Treatment Of Bone Marrow Failure Research Articles

Impaired Efficacy and Durability of Hematopoietic Stem Cell Engraftment Caused By Samd9l-Deficiency within Bone Marrow Niches

Germline mutations in the SAMD9 and SAMD9L (SAMD9/L) genes on chromosome 7q drive both childhood myelodysplastic syndrome (MDS)/secondary acute myeloid leukemia (sAML) associated with monosomy 7, and adult-onset MDS/sAML associated with distinct somatic genetic alterations. The ortholog for SAMD9/L in mice is Samd9l. Gain of Function (GoF) and Loss of Function (LoF) Samd9l alleles predispose to MDS at distinct ages. LoF models show that Samd9l-deficient mice, like humans, are more likely to develop MDS during adulthood, while GoF models result in early onset bone marrow (BM) dysfunction and MDS/sAML. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure (BMF), MDS, and AML arising from germline genetic syndromes. Our previous research demonstrated that deficits in host BM microenvironment (ME) niches contribute to hematopoietic failure and poor HSC engraftment in murine models of many MDS predisposition syndromes associated with BMF. As we have observed clinical cases where poor engraftment impaired outcomes in patients with SAMD9/L mutations, we recently hypothesized, BM niche functions critical to facilitating HSC engraftment may be impaired due to niche-intrinsic deficits caused by SAMD9/L mutations. Herein, we present data testing this hypothesis, demonstrating Samd9l deficiency alters BM ME composition, extracellular matrix (ECM) regulation, and proinflammatory cytokine signaling resulting in impaired donor HSC engraftment after HSCT. We performed primary (1 ry) and secondary (2 ry) HSCT to model impacts of SAMD9/L deficiency on BM niche function during HSCT using WT ( Samd9l+/+), Samd9l+/-, and Samd9l-/- mice as HSCT recipients of healthy WT GFP + donor BM to measure the capacity of Samd9l LoF niches to engraft donor HSCs. To assess changes in niche composition and gene expression during HSCT, flow cytometry, multiplex ELISA, and RNASeq analysis were performed on WT, Samd9+/- and Samd9-/- mice. Compared to WT control niches, Samd9+/- and Samd9-/- niches exhibited impaired efficiency of donor HSC engraftment at 3 weeks post-1 ry HSCT, as measured by competitive 2 ry-HSCT performed with 1 ry recipient BM harvested at this timepoint. Beyond 30 weeks post-2 ry-HSCT in this model, GFP + reconstitution of GR1 + myeloid, RBC's, and platelets was reduced by 25%, 18%, and 35% in 2 ry recipients of Samd9+/-1 ry recipient BM and by 26%, 28%, and 32% in 2 ry recipients of Samd9-/- 1 ry recipient BM. Durability of HSC engraftment by 8 months was even more significantly impaired in Samd9+/- and Samd9-/- niches, with Samd9-/- mice exhibiting low survival by this timepoint. Competitive 2 ry HSCT performed 8 months post-1 ry HSCT showed reduction in trilineage GFP + blood cells out to 24 weeks post 2 ry-HSCT, including reductions in GFP + Gr1 + myeloid cells by 50% and 83% in 2 ry recipients of Samd9l+/-and Samd9l-/-1 ry recipient BM, respectively, compared to control 1 ry recipient BM. Similar reductions in BM long-term HSC and Lin -Sca-1 +cKit + HSC/progenitor cells in 2 ry recipients of Samd9l-/-compared to Samd9l+/+ 1 ryrecipient BM 8 months post 1 ry-HSCT. Following TBI, Samd9l+/-and Samd9l-/- BM niches contained similar percentages of osteoprogenitors, mesenchymal stem cells, and endothelial cells compared to WT controls. Multiplex ELISA on BM plasma collected 48hrs post-TBI revealed that Samd9l+/-and Samd9l-/-deletion in the BM ME led to markedly reduced expression of IFN-γ (43% and 55% reduction in Samd9l+/-and Samd9l-/-), Eotaxin (33%/36%), GM-CSF (47%/47%), and IL-1a (48%/65%). In contrast, expression of niche cell extracts showed markedly increased levels of IL-17 (4 fold) and CXCL11 (1.4 fold) in Samd9l+/-and of IL-17 (5.3 fold) IL-12p70 (1.3 fold), and CXCL11 (1.5 fold) in Samd9l-/-, suggesting that aberrantly active Th17 responses caused by niche Samd9l deficiency may be responsible for impairing HSC function and maintenance post-HSCT. RNASeq analysis showed downregulation of ECM genes in both Samd9l-/-and Samd9l+/-models. Our data demonstrates BM niche function is impaired by Samd9l deficiency in a gene dosage-dependent manner. These deficits are associated with increased expression of a specific inflammatory cytokine pathway, suggesting that aberrant growth factor receptor recycling due to Samd9l deficiency may drive stress hematopoiesis and alter HSC cell fate decisions leading to failure of durable donor HSC maintenance post-HSCT.

