Fanconi Anemia Bone Marrow Research Articles

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-β pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Structural insight into FANCI-FANCD2 monoubiquitination.

The Fanconi anemia (FA) pathway coordinates a faithful repair mechanism for DNA damage that blocks DNA replication, such as interstrand cross-links. A key step in the FA pathway is the conjugation of ubiquitin on to FANCD2 and FANCI, which is facilitated by a large E3 ubiquitin ligase complex called the FA core complex. Mutations in FANCD2, FANCI or FA core complex components cause the FA bone marrow failure syndrome. Despite the importance of these proteins to DNA repair and human disease, our molecular understanding of the FA pathway has been limited due to a deficit in structural studies. With the recent development in cryo-electron microscopy (EM), significant advances have been made in structural characterization of these proteins in the last 6 months. These structures, combined with new biochemical studies, now provide a more detailed understanding of how FANCD2 and FANCI are monoubiquitinated and how DNA repair may occur. In this review, we summarize these recent advances in the structural and molecular understanding of these key components in the FA pathway, compare the activation steps of FANCD2 and FANCI monoubiquitination and suggest molecular steps that are likely to be involved in regulating its activity.

Reduced Cell Division Control Protein 42 Activity Compromises Hematopoiesis-Supportive Function of Fanconi Anemia Mesenchymal Stromal Cells.

Hematopoietic stem cells preserve their ability to self-renew and differentiate to different lineages in the bone marrow (BM) niche, which is composed in large part by BM stromal cells. Studies have shown that altered signaling in the BM niche results in leukemia initiation or progression. Fanconi anemia (FA) is an inherited BM failure syndrome associated with extremely high risk of leukemic transformation. By using two FA mouse models, here we have investigated the hematopoiesis-supportive function of FA BM mesenchymal stroma cells (MSCs). We found that MSCs deficient for Fanca or Fancc gene are defective in proliferation and prone to undergo senescence in vitro. Mechanistically, we show that the activity of cell division control protein 42 (Cdc42), a Rho GTPase known to be a critical regulator for cytoskeleton organization, is significantly reduced in FA MSCs. Furthermore, we demonstrate that this reduction in Cdc42 activity plays a causal role in defective hematopoiesis-supportive function of the FA MSCs. The progenies of wild-type hematopoietic stem and progenitor cells cocultured on FA MSCs exhibit compromised self-renewal capacity both in vitro and in vivo. Genetic correction of FA deficiency restores Cdc42 activity and improves the hematopoiesis-supportive capacity of FA MSC. Finally, ectopic expression of a constitutively active Cdc42 mutant, Cdc42F28L, or pretreatment with Wnt5a, increases the active Cdc42 level and rescues the hematopoietic supportive defects of FA MSCs. Taken together, our results identify a novel link between Cdc42 activity and the hematopoiesis-supportive function of MSCs and suggest that a niche-specific increase of Cdc42 activity may be beneficial for FA therapy. Stem Cells 2018;36:785-795.

Hyperactive Non-Canonical TGF-β Pathway Signaling in Fanconi Anemia Bone Marrow Stromal Cells Contributes to Growth Suppression

