The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.

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Xeroderma Pigmentosum as a Model for Treatment of Bone Marrow Failure in Fanconi Anemia and Aplastic Anemia.

Blood ◽

10.1182/blood.v104.11.4235.4235 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4235-4235

Author(s):

W. Clark Lambert ◽

Santiago A. Centurion

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Fanconi Anemia ◽

Xeroderma Pigmentosum ◽

Bone Marrow Failure ◽

S Phase ◽

White Female ◽

Cross Linking

Abstract We have previously shown that the primary cell cycle defect in the inherited, cancer-prone, bone marrow failure associated disease, Fanconi anemia (FA), is not in the G2 phase of the cell cycle, as had been thought for many years, but rather in the S phase. FA cells challenged with the DNA cross-linking agent, psoralen coupled with long wavelength, ultraviolet (UVA) radiation (PUVA), fail to slow their progression through the S phase of the subsequent cell cycle, as do normal cells. FA cells are extremely sensitive to the cytotoxic and clastogenic effects of DNA cross-linkers, such as PUVA, so much so that the diagnosis of FA is based on an assay, the “DEB test”, in which cells are examined for clastogenic and cytotoxic effects of diepoxybutane (DEB), a DNA cross-linking agent. More recently, we have shown that artificially slowing the cell cycle of FA cells exposed to PUVA by subsequent treatment with agents which slow their progression through S phase leads to markedly increased viability and reduced chromosome breakage in vitro. We now show that similar results can be obtained in vivo in patients with another DNA repair deficiency disease, xeroderma pigmentosum (XP), a recessively inherited disorder associated with defective repair of sunlight induced adducts in the DNA of sun-exposed tissues followed by development of numerous mutations causing large numbers of cancers in these same tissues. We treated two patients with XP, a light complected black male and a white female, both 14 years of age, in sun-exposed areas with 5-fluorouracil, an inhibitor of DNA synthesis, daily for three months. In contrast to normal patients, who only show clinical results if an inflammatory response is invoked, marked improvement in the clinical appearance of the skin was seen with no inflammation observed. This effect was confirmed histologically by examining epidermis adjacent to excised lesions in sun-exposed areas and further verified by computerized image analysis. Treatment with agents that slow progression through S phase, such as hydroxyurea, may similarly improve clinical outcomes in patients with FA or others who are developing bone marrow failure.

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The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.01521-08 ◽

2008 ◽

Vol 29 (5) ◽

pp. 1116-1122 ◽

Cited By ~ 16

Author(s):

Laurent Malivert ◽

Isabelle Callebaut ◽

Paola Rivera-Munoz ◽

Alain Fischer ◽

Jean-Paul Mornon ◽

...

Keyword(s):

Dna Repair ◽

Nonhomologous End Joining ◽

Dna Ligase ◽

Repair Pathway ◽

End Joining ◽

Functional Assays ◽

The Core ◽

Ligase Iv

ABSTRACT The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

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The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC

Blood ◽

10.1182/blood-2002-03-0860 ◽

2002 ◽

Vol 100 (7) ◽

pp. 2457-2462 ◽

Cited By ~ 55

Author(s):

Toshiyasu Taniguchi ◽

Alan D. D'Andrea

Keyword(s):

Mitomycin C ◽

Fanconi Anemia ◽

Cancer Susceptibility ◽

Autosomal Recessive Disorder ◽

Nuclear Accumulation ◽

Retroviral Transduction ◽

Nuclear Foci ◽

Fancd2 Monoubiquitination

Fanconi anemia is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. The 6 known Fanconi anemia gene products (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG proteins) interact in a common pathway. The monoubiquitination and nuclear foci formation of FANCD2 are essential for the function of this pathway. FANCA, FANCC, FANCG, and FANCF proteins form a multisubunit nuclear complex (FA complex) required for FANCD2 monoubiquitination. Because FANCE and FANCC interact in vitro and FANCE is required for FANCD2 monoubiquitination, we reasoned that FANCE is a component of the FA complex in vivo. Here we demonstrate that retroviral transduction of Fanconi anemia subtype E (FA-E) cells with the FANCE cDNA restores the nuclear accumulation of FANCC protein, FANCA–FANCC complex formation, monoubiquitination and nuclear foci formation of FANCD2, and mitomycin C resistance. Hemagglutinin (HA)-tagged FANCE protein localizes diffusely in the nucleus. In normal cells, HA-tagged FANCE protein coimmunoprecipitates with FANCA, FANCC, and FANCG but not with FANCD2. Our data indicate that FANCE is a component of the nuclear FA complex in vivo and is required for the monoubiquitination of FANCD2 and the downstream events in the FA pathway.