Phenotypic HSPC Rescue By RNA Lipid Nanoparticles in a Murine Model of Fanconi Anemia

Background: Fanconi Anemia (FA) is a recessively heritable multisystem disorder that manifests with bone marrow failure in early childhood. Current gene therapy trials for FA rely on transplantation of ex vivo transduced autologous hematopoietic stem cells (HSCs). This approach is limited by poor mobilization efficiency and a depleted HSC pool in FA patients. The recent use of mRNA encapsulated within lipid nanoparticles (LNP mRNA) for cancer immunotherapy and vaccines has created an interest in adapting mRNA delivery for protein replacement therapies. Phase 1/2 trials have demonstrated both safety and efficacy for in vivo treatment of multiple monogenic metabolic disorders. We hypothesized that LNP Fancc could be developed as an in vivo protein replacement therapy for FA. Methods: Custom mRNA transcripts modified with N1-methylpseudouridine and packaged into LNPs (diameter 80 ± 5nm; PDI 0.01), their performance was tested ex vivo and in vivo. LNP mRNA were delivered either intrafemorally or by tail vein injection into Fancc -/- mice, a mouse model of FA or Ai14 mice, a Cre-sensitive reporter mouse model. We also tested LNP performance using ex vivo expanded hematopoietic stem and progenitor cell (HSPC) populations and fresh CD150 +CD48 -Lin -Sca1 +ckit + (long-term hematopoietic stem cells; LT-HSC) cells obtained from wildtype (WT, Fancc +/+) and knockout (KO, Fancc -/-) mice. LNP formulations delivered mRNA transcripts of the fluorescent reporter mCherry (LNP mCherry), cre-recombinase (LNP Cre), luciferase bioluminescent reporter gene (LNP Luc) or a truncated murine FANCC gene (LNP Fancc). Results: To characterize access to the bone marrow compartment in vivo, we delivered LNP mCherry or LNP Cre via systemic (intravenous) and direct (intraosseous) routes to healthy WT and Ai14 mice. Using LNP Cre, we achieve up to 92.6% ± 5% tdTomato reporter expression in Lin -Sca1 +ckit + (LSK) cells 5 days following intraosseous delivery. Consistent with reports by others, we demonstrate lower average transfection rate of 24.8% ± 15.9% fluorescent positivity rates in LSK cells after intraosseous LNP mCherry delivery, and 9.5% ± 6.7% after intravenous delivery. mCherry fluorescent signal was detectable by FACS for up to 72 hours following LNP mCherry injection. We reasoned that meaningful FA HSC correction would require more extended protein expression. Circular mRNA shows improved biostability and durability of expression. Comparing both linear and circular mRNA performance, we observed that circularized LNP Luc is expressed for up to 7 days post ex vivo exposure in LT-HSC populations as compared to 3 days when using linear LNP Luc. Functionally, ex vivo LNP Fancc treatment improves proliferation rates and Mitomycin C (MMC) resistance in colony forming unit (CFU) assays of Fancc -/-HSPC. Here, LNP Fancc treated HSPC demonstrate improved MMC survival rates reaching 37.2% ± 10.5% up from 16.1% ± 8.2%. Ex vivo expanded Fancc -/- HSPC demonstrate an even greater benefit from LNP Fancc treatment, with CFU survival rates increasing to 62.7% from a baseline of 3.3%. Finally, we demonstrate improved repopulation of LNP Fancc treated HSPC 2 weeks after intravenous delivery to myeloablated recipients, measuring up to 73.9% donor chimerism after treatment with circular LNP Fancc compared to 50% among untreated (no LNP) controls. Conclusion: Our studies show that LNP Fancc can rescue Fancc -/- HSPC in vitro, and ongoing experiments reveal improved repopulation in vivo. Along with evidence for efficient in vivo delivery to the bone marrow, our data support the use of LNP mRNA as a potential in vivo treatment of bone marrow failure in FA patients.

Genome Wide siRNA Screen Identifies CGAS-Sting Pathway As a Pharmacological Target That Promotes Survival of Hematopoietic Stem Cells Deficient in Fanconi Genes