Introduction: 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 cells (HSCs). FA is caused by autosomal recessive or X-linked mutations in one of nineteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to genotoxic DNA cross-linking agents. Although its mechanism is unknown, bone marrow failure in FA may be the result, directly or indirectly, of hyperactivation of cell-autonomous or microenvironmental growth-suppressive pathways induced, in part, due to genotoxic stress. We have recently identified canonical transforming growth factor-β (TGF-β) pathway-mediated growth suppression of HSCs as a cause of bone marrow failure in FA (Zhang H et al, Cell Stem Cell, 2016). We have shown that TGF-β pathway inhibition rescues genotoxic stress, proliferation defects and engraftment defects of FA-deficient HSCs, and ameliorates bone marrow failure in FA mice. Previous studies have suggested that bone marrow stromal fibroblasts from human FA patients and FA pathway-deficient mouse models, like HSCs, are hypersensitive to genotoxic stress and have impaired growth. Here, we therefore investigated the possible suppressive function of the TGF-β pathway in bone marrow stromal cells derived from FA mice and patients with FA.Methods: We established primary stromal cell lines from bone marrow of FA-deficient mice (Fancd2-/- mice) or wild-type sibling control mice. Primary bone marrow stromal cultures were also established from FA patients or normal healthy donors. The stromal cells were characterized and evaluated for growth kinetics, mitomycin C (MMC) sensitivity, chromosome breakage, inflammatory signals and response to the TGF-β inhibitors. CRISPR/Cas-9 technology was used to knockdown specific genes in stromal cells.Results: As expected,the primary bone marrow stromal cells from Fancd2-/- mice exhibited classical FA phenotypes, including hypersensitivity to a DNA cross-linking agent, MMC, and increased MMC-induced chromosomal radials. Fancd2-/- stromal cells also demonstrated a growth defect characterized by an enrichment of cells in G1 and elevated p21 expression. Interestingly, the FA stromal cells derived from FA patients or from Fancd2-/- mice expressed constitutively elevated levels of phosphorylated (activated) ERK1/2 (pERK) compared to control cells. In order to determine whether the factor responsible for inducing ERK1/2 phosphorylation in the murine FA stromal cells was cell-intrinsic or cell-extrinsic, we examined the conditioned media from the stromal cells. Indeed the FA stromal cells secreted a high level of TGF-β cytokine responsible for increased pERK levels, and expressed a high level of secreted TGF-β mRNA. The high level of pERK indicated that the TGF-β non-canonical pathway was hyperactive in the FA stromal cells. Interestingly, CRISPR/Cas9-mediated knockdown of Tgfbr1 or inhibition of the TGF-β pathway by a treatment with a small molecule inhibitor of TGFβR1 or a neutralizing antibody against TGF-β in these cells reduced pERK levels, promoted DNA repair and rescued MMC sensitivity. In addition, a MEK inhibitor also significantly improved the clonogenic growth of Fancd2-/- stromal cells. However, CRISPR/Cas9-mediated knockdown of Smad3, a downstream target of the canonical TGF-β pathway, did not rescue the growth inhibition of FA stromal cells in MMC, further indicating that hyperactivation of the canonical pathway is less relevant to their growth defect. Collectively, these results demonstrated that the hyperactive TGF-β pathway increases phosphorylation of ERK1/2 in FA stromal cells through the non-canonical signaling pathway and impairs their growth after genotoxic stress.Conclusions: The primary FA bone marrow stromal cells exhibit hyperactive non-canonical TGF-β pathway signaling and blocking this pathway improves their growth under genotoxic stress. The TGF-β signaling pathway-mediated growth suppression in bone marrow stromal cells may account, at least in part, for defective microenvironment, impaired HSC function and bone marrow failure in FA. This work suggests that the TGF-β signaling pathway may be a potential therapeutic target for the treatment of bone marrow failure in FA. DisclosuresShimamura:TransCellular Therapeutics: Other: Husband is founder. No revenue to date.; Novartis: Other: In discussion regarding possible clinical trial for aplastic anemia; Glaxo Smith Kline: Honoraria.

TGF-β Pathway Inhibition Rescues the Function of Hematopoietic Stem and Progenitor Cells Derived from Patients with Fanconi Anemia

Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life, and frequently require an allogeneic or unrelated donor bone marrow transplant. 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 mutation in one of eighteen 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 is attributable to an impaired hematopoietic stem and progenitor cell (HSPC) pool. HSPCs in FA patients and FA mice exhibit reduced cell number and compromised stem cell function. Recent studies suggest that bone marrow failure in FA and impaired HSPC function result from the genotoxicity of endogenous cross-linking agents or from physiological stress. A greater understanding of the mechanisms of impairment of HSPC function could improve the therapeutic options for FA patients. Using a whole genome-wide shRNA screen, we have recently identified that the canonical transforming growth factor-β (TGF-β) pathway plays an important growth suppressive role in FA and targeting this pathway can reduce the genotoxic stress-induced growth inhibition of FA cells. Here, we investigated the possible suppressive function of the TGF-β pathway in HSPCs derived from patients with FA.Methods: We performed in vitro colony-forming assays using primary FA patient- derived bone marrow CD34+ cells which were either transduced with shRNA targeting SMAD3 or treated with the anti-human TGF-β neutralizing antibody GC1008. FA-like HSPCs were generated by stably knocking down FANCD2 with lentivirus encoded shRNA in primary human cord blood CD34+ cells. An in vivo engraftment assay was performed by transplanting the FA-like HSPCs into irradiated NSG mice.Results: The primary human FA bone marrow cells displayed elevated mRNA expression of multiple TGF-β pathway components. The TGF-β pathway inhibition, by knockdown of SMAD3 or anti-human TGF-β neutralizing antibody GC1008, rescued the in vitro clonogenic defects of primary CD34+ cells from bone marrow of five different FA patients. Similarly, the TGF-β pathway disruption by depletion of SMAD3 or GC1008 antibody in primary FA-like HSPCs, also rescued their clonogenic defect, and partially restored genotoxic stress-induced growth inhibition. Further, as the very low number of CD34+ cells in FA patients did not allow efficient xenograft assay to analyze in vivo clonogenicity, we performed a surrogate in vivo xenograft assay using FA-like primary CD34+ cells. Importantly, blockade of the TGF-β pathway by GC1008 antibody treatment enhanced the engraftment potential of primary FA-like CD34+ cells in vivo. Collectively, these results demonstrated that increased TGF-β pathway signaling impairs the hematopoietic function of primary human FA HSPCs.Conclusions: The TGF-β pathway signaling is increased in primary FA patient-derived hematopoietic cells and blockade of this pathway can restore the function of human FA-deficient primary HSPCs. The TGF-β signaling pathway-mediated growth suppression may account, at least in part, for bone marrow failure in FA. This work suggests that the TGF-β signaling pathway provides a novel therapeutic target for the treatment of bone marrow failure in FA. DisclosuresNo relevant conflicts of interest to declare.

KIT Blockade Is Sufficient to Sustain Donor Hematopoietic Stem Cell Engraftment in Fanconi Anemia Mice

Background: Fanconi anemia (FA) is the most common cause of inherited bone marrow failure (BMF). Currently, the only curative option for the BMF in FA is an allogenic hematopoietic stem cell transplant (HSCT). However, due to the underlying DNA repair defect, FA patients poorly tolerate alkylating chemotherapy or irradiation based conditioning, which is necessary for donor engraftment. However, this results in significant short and long term morbidity/mortality and augments the inherent increased risk of malignancies in FA patients. To overcome the adverse effects associated with alkylating conditioning agents, alternate experimental approaches exploiting the inherent hematopoietic stem cell (HSC) defect in FA are of utmost clinical necessity.Objective: To develop a safe KIT blocking antibody (KIT-Ab) based HSCT conditioning regimen for FA that does not involve chemotherapy or irradiation.Method: High purity KIT-Ab was made from the ACK2 hybridoma and its specificity to KIT binding was validated using mast cell assay. Baseline peripheral blood cells and the bone marrow hematopoietic stem and progenitor cell (HSPC) compartment (Lin-Kit+Sca+ and Lin-Kit+Sca+CD150+CD48- cells) of FANCA-/- and FANCD2-/- murine models were analyzed. Mechanistic studies using sorted FA bone marrow HSPC were performed ex vivo. This was followed by definitive primary and secondary transplants experiments following injection of KIT-Ab.Results: Several features of FA hematopoietic stem/progenitor cells (HSPC) suggested their susceptibility to KIT-Ab blockade-mediated killing: (a) Expression of KIT was significantly lower in FANCA-/- HSPC, while expression of its ligand was higher in bone marrow stroma; (b) Moreover, genes associated with apoptosis/senescence, stress and inflammatory signaling that were upregulated in WT-HSPC following KIT-Ab blockade, were upregulated in FANCA-/- HSPC at baseline; (c) Furthermore, FANCA-/- HSPC demonstrated increased susceptibility to KIT-Ab mediated apoptosis and had a reduced proliferative capacity. In-vivo studies following ACK2 injection showed a marked reduction of colony-forming units (CFU-C) from both FANCA-/- and FANCD2-/- mice one week following injection, when compared to WT mice (48% and 76% decrease in CFU-C, respectively). Based on these findings, we evaluated the role of ACK2 as a sole HSCT conditioning regimen in FANCA-/- and FANCD2-/- mice. Indeed, definitive HSCT in both FANCA-/- and FANCD2-/- mice using KIT-Ab based conditioning resulted in donor HSC engraftment with multi-lineage chimerism, which progressively increased to 22-24% by 4-months, and was sustained in secondary transplants. Overall, we show that KIT-blockade alone is an adequate non-genotoxic HSPC-targeted conditioning in FA mice, and its clinical translation could circumvent the extensive transplant-related morbidity/mortality in this disease. DisclosuresNo relevant conflicts of interest to declare.

