Andrew F. Wilson

Fancd2 is required for nuclear retention of FOXO3a in hematopoietic stem cell maintenance

Background: Maintenance of HSCs is challenged by DNA damage and oxidative stress. Results: Fancd2 deficiency promoted cytoplasmic localization of Foxo3a in HSCs. Re-expression of Fancd2 restored nuclear Foxo3a localization and prevented HSC exhaustion. Conclusion: Fancd2 is required for nuclear retention of Foxo3a and maintaining hematopoietic repopulation of HSCs. Significance: Our results implicate an interaction between FA DNA repair and FOXO3a pathways in HSC maintenance. Functional maintenance of hematopoietic stem cells (HSCs) is constantly challenged by stresses like DNA damage and oxidative stress. Here we show that the Fanconi anemia protein Fancd2 and stress transcriptional factor Foxo3a cooperate to prevent HSC exhaustion in mice. Deletion of both Fancd2 and Foxo3a led to an initial expansion followed by a progressive decline of bone marrow stem and progenitor cells. Limiting dilution transplantation and competitive repopulating experiments demonstrated a dramatic reduction of competitive repopulating units and progressive decline in hematopoietic repopulating ability of double-knockout (dKO) HSCs. Analysis of the transcriptome of dKO HSCs revealed perturbation of multiple pathways implicated in HSC exhaustion. Fancd2 deficiency strongly promoted cytoplasmic localization of Foxo3a in HSCs, and re-expression of Fancd2 completely restored nuclear Foxo3a localization. By co-expressing a constitutively active CA-FOXO3a and WT or a nonubiquitinated Fancd2 in dKO bone marrow stem/progenitor cells, we demonstrated that Fancd2 was required for nuclear retention of CA-FOXO3a and for maintaining hematopoietic repopulation of the HSCs. Collectively, these results implicate a functional interaction between the Fanconi anemia DNA repair and FOXO3a pathways in HSC maintenance.

Hyper-active non-homologous end joining selects for synthetic lethality resistant and pathological Fanconi anemia hematopoietic stem and progenitor cells.

The prominent role of Fanconi anemia (FA) proteins involves homologous recombination (HR) repair. Poly[ADP-ribose] polymerase1 (PARP1) functions in multiple cellular processes including DNA repair and PARP inhibition is an emerging targeted therapy for cancer patients deficient in HR. Here we show that PARP1 activation in hematopoietic stem and progenitor cells (HSPCs) in response to genotoxic or oxidative stress attenuates HSPC exhaustion. Mechanistically, PARP1 controls the balance between HR and non-homologous end joining (NHEJ) in double strand break (DSB) repair by preventing excessive NHEJ. Disruption of the FA core complex skews PARP1 function in DSB repair and led to hyper-active NHEJ in Fanca−/− or Fancc−/− HSPCs. Re-expression of PARP1 rescues the hyper-active NHEJ phenotype in Brca1−/−Parp1−/− but less effective in Fanca−/−Parp1−/− cells. Inhibition of NHEJ prevents myeloid/erythroid pathologies associated with synthetic lethality. Our results suggest that hyper-active NHEJ may select for “synthetic lethality” resistant and pathological HSPCs.

Fanconi Anemia Mesenchymal Stromal Cells‐Derived Glycerophospholipids Skew Hematopoietic Stem Cell Differentiation Through Toll‐Like Receptor Signaling

Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)‐derived metabolites, glycerophospholipids, and their endogenous inhibitor, 5‐(tetradecyloxy)−2‐furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells. We provided two pieces of evidence that TOFA could improve hematopoiesis‐supporting function of FA MSCs: (a) limiting‐dilution cobblestone area‐forming cell assay revealed that TOFA significantly increased cobblestone colonies in Fanca−/− or Fancd2−/− cocultures compared to untreated cocultures. (b) Competitive repopulating assay using output cells collected from cocultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca−/− or Fancd2−/− cocultures. Furthermore, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation. Stem Cells 2015;33:3382–3396

