Mesodermal Development From Reprogrammed Fanconi Anemia Cells Is Affected by ALDH2 Enzymatic Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 648-648
Author(s):  
Naoya Suzuki ◽  
Asuka Hira ◽  
Akira Niwa ◽  
Megumu Saito ◽  
Keitaro Matsuo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Developmental Stage ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
A Genome ◽  
The Impact ◽  
Mesodermal Development

Abstract Abstract 648 Introduction Fanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination. Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes. It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients. Results and discussion In FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study. To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway. To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues. Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage. 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 Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3453-3462 ◽  
Author(s):  
Asmin Tulpule ◽  
M. William Lensch ◽  
Justine D. Miller ◽  
Karyn Austin ◽  
Alan D'Andrea ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Embryonic Stem ◽  
Developmental Defects ◽  
Hematopoietic Development

Abstract Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

scholarly journals Genetic background and diagnosis of Fanconi anemia

2020 ◽  
Vol 74 ◽  
pp. 589-600
Author(s):  
Anna Repczyńska ◽  
Olga Haus
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Cell Cycle Control ◽  
Skin Pigmentation ◽  
Chromosome Instability ◽  
Molecular Techniques ◽  
Genetic Diagnostics ◽  
Risk Of Cancer

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).

scholarly journals Loss of the homologous recombination generad51leads to Fanconi anemia-like symptoms in zebrafish

2016 ◽  
Author(s):  
Jan Gregor Botthof ◽  
Ewa Bielczyk-Maczyńska ◽  
Lauren Ferreira ◽  
Ana Cvejic
Keyword(s):  
Bone Marrow ◽  
Homologous Recombination ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Tumor Development ◽  
Embryonic Stem ◽  
Dominant Negative ◽  
Hematopoietic Stem ◽  
Recombination Protein

AbstractRAD51is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterized by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. In this paper, we describe the first viable vertebrate model ofRAD51loss. Zebrafishrad51loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Co-mutation ofp53was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment ofRAD51as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.Significance statementThe homologous recombination protein RAD51 has been extensively studied in prokaryotes and lower eukaryotes. However, there is a significant lack of knowledge of the role of this protein and its regulation in anin-vivocontext in vertebrates. Here we report the first viable vertebrate mutant model ofrad51in zebrafish. These mutant fish enabled us to confirm for the first time the recently discovered role ofRAD51in Fanconi anemia pathogenesis. We report that p53 linked embryonic stem cell defects directly lead to hematological impairments later in life. Co-mutation ofrad51withp53rescues the observed hematological defects, but predisposes the fish to early tumor development. The application of this model opens new possibilities to advance Fanconi anemia drug discovery.

Mobilization and collection of peripheral blood CD34+ cells from patients with Fanconi anemia

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 2917-2921 ◽  
Author(s):  
James M. Croop ◽  
Ryan Cooper ◽  
Christine Fernandez ◽  
Vicki Graves ◽  
Susan Kreissman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Peripheral Blood Stem Cells ◽  
Target Weight ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Blood Stem Cells

Abstract A potential therapeutic option for patients with Fanconi anemia is collection of peripheral blood stem cells prior to the development of severe pancytopenia. These hematopoietic cells potentially could be infused when symptomatic bone marrow failure develops, as autologous rescue after chemotherapy in the event of leukemic transformation, or as targets for gene therapy. Eight patients with Fanconi anemia were mobilized with 10 μg/kg per day of granulocyte colony-stimulating factor (median, 10 ± 4 days) to determine the feasibility of collecting peripheral blood stem cells for future use. Six patients achieved a peripheral blood CD34+ count of ≥ 6/μL and underwent apheresis. The collection goal was 2 × 106 CD34+ cells/kg based on a predicted weight 5 years from the date of collection. A mean of 2.6 ± 0.9 × 106 CD34+ cells/kg of the weight at the time of collection were collected, which corresponded to 1.9 ± 0.4 × 106 CD34+cells/kg of the target weight. The collections required a mean of 4 ± 3 days (range, 2-8 days) of apheresis. Six of the 8 subjects had ≥ 1 × 106 CD34+ cells/kg cryopreserved based on both actual and target weights, and 4 subjects had ≥ 2 × 106 CD34+ cells/kg cryopreserved based on the target weight. These results suggest that some patients with Fanconi anemia can have adequate numbers of CD34+ cells mobilized and collected from the peripheral blood prior to the onset of severe bone marrow failure, but they may require an extended mobilization and multiple days of collection.

Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2221-2221
Author(s):  
Cyrus Khandanpour ◽  
Ulrich Duehrsen ◽  
Tarik Möröy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

TNFα Overproduction in FANCC-Deficient Cells Is TLR8 Dependent.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 494-494
Author(s):  
Scott Vanderwerf ◽  
Johanna Svahn ◽  
Praveen Anur ◽  
Ricardo Pasquini ◽  
Grover C. Bagby
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Hematopoietic Cells ◽  
Mononuclear Phagocytes ◽  
C Cells ◽  
Mutant Cells

