Chaperone-Mediated Autophagy Ensures Hematopoietic Stem Cell Maintenance

Hematopoietic stem cells (HSC) harbor extensive self-renewal capabilities along with multilineage differentiation plasticity to sustain blood production over a lifetime. Maintenance of a fully functional proteome that can rapidly change to adjust to the status of HSC activation is facilitated in part by two major intracellular proteolytic pathways, the ubiquitin proteasome system and the autophagy/lysosomal system. Alterations in HSC proteostasis have been associated with a number of degenerative and malignant diseases, underscoring the importance of elucidating the precise contribution of components of the cellular proteostasis network to HSC maintenance. In this work, we have focused in a highly selective type of autophagy, known as chaperone-mediated autophagy (CMA), whereby individual proteins bearing a unique pentapeptide motif (KFERQ-like) are targeted for degradation in lysosomes upon binding to the heat shock cognate protein of 70 kDa (HSC70). Substrate proteins are directly translocated into the lysosomal lumen through a dedicated multiprotein translocation complex. The main component of this complex is the lysosome-associated membrane protein type 2A (LAMP-2A) that also serves as substrate receptor and is the limiting component of CMA. Using novel mouse models that allow for CMA tracking (KFERQ-Dendra mice) and for selective depletion of LAMP-2A in hematopoietic cells (Vav-iCre:LAMP2Af/f mice), we have investigated the physiological role of CMA in HSCs during steady-state and upon activation and gained novel insights on the consequences of CMA failure in these cells in aging. Our work revealed that the basal CMA activity detected in quiescent HSC under steady-state conditions is significantly stimulated upon HSC activation following 5-fluorouracil (5-FU) in vivo exposure. This upregulation of CMA is necessary to ensure HSCs persistence during activation because, upon serial 5-FU injections, CMA-deficient HSCs had a significantly reduced multilineage reconstitution ability with premature bone marrow failure. We found reduced long-term colony formation of CMA-deficient HSCs in serial colony formation assays and demonstrated that these cells had a significant and progressive disadvantage of repopulating lethally-irradiated congenic recipient mice upon serial bone marrow transplantation. We also found that CMA becomes increasingly important for the maintenance of functional HSCs in aging, since as mice age, CMA-deficient HSCs showed an even greater functional defect compared to age-mated control-derived HSCs. Using comparative transcriptomics and metabolomics on HSCs from control and LAMP-2A-deficient mice, we found evidence for metabolic alterations and dysfunctional redox signaling. We confirmed that CMA-deficient HSCs have reduced rates of glycolysis, lower ATP production and higher reactive oxygen species levels than control cells. Deficient cellular energetics and increased oxidative stress are important consequences of CMA failure in HSC, since supplementation with pyruvate or treatment with anti-oxidant agents (i.e. N-acetyl-cysteine) was sufficient to restore CMA-deficient HSC function, as measured by serial colony formation. Proteomic analysis of CMA-deficient Lin-Scal+c-Kit+ (LSK) cells revealed an overall increase of acetylated and oxidized proteins, including key metabolic enzymes and proteins required for the cellular response to oxidative stress. We propose that, upon CMA failure, the inability of HSC cells to timely turning over these regulatory proteins, favors accumulation of unwanted post-translational modifications that interfere with their normal functioning. Together, our findings suggest that CMA upregulation during HSCs activation is required for the proteome remodeling that facilitates transition from quiescent to activated cells. By assuring timely turnover of selected proteins, CMA sustains the metabolic adaptation required to meet these cells' energetic needs and assures an efficient cellular response to stress. Disclosures No relevant conflicts of interest to declare.

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23 Years of Management: A Retrospective Review of Treatment for Aplastic Anemia.