Fanconi Anemia Bone Marrow Stem Cells: Innately Complex

Fanconi Anemia Bone Marrow Stem Cells: Innately Complex

Micro RNA Regulate Developmental Deficits In The Hematopoietic Stem Cell Compartment In Fanconi Anemia

Hematopoietic failure is the predominant manifestation of Fanconi anemia (FA), a recessively inherited disorder in DNA double strand break (DSB) repair. The etiology underlying the progressive loss of hematopoietic stem and progenitor cells (HSPC) in FA remains to be fully clarified, but is widely considered to be mediated by the “stress activation” of p53. In a murine model of FA (Fancc), we recently showed that the onset of hematopoietic failure precedes the developmentally timed HSPC migration to the bone marrow (BM), corroborating prior reports of reduced cord blood progenitor frequency and work in human ES cell lines. Comparison of genotype frequencies at E14.5 and postnatally (25% vs 19%; p<0.004) further indicated that hematopoietic failure coincides with fetal loss during late gestation. This data has now been replicated in Fancd2 animals. The quantitative and qualitative severity of fetal HSPC pool defects in Fancc and Fancd2 mice suggested to us the possibility of a unique developmental phenotype. Fetal liver HSPC are characterized by specific molecular and functional features to support the high self-renewal capacity during developmental pool expansion. To determine the molecular basis of fetal HSPC losses in FA, we systematically studied progenitor cell populations from Fancc E14.5 fetal liver and postnatal bone marrow for the expression of candidate genes involved in HSPC function. Results confirmed in utero genotype-specific p53 pathway activation and differences in key cell cycle regulators (p21, p27 and p57). Among a group of developmentally dysregulated genes, we found the non-histone chromatin remodeling factor Hmga2 substantially upregulated in FA versus WT E14.5 cells. As an important developmental regulator of self-renewal activity, Hmga2 is not expressed postnatally, which we confirmed in FA bone marrow cells. To dissect the FA phenotype further, we investigated the expression of other components of the developmental signaling cascade, in particular micro RNA (miR) let7b, the major let7 family member accounting for translational suppression of Hmga2 during late gestation. Results indicate that let7b is downregulated in E14.5 Fancc and Fancd2 progenitor cells. To reconcile the in utero activation of a major pathway promoting self-renewal and HSPC expansion with the functional and numeric defects we found in fetal FA HSPC, we investigated other upstream components. Systematic dissection of the signaling cascade revealed an 80% decline by q RT-PCR of miR-125b, a stem cell regulatory miRNA and nodal point that targets let7b (indirectly via Lin28 suppression), but also prevents inappropriate p53 activation. A relatively more profound decrease in miR-125b expression in Sca-1+ cells substantiate this as a stem/progenitor cell phenotype. The data are further consistent with our results showing the upregulation of p21 in E14.5 FL cells (as an indicator of p53 pathway activation), suggesting that the loss of miR-125b expression is a proximal mechanism for p53 upregulation in FA HSPC. Finally, when we tested for evidence of DNA damage, we observed gamma-H2AX foci in sorted HSPC as well as increased transcriptional activation of DNA repair via non-homologous end-joining (Ku70, Ku80, Prkdc transcripts) in Sca-1 enriched Fancc and Fancd2 FL cells. Based on the results to-date, we propose a model whereby relatively decreased expression of miR-125b serves as a key regulatory step for the activation of a critical self-renewal pathway (let7b-Hmga2), but also dysregulates p53 signaling during FA hematopoietic development. Accruing and unresolved DNA damage in repair-deficient fetal FA HSPC curtails their net expansion. The functional compromise in the murine HSPC pool reveals a regulatory role for miRNA in FA HSPC failure and provides a unique model for the mechanistic study of HSPC loss under physiologic conditions and without the experimental induction via exogenous cytokines, aldehydes or alkylating agents. Disclosures:No relevant conflicts of interest to declare.