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Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences

Genes ◽

10.3390/genes11121528 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1528

Author(s):

Benilde García-de-Teresa ◽

Alfredo Rodríguez ◽

Sara Frias

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Chromosomal Instability ◽

Bone Marrow Failure ◽

Chromosome Instability ◽

Premature Aging ◽

Dna Double Strand Breaks ◽

Interstrand Crosslinks ◽

Pathogenic Variants ◽

Extreme Risk

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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Association of clinical severity with FANCB variant type in Fanconi anemia

Blood ◽

10.1182/blood.2019003249 ◽

2020 ◽

Vol 135 (18) ◽

pp. 1588-1602 ◽

Cited By ~ 8

Author(s):

Moonjung Jung ◽

Ramanagouda Ramanagoudr-Bhojappa ◽

Sylvie van Twest ◽

Rasim Ozgur Rosti ◽

Vincent Murphy ◽

...

Keyword(s):

Bone Marrow ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Complementation Group ◽

Clinical Severity ◽

Missense Variants ◽

Interstrand Crosslinks ◽

Pathogenic Variants ◽

Enzymatic Activation ◽

Fancd2 Monoubiquitination

Abstract Fanconi anemia (FA) is the most common genetic cause of bone marrow failure and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry. Those with FANCB deletion or truncation demonstrate earlier-than-average onset of bone marrow failure and more severe congenital abnormalities compared with a large series of FA individuals in published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization were associated with 2 missense variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays tended to show earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is associated with level of residual FANCD2 monoubiquitination.

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

Genes ◽

10.3390/genes12111763 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1763

Author(s):

Masamichi Ishiai

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Genome Stability ◽

Short Review ◽

Repair Pathway ◽

Binding Partner ◽

Interstrand Crosslinks ◽

Pathway Activation ◽

Dna Interstrand Crosslinks

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

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Germline MPO Variants Predispose to Myeloid Neoplasia: Potential Mechanisms Suggested By In Vivo and in Vitro Studies

Blood ◽

10.1182/blood-2021-149598 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3662-3662

Author(s):

Sunisa Kongkiatkamon ◽

Laila Terkawi ◽

Vera Adema ◽

Yihong Guan ◽

Metis Hasipek ◽

...

Keyword(s):

Bone Marrow ◽

Dna Repair ◽

Murine Model ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Human Leukemia ◽

Risk Alleles ◽

Myeloid Neoplasia

Abstract In recent decades, there has been a substantial increase in recognition of germline (GL) predisposition to MN. Some congenital blood disorders predispose to subsequent myeloid neoplasia (MN). Severe congenital neutropenia (SCN) and Fanconi anemia serving as prominent examples. There is emerging evidence that heterozygous variants of disease recessive conditions may constitute risk alleles for late developing MN. However, their low penetrance may preclude identification of such alterations. Our previous analysisof allelic burden among selected combined potential GL predisposition genes to MN identified that the myeloperoxidase (MPO) gene (17q22-23) carried the highest risk of pathogenic alterations (Li, S.T, Leukemia, 2020). GL MPO mutations cause MPO deficiency syndrome, one of the most common inherited disorder associated with phagocyte defects and variable clinical penetrance. Despite the high prevalence, inherited MPO deficiency patients are asymptomatic and may remain unrecognized. We investigated 3280 MN and bone marrow failure syndromes (BMF) pts including 1138 MDS, 260 MDS/MPN, 1661 AML and 221 AA for presence of MPO mutations. In total, 38 different germline MPO mutations were identified in 143 cases. With a stringent bioanalytic pipeline, 28 pathogenic/likely pathogenic variants from 100 MN patients were included in the study. Germline MPO variants were significantly enriched in MN compared to control population (294 vs. 125 per 10 4 individuals; P<.0001) with an odds ratio of 2 (95%CI=1.6-2.5, P<.0001) for AML and 1.8 (95%CI=1.4-2.4, P<.0001) for MDS. The most common pathogenic/likely pathogenic variant (46%; 46/100) was a 3' splice site of intron11 (c.2031-2A>C) followed by R569W (13/100), M519fs* (13/100) and Y173C (6/100) with none being biallelic. While no differences were found in distribution of -7/7q, del5q, or del20q, MPO variants carriers harbored less tri-8 compared to wild type counterparts (2% vs. 12%; P=.008). Only FLT3 and NRAS were significantly associated with MPO mutations. We then investigated the effects of MPO deficiency on hematopoietic function in murine model using competitive repopulation assays, whereby the difference in CD45.1/CD45.2 isotypes transplanted in to ROSA26 (tdTomato-EGFP) recipients assayed by flow cytometry allowed distinction of the 2 grafts. Mpo-/- cells gained over time proliferative advantage over normal murine bone marrow cells. Reverse combinations (graft mix vs recipient) also replicated this result. This effect was not due to increased HSC content in Mpo-/- marrow as there were no significant differences in LSK, CMP and MEPs cells. We then investigated the clonogenic consequences of Mpo-/- cells in the setting of H 2O 2 induced oxidative stress, after exposing to hydrogen peroxide. Mpo-/- cells increased clonogenic potential after second serially replating. This prompted us to investigate an MPO inhibitor, AZD5904 in human leukemia cells with different MPO expression (HL-60, high; K562, low). High MPO expressors retained higher cell viability following H 2O 2 and MPOi addition compared to those without MPOi (85±10 vs.59±8, P<.0001) while no change was observed in low MPO stage cells with or without MPOi (84±2 vs.89±10, P=0.7). In conclusion, for the first time we demonstrated that germline MPO variants constitute risk alleles for MN evolution and replicated the potential replicative advantage of MPO deficient cells in murine model and potential mechanisms of the MPO deficiency in vitro including activation of non-homologous DNA repair response and error-prone DNA repair favoring replication. Repeated cycles of stress hematopoiesis e.g., due to increased infection rate may provide conditions in which MPO variants contribute to the risk of MN. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy.