Blood ◽

10.1182/blood.v114.22.1091.1091 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1091-1091

Author(s):

Connie M Piccone ◽

Marie Boorman Martin ◽

Zung Vu Tran ◽

Kim Smith-Whitley

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Retrospective Review ◽

Pediatric Patients ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Complete Response ◽

Primary Objective ◽

Hematopoietic Stem ◽

Transfusion Independence

Abstract Abstract 1091 Poster Board I-113 Introduction Aplastic anemia (AA) is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. In the past, AA was considered to be a fatal disease; however, current therapies, including bone marrow transplantation or immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CSA), are curative in the majority of patients. IST is effective at restoring hematopoietic stem cell production, but relapse and evolution to myelodysplastic syndromes remain clinical challenges. Additionally, there is no real consensus regarding optimal CSA levels, duration of CSA treatment, or the optimal use of growth factors and their relationship to the development of clonal disease. Objectives The primary objective was to review treatment management for severe AA in pediatric patients in order to elucidate treatment differences and review morbidity and mortality as they relate to treatment variation. Study Design/Methods A retrospective review of pediatric patients treated at the Children's Hospital of Philadelphia for AA (both severe and moderate) over a 23 year period was performed. Results A total of 70 patients with AA were treated at our institution from 1985 to July 2008. Exclusions included: 6 patients who received some type of initial treatment at outside institutions, 4 patients who had missing records, and 2 patients who had a diagnosis of moderate AA. Thus, a total of 58 patient records were included in the analysis. Of the total patients reviewed, 60% were male and 40% were female. 34.5% of patients were African-American, and 57% were diagnosed in 2000 or later. The mean age at diagnosis was 9.5±5.8 years. 52% fell into the category of very severe AA based on published diagnostic criteria, 45% had severe AA, and 2 patients (3%) had moderate AA. 15.5% of patients developed AA in the setting of acute hepatitis. More than half of the patients treated with IST had a complete response (CR). The average time to CR was 15±15 months. Average duration of CSA treatment was 15±13 months and 8.6±10.7 months for growth factor. Two patients (3.5%) died, one from complications unrelated to AA and one from infectious complications post-BMT after initial IST failure. Average time to transfusion independence for all patients was 8±11 months (with a range of 0-54 months). Not surprisingly, the time to transfusion independence was significantly associated with IST failure (p=0.010). Patients who failed treatment had an average time to transfusion independence of 17±16 months as compared to those who were complete responders who had an average time to transfusion independence of 3±3 months. Additionally, there was a significant association between IST failure and CSA levels (p=0.014). Patients who had nontherapeutic CSA levels overall had an increased rate of treatment failure. Of those patients who were nontherapeutic, 56% were noncompliant with CSA administration. There was no significant association between IST failure and bone marrow cellularity (p=0.251). PNH was diagnosed in 5% of patients; there were no patients with evidence of myelodysplastic syndrome (MDS). Two of the 3 patients with PNH failed initial IST. Another 2 patients had evidence of a cytogenetic abnormality (16q deletion), but never progressed to MDS. (Note: averages presented as mean±SD) Conclusions/Methods With current IST regimens, AA is curative in the majority of pediatric patients. IST failure was associated with nonadherence to CSA treatment. For patients with confirmed clonal disease, it is possible that IST failure and the ultimate development of clonal disease are related. Disclosures No relevant conflicts of interest to declare.

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Apoptosis and the DNA Damage Response: The Role of the RB Tumor Suppressor Pathway in Oxidative Stress Responses in the Hematopoietic System.

Blood ◽

10.1182/blood.v114.22.sci-15.sci-15 ◽

2009 ◽

Vol 114 (22) ◽

pp. SCI-15-SCI-15

Author(s):

Kay F. Macleod

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Stem Cell ◽

Tumor Suppressor ◽

Bone Marrow Failure ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Protein Levels ◽