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.

Immunology and Gene Therapy: Shoulder to Shoulder Into the Fray: International Symposium: Critical Frontiers Between Immunity and Gene Therapy Pamplona, Spain, 27–28 October 2009

Functions of the immune system are critical for the outcome of gene therapy. Immunologists, virologists, and gene therapists met in Pamplona, Spain, in October 2009 to present and analyze common frontiers in these fields under the auspices of the Ramon Areces Foundation (http://www.fundacionareces.es). Antibodies and cell-mediated immune mechanisms can be either friends or foes in gene therapy. A form of Murphy’s law seems to apply: “If an immune response is to be induced, it will usually be weak. If an immune response is to be avoided, it will be almost always neutralizing.”

TLR8-dependent TNF-α overexpression in Fanconi anemia group C cells

AbstractTumor necrosis factor alpha (TNF-α) production is abnormally high in Fanconi anemia (FA) cells and contributes to the hematopoietic defects seen in FA complementation group C–deficient (Fancc−/−) mice. Applying gene expression microarray and proteomic methods to studies on FANCC-deficient cells we found that genes encoding proteins directly involved in ubiquitinylation are overrepresented in the signature of FA bone marrow cells and that ubiquitinylation profiles of FA-C and complemented cells were substantially different. Finding that Toll-like receptor 8 (TLR8) was one of the proteins ubiquitinylated only in mutant cells, we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells and that TNF-α production in mutant cells depended upon TLR8 and the canonical downstream signaling intermediates interleukin 1 receptor–associated kinase (IRAK) and IκB kinase-alpha/beta. FANCC-deficient THP-1 cells and macrophages from Fancc−/− mice overexpressed TNF-α in response to TLR8 agonists but not other TLR agonists. Ectopically expressed FANCC point mutants were capable of fully complementing the mitomycin-C hypersensitivity phenotype of FA-C cells but did not suppress TNF-α overproduction. In conclusion, FANCC suppresses TNF-α production in mononuclear phagocytes by suppressing TLR8 activity and this particular function of FANCC is independent of its function in protecting the genome from cross-linking agents.

Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

A Cell-Autonomous Defect of Bone Marrow Hematopoietic Stem/Progenitor Cells and Impaired Stromal Function in FA-a Patients

Fanconi anemia (FA) is a devastating blood disease associated with bone marrow (BM) failure. Currently the effective treatment for FA is BM transplantation. We and others showed that FA murine hematopoietic stem and progenitor cells (HSC/Ps) have defective hematopoietic reconstitution after BM transplantation. In addition, several clinical gene therapy trials in FA patients have failed to show sustained engraftment of FA HSC/Ps complemented with a functional FA gene. The mechanism underlying this engraftment defect remains unknown. To determine whether the poor engraftment of FA HSC/Ps observed in mouse knockout models and clinical gene therapy trials in FA patients results from an intrinsic defect of the FA HSC/Ps or an impaired microenvironment in the FA bone marrow, we have assessed the self-renewal ability of HSC/Ps and hematopoietic supportive capacity of BM stromal cells from FA-A patients. First, we performed quantitative cobblestone area-forming cell (CAFC) assays using MS-5 stromal layer. CAFCs were enumerated at 2 and 5 weeks of cocultures between BM mononuclear cells from a normal donor and three FA-A patients. Early-appearing CAFCs (week 2) represent transient repopulating cells equivalent to progenitor colony-forming units (CFUs), whereas late-appearing CAFCs (week 5) are representative of long-term repopulating stem cells. The frequency of FA-A CAFCs was 15–20 folds decreased compared with that of the normal donor at both week 2 and week 5. Second, we carried out long-term culture-initiating cell (LTC-IC) assays using BM mononuclear cells and BM-derived stromals from normal donors or FA-A patients. Compared to coculture with normal stromal cells, the recovery of colony forming cells (CFCs) that grew on FA-A stromal layer after 5–6 weeks incubation was significantly decreased for either normal or FA-A HSC/Ps (41.4% ± 3.1 CFC in normal HSC/Ps - normal stroma compared to 14.3% ± 1.6 CFC in normal HSC/Ps – FA-A stroma and 2.4 ± 0.6 CFC in FA-A HSC/Ps- FA-A stroma). Finally, we conducted CFC and cell-cell adhesion assays using BM-derived stromal cells from normal donors and FA-A patients. We observed that FA-A BM stroma failed to support HSC/P cells from either normal donors or FA-A patients. Specifically, severe adhesion defect was found in adhesion of normal and FA-A HSC/Ps with FA-A stromal layer (5.5% ± 0.6 CFC adhesion in normal HSC/Ps - normal stroma compared to 2.4% ± 0.5 CFC adhesion in normal HSC/Ps – FA-A stroma and 1.0 ± 0.7 CFC adhesion in FA-A HSC/Ps- FA-A stroma). These results suggest that both a cell-autonomous defect of FA HSC/Ps and an impaired FA BM microenvironment may contribute to the abnormal hematopoiesis in FA.

