scholarly journals Holding All the Cards—How Fanconi Anemia Proteins Deal with Replication Stress and Preserve Genomic Stability

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 170 ◽  
Author(s):  
Arindam Datta ◽  
Robert M. Brosh Jr.
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Excision Repair ◽  
Double Helix ◽  
Bone Marrow Failure ◽  
Molecular Genetic ◽  
Replication Stress ◽  
Gene Products ◽  
Hematopoietic Stem ◽  
Dna Double Helix

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.

scholarly journals LNK (SH2B3) Deficiency Ameliorates Hematopoietic Stem Cell Defects in Fanconi Anemi

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2670-2670
Author(s):  
Joanna Balcerek ◽  
Jing Jiang ◽  
Qinqin Jiang ◽  
Kaosheng Lyu ◽  
Roger Greenberg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Genome Instability ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Replication Stress ◽  
Cytokine Signaling ◽  
Hematopoietic Stem

Abstract Fanconi Anemia (FA) is one of the most common inherited bone marrow failure (iBMF) syndromes. Although initially identified over 85 years ago, FA remains a fatal genetic disease. Nineteen FA genes cooperate in a genome stability pathway that is essential for repair of DNA damage and tolerance of replication stress. Cells derived from FA patients are hypersensitive to DNA interstrand crosslink (ICL)-inducing agents, such as Mitomycin C (MMC), and exhibit DNA damage checkpoint and mitosis defects. Mutations in FA genes result in hematopoietic stem cell (HSC) defects, bone marrow failure and cancer predisposition. Importantly, interventions to mitigate HSC defects do not exist, aside from allogeneic bone marrow transplantation (BMT). HSCs deficient for FancD2, a central component of the FA signaling pathway, are markedly compromised in reconstituting the hematopoietic system in murine BMT models. Remarkably, we found that loss of the adaptor protein Lnk (also called Sh2b3) restores HSC function in FancD2 knockout mice without accelerating neoplastic transformation. LNK negatively regulates HSC self-renewal, in part by attenuating cytokine signaling-activated JAK2 signaling in HSCs (J Clin Invest. 2008;118(8):2832-2844). Fancd2-/-;Lnk-/- (DKO) mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals, and DKOHSC function is also largely restored to WT levels in serial transplantation assays. Primary DKOHSC and progenitors (HSPCs) are still sensitive to MMC, indicating that LNK does not play an overt role in ICL repair. Instead, Lnk deficiency notably reduces spontaneous DNA damage and genome instability. This is in agreement with recent studies that reveal a requirement for FA proteins in replication stress, which is a separable function from their role in DNA repair (Cell. 2011;145(4):529-542; Cancer Cell. 2012;22(1):106-116). Strikingly, we demonstrated that Lnk deficiency mitigates replication stress by stabilizing stalled replication forks, and that this effect is dependent upon cytokine signaling. Together, our data demonstrate that Lnk deficiency ameliorates FA HSPC defects by alleviating replication stress associated DNA damage and genome instability. To our knowledge, this is one of the first examples of in vivo genetic suppression of FA-associated HSPC defects. Our work sheds light on mechanisms underlying the origin of bone marrow failure in FA patients and has implications for new therapeutic strategies to treat FA associated bone marrow failure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fanconi Anemia: A Paradigm for Understanding DNA Repair During Replication.

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-32-SCI-32 ◽  
Author(s):  
Agata Smogorzewska
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Dna Repair ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Chromosome Breakage ◽  
Dna Lesions ◽  
Hematopoietic Stem

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.

scholarly journals Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair

2014 ◽  
Vol 42 (15) ◽  
pp. 9807-9820 ◽  
Author(s):  
Meghan Larin ◽  
David Gallo ◽  
Laura Tamblyn ◽  
Jay Yang ◽  
Hudson Liao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Congenital Abnormalities ◽  
Bone Marrow Failure ◽  
In Utero ◽  
Critical Threshold ◽  
Growth Defects ◽  
Repair Defect ◽  
In Utero Development ◽  
Crosslink Repair

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.

scholarly journals ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ADULT PATIENTS WITH FANCONI ANEMIA.