Replicative Stress

Abstract Abstract SCI-15 Exposure to pro-oxidants and defects in repair of oxidative base damage is associated with disease and aging and also contributes to the development of anemia, bone marrow failure and hematopoietic malignancies. Our work examines the role of the RB tumor suppressor pathway in the response of the hematopoietic system to oxidative stress and DNA damage. Evidence from mouse models has identified a role for the Rb protein (pRb) in the regulation of hematopoiesis through cell intrinsic functions in blood cell types but also through effects on the bone marrow microenvironment (Spike et al, 2004; Walkley et al, 2007; Daria et al, 2008). Such models have also demonstrated that pRb is required under stress conditions but not under conditions of steady state hematopoiesis (Spike et al, 2004; Spike et al, 2007; Daria et al, 2008). In particular, pRb was required to modulate the response of the hematopoietic system to replicative stress and hypoxia (Spike et al, 2007; Daria et al, 2008). To explain the mechanisms underlying these unique properties of pRb in hematopoiesis, we hypothesized that pRb protein levels are regulated by oxidative stress, including hypoxia and ROS generated as a consequence of stem cell location in the bone marrow niche or in response to replicative stress induced by agents such as 5-fluorouracil. Notably, hypoxia within the bone marrow niche has been reported to promote stem cell expansion and we postulated that this may be due to reduced pRb protein levels in response to hypoxia. We present evidence that pRb protein levels are regulated in wild-type bone marrow in response to replicative stress and that this in turn modulates expansion of stem cells and myeloid progenitors and also impacts end-stage differentiation in the erythroid lineage. Acetylation of pRb stabilized the protein in an active conformation while de-acetylation de-stabilized the protein and promoted pRb protein turnover and increased progenitor cell proliferation. We will present on-going studies that examine how hypoxia and/or ROS affects hematopoietic stem cell proliferation, self-renewal and differentiation in vivo as a function of pRb protein levels using conditional mouse models. The significance of our findings for bone marrow failure in human patients will be discussed. References Spike, B.T. et al. The Rb tumor suppressor is required for stress erythropoiesis. The EMBO J. 2004: 23, 4319-29. Spike, B.T., Dibling, B.C. & Macleod, K.F. Hypoxic stress underlies defects in erythroblast island formation in the Rb null mouse. Blood 2007; 110, 2173-81. Walkley, C.R., Shea, J.M., Sims, N.A., Purton, L.E. & Orkin, S.H. Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 2007; 129, 1081-95. Daria, D. et al. The retinoblastoma tumor suppressor is a critical intrinsic regulator for hematopoietic stem and progenitor cells under stress. Blood 2008; 111, 1894-902. Funding: The author is grateful to the J.P. McCarthy Foundation, the Aplastic Anemia and MDS International Foundation and the National Heart Lung & Blood Institute (RO1 HL080262) for funding of work in her laboratory relating to oxidative stress, erythropoiesis and hematopoietic diseases. Disclosures No relevant conflicts of interest to declare.

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Alterations In the Bone Marrow Microenvironment Contribute to Oxidative Stress and DNA Damage In Hematopoietic Stem/Progenitors Carrying a Csf3r Truncation Mutation

Blood ◽

10.1182/blood.v116.21.387.387 ◽

2010 ◽

Vol 116 (21) ◽

pp. 387-387

Author(s):