Non-Random Lentiviral Vector Insertions in Bone Marrow Progenitors from Fanconi Anemia Patients

Improved protocols using lentiviral vectors have been established with minimal cytokine exposure and short transduction times proving more suitable for overcoming the disease-specific challenge in correcting functionally defective hematopoietic stem cells (HSCs) of Fanconi Anemia (FA) patients. Bone marrow (BM) cells from FA patients were transduced ex vivo with lentiviral vectors (LVs) expressing FANCA and/or EGFP using optimized conditions to preserve the repopulating properties of the primitive hematopoietic stem cells (manuscript submitted). In a forward preclinical screening of possible LV-induced side effects we analyzed the insertional inventory in colonies generated by FA BM cells previously transduced with the LVs. We have established and optimized DNA and RNA isolation procedures for minimal cell numbers, suitable for large scale screening of colony forming cell (CFC) derived colonies by linear amplification-mediated PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; Roche). This approach is applicable for detecting early indicators of clonal selection, and is based on the analysis of common integration sites (CIS) and non-random distribution of vector insertions in particular genomic loci. From a total of 180 CFC-derived colonies expressing the EGFP LV marker gene, 298 vector insertions could be sequenced and mapped to the human genome. The analysis of vector targeted gene coding regions showed a non-random genomic distribution of LV insertions, with a significant overrepresentation of RefSeq genes that are part of distinct functional categories. Accordingly vector associated genes are predominantly involved in cellular signal cascades regulated by the MAP Kinase family known to be involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Apart from the observed high integration frequency in genes (>80%), partial loss of vector LTR nucleotides was detected in >10% of the integrants (3–25bp). Notably, >20% of the lentiviral insertions were found to be located in CIS of predominantly 2nd order. Further screening assays of LV transduced CFC-derived colonies will allow a deeper investigation in the functional consequences of such CIS targeting in gene therapy protocols of FA. However our results suggest that the LV transduction of FA BM progenitors leads to a relatively high frequency of insertions in CIS which may be indicative of an insertion based (specific) selection mechanism. We herby show that the ex vivo large scale integration site analyses of CFC-derived colonies from patients considered to undergo gene therapeutic treatments constitutes a robust approach, which combined with mouse preclinical biosafety studies will help to improve the safety of clinical gene therapy protocols. The non-random distribution of LV integrations in CIS associated genes and in genes involved in particular cellular pathways may be indicative for the altered biochemical pathways characteristic of FA stem cells, with reported defects in DNA repair and self-renewal.

Correction of Fanconi Anemia Group A in Primary Human and Murine Hematopoietic Progenitors with a Clinical Lentiviral Vector