Abstract Abstract 494 The Fanconi anemia (FA) proteins play a role in regulating genome stability but it is not clear that loss of genoprotection in FA hematopoietic cells accounts for the molecular pathogenesis of bone marrow failure so characteristic of this disease. Other factors are known to influence survival and replication of FA stem cells. For example, not only are FA progenitors and stem cells hypersensitive to the apoptotic effects of TNFα, FA cells over-produce TNFα. Most importantly over-production of and hypersensitivity to TNFα in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia 1;2 and long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFα results in the evolution of cytogenetically marked preleukemic clones.3 Therefore, the hematopoietic phenotype of FA is likely multifactorial and may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. Methods: We sought to clarify the molecular basis of aberrant TNFα-production. We conducted gene expression microarray experiments using RNA samples from low density marrow cells obtained from 11 normal volunteers and 22 Fanconi anemia patients with uncomplicated marrow hypoplasia without clonal cytogenetic defects. Because the FA complex is known to enhance ubiquitinylation of FANCD2, we reasoned that the ubiquitinylation state of proteins involved in the TNF pathways might also be influenced by core FA proteins. Therefore, we conducted in vitro ubiquitinylation assays using hexahistidine-tagged ubiquitin and an ATP-recycling system added to lysates of FANCC-deficient lymphoblasts (HSC536) and control cells (isogenic cells complemented with WT FANCC cDNA). Following the ubiquitinylation reaction, ubiquitinylated proteins were affinity purified, digested and analyzed by 2D capillary LC-MS/MS. Mass spectra were obtained and peptide precursor-MS/MS spectrum pairs were analyzed using SEQUEST and support vector machine learning.4 Peptides identified only in one or the other cell line were considered. Results: Initially we anticipated focusing on the set of proteins uniquely ubiquitinated in normal cells. However, the transcriptomal results indicated that genes encoding proteins in the ubiquitin pathway were over-represented in the list of genes that were over-expressed in FA samples. Consequently, we examined both differential ubiquitination lists and found that a major regulator of TNF-gene expression, TLR8, appeared in the ubiquitinylated fraction only in mutant cells. In co-immunoprecipitation studies we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells, and using RNAi determined that high level TNFα synthesis in mutant cells depended upon TLR8 and its downstream signaling intermediates IRAK-1 and IKK-alpha/beta. FANCC deficient THP1 blue cells were created using lentiviral shRNA targeting FANCC. These cells exhibited the MMC hypersensitive phenotype and over-expressed both TNFα and an NF-kappaB reporter gene (secreted embryonic alkaline phosphatase) in response to TLR8 agonists but not to other TLR agonists. Primary splenic macrophages from Fancc-/- mice were also hypersensitive to the TLR8 agonist R848. TNFα production in FA-C cells was suppressed by inhibitors of TLR8, p38 MAPK, IRAK, and IKK. Engineered point mutants of FANCC were capable of complementing the mitomycin C hypersensitivity phenotype of FANCC mutant cells but did not suppress TNFα overproduction in FANCC mutant cells. In conclusion, TNF over-expression in FANCC-deficient cells reflects the loss of FANCC function as a suppressor of TLR8 activation. In addition, FANCC suppresses TLR8 dependent production of TNFα in normal mononuclear phagocytes at least in part by suppressing either TLR8 ubiquitinylation or by inhibiting its association with an ubiquitinylated protein. Finally, this function of FANCC is independent of its function in protecting the genome from cross-linking agent-induced damage. In light of the role of TNFα in bone marrow failure and clonal evolution in this disease, control of TNF-production by targeting the TLR8 pathway might provide an opportunity to enhance hematopoietic activity and forestall clonal evolution in patients with this disorder. 1. Sejas DP, et al, J Immunol 2007;178:5277-5287. 2. Zhang × et al, J.Cell Sci. 2007;120:1572-1583. 3. Li J, et al, J.Clin.Invest. 2007;117:3283-3295, 4. Anderson DC, et al, J Proteome.Res 2003;2:137-146. Disclosures: No relevant conflicts of interest to declare.

Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 797-797
Author(s):  
Krasimira Rozenova ◽  
Jing Jiang ◽  
Chao Wu ◽  
Junmin Wu ◽  
Bernadette Aressy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Sandra Petrovic ◽  
Andreja Leskovac ◽  
Jelena Kotur-Stevuljevic ◽  
Jelena Joksic ◽  
Marija Guc-Scekic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Significant Difference ◽  
Heterozygous Carriers ◽  
Male Carriers ◽  
Female Carriers

Abstract Fanconi anemia (FA) is a rare cancer-prone genetic disorder characterized by progressive bone marrow failure, chromosomal instability and redox abnormalities. There is much biochemical and genetic data, which strongly suggest that FA cells experience increased oxidative stress. The present study was designed to elucidate if differences in oxidant state exist between control, idiopathic bone marrow failure (idBMF) and FA cells, and to analyze oxidant state of cells in FA heterozygous carriers as well. The results of the present study confirm an in vivo prooxidant state of FA cells and clearly indicate that FA patients can be distinguished from idBMF patients based on the oxidant state of cells. Female carriers of FA mutation also exhibited hallmarks of an in vivo prooxidant state behaving in a similar manner as FA patients. On the other hand, the oxidant state of cells in FA male carriers and idBMF families failed to show any significant difference vs. controls. We demonstrate that the altered oxidant state influences susceptibility of cells to apoptosis in both FA patients and female carriers. The results highlight the need for further research of the possible role of mitochondrial inheritance in the pathogenesis of FA.

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