Ghada M Kunter ◽

Jill Woloszynek ◽

Daniel C. Link

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Dna Damage ◽

Single Dose ◽

Bone Marrow Failure ◽

Bone Marrow Microenvironment ◽

Wild Type ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Increased Risk

Abstract Abstract 387 A shared feature of many bone marrow failure syndromes is their propensity to develop myelodysplasia (MDS) or acute myeloid leukemia (AML). The molecular mechanisms that underlie this susceptibility are largely unknown. Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a marked increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), that truncate the carboxy-terminal tail are associated with the development of MDS/AML in SCN. Transgenic mice carrying a ‘knock-in’ mutation of their Csf3r (termed d715 G-CSFR) reproducing a mutation found in a patient with SCN have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We previously reported that the d715 G-CSFR is able to cooperate with the PML-RARƒÑ oncogene to induce AML in mice. Herein, we summarize data supporting the hypothesis that alterations in the bone marrow microenvironment induced by G-CSF contribute to oxidative DNA damage in hematopoietic stem/progenitors cells (HSPCs) and possibly leukemic transformation. We previously showed that G-CSF treatment is associated with a marked loss of osteoblasts in the bone marrow, thereby potentially disrupting the osteoblast stem cell niche (Semerad, Blood 2005). Of note, patients with SCN chronically treated with G-CSF are prone to develop osteopenia, suggesting that osteoblast suppression by G-CSF also may occur in humans. We first asked whether the d715 G-CSFR was able to mediate this response. Wild-type or d715 G-CSFR were treated with G-CSF for 1–7 days and osteoblast activity in the bone marrow measured by expression of CXCL12 and osteocalcin. Consistent with previous reports, a decrease in osteocalcin and CXCL12 was not apparent until after 3 days of G-CSF treatment and reached a maximum after 7 days. Surprisingly, the magnitude of osteoblast suppression was greater in d715 G-CSFR compared with wild-type mice. The fold-decrease in osteocalcin mRNA from baseline in wild-type mice was 147 ± 70.1 versus 1,513 ± 1091 in d715 G-CSFR mice (p < 0.001). Likewise, a greater fold-decrease in CXCL12 mRNA was observed. We next assessed oxidative stress in c-KIT+ Sca+ lineage− (KSL) progenitors after G-CSF treatment. In both wild-type and d715 G-CSFR KSL cells no increase in reactive oxygen species (ROS) was observed at baseline or 12 hours after a single dose of G-CSF. However, after 7 days of G-CSF, a significant increase (3.4 ± 0.1 fold; p = 0.009) in ROS was observed in d715 G-CSFR but not wild-type KSL cells. To determine whether oxidative stress contributed to DNA damage, histone H2AX phosphorylation (pH2AX) was measured by flow cytometry. No increase in pH2AX was observed after short-term (less than 24 hour) G-CSF treatment. However, a modest but significant (1.9 ± 0.1 fold; p = 0.0007) increase in pH2AX was observed in d715 G-CSFR but not wild-type KSL cells after 7 days of G-CSF. To determine whether increased oxidative stress was casually linked to DNA damage, we co-administered the antioxidant N-acetyl cysteine (NAC) during G-CSF treatment. As expected, induction of ROS in KSL cells was markedly suppressed by NAC administration. Importantly, the increase in pH2AX levels in d715 G-CSFR KSL cells induced by G-CSF was completely blocked by NAC administration. Finally, to determine whether alterations in the bone marrow microenvironment, specifically decreased CXCL12 expression, contributed to DNA damage, we treated mice with AMD3100, a specific antagonist of CXCR4 (the major receptor for CXCL12). Treatment of wild-type or d715 G-CSFR mice with a single dose of G-CSF (3 hour time point) or with AMD3100 alone did not induce H2AXp. However, co-administration of AMD3100 with a single dose of G-CSF induced modest but significant H2AXp in d715 G-CSFR KSL cells (5.74 ± 1.06 fold; P<0.001). Collectively, these data suggest a model in which alterations in the bone marrow microenvironment induced by G-CSF may contribute to genetic instability in HSPCs and ultimately leukemic transformation. The mutant CSF3R may contribute to leukemogenesis through both increased ROS production in HSPCs and increased suppression of osteoblasts. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Failure with 13q Deletion: A Distinct Clinicopathological Entity with Immune Pathophysiology,

Blood ◽

10.1182/blood.v118.21.3420.3420 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3420-3420

Author(s):

Kohei Hosokawa ◽

Takamasa Katagiri ◽

Naomi Sugimori ◽

Ken Ishiyama ◽

Yumi Sasaki ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Survival Rates ◽