Introduction Fanconi anemia (FA) is a homozygous recessive genetic disorder due to mutation in the Fanconi family of genes. The disease is characterized by early onset bone marrow failure and a predisposition to cancer. The FA genes are involved in DNA damage sensing and repair. Mutation in these genes leads to heightened DNA damage and extreme sensitivity to cross-linking agents. Hematopoietic stem cells of FA patients are defective in differentiation and self-renewal, leading to stem cell exhaustion and bone marrow failure early in life. The current treatment for bone marrow failure is allogeneic stem cell transplant from healthy donors. Due to difficulties in finding matching donors and the inherent problems associated with allogeneic transplants, pharmacological therapies that delay or prevent bone marrow failure are of clinical importance. Other proposed therapies including TGF-b, metformin, and resveratrol have met with limited clinical success. Methodology: To identify genes that promote survival of FA cells we performed a genome wide siRNA screen using isogenic pair of cell line with FANCD2 deletions and its corrected counterpart using transfection of siRNA libraries targeting 18,119 genes from Human Drug Targets, Human Druggable Genome, and Human Genome libraries at 2000 cells/well in 384-well plates followed by cell viability measurement after 72 hrs. We measured IRF3 phosphorylation and expression of immune related genes to analyze the CGAS-STING pathway. Using peripheral blood and bone marrow derived CD34+ cells transduced with lentivirus expressing shRNA against FANCD2 we tested the effect of CGAS inhibitor in promoting CFU-GM and CFU-E. Results: A genome wide siRNA screen identified IRF7 as one of the top 5 hits whose diminished expression resulted in increased viability of F-D2 mutant PD20 cells. IRF7 along with IRF3 serve as downstream transcription factors including various Toll-like receptors, CGAS-STING pathways, and other pattern recognition innate immune receptors. Using pIRF3 as an activation readout for CGAS-STING pathway we identified that both FANCD2 and FANCA mutant cells have elevated pIRF3 levels. The elevated pIRF3 levels were further increased upon addition of mitomycin C (MMC) in FA mutant cells as compared to gene corrected cells. The pIRF3 levels decreased upon both genetic KD and small molecule inhibition of both CGAS and STING. In line with increased pIRF3 FA mutant cells showed increased gene expression of various interferon-regulated genes, which were then also reduced by CGAS inhibitor. Overexpression of RNAseH1 partially reduced pIRF3 levels in FA mutant cells indicating that the RNA:DNA hybrids whose levels are elevated in FA mutant cells is one of the ligands of CGAS. Most importantly 10µM of CGAS inhibitor RU.51 was able to rescue FA-A mutant cell line 6914 cells from low dose MMC-induced cell death showing that the sensitivity of FA mutant cells to DNA damaging agents was at least partially driven by CGAS-STING pathway. We then tested if the protective effect of CGAS inhibitor extended to human CD34+ cells deficient in FA genes. 20µM of RU.51 was able to rescue the CFU-GM and CFU-E defects of CD34+ cells carrying lentiviral integrated shRNA against FANCD2 to a level to that in cells expressing non-silencing shRNAs. Interferon-regulated gene expression was also elevated in the FANCD2-deficient CD34+ cells, which was reversed by treatment with cGAS inhibitor. Conclusions: It has previously been shown that FA patients have elevated TNF-alpha, IFN-gamma levels, and NFkbactivation, and interferon alpha administration hastens the onset of bone marrow failure in FA patients. Our results show that FA mutant cells have elevated CGAS-STING signaling resulting in heightened interferon gene expression. Elevated R-loop levels in FA cells act as trigger for the CGAS-STING pathway. CGAS inhibitor is able to rescue low dose MMC-induced cell death in FA mutant fibroblasts and also restores the CFU-GM and CFU-E forming ability of CD34+ cells. These results indicate that CGAS-STING pathway is a pharmacologically tractable target to reverse the hematopoietic stem cell exhaustion seen in FA patients.

Clinical case: allogeneic transplantation of hematopoietic stem cells for Fanconi anemia in the onset of refractory anemia with excess blasts

Fanconi anemia (AF) is a hereditary genetic disease characterized by developmental abnormalities, progressive bone marrow failure, hypersensitivity to alkylating agents, and a tendency to hematological and solid tumors throughout life. The only curative option in the treatment of bone marrow failure in patients with AF is allogeneic hematopoietic stem cell transplantation (allo-HSCT). There are no detailed descriptions of allo-HSCT in patients with AF in the Russian-language literature. On the example of a clinical case with AF at the onset of myelodysplastic syndrome, a choose of method for treating bone marrow failure is presented.

Successful Ex Vivo Telomere Elongation with Exg-001 in a Patient with a Dyskeratosis Congenita

We report a first-in-human gene therapy strategy to extend telomere length in patients with telomere biology disorders. Patients with telomere biology disorders, including dyskeratosis congenita, have extremely short telomeres leading to premature cellular senescence and increased risk of bone marrow failure. Zinc Finger and SCAN Domain Containing 4 (ZSCAN4) is a protein that regulates telomere elongation, enhances genome stability, and is expressed transiently in preimplantation embryos, germ cells during meiosis, embryonic stem cells, and rare adult tissue stem cells. EXG-001 is a non-integrating temperature-sensitive non-transmissible Sendai virus vector encoding for human ZSCAN4. We demonstrated that the transient exposure to ZSCAN4 via EXG-001 extended telomeres of primary fibroblast cells derived from dyskeratosis congenita patients as well as healthy human CD34+ cells. However, we did not know whether EXG-001 can extend short telomeres of CD34+ cells of dyskeratosis congenita patients. We translated this work in a first-in-human clinical trial (NCT04211714), in which autologous CD34+ hematopoietic stem cells (HSC) were collected by pheresis from an adult subject with dyskeratosis congenita over two days after mobilization with plerixafor and filgrastim with IRB approval and consent. 2.8x106 CD34/kg were collected and treated with EXG-001 ex vivo in a functionally closed tubing system using the CliniMACS Prodigy (Miltenyi Biotec). EXG-001 treatment increased telomere length of the CD34+ cells of study subject by 1.24-fold, bringing them into the healthy normal control range of CD34+ cell telomere length (Figure 1A). To address the concern of effects on terminal colony forming unit studies were performed and demonstrated maintenance of normal differentiation potential after treatment (Figure 1B; EXG34217). 0.85x106 CD34/kg autologous treated HSC (EXG34217) were reinfused into the subject in February 2022 without conditioning regimen and further in vivo studies are in progress. This novel approach is genotype and mutation independent and uses modified autologous stem cells in the absence of conditioning regimen or immunosuppression which is highly desirable in this radiation and chemotherapy sensitive population. These data support further investigation and translation of telomere elongation via ZSCAN4 exposure by EXG-001 in patients with telomere biology disorders as a potential therapeutic intervention for the prevention or treatment of bone marrow failure. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prospective Phase I/II Study of Eltrombopag for the Treatment of Bone Marrow Failure in Fanconi Anemia