Fancd2 in vivo interaction network reveals a non-canonical role in mitochondrial function

Fancd2 is a component of the Fanconi anemia (FA) DNA repair pathway, which is frequently found defective in human cancers. The full repertoire of Fancd2 functions in normal development and tumorigenesis remains to be determined. Here we developed a Flag- and hemagglutinin-tagged Fancd2 knock-in mouse strain that allowed a high throughput mass spectrometry approach to search for Fancd2-binding proteins in different mouse organs. In addition to DNA repair partners, we observed that many Fancd2-interacting proteins are mitochondrion-specific. Fancd2 localizes in the mitochondrion and associates with the nucleoid complex components Atad3 and Tufm. The Atad3-Tufm complex is disrupted in Fancd2−/− mice and those deficient for the FA core component Fanca. Fancd2 mitochondrial localization requires Atad3. Collectively, these findings provide evidence for Fancd2 as a crucial regulator of mitochondrion biosynthesis, and of a molecular link between FA and mitochondrial homeostasis.

SCO2 Mediates Oxidative Stress-Induced Glycolysis to Oxidative Phosphorylation Switch in Hematopoietic Stem Cells.

Fanconi anemia (FA) is an inherited bone marrow (BM) failure syndrome, presumably resulting from defects in hematopoietic stem cells (HSCs). Normal HSCs depend more on glycolysis than on oxidative phosphorylation (OXPHOS) for energy production. Here, we show that FA HSCs are more sensitive to the respiration inhibitor NaN3 treatment than to glycolytic inhibitor 2‐deoxy‐d‐glucose (2‐DG), indicating more dependence on OXPHOS. FA HSCs undergo glycolysis‐to‐OXPHOS switch in response to oxidative stress through a p53‐dependent mechanism. Metabolic stresses induce upregulation of p53 metabolic targets in FA HSCs. Inactivation of p53 in FA HSCs prevents glycolysis‐to‐OXPHOS switch. Furthermore, p53‐deficient FA HSCs are more sensitive to 2‐DG‐mediated metabolic stress. Finally, oxidative stress‐induced glycolysis‐to‐OXPHOS switch is mediated by synthesis of cytochrome c oxidase 2 (SCO2). These findings demonstrate p53‐mediated OXPHOS function as a compensatory alteration in FA HSCs to ensure a functional but mildly impaired energy metabolism and suggest a cautious approach to manipulating p53 signaling in FA. Stem Cells 2016;34:960–971

Loss of Fancc Impairs Antibody-Secreting Cell Differentiation in Mice through Deregulating the Wnt Signaling Pathway

Fanconi anemia (FA) is characterized by a progressive bone marrow failure and an increased incidence of cancer. FA patients have high susceptibility to immune-related complications such as infection and posttransplant graft-versus-host disease. In this study, we investigated the effect of FA deficiency in B cell function using the Fancc mouse model. Fancc−/− B cells show a specific defect in IgG2a switch and impaired Ab-secreting cell (ASC) differentiation. Global transcriptome analysis of naive B cells by mRNA sequencing demonstrates that FA deficiency deregulates a network of genes involved in immune function. Significantly, many genes implicated in Wnt signaling were aberrantly expressed in Fancc−/− B cells. Consistently, Fancc−/− B cells accumulate high levels of β-catenin under both resting and stimulated conditions, suggesting hyperactive Wnt signaling. Using an in vivo Wnt GFP reporter assay, we verified the upregulation of Wnt signaling as a potential mechanism responsible for the impaired Fancc−/− B cell differentiation. Furthermore, we showed that Wnt signaling inhibits ASC differentiation possibly through repression of Blimp1 and that Fancc−/− B cells are hypersensitive to Wnt activation during ASC differentiation. Our findings identify Wnt signaling as a physiological regulator of ASC differentiation and establish a role for the Wnt pathway in normal B cell function and FA immune deficiency.