Fanconi Anemia (FA) is a rare genetic disorder of DNA repair that typically manifests with bone marrow failure. Allogeneic stem cell transplant is the only cure known for the bone marrow disorder. Unfortunately, many patients do not have matched family donors, and alternative donor transplant has been associated with considerable morbidity and mortality. For individuals without an appropriate donor, ex vivo genetic modification of autologous stem cells is a potential therapeutic strategy. However, one of the major hurdles in gene therapy for this condition is the increased sensitivity of FA stem cells to free-radical induced DNA damage during ex vivo culture and manipulation. To minimize this damage we have developed a brief transduction procedure for lentivirus vector mediated gene transfer for Fanconi Anemia complementation group A (FancA), and evaluated this method in bone marrow progenitors from patients with FA, and murine progenitors from FancA deficient mice. The lentiviral vector, RSCPFancA-sW (FancA-sW), was specifically developed for clinical studies, and has a synthetic wpre which has been modified for safety (does not express a partial woodchuck hepatitis virus X protein open reading frame) and most of the 3′ untranslated region of the FancA gene was removed. FancA −/− mice were preconditioned with cyclophosphamide (120 mg/kg) prior to injection of transduced syngeneic bone marrow. After two subsequent rounds of the same dose of cyclophosphamide, 3/5 mice demonstrated mitomycin C (MMC) resistance in cells from bone marrow or spleen via colony assay. Two mice had peripheral blood marking with the vector seen by quantitative PCR. In an initial mouse experiment using similar conditions, the estimated transduction efficiency by colony PCR was determined to be 31% with the average number of integrations per cell determined to be 1.8 by LAM-PCR. Bone marrow mononuclear cells (BM MNCs) from a patient with FancA were also transduced with FancA-sW. Very few colonies were obtained on methylcellulose, although cell clusters were observed in MMC-treated cultures derived from FancA-sW transduced BM MNCs, while no clusters or colonies formed from cells transduced with the control vector. In an attempt to increase progenitor survival by reducing oxidative stress, primary human FA bone marrow was transduced and placed in 5% oxygen in the presence of 1 nM NAC (N-Acetyl-L-Cysteine). Cells plated in methylcellulose under these conditions had 2–3 fold increased colony formation compared to conditions without NAC or in 21% oxygen (P < 0.03). FancA-sW transduced BM MNCs from a second FancA patient were plated under these conditions formed colonies and numerous cell clusters and demonstrated resistance to MMC compared to GFP transduced controls (P < 0.007). Furthermore, transduced BM MNCs in culture had increased survival in culture when exposed to MMC ( P < 0.004). In summary, a lentiviral vector with a functional FancA transgene was developed which achieves phenotypic correction of human and murine hematopoietic progenitors with the brief period of prestimulation and transduction that would be required to maintain survival of FA cells ex vivo in culture. Reduction of oxidative stress may enhance the viability and engraftment of fragile FA stem cells.

Defective Adhesion, Migration and Homing Are Associated with Altered Rho GTPase Activity in Cells from Fanconi Anemia Patients.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and predisposition to malignancy. One of the potential therapeutic options for patients with FA is collection of autologous multipotent hematopoietic progenitors prior to the development of severe pancytopenia for autologous transplantation and gene therapy. However, poor engraftment of FA hematopoietic cells represents a major obstacle for effective transplantation. Our current study attempted to investigate the mechanism underlying defective engraftment of FA bone marrow (BM) cells. Using BM cells from patients carrying mutations in the FA complementation group A (FA-A), we demonstrate that SDF (Stromal cell-derived factor)-1alpha – and integrin-mediated migration and adhesion, respectively, is defective in FA primary BM cells compared to those from normal donors (more than 2-fold decrease in both migration and adhesion compared to normal BM cells). Complementation of the FA-A defect by retrovirus gene transfer of FANCA gene almost completely restores the ability of the BM cells to migrate towards the chemokine SDF-1alpha. Similar results are obtained with primary fibroblast cells derived from a FA-A patient, which show 3-fold and 35% decrease in adhesion and migration, respectively, compared to FANCA-corrected cells. Furthermore, when transplanted into immunodeficient Nod/scid recipient mice, the FA-A BM cells exhibited significantly impaired homing function whereas normal cells were efficiently homed in the bone marrow. A significant decrease in the activity of the Rho GTPase Cdc42 in FA-A cells is found associated with the patient cell defective functions. Taken together, these data suggest that the FA proteins play a role in the regulation of cell adhesion and migration in addition to maintaining genomic stability, influencing homing and engraftment, possibly via the small GTPase signaling pathway. These findings may have implications in development of strategies for restoring engraftment function of FA hematopoietic cells.

In vivo repopulation ability of genetically corrected bone marrow cells from Fanconi anemia patients

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo, knockout animal models are not sufficient to guide preclinical steps, and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA, we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for >120 days, and after cultured cell transplantation into NOD/SCID mice, clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison, untransduced cells died in culture by 15 days. Of necessity for ethical reasons, experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements, a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition, we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA.