Fanconi anemia (FA) is an inherited disorder associated with loss of function mutations in gene members of the FA-BRCA pathway involved in interstrand cross-link DNA damage repair during cell division. Progressive bone marrow failure (BMF) is a primary cause of morbidity and mortality in patients with FA. Allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation is the only curative treatment for FA-associated BMF. However, donor availability, graft failure, and FA-specific transplant toxicities remain significant hurdles. Attempts at genetic correction of FA are underway but collection of sufficient numbers of autologous HSPCs is challenging in subjects with advanced BMF. Androgens have been successfully used but side effects often prevent prolonged therapy. The orally bioavailable small molecule mimetic of thrombopoietin, eltrombopag (EPAG), was recently shown to stimulate multipotent long-term repopulating HSPCs in patients with acquired BMF, resulting in persistent trilineage hematopoiesis. EPAG promotes DNA repair (Guenther, Exp Hematol 2019) and maintains HSPCs under inflammatory conditions (Alvarado, Blood 2019), indicating potential relevance in FA. In this study, we investigated whether EPAG may offer a novel therapeutic modality for FA-associated BMF.We have enrolled 10 patients (FANCA, n=9 and FANCC, n=1) to date in a prospective phase I/II study of EPAG in Fanconi anemia (NCT03204188). All subjects received EPAG at an oral daily dose adjusted for age and ethnicity. The primary efficacy endpoint was the proportion of subjects with at least 2-fold increase in marrow cellularity or CD34+ HSPC frequency (marrow response), or clinically significant improvement in peripheral blood (PB) counts at 6 months (blood response). The primary safety endpoint was the global toxicity profile assessed at 6 months using CTCAE criteria. Responders were invited to continue on the extension phase of the study for an additional 3 years.Four of 10 patients have reached the 6-month assessment endpoint, and a pre-specified 3-month interim analysis is available on two additional subjects. A marrow response was observed in all 4 subjects at 6 months. Mean marrow cellularity increased by a factor of 4 (Fig. A/B), and CD34+ HSPC frequency improved 2.5-fold at 6 months relative to pre-treatment values (Fig. C/D). Marrow response criteria were also met in the other two subjects at the 3-month interim assessment. Primary PB response was observed in 2 of 4 patients in one (hemoglobin) or two lineages (hemoglobin and platelets) at 6 months of treatment. Responders continue to receive EPAG at 10 or 25 months with sustained improvements in blood counts and transfusion independence. The subject treated for 25 months recently met response criteria in a third lineage (neutrophils) (Fig. E). Both patients who had a marrow but no PB response are clinically well; one subject (FANCA) underwent an unrelated allogeneic HSPC transplantation one year ago, and the other subject (FANCC) recently entered the extension phase of this study. Unilineage PB response criteria were also met in one of two subjects at the 3-month interim assessment.No drug-related serious adverse events have occurred during EPAG treatment. All patients tolerated the maximum dose of EPAG and no interruption or dose reduction was required for adverse events attributable to EPAG. Patients without transfusional iron overload at study entry developed progressive depletion of iron stores attributable to the known potent iron chelating and mobilizing properties of EPAG. At a median of 3 months after initiating EPAG (range, 1 to 6 months), affected patients initiated daily oral iron supplementation with gradual amelioration of iron store levels. To date, there has been no occurrence of marrow fibrosis, solid malignancies or clonal evolution, defined as abnormalities on standard metaphase analysis of bone marrow or overt clinical transformation to MDS/AML.In sum, treatment with EPAG was associated with increased marrow HSPCs in all subjects and PB responses in a subset of patients with Fanconi anemia. The overall safety profile was favorable. Further follow-up and expansion of the ongoing patient cohort will confirm EPAG's potential as a safe and efficient long-term therapeutic modality for FA-associated BMF. Alternatively, EPAG could also be used safely to boost marrow CD34+ cell numbers prior to autologous gene therapy applications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TINF2 Mutations Are Associated with Poor Outcome Post Hematopoietic Stem Cell Transplantation for Dyskeratosis Congenita