2016 ◽  
Vol 8 ◽  
pp. 2016054 ◽  
Author(s):  
Hosein Kamranzadeh fumani ◽  
Mohammad Zokaasadi ◽  
Amir Kasaeian ◽  
Kamran Alimoghaddam ◽  
Asadollah Mousavi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Term Survival ◽  
Hematopoietic Stem

Background & objectives: Fanconi anemia (FA) is a rare genetic disorder caused by an impaired DNA repair mechanism which leads to an increased tendency toward malignancies and progressive bone marrow failure. The only curative management available for hematologic abnormalities in FA patients is hematopoietic stem cell transplantation (HSCT). This study aimed to evaluate the role of HSCT in FA patients.Methods: Twenty FA patients with ages of 16 or more who underwent HSCT between 2002 and 2015 enrolled in this study. All transplants were allogeneic and the stem cell source was peripheral blood and all patients had a full HLA-matched donor.Results: Eleven patients were female and 9 male (55% and 45%). Mean age was 24.05 years. Mortality rate was 50% (n=10) and the main cause of death was GVHD. Survival analysis showed an overall 5-year survival of 53.63% and 13 year survival of 45.96 % among patients.Conclusion: HSCT is the only curative management for bone marrow failure in FA patients and despite high rate of mortality and morbidity it seems to be an appropriate treatment with an acceptable long term survival rate for adolescent and adult group.

scholarly journals Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

Blood ◽  
2017 ◽  
Vol 130 (13) ◽  
pp. 1523-1534 ◽  
Author(s):  
Ana Martín-Pardillos ◽  
Anastasia Tsaalbi-Shtylik ◽  
Si Chen ◽  
Seka Lazare ◽  
Ronald P. van Os ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mitochondrial Dysfunction ◽  
Replication Stress ◽  
Precursor Cells ◽  
Dna Lesions ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Functional Integrity ◽  
Key Points

Key Points Tolerance of oxidative DNA lesions ensures the genomic and functional integrity of hematopoietic stem and precursor cells. Endogenous DNA damage–induced replication stress is associated with mitochondrial dysfunction.

scholarly journals Replication Stress Response and CDKN1A Engagement Constrain Fetal Hematopoietic Stem Cell Pool Size in Fanconi Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 107-107
Author(s):  
Makiko Mochizuki-Kashio ◽  
Young Me Yoon ◽  
Theresa N Menna ◽  
Markus Grompe ◽  
Peter Kurre
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Nuclear Localization ◽  
Fanconi Anemia ◽  
Fetal Liver ◽  
Replication Stress ◽  
Pool Size ◽  
Hematopoietic Stem ◽  
Independent Functions

Bone marrow (BM) failure is the principal source of morbidity and mortality in Fanconi Anemia (FA) patients. Recessively inherited germline mutations in one of 25 genes lead to deficits by in a pathway central to DNA crosslink repair. Functionally, FA proteins protect adult hematopoietic stem cells (HSC) from p53 mediated apoptosis elicited by alkylating agents, a range of experimental inflammatory cues or aldehyde exposure. However, these mechanisms do not seem to account for depleted hematopoietic stem and progenitor cell pools in very young FA patients, or the spontaneous, non-apoptotic and p53-independent fetal HSC deficits observed in murine models. Building on our previous observation of a quantitatively constrained fetal HSC pool in FA mice (Fancd2-/-), the current experiments reveal the specific developmental timeframe for the onset of stem cell deficits during HSC expansion in the fetal liver (FL). Cell cycle studies using an EdU/BrdU pulse chase protocol reveal delays in S-phase entry and progression at E13.5. Building on the role of FA proteins (FANCM, FANCI and FANCD2) in countering experimental replication stress (RS) in cell line models, we reasoned that rapid rates of proliferation required during expansion in the FL may similarly confer RS on the FA HSC pool. Experiments in E13.5 FL HSC confirmed the predicted increase in single stranded DNA and accumulation of nuclear replication associated protein (pRpa), along with activation of pChk1, a critical cell cycle checkpoint in cells under RS. For comparison, pChk1 in unperturbed adult cells was no different between Fancd2-/- and WT. The data are also consistent with gains in RAD51 and alkaline comet assays we previously published (Yoon et al., Stem Cell Reports 2016). The cell cycle regulator Cdkn1a (p21) is considered a canonical downstream component of the p53 response in adult FA HSC, but it also performs p53 independent functions in the RS response that coincide with its role in the nucleus. Here, we observed an increase in nuclear localization of p21 in Fancd2-/- FL HSC. TGF-β is a critical developmental morphogen that regulates p21 activity, and TGF-β inhibitors can partially reverse adult FA HSC function along with suppression of NHEJ mediated DNA repair. To test regulation of p21 in fetal HSC under RS, we first treated WT FL HSC with aphidicolin to experimentally simulate RS and found that SD208, a small molecule TGF-β-R1 inhibitor, completely rescued the p21 nuclear localization. We then went on to demonstrate that pharmacological inhibition of TGF-β signaling also reversed the nuclear p21 translocation in FA FL HSC (under physiological RS) and functionally rescued the primitive myeloid progenitor colony formation (CFU-GEMM) in vitro. Altogether, our data show that HSC deficits in FA first emerge in the fetal liver, where rapid fetal expansion causes RS that elicits pChk1 activation and nuclear p21 translocation, which restrain cell cycle progression and act as principal mechanisms limiting fetal HSC pool size in FA. Our experiments suggest a central and p53-independent role for p21 in fetal FA HSC regulation. Detailed knowledge of the physiological role of FA proteins in fetal phenotype HSC has the potential to lead to new therapies that delay or rescue hematopoietic failure in FA patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals New Bone Marrow Failure Genes: DNAJC21 and ERCC6L2