Immune Defects in Fanconi Anemia

Fanconi anemia (FA) is a genetic disorder characterized by sensitivity to DNA cross-linking agents, multiple congenital anomalies, progressive bone marrow failure, and an increased prevalence of malignancy. The nature of chromosomal instability in FA is better understood today than in the past, but the molecular pathogenesis of bone marrow failure in this disease has not been clarified. Although there is documented evidence that FA hematopoietic stem cells (HSC) have inherent defects that reduce their survival, the potential influence of auxiliary cells on the ability of the FA bone marrow microenvironment to maintain and support HSC in unknown. Historically, FA has not been represented as a disease that affects the lymphoid compartment. In this article we review the results of studies that suggest that the FA immune system is dysfunctional and may contribute to the pathogenesis of both FA bone marrow failure and neoplastic disease.

Clonal Evolution of Fanconi Anemia (FA) Stem Cells Results from Clonal Adaptation and Clonal Selection. A Report from the International Fanconi Anemia Transcriptome Consortium.

FA stem cells and progenitor cells are apoptotic in the ground state and hypersensitive to a variety of extracellular apoptotic cues. The relative lifetime risk of AML or MDS in FA is high and clonal cytogenetic defects are universally found in evolved clones, but the mechanisms involved in leukemogenesis are unknown. The combined influences of genetic instability and high level stem cell apoptosis represents a de-facto selective pressure that in our view favors the emergence of more resistant stem cell clones. Predictions of this model are: (1) Non clonal FA progenitor cells (FAwt) are hypersensitive to apoptotic cues but FA progenitors derived from cytogenetically abnormal clones (FAclon) will be resistant, (2) anti-apoptotic events that attend cytogenetic clonal evolution should interdict precisely those apoptotic pathways activated by FA gene mutations, and (3) Key transcripts differentially expressed in the normal (N) vs. FAwt comparison should normalize (ie. should register as "no change") in the N vs. FAclon comparison. Preliminary studies support the first 2 predictions (Lensch et al, Leukemia, 1999 and Haneline et al, Blood, 2003). To further test the adaptive model, we compared transcriptomes of 41 low density bone marrow cell samples: (11 normal volunteers [N], 9 FAclon [marrow replaced [>75%] by cells bearing clonal cytogenetic defects] and 21 FAwt [no cytogenetic abnormalities]). RNA was prepared in the participating institutions and shipped to Portland on dry ice. Complementary DNA and cRNA was prepared, cRNA fragments labeled for use as targets of the probes in the human U133A Affymetrix chip. MAS 5.0 was utilized to process images, quantify signals, adjust background, and to scale the data. A linear mixed model was used for inter-chip normalization. Unsupervised hierarchical clustering and multidimensional scaling (MDS) distinguished N from FAwt samples but FAclon fell within the mathematical space defined by the N RNA. Of 17,044 genes tested, 1,430 were expressed differentially (FDR adjusted p <0.05, fold change ≥2) between N and FAwt samples. Differences did not derive from changes in cell lineages from case-to-case, and did not vary between geographical sites and were not linked to particular complementation groups. 659 transcripts were UP in FAwt (vs. N), but 587 of these were not UP in FAclon (vs N). The two dominant over-represented (p<0.01) ontologies (biological process) of the 659 genes were apoptosis and transcriptional control. 771 transcripts were DN in FAwt (vs. N) but 734 of these were not DN in the FAclon vs. N comparison. Dominant ontologies included cell proliferation, and responses to biotic stimuli. Our state assessment of FA bone marrow cells has defined a transcriptomal difference between and FAwt and normal cells and reveals that only 5-10% of these differentially expressed genes are differentially expressed between normal and FAclon cells. These observations support the notion that cytogenetically marked clones evolve adaptively from initially pro-apoptotic stem cells.

The Fanconi Anemia Proteins Functionally Interact with the Protein Kinase Regulated by RNA (PKR)

Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimer's, Parkinson's, and Huntington's diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-gamma and tumor necrosis factor-alpha. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-gamma and tumor necrosis factor-alpha. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.