Background. Dyskeratosis congenita (DC) is a telomere biology disorder with a high risk of bone marrow failure, cancer, pulmonary and liver disease. Mutation in multiple telomere related genes including DKC1, TINF2, RTEL1, TERC, TERT, WRAP53, CTC1, NOP10, NHP2 and TPP1 have been reported. Patients with mutations in several DC genes (e.g. heterozygous TINF2, hemizygous DKC1 and biallelic RTEL1 mutations) typically tend to have a particularly serious disease with severe bone marrow failure (SBMF) at a young age, non-hematological manifestations and very short telomeres. Currently, allogeneic hematopoietic stem cell transplantation (HSCT) is the only available curative treatment for bone marrow failure and leukemia in DC patients. Relatively few case series of patients with DC undergoing HSCT have been reported, which generally suggested a poor outcome. The present study aimed to characterize the outcome of HSCT in a Canadian cohort of patients with DC and determine potential relationships between outcome and genotype. Methods. The Canadian Inherited Marrow Failure Registry (CIMFR) is a multicenter registry that includes tertiary centers that care for IBMFS patients across all Canadian provinces. Patients with DC who had been enrolled in CIMFR and underwent HSCT between January 2001 and December 2019 were included. Data were extracted from the CIMFR database. Results. Among 35 patients with DC enrolled in the CIMFR, 11 underwent HSCT. Seven patients were male. Median age at presentation, diagnosis and HSCT was 2.1 years (range: 0 to 9.13s), 5.5 years (range: 1.94 to 35.25), and 7.0 years (range; 0.5-37), respectively. The diagnosis of 3 patients was made after HSCT. Median follow up time from HSCT was 5.89 years (range; 0.2-14.0 years). Among transplanted patients, five had TINF2 mutations, two had RTEL1 mutations, and one patient had DKC1 mutation. Eight patients underwent HSCT for severe bone marrow failure, and three patients for single or multilneage cytopenia. All patients had normal bone marrow karyotype before HSCT. All patients had a full matched donor; two were related and nine were unrelated. Ten patients received reduced-intensity conditioning, and one received myeloablative conditioning. Two patients experienced engraftment failure and underwent a second HSCT. Five years and ten years overall survival after HSCT were 90.9% (95% CI 73.9-100%) and 80% (95% CI 27.2- 97.5%), respectively; however, complications and deaths started appearing thereafter, mainly in patients with TINF2 mutation. All five patients with TINF2 mutation died, and other patients were alive. The causes of death were: 1) pulmonary fibrosis (N=2), 2) gastrointestinal bleeding (N=2), and 3) EBV infection (N=1). Two patients were diagnosed with pulmonary fibrosis after 8 and 11 years from HSCT and died 13.7 and 14 years post-transplant. Two patients had gastrointestinal bleeding after 3.9 years and 4.8 years from HSCT and died 6.6 and 5.7 years post-transplant. Of the patients with GI bleeding, both had hepatic fibrosis and one had pulmonary fibrosis. Summary: In this series, most patients with DC had resolution of the bone marrow failure and relatively good quality of life in the first few years post HSCT. However, longer outcome in the patients with TINF2 mutation was dismal because of DC-related complications, especially pulmonary fibrosis and gastrointestinal bleeding. Effective therapies to prevent these complications are critically needed. Additional reports about HSCT outcome of patients with DC are necessary to characterize HSCT in patients with other genetic groups and to replicate the above results in TINF2 patients. Disclosures No relevant conflicts of interest to declare.

Efficacy of thrombopoietin in bone marrow failure

Thrombopoietin (Thpo) is a hematopoietic cytokine that regulates the production of megakaryocyte/platelet lineage cells and maintains hematopoietic stem and progenitor cells (HSPCs). While Thpo directly stimulates the proliferation of HSPCs, it also maintains HSCs in quiescence to form a reserve pool of HSCs in the bone marrow. Moreover, Thpo activates mitochondria and induces HSC differentiation to megakaryocyte/platelet lineage cells. Being void of instigating anti-Thpo antibody formation in vivo, the use of Thpo receptor agonists (Mpl agonists) transcends the use of recombinant Thpo in the treatment of immune thrombocytopenia. Since its invention, the therapeutic indication of Mpl agonists has extended to the treatment of bone marrow failure in aplastic anemia. As the clinical application of Mpl agonists expands, a detailed investigation of the function and effect of Mpl agonists on physiological HSCs and bone marrow failure is necessary.

Efficacy and Safety of a FLAG-Sequential Regimen Followed By Hematopoietic Stem Cell Transplantation for Myelodysplasia or Acute Leukemia in Fanconi Anemia: A Franco-Brazilian Report

Background: Fanconi anemia (FA) is the most frequent genetic cause of bone marrow failure (BMF) due to a DNA repair mechanism defect. The natural history of FA is marked by progressive BMF during early childhood. Throughout life, the hematopoietic situation may change by clonal evolution toward myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure in FA patients. The role of HSCT for FA patients with AML or advanced MDS is less defined. Currently, HSCT first line result offers 50% Overall Survival (OS) for patients with cytogenetic abnormalities only and 30% OS for patients with advanced MDS or AML in FA (Ayas et al., JCO 2013; Mitchell et al., BJH 2014). We previously reported a FLAG-sequential approach in 6 patients with FA (5 AML and 1 advanced MDS), all alive at a median follow-up of 28 months (Talbot et al., Hematologica 2014). We update here those patients and report 12 more patients treated by FLAG-sequential since then. Materials & Methods: This retrospective study (2006-2019) was conducted in 7 centers in France and Brazil on behalf of the French Reference Center for Aplastic Anemia to evaluate FLAG-sequential in FA patients with morphological clonal evolution (no patients with cytogenetic abnormalities only). The study was conducted in accordance with the Declaration of Helsinki. Anonymous data collection was declared to the appropriate authorities. The FLAG-sequential treatment consisted of FLAG, Fludarabine 30 mg/m²/d for five days and Cytarabine 1 g/m²x2/d with G-CSF for five days, which was followed three weeks later by Cyclophosphamide 10 mg/kg/d for four days, Fludarabine 30 mg/m²/d for four days and TBI 2 Gy (Fig 1A). In a haploidentical setting, Cyclophosphamide at 30 mg/kg/d was performed only in post-transplantation, at Days +4 and +5 (Fig 1B). Results: Eighteen patients were included with 14 AML, 1 acute lymphoblastic leukemia (ALL), and 3 RAEB-2 (Table 1). The median age at the time of HSCT was 22 years (4-37 years). Fifteen patients (83%) were older than 10 years at the time of HSCT. The median follow-up was 31 months (3- 153 months). Eight patients (44%) had complex karyotype. None of the included patients had a history of solid malignancies before HSCT. All patients engrafted. The cumulative incidence of neutrophil engraftment at Day 60 was 94% (95% CI 63-100%) with a median of 18 days (12-343 days). The cumulative incidence of platelet engraftment at Day 60 was 83% (95% CI 50%-96%) with a median of 25 days (17-245 days). The donor chimerism was complete at Day +100 for 15 patients. The three patients without full donor chimerism at Day +100 either had a relapse (n=1) and 2 early deaths before Day+100 from steroid-refractory aGVHD (n=1) or septic shock (n=1). None of the patients received a second HSCT. Non-relapse mortality (NRM) at 3 years was 32% (95% CI 6-58%) (Fig 2). Cumulative incidence of grades II to IV aGVHD was 56% (35% grades III to IV). Cumulative incidence of extensive cGVHD was 16%. Infectious complications during HSCT include the following: CMV (n=8), EBV (n=2), adenovirus (n=4), BK virus (n=7), respiratory syncytial virus (n=1), candidaemias (n=2) and invasive aspergillosis (n=3). Progression free survival (PFS) and OS at 3 years were 53% (95%CI 32-89%) and 53% (95%CI 32-89%), respectively (Fig 2). Cumulative incidence of relapse at 3 years was 13% (95%CI 0-31%) (Fig 2). Seven patients died during the study. Causes of death were relapse (n=2), aGVHD (n=2), cGVHD (n=1), septic shock (n=1), and respiratory syncytial virus associated with invasive aspergillosis (n=1). GVHD-relapse free survival (GRFS) at 3 years was 48% (95%CI 29-78%). One patient had anal epidermoid carcinoma at 4 years after HSCT, which required multiple surgical ablations. Conclusion: With almost 3 years follow-up, which is long enough for our results to be considered robust, we report an OS and PFS of 53%, which compares favorably to historical controls since all of our 18 patients were treated with florid disease at time of HSCT (and not with cytogenetic abnormality only, known to be associated with a better prognosis). Toxicity is still a concern in this particular population of FA patients with notably a high rate of infectious complications. Future well designed prospective clinical trials will refine this sequential strategy, which appears promising in this particular difficult clinical situation. Disclosures Socie: Alexion: Consultancy. Peffault de Latour:Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Transcriptomic and Epigenomic Profiling of Histone Deacetylase Inhibitor Treatment Reveals Distinct Gene Regulation Profiles Leading to Impaired Neutrophil Development