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Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences

10.20944/preprints202011.0563.v2 ◽

2020 ◽

Author(s):

Benilde García-de-Teresa ◽

Alfredo Rodríguez ◽

Sara Frias

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Chromosomal Instability ◽

Bone Marrow Failure ◽

Chromosome Instability ◽

Premature Aging ◽

Dna Double Strand Breaks ◽

Interstrand Crosslinks ◽

Pathogenic Variants ◽

Extreme Risk

Abstract: Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, that 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. In order to repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, that 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 like 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, that results in tissue attrition, selection of malignant clones and cancer onset. Moreover, the effect of the FA/BRCA pathway in germinal cells, evidenced by infertility in patients with FA attests of chromosomal instability and cell death also occurring in the germinal compartment.

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Impaired Bone Marrow Microenvironment in Fanconi Anemia Murine Model Is Responsible for the Defective Hematopoietic Stem/ Progenitor Cell Mobilization.

Blood ◽

10.1182/blood.v114.22.3629.3629 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3629-3629

Author(s):

Yan Li ◽

Shi Chen ◽

Yongzheng He ◽

Xiaohong Li ◽

Fengchun Yang

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fanconi Anemia ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Bone Marrow Microenvironment ◽

Hematopoietic Stem

Abstract Abstract 3629 Poster Board III-565 Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by progressive bone marrow failure (BMF) and acquisition of malignancies. The only cure for BMF is a human leukocyte antigen (HLA)-matched BM transplantation from a family member or autologous stem cells before BMF develops. Therefore, mobilization of hematopoietic stem/progenitor cells (HSPCs) from BM into peripheral blood (PB) for collection has been a prerequisite for the therapy. However, patients with FA show a markedly decreased HSPC mobilization in response to the traditional mobilizing drug G-CSF and the mechanism(s) underlying the defect remains unknown. Mesenchymal stem/progenitor cells (MSPCs) have been known to be the common progenitor of a variety of cellular components in the bone marrow microenvironment. MSPCs express/secrete cytokines, extracellular matrix proteins and cell adhesion molecules, which regulate the homing, migration, proliferation and survival of HSPCs in vitro and in vivo. Recently, we reported that Fancg-/- MSPCs have a defect in hematopoietic supportive activity both in vitro and in vivo (Li et al. Blood, 2009). In the current studies, we show that Fancg-/- MSPCs have significant reduction in HSPC recruitment as compared to WT MSPCs in a transwell assay. Furthermore, Fancg-/- MSPCs have an alteration in the production of multiple cytokines/chemokines. Application of a neutralizing antibody to the cytokine blocked WT MSPC mediated HSPC migration in vitro. Furthermore, administration of the specific cytokine significantly increased HSPC mobilization in the Fancg-/- mice in vivo. These results demonstrated that an impaired BM microenvironment, specifically MSPCs in Fancg-/- mice, is contributory to defective HSPC mobilization. This study provides evidence of alternative clinical therapeutics for the mobilization of HSPCs in FA patients. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Failure in Fanconi Anemia from Hyperactive TGF-β Signaling

Blood ◽

10.1182/blood.v124.21.356.356 ◽

2014 ◽

Vol 124 (21) ◽

pp. 356-356

Author(s):

Haojian Zhang ◽

David Kozono ◽

Kevin O'Connor ◽

Sofia Vidal-Cardenas ◽

Abigail Hamilton ◽

...

Keyword(s):

Bone Marrow ◽

Dna Repair ◽

Signaling Pathway ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Genotoxic Stress ◽

Cellular Growth ◽

Cross Linking ◽

Repair Pathway ◽

Growth Defects

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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