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-22-SCI-22
Author(s):  
Inderjeet Dokal
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Nuclear Export ◽  
Conflicts Of Interest ◽  
Excision Repair ◽  
Bone Marrow Failure ◽  
Retinal Dystrophy ◽  
Ribosomal Subunit ◽  
Genotoxic Stress ◽  
Biallelic Mutations

A significant number of cases with bone marrow failure present with one or more extra-hematopoietic abnormality. This suggests a constitutional or genetic basis, and yet many of them remain uncharacterized. Through exome sequencing, we have recently identified two sub groups of these cases, one defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21) and the other in ERCC6L2 (excision repair cross complementing 6 like-2). Patients with DNAJC21 mutations are characterized by global bone marrow failure in early childhood. They can also have a variable number of extra-hematopoietic abnormalities such as short stature and retinal dystrophy. The encoded protein associates with ribosomal RNA (rRNA) and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid patient cells exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced levels of rRNA. Characterisation of mutations has revealed impairment in interactions with cofactors (PA2G4, HSPA8 and ZNF622) involved in 60S maturation. DNAJC21 deficiency results in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles and increased cell death. Collectively these findings demonstrate that biallelic mutations in DNAJC21 cause disease due to defects in early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit. Patients harbouring biallelic ERCC6L 2 mutations are characterized by bone marrow failure (in childhood or early adulthood) and one or more extra-hematopoietic abnormality such as microcephaly. Knockdown of ERCC6L2 in human cells significantly reduces their viability upon exposure to the DNA damaging agent irofulven but not etoposide and camptothecin suggesting a role in nucleotide excision repair. ERCC6L2 knockdown cells and patient cells harbouring biallelic ERCC6L2 mutations also display H2AX phosphorylation that significantly increases upon genotoxic stress, suggesting an early DNA damage response. ERCC6L2 is seen to translocate to mitochondria as well as the nucleus in response to DNA damage and its knockdown induces intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger, N-acetyl-cysteine, attenuates the irofulven-induced cytotoxicity in ERCC6L2 knockdown cells and abolishes its traffic to mitochondria and nucleus in response to this DNA damaging agent. Collectively, these observations suggest that ERCC6L2has a pivotal rolein DNA repair and mitochondrial function. In conclusion, ERCC6L2 and DNAJC21 have an important role in maintaining genomic stability and ribosome biogenesis, respectively. They bring into focus new biological connections/pathways whose constitutional disruption is associated with defective hematopoiesis since patients harbouring germline biallelic mutations in these genes uniformly have bone marrow failure. Disclosures No relevant conflicts of interest to declare.

scholarly journals DNA Repair: Exploiting the Fanconi Anemia Pathway As a Potential Therapeutic Target

2011 ◽  
pp. 453-465 ◽  
Author(s):  
T. HUCL ◽  
E. GALLMEIER
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Synthetic Lethality ◽  
Bone Marrow Failure ◽  
Long Term Effects ◽  
Alternative Pathways ◽  
Repair Mechanisms ◽  
Relationship Of

DNA repair is an active cellular process to respond to constant DNA damage caused by metabolic processes and environmental factors. Since the outcome of DNA damage is generally adverse and long term effects may contribute to oncogenesis, cells have developed a variety of DNA repair mechanisms, which operate depending on the type of DNA damage inflicted. At least 15 Fanconi anemia (FA) proteins interact in a common pathway involved in homologous recombination. Inherited homozygous mutations in any of these FA genes cause a rare disease, Fanconi anemia, characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility. Heterozygous germline FA mutations predispose to various types of cancer. In addition, somatic FA mutations have been identified in diverse cancer types. Evidence exists that cells deficient in the FA pathway become dependent on alternative pathways for survival. Additional inhibition of such alternative pathways is thus expected to result in cell death, creating a relationship of synthetic lethality. Identifying these relationships can reveal yet unknown mechanisms of DNA repair and new targets for therapy.

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