Faculty Opinions recommendation of Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia.

Abstract 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.

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Regulation of FANCL by Glycogen Synthase Kinase-3beta Links the Fanconi anemia pathway to Self Renewal and Survival Signals

Blood ◽

10.1182/blood.v120.21.1263.1263 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1263-1263

Author(s):

Kim-Hien T. Dao ◽

Michael D. Rotelli ◽

Jane E. Yates ◽

Brieanna Brown ◽

Juha Rantala ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Glycogen Synthase ◽

Tnf Alpha ◽

Beta Catenin ◽

Self Renewal ◽

Hematopoietic Stem ◽

Glycogen Synthase Kinase 3Beta ◽

N Terminus

Abstract Abstract 1263 The molecular basis for how a Fanconi anemia (FA) genetic background contributes to hematopoietic stem cell defects and hypoplastic organ development remains poorly understood. Protein modification by ubiquitination is a mechanism that diversifies the function and regulation of proteins. In light of this, we focus on the dysfunction of FANCL, the E3 ubiquitin ligase of the FA pathway, as a key molecular defect in Fanconi anemia. Here we report our studies investigating mechanisms of post-translational regulation of FANCL. We view these mechanisms as potential targets to augment the function of the FA core complex and correct hematopoietic stem cell defects. We provide evidence that FANCL is exquisitely regulated by ubiquitin-proteosome degradation. Ligase-inactive mutants (FANCL-C307A and -W341G) are less sensitive to this regulation, suggesting a role for auto-ubiquitination in directing lysine-48 polyubiquitination. This constitutive negative regulation of FANCL is partially reversed with an ATP-competitive glycogen synthase kinase-3beta (GSK-3beta) inhibitor. GSK-3beta is a serine/threonine kinase that phosphorylates proteins and marks them for ubiquitin-mediated proteolysis. Mitogenic and survival pathways, including Ras/MAPK and PI3K/Akt, negatively regulate GSK-3beta by serine-9 phosphorylation. We show that the regulation of FANCL by GSK-3beta is likely direct because FANCL and GSK-3beta co-immunoprecipitate in cell lysates and as GST-fusion proteins. To define the biochemical mechanisms of FANCL regulation, we generated N-terminal deletion mutants of FANCL and we show that the regulation of FANCL is dictated by a region at the N-terminus (aa1-78). Mutational analysis of FANCL (lysine to arginine) in this N-terminus region does not affect the overall protein level or ubiquitination of FANCL, suggesting that FANCL may be targeted for degradation by phosphorylation and/or in a complex with other proteins. The potential biological relevance of our findings, that FANCL is regulated by GSK-3beta is revealed in studies overexpressing constitutively active, myristoylated-Akt. This experimental condition increases FANCL protein levels and suggests a role for FANCL as a downstream effector of PI3K/Akt signaling. In turn, FANCL likely regulates non-canonical targets that alter the transcriptome profile favoring self-renewal and survival of hematopoietic stem cells. We recently published our studies identifying beta-catenin as one such downstream target (Blood 2012 Jul 12;120:323). Suppression of FANCL expression severely disrupts Wnt/beta-catenin signaling and expression of downstream Wnt-responsive targets MYC and CCND1. We also identified that GSK3B gene expression is approximately 5-fold higher in Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared to untreated cells or to wildtype cells with or without TNF-alpha. Our current studies show that inhibition of GSK-3beta preserves the number of murine Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared with no GSK-3beta inhibition. Taken together, we have accumulated evidence suggesting that GSK-3beta is a promising molecular target to improve the self-renewal and survival of FA hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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In Vivo Selection of Wild-Type Hematopoietic Stem Cells in a Murine Model of Fanconi Anemia

Blood ◽

10.1182/blood.v94.6.2151.418a22_2151_2158 ◽

1999 ◽

Vol 94 (6) ◽

pp. 2151-2158 ◽

Cited By ~ 1

Author(s):

Kevin P. Battaile ◽

Raynard L. Bateman ◽

Derik Mortimer ◽

Jean Mulcahy ◽

R. Keaney Rathbun ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Wild Type ◽

Hematopoietic Stem ◽

In Vivo Selection ◽

Serial Transplantation

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.

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SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells

Blood ◽

10.1182/blood.2019003782 ◽

2020 ◽

Author(s):

Yusuke Okamoto ◽

Masako Abe ◽

Anfeng Mu ◽

Yasuko Tempaku ◽

Colette B Rogers ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Degradation Process ◽

Chromosome Breakage ◽

Replication Forks ◽

Hematopoietic Stem ◽

Chemotherapeutic Treatment ◽

Underlying Mechanisms ◽

Stalled Replication Forks

Fanconi anemia (FA) is a hereditary disorder caused by mutations in any one of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death upon chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Since SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival upon treatment with ICL inducers. FANCD2-/-SLFN11-/- HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared to FANCD2-/- cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2-/- cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.

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Aldehyde dehydrogenase 2 in aplastic anemia, Fanconi anemia and hematopoietic stem cells

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2016.07.004 ◽

2016 ◽

Vol 119 (1-2) ◽

pp. 28-36 ◽

Cited By ~ 13

Author(s):

Lauren D. Van Wassenhove ◽

Daria Mochly-Rosen ◽

Kenneth I. Weinberg

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Aplastic Anemia ◽

Aldehyde Dehydrogenase ◽

Fanconi Anemia ◽

Aldehyde Dehydrogenase 2 ◽

Hematopoietic Stem

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The PI3K/Akt1 pathway enhances steady-state levels of FANCL

Molecular Biology of the Cell ◽

10.1091/mbc.e13-03-0144 ◽

2013 ◽

Vol 24 (16) ◽

pp. 2582-2592 ◽

Cited By ~ 5

Author(s):

Kim-Hien T. Dao ◽

Michael D. Rotelli ◽

Brieanna R. Brown ◽

Jane E. Yates ◽

Juha Rantala ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Cell Function ◽

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3Β ◽

Self Renewal ◽

Hematopoietic Stem

Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48–linked chains. Evaluation of a series of N-terminal–deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3β is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3β. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function.

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Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer

Anemia ◽

10.1155/2010/947816 ◽

2010 ◽

Vol 2010 ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Ouassila Habi ◽

Johanne Girard ◽

Valérie Bourdages ◽

Marie-Chantal Delisle ◽

Madeleine Carreau

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Ex Vivo ◽

Direct Injection ◽

Negative Effects ◽

Hematopoietic Stem

The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM) failure; thus correction of hematopoietic stem cells (HSCs) through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs ofFancC−/−mice. Using this approach, we show thatFancC−/−HSCs were efficiently corrected. Intrafemoral gene transfer of theFancCgene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

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