Snp Array ◽

Normal Karyotype ◽

Genetic Alterations ◽

Fish Analysis ◽

Hematopoietic Stem ◽

Who 2008

Abstract Abstract 3420 Background: Numerical karyotypic abnormalities such as −7/del(7q) and del(13q) are occasionally seen in patients with bone marrow (BM) failure who do not have typical signs of myelodysplasia. The WHO 2008 defined this subset of BM failure as MDS-U because of its likely association with a risk of evolving into leukemia, while the presence of isolated abnormalities including +8, del(20q), and -Y was not considered to be presumptive evidence of MDS. Previous studies showed that BM failure patients with del(13q) responded to immunosuppressive therapy (IST) and had a favorable prognosis (Ishiyama K et al, Br J Haematol; 117: 747. 2002; Sloand, JCO 2010). However, the clinical features of del(13q) BM failure remain unclear due to its low incidence as well as the frequently associated karyotypic abnormalities. Objectives/Methods: To characterize the clinicopathological features of patients with BM failure with del(13q), this study reviewed the clinical data of 1705 BM failure patients (733 with AA, 286 with MDS-RCUD, 149 with RCMD, 60 with MDS-U) whose blood was examined for the presence of glycosylphosphatidyl-inositol anchored protein (GPI-AP)-deficient granulocytes and erythrocytes from May 1999 through July 2010. Genomic DNA was isolated from the peripheral blood cells of 7 patients with 13q- and was subjected to SNP array-based genome-wide analysis for genetic alterations using GeneChip® 250K arrays to identify the gene locus that is commonly deleted as a result of 13q-. Results: The 13q- clone was found in 25 (1.5%) of the 1705 patients. All the 13q- patients were classified as MDS-U, due to the absence of significant dysplasia to fulfill the criteria for MDS defined by the WHO 2008 classification. BM was hypocellular in 17 patients and normocellular in 6. Seventeen patients had a clone with 13q- alone, while the remaining 8 patients had a clone with 13q- and other numerical abnormalities including –Y, +mar in 2, and −20, del(7q), +8, der(1;7) in 1. A significant increase in the percentage of GPI-AP- granulocytes was detected in 366 (50%) of 733 patients with AA and 115 (23%) of 495 patients with MDS. GPI-AP- cells were detected in all (100%) of the 17 patients with 13q- alone. On the other hand, the prevalence of increased GPI-AP− cells in patients with 13q- plus other abnormalities and in those with the normal karyotype was 38% (3/8) and 43% (405/937), respectively. Fifteen patients with 13q- alone were treated with IST (ATG + cyclosporine in 6 and cyclosporine ± anabolic steroid in 9) and all of them achieved either a PR or a CR, while in the patients with 13q- plus other abnormalities, the response rate to IST was 40%. A total of 106 patients with the normal karyotype were treated with ATG+CsA (48) or CsA±AS (58) and the response rates were 73% and 85%, respectively. None of the 17 13q- patients progressed to advanced MDS or AML during the follow-up period of 3 to 108 months (median: 52 months) while 2 of 8 patients with 13q- plus other abnormalities developed AML. The 5-year overall survival rates of the patients with 13q-, those with 13q- plus other abnormalities, and patients with a normal karyotype were 84%, 45%, and 91%, respectively. The percentage of 13q- clones increased in 5 patients, and decreased in 3 patients after successful IST. When GPI-AP- and GPI-AP+ granulocytes were subjected to a FISH analysis using a 13q probe (13q14.3), the 13q- clones were detected only in of GPI-AP+ granulocytes, suggesting that 13q- cells are derived from non-PIG-A mutant HSCs. SNP arrays identified 13q13.3 to 13q14.3 regions in all cases. Conclusions: MDS-U with 13q- is a benign BM failure syndrome characterized by a good response to IST and a markedly high prevalence of GPI-AP cells. Patients with this type of BM failure may be inappropriately treated with hypomethylating agents or hematopoietic stem cell transplantation from unrelated donors, which is associated with high therapy-related mortality. Therefore, del(13q) should be eliminated from the intermediate prognosis group defined by the IPSS, and BM failure with del(13q) should be managed as idiopathic AA. Disclosures: No relevant conflicts of interest to declare.

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Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽

10.1182/blood.v120.21.1224.1224 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1224-1224

Author(s):

Junke Zheng ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Actin Binding ◽

Wild Type ◽

Cell Fates ◽

Hematopoietic Stem ◽

Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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G-CSF Treatment Induces Toll-Like Receptor Signaling and Regulates Hematopoietic Stem Cell Function

Blood ◽

10.1182/blood.v122.21.1181.1181 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1181-1181 ◽

Cited By ~ 1

Author(s):

Laura G. Schuettpelz ◽

Joshua N. Borgerding ◽

Priya Gopalan ◽

Matt Christopher ◽

Molly Romine ◽

...