The clinical use of histone deacetylase inhibitors (HDACi) for the treatment of bone marrow failure and hematopoietic malignancies has increased dramatically over the last decades. Nonetheless, their effects on normal myelopoiesis remain poorly evaluated. Here, we treated cord blood derived CD34+ progenitor cells with two chemically distinct HDACi inhibitors MS-275 or SAHA and analyzed their effects on the transcriptome (RNA-seq), epigenome (H3K27ac ChIP-seq) and functional and morphological characteristics during neutrophil development. MS-275 (entinostat) selectively inhibits class I HDACs, with a preference for HDAC1, while SAHA (vorinostat) is a non-selective class I/II HDACi. Treatment with individual HDACi resulted in both overlapping and distinct effects on both transcriptome and epigenome, whereas functional effects were relatively similar. Both HDACi resulted in reduced expansion and increased apoptosis in neutrophil progenitor cells. Morphologically, HDACi disrupted normal neutrophil differentiation what was illustrated by decreased percentages of mature neutrophils. In addition, while SAHA treatment clearly showed a block at the promyelocytic stage, MS-275 treatment was characterized by dysplastic features and skewing towards the monocytic lineage. These effects could be mimicked using shRNA-mediated knockdown of HDAC1. Taken together, our data provide novel insights into the effects of HDAC inhibition on normal hematopoietic cells during neutrophil differentiation. These findings should be taken into account when considering the clinical use of MS-275 and SAHA, and can be potentially utilized to tailor more specific, hematopoietic-directed HDACi in the future.

Results of Allogenic Hematopoietic Stem Cell Transplantation in Fanconi Anemia Caused by Bone Marrow Failure: Single-Regimen, Single-Center Experience of 14 Years.

Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure (BMF) in patients with Fanconi anemia (FA). We retrospectively analyzed the records of patients with FA who underwent HSCT with a radiation-free, reduced-intensity conditioning regimen (fludarabine, cyclophosphamide, and antithymocyte globulin) along with an unmanipulated graft infusion between 2004 and 2018. A total of 44 patients underwent HSCT during the study period. Median age at transplantation was 121 months. Regarding the donor source, 22 transplants (50%) were collected from matched related donors (MRDs), and 22 transplants (50%) were collected from alternative donors (ADs). The median infused CD34+ cell dose was 4.7 × 106/kg (range, 0.8 to 23) in bone marrow or peripheral blood stem cell recipients and 1.2 × 105/kg (range, 1.1 to 3.6) in umbilical cord blood recipients. All but 2 patients achieved primary neutrophil engraftment (95%). In a median follow-up of 36 months (range, 1 to 159), 3-year overall survival was 70.5% in the entire group and 91% in the MRD recipients. Primary causes of death were infections (n = 5), acute grade 3 to 4 graft-versus-host disease (n = 4), and hemorrhagic cystitis (n = 3). All surviving patients have full (n = 29) and acceptable mixed (n = 2) donor chimerism and good clinical status. Our results showed an excellent outcome with unmanipulated grafts using a fludarabine-based, radiation-free preparative regimen for MRD recipients. Even though primary neutrophil engraftment rates were good in AD recipients, intervening complications increased mortality in these patients. In clinics where T cell depletion is not feasible, more effort is warranted to improve outcomes for AD recipients.

