of bone marrow failure (BMF) and leukemia in patients with FA. Safe, curative options are currently unavailable. Recent investigations have uncovered a specific function of thrombopoietin (TPO), a key regulator of hematopoietic stem/progenitor cell (HSPC) self-renewal and survival, in promoting DNA damage response in these cells. TPO was shown to specifically increase the efficiency of non-homologous end joining (NHEJ), the predominant repair mechanism for DSBs in HSPCs; however, recombinant TPO is no longer approved for clinical applications. The alternative orally bioavailable small molecule TPO receptor agonist, eltrombopag (epag), was recently used in clinical trials to successfully treat life-threatening cytopenias in patients with acquired BMF. In this study, we investigate whether epag can promote NHEJ DSB repair in human HSPCs and thus provide a potential new therapeutic modality for patients with FA. To assess DNA repair activity of epag in FA CD34+ HSPCs, a population of cells that is markedly reduced in these patients, CD34+ HSPCs from 6 healthy individuals were subjected to CRISPR/Cas9-induced knockout mutations in FANCA, the most commonly mutated gene in FA. These FA HSPCs were cultured for 24 hr in the presence of early-acting cytokines, SCF and Flt3-L (designated "SF"), or SF supplemented with epag ("SFE") or TPO ("SFT") prior to induction of DSBs by exposure to 2Gy γ-irradiation (IR). Cells were then cultured for an additional 1, 5, or 24 hr to assess the kinetics of DNA repair, as measured by decreases in H2AX phosphorylation (γH2AX), an indicator of IR-induced DSBs. Maximal H2AX phosphorylation was observed 1 hr after IR of FA HSPCs and was similar for all culture conditions (>90% γH2AX+ cells), indicating that epag and TPO do not prevent DSB formation or phosphorylation of H2AX. Five hours after IR, most FA HSPCs cultured with epag (Fig. A) or TPO (Fig. B) had resolved the IR-induced DSBs, while much higher percentages of γH2AX+ cells persisted in the control SF group. The observed effect was specific to epag and TPO; removal of SCF had no significant impact on DNA repair. Regardless of culture condition, the majority of FA HSPCs either resolved DSBs or progressed through apoptosis by 24 hr post-IR. These findings indicate that epag and TPO increase the kinetics of DSB repair in FA HSPCs. To gain insight into the primary mechanism of DNA repair promoted by epag and TPO, we inhibited DNA-PK, an essential component of the classical NHEJ (C-NHEJ) pathway. Addition of a DNA-PK inhibitor (NU7441) had no impact on DSB formation measured at 1 hr or on DNA repair at 24 hr, but completely abrogated the enhanced kinetics of DSB repair observed at 5 hr with epag (Fig. A) and TPO (Fig. B). These data indicate that culturing FA HSPCs with epag or TPO promotes the fast-acting DNA-PK-dependent C-NHEJ DNA repair mechanism, a pathway known to promote genomic stability. In contrast, cells cultured without epag or TPO resolved DSBs using a slower, DNA-PK-independent alternative NHEJ (alt-NHEJ) mechanism, a pathway associated with genomic instability. Shunting of DSB repair in rapid C-NHEJ with epag (Fig. C) or TPO (Fig. D) was associated with substantial increase in survival of γ-irradiated FA HSPCs compared with control (SF) groups. In contrast, when C-NHEJ DNA repair was inhibited with NU7441, the cell survival benefit observed with epag (Fig. C) or TPO (Fig. D) was abolished. In colony forming unit (CFU) progenitor assays, γ-irradiated HSPCs cultured with epag or TPO yielded 4- to 6-fold more CFUs than control SF groups (Fig. E). When γ-irradiated HSPCs were transplanted into immunodeficient NSG mice, a 2-fold increase in human cell engraftment was observed in cultures containing epag or TPO compared to controls (p<0.01), suggesting activation of DNA repair activity by these cytokines in cells with long-term repopulating capacity (Fig. F). Overall, our data indicate that epag and TPO enhance DSB repair in human HSPCs by promoting the fast-operating and faithful C-NHEJ pathway. A phase II clinical trial is in development to assess safety and efficacy of epag in the treatment of hematological manifestations of FA. Figure Figure. Disclosures Guenther: Agilent Laboratories: Other: Stipend, Research Funding; Norvartis: Research Funding. Cheruku: Novartis: Other: Stipend, Research Funding. Smith: Novartis: Research Funding; Agilent Laboratories: Research Funding. Larochelle: StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit; Novartis: Research Funding; Novartis: Research Funding.
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Ghosh, Soma, and Tapas Saha. "Central Role of Ubiquitination in Genome Maintenance: DNA Replication and Damage Repair." ISRN Molecular Biology 2012 (February 8, 2012): 1–9. http://dx.doi.org/10.5402/2012/146748.
Full textAbstract:
Faithful transmission of genetic information through generations ensures genomic stability and integrity. However, genetic alterations occur every now and then during the course of genome duplication. In order to repair these genetic defects and lesions, nature has devised several repair pathways which function promptly to prevent the cell from accumulating permanent mutations. These repair mechanisms seem to be significantly impacted by posttranslational modifications of proteins like phosphorylation and ubiquitination. Protein ubiquitination is emerging as a critical regulatory mechanism of DNA damage response. Non-proteolytic, proteasome-independent functions of ubiquitin involving monoubiquitination and polyubiquitination of DNA repair proteins contribute significantly to the signaling of DNA repair pathways. In this paper, we will particularly highlight the work on ubiquitin-mediated signaling in the repair processes involving the Fanconi anemia pathway, translesional synthesis, nucleotide excision repair, and repair of double-strand breaks. We will also discuss the role of ubiquitin ligases in regulating checkpoint mechanisms, the role of deubiquitinating enzymes, and the growing possibilities of therapeutic intervention in this ubiquitin-conjugation system.
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Esteban-Jurado, Clara, Sebastià Franch-Expósito, Jenifer Muñoz, Teresa Ocaña, Sabela Carballal, Maria López-Cerón, Miriam Cuatrecasas, et al. "The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer." European Journal of Human Genetics 24, no. 10 (May 11, 2016): 1501–5. http://dx.doi.org/10.1038/ejhg.2016.44.
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