Keyword(s):

Bone Marrow ◽

Cell Function ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Intestinal Flora ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Tlr Signaling ◽

Hematopoietic Stem

Abstract Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

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Utility of Telomere in Diagnosis of Bone Marrow Failure Syndromes

Blood ◽

10.1182/blood.v126.23.4793.4793 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4793-4793

Author(s):

Hasan Ahmed Abdel-ghaffar ◽

Hosam Zaghloul ◽

Ahmed El-Waseef ◽

Mohamed El-Naggar ◽

Mohamed Mabed ◽

...

Keyword(s):

Bone Marrow ◽

Telomere Length ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Quantitative Polymerase Chain Reaction ◽

Control Group ◽

Hematopoietic Stem ◽

Relative Telomere Length ◽

Bone Marrow Failure Syndromes ◽

Significant Difference

Abstract Background and aim of the work: Bone marrow failure syndromes (BMFS) includes inherited and acquired conditions. Inherited bone marrow failure includes a number of syndromes; with Fanconi anemia (FA) being the most common one of them. Telomeres are eroded with cell division, but in hematopoietic stem cell, maintenance of their length is mediated by telomerase. Short telomeres can result in instability of cell function where diseases occur. Bone Marrow Failure might be developed due to low telomerase activity or short telomeres. Our study is aiming to evaluate the utility of Real Time Quantitative-Polymerase Chain Reaction (RT-qPCR) in measuring the relative telomere length and its significance in diagnosis and prognosis of patients with BMFS. Materials and methods: The study includes 3 groups: A group of congenital BMF (29 patients), a group of acquired BMF (10 patients) and a third control group (15 cases). The relative telomere length is evaluated for them using RT-qPCR. Results: We have found that there is a significant difference in relative telomere length between congenital group and controls (p=0.001), also a significant difference between acquired group and controls (p= 0.029). However, there is no significant difference between congenital and acquired groups (p= 0.479). There is no significant correlation between the telomere length and the overall survival or prognosis of the patients of BMFS. Conclusion: We conclude that the telomere length is significantly altered in patients with BMFS whether being congenital or acquired compared to the control group. Disclosures No relevant conflicts of interest to declare.

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Excelent Option Therapy of BONE Marrow Failure in Fanconi Anemia Patients Withouth Full Match Donnor

Blood ◽

10.1182/blood.v128.22.5075.5075 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5075-5075 ◽

Cited By ~ 1

Author(s):

Lisandro L Ribeiro ◽

Samantha Nichele ◽

marco Antonio Bitencourt ◽

Ricardo Petterle ◽

Gisele Loth ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Hematological Parameters ◽

Severe Neutropenia ◽

Cell Transplant ◽

Variable Degree ◽

Duration Of Treatment ◽

Hematopoietic Stem ◽

Hematological Response

Abstract The main cause of morbidity and mortality of FA pts is bone marrow failure (BMF), which usually arises in the first decade of life and progresses to transfusion dependence and severe neutropenia. Androgen treatment has been recommended for FA pts with BMF for whom there is no acceptable hematopoietic stem cell transplant donor. Oxymetholone and Danazol are frequently used in these pts. We retrospectively analyzed data on 67 FA pts who received oxymetholone or danazol for the treatment of their BMF. The starting dose was approximately 1mg/kg for oxy and 2-4mg/kg for danazol. The hematological parameters at the initiation of treatment were hemoglobin (Hb) < 8 g/dL and/or thrombocytes < 30.000/μl. Patients were diagnosed between 01.2005 and 01.2016. The median age was 10.5 ys (2.9 - 40ys). Gender: 39M/27F. The median duration of treatment was 18m (3m - 95m). Fifty-three patients (79%) showed hematological response and became transfusion independence at a median of 3 months after beginning oxymetholone (2-9m) and 5 months after danazol (4-7m). Two adult pts treated with danazol achieved total hematological response with 2.5mg/kg. Seven pts are stable after tapering and stopping androgen with a median follow up of 4 ys (6m-8.5ys). Fourteen pts did not respond to treatment (21%). Eleven pts received an HSCT and seven are alive and well. Three pts were not transplanted and two are alive