A High-Throughput Screening Approach to Identify Therapeutics for the Treatment of Diamond-Blackfan Anaemia

Diamond-Blackfan Anaemia (DBA) is a rare blood cell aplasia that presents clinically at approximately 2-3 months of age and its main characteristic is reduced erythroid precursors in the bone marrow, i.e. anemia. Mutations in different ribosomal protein (RP) genes have been associated with DBA, with mutations in RPS19 accounting for 20-25% of all cases. It has been proposed that RPS19 deficiency causes perturbations in ribosome biogenesis, thus activation of the p53-dependent Nucleolar Surveillance Pathway (NSP). In this context free RPs (predominantly L5 and L11) in a complex with 5S rRNA sequester the E3 ubiquitin ligase murine double minute 2 (MDM2), leading to the accumulation of p53 and subsequent activation of its transcriptional targets mediating cell cycle arrest or apoptosis. In DBA, one of the molecular mechanisms impairing the proliferation and thus reducing the number of erythroid progenitors that can progress to mature red blood cells is an elevation of p53 protein mediating activation of the NSP.In order to identify potential therapeutics that could be repurposed to prevent the activation of NSP in DBA patients, we have screened compound libraries of clinically approved therapeutics to identify pathways implicated in the p53-dependent NSP due to RPS19 deficiency. We quantitated both cell number and the level of p53 expression, identifying compounds that can result in low and high expression of p53, the latter for potential use in cancer therapy. Using an RPS19 depleted A549 cell line as a model system, the screen successfully identified different therapeutic groups. In the DBA context, we were most interested in the compounds that reduced p53 and had no negative effect on cell number. A selection of 22 molecules were re-evaluated in vitro, again using RPS19 depleted A549 cells, through the quantification of p53 protein expression and densitometry analysis. From this, 10candidates were evaluated ex vivofor their effects on proliferation using bone marrow obtained from an inducible Rps19 knockdown (DBA) mouse model.While we are currently testing a number of compounds in vivo using the Rps19 DBA mouse model (as described), one of the compounds tested thus far has demonstrated a partial rescue of the cKit+ population, no changes in erythroid precursors but interestingly a reversal of the defect in the Granulocyte-Monocyte Progenitor (GMP) population. Impairment in lineage progression in the GMP compartment has also been reported to present in bone marrow failure Shwachman-Diamond Syndrome. We are currently evaluating the mechanism by which this drug is rescuing the c-Kit+ and GMP populations in these mice.In summary, our high-throughput screening approach and follow up studies have identified a suite of novel therapies that may be beneficial for repurposing for the treatment of bone marrow failure by increasing hematopoietic progenitor cells. We plan to evaluate this, and potentially other therapies, in a clinical trial with DBA patients. DisclosuresFlygare:LU Holding: Patents & Royalties: Patent.

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.

Allogeneic hematopoietic stem cell transplantation for dyskeratosis congenita

Dyskeratosis congenita is an inherited bone marrow failure syndrome caused by defects in telomere maintenance. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure because of dyskeratosis congenita. The present review summarizes the literature with respect to the diagnosis and treatment of patients with dyskeratosis congenita who received HSCT, and discusses the recent progress in the management of dyskeratosis congenita. The recent systematic review of the literature showed poor long-term outcome, with 10-year survival estimates of only 23% in 109 patients with dyskeratosis congenita who received HSCT. Multivariate analysis identified age greater than 20 years at HSCT, HSCT before 2000, and alternative donor source to be poor prognostic markers. HSCT for dyskeratosis congenita is characterized by a marked decline in long-term survival because of late deaths from pulmonary complications. However, a prospective study using danazol showed promising results in gain in telomere length and hematologic responses. A recent prospective study may support the recommendation that HSCT is not indicated for patients with dyskeratosis congenita; instead, they should receive androgen, particularly danazol, as a first-line therapy. Another option may be routine use of androgen after HSCT for the prophylaxis of pulmonary fibrosis.

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.

Fanconi Anemia Pathway Suppression of R Loops Is a Mechanism of Maintenance of Genomic Stability

Background The FA pathway involvement in homologous recombination and translesion synthesis repair requires binding to DNA by central proteins of the pathway, but interaction with RNA and modulation of genotoxic intermediates such as R loops has not been explored. In this study we explore the incidence of R loops in FA mutant cells. Methods DRIP: duplex RNA hybrid immunoprecipitation-genomic DNA was extracted, sonicated, and subjected to immunoprecipitation using S9.6 DNA:RNA hybrid antibody. Resulting immunoprecipitates were run on agarose gel and stained using ethidium bromide. Bisulfite modification/DNA Sequencing: genomic DNA was extracted and a portion was denatured. After bisulfite modification, coding sequence in GAPDH was amplified by PCR, and the resulting products sequenced. Similarly prepared genomic DNA was prepared, exomic sequencing was performed. Bioinformatic analysis was undertaken on each sample. Immunofluorescence microscopy: cells were cultured in chamber slides, treated or not with mitomycin C, fixed, and stained. Cell culture: mutant and complemented versions of each Results Using anti-RNA:DNA hybrid antibodies, we used a series of assays to test for the presence of R loops in FA mutant cells. Agarose gel electrophoresis demonstrated increased pulldown of RNA:DNA hybrids in FA-D2 and FA-A mutant cells but not FA-J. Similar results were seen in qPCR data designed to detect R loops in multiple sites in G-C rich areas of beta-actin and GAPDH. Immunofluorescence revealed increased signal using the S9.6 antibody in a DNA damage inducible manner in mutant FA-D2 and FA-A cells but not FA-J mutant cells. BRCA2 mutant cells also demonstrated increases in R loops. Exome sequencing revealed that R loops were globally present in far greater amounts in FA-D2 mutant cells. Conclusions RNA:DNA hybrids as represented by R loops are present in greater amounts in FA-D2, FA-A, and BRCA2/FA-D1 mutant cells but not in FA-J mutant cells. Suppression of R loops represents a non-canonical function for the FA pathway in maintenance of genomic stability. Translational potential statement The net cause of genomic instability in FA may be formation of DNA:RNA hybrids, which could be prevented by means of small molecules. Understanding the biology can lead to enhanced treatment of bone marrow failure as well as of FA-associated tumors. Disclosures No relevant conflicts of interest to declare.