In Vivo RNAi Screen Unveils PPARγ as a Regulator of Hematopoietic Stem Cell Homeostasis

Summary Hematopoietic stem cell (HSC) defects can cause repopulating impairment leading to hematologic diseases. To target HSC deficiency in a disease setting, we exploited the repopulating defect of Fanconi anemia (FA) HSCs to conduct an in vivo short hairpin RNA (shRNA) screen. We exposed Fancd2−/− HSCs to a lentiviral shRNA library targeting 947 genes. We found enrichment of shRNAs targeting genes involved in the PPARγ pathway that has not been linked to HSC homeostasis. PPARγ inhibition by shRNA or chemical compounds significantly improves the repopulating ability of Fancd2−/− HSCs. Conversely, activation of PPARγ in wild-type HSCs impaired hematopoietic repopulation. In mouse HSCs and patient-derived lymphoblasts, PPARγ activation is manifested in upregulating the p53 target p21. PPARγ and co-activators are upregulated in total bone marrow and stem/progenitor cells from FA patients. Collectively, this work illustrates the utility of RNAi technology coupled with HSC transplantation for the discovery of novel genes and pathways involved in stress hematopoiesis.

Persistent response of Fanconi anemia hematopoietic stem and progenitor cells to oxidative stress

ABSTRACT Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G2/M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.

Loss of Faap20 Causes Hematopoietic Stem and Progenitor Cell Depletion in Mice Under Genotoxic Stress

20‐kDa FANCA‐associated protein (FAAP20) is a recently identified protein that associates with the Fanconi anemia (FA) core complex component, FANCA. FAAP20 contains a conserved ubiquitin‐binding zinc‐finger domain and plays critical roles in the FA‐BRCA pathway of DNA repair and genome maintenance. The function of FAAP20 in animals has not been explored. Here, we report that deletion of Faap20 in mice led to a mild FA‐like phenotype with defects in the reproductive and hematopoietic systems. Specifically, hematopoietic stem and progenitor cells (HSPCs) from Faap20−/− mice showed defects in long‐term multilineage reconstitution in lethally irradiated recipient mice, with milder phenotype as compared to HSPCs from Fanca−/− or Fancc−/− mice. Faap20−/− mice are susceptible to mitomycin C (MMC)‐induced pancytopenia. That is, acute MMC stress induced a significant progenitor loss especially the erythroid progenitors and megakaryocyte–erythrocyte progenitors in Faap20−/− mice. Furthermore, Faap20−/− HSPCs displayed aberrant cell cycle pattern during chronic MMC treatment. Finally, using Faap20−/− Fanca−/− double‐knockout mice, we demonstrated a possible dominant effect of FANCA in the interaction between FAAP20 and FANCA. This novel Faap20 mouse model may be valuable in studying the regulation of the FA pathway during bone marrow failure progress in FA patients. Stem Cells 2015;33:2320–2330

Transcriptional profiling of Foxo3a and Fancd2 regulated genes in mouse hematopoietic stem cells.

Functional maintenance of hematopoietic stem cells (HSCs) is constantly challenged by stresses like DNA damage and oxidative stress. Foxo factors particularly Foxo3a function to regulate the self-renewal of HSCs and contribute to the maintenance of the HSC pool during aging by providing resistance to oxidative stress. Fancd2-deficient mice had multiple hematopoietic defects including HSC loss in early development and in response to cellular stresses including oxidative stress. The cellular mechanisms underlying HSC loss in Fancd2-deficient mice include abnormal cell cycle status loss of quiescence and compromised hematopoietic repopulating capacity of HSCs. To address on a genome wide level the genes and pathways that are impacted by deletion of the Fancd2 and Foxo3a we performed microarray analysis on phenotypic HSCs (Lin−ckit+Sca-1+CD150+CD48−) from Fancd2 single knockout Foxo3a single knockout and Fancd2 −/− Foxo3a −/− double-knockout (dKO) mice. Here we provide detailed methods and analysis on these microarray data which has been deposited in Gene Expression Omnibus (GEO): GSE64215.