Outcome of BONE Marrow Transplantation in Congenital Diskeratosis: Preliminary DATA from the European Group for BONE Marrow Transplantation (EBMT)

▪Hematopoietic Stem cell Transplantation (HSCT) is the definitive treatment for bone marrow failure in diskeratosis congenita (DC). Organ dysfunction (mainly lung and gastrointestinal tract) are often part of the disease and may be a limiting factor for or may affect the final outcome of HSCT. Scarce data or relatively small cohort studies are available in current literature on outcome of HSCT in this disease. We analyzed the outcome of 87 patients diagnosed with DC reported in the data base of the European Society for Blood an Bone Marrow Transplantation (EBMT) who underwent HSCT from 1979 to 2014. In particular we analyzed data on type of HSCT, characteristic of donors, source of cells, incidence of acute and chronic GvHD. Males were 76%. Median age at diagnosis of DC was 5.7 years (0-33 yrs), median age at HSCT was 11.2 years (range 0.56-41 yrs). Median time from diagnosis to HSCT was 19 months (0.62-304 mo). The cell source was bone marrow (BM) in 67%, peripheral blood (PB) in 22% and cord blood (CB) 11%. Twenty six percent of patients were engrafted from a matched related and 53% from a matched unrelated donor. Twenty one percent of subjects were engrafted from a mismatched (both related and unrelated) HSCT. Engraftment was documented in 95% of subjects: 4 % had primary graft failure and 10% lost the graft. Overall 35 patients (40%) died, and 52 were alive (60%) at last follow-up. Causes of death were: infections (43%), multi-organ failure (26%), rejection/oss of graft (14%), GvDH (8.5%) undefined (8.5%). In 32% of cases death was related to transplant. Lung injury was present in 17% of subjects who died. Acute GVDH (mainly grade 2-4) and chronic GvHD occurred in 39% and 30% of cases. Five year OS was 60 %. OS was not significantly different by calendar period (before and after year 2000: 50% vs 67% respectively, p=0.424), by age at transplant (below and above age of 12 years: 64% vs 55% respectively ; p= 0.564) and by source of cells (marrow 60%, cord blood 68%, and peripheral cells 46%; p= 0.687). Conversely, OS in HSCT from mismatched donor was inferior to that of transplants from matched donor (38% vs 65% respectively; p= 0.045). The use of radiotherapy or busulfan (any dose) in the conditioning vs other regimens did not impact on 5 y-OS ( 58% vs 62% respectively; p=0.898). HSCT from HLA matched donor can be considered a treatment option in DC. Transplant related mortality looks rater high. Multi organ failure and lung injury are amongst the main causes of death thus pointing to pre-transplant organ status as important determinant of HSCT outcome. DisclosuresDufour:Pfizer: Consultancy.

Oxymetholone Therapy of Fanconi Anemia Induces Hematopoietic Stem Cell Cycling By Suppressing Osteopontin Transcription

Androgens are widely used for treating Fanconi anemia and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2-/- mice were used to assess the therapeutic efficacy of oxymetholone and its mechanism of action. 18-month old Fancd2-/- mice recapitulated key human Fanconi anemia phenotypes including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic oxymetholone treatment significantly improved these hematological parameters by stimulating the proliferation of hematopoietic stem and progenitor cells. RNAseq analysis implicated down-regulation of osteopontin as an important mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic oxymetholone therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Oxymetholone Therapy of Fanconi Anemia Suppresses Osteopontin Transcription and Induces Hematopoietic Stem Cell Cycling

SummaryAndrogens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2−/− mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2−/− mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.

Susan R. Rose, Mi‐Ok Kim, Leslie Korbee, Kimberly A. Wilson, M. Douglas Ris, Ori Eyal, Rosa Sherafat‐Kazemzadeh, Sureka Bollepalli, Richard Harris, Michael R. Jeng, David A. Williams and Franklin O. Smith. Oxandrolone for the treatment of bone marrow failure in Fanconi anemia. Pediatr Blood Cancer 2014;61:11–19

Susan R. Rose, Mi‐Ok Kim, Leslie Korbee, Kimberly A. Wilson, M. Douglas Ris, Ori Eyal, Rosa Sherafat‐Kazemzadeh, Sureka Bollepalli, Richard Harris, Michael R. Jeng, David A. Williams and Franklin O. Smith. Oxandrolone for the treatment of bone marrow failure in Fanconi anemia. Pediatr Blood Cancer 2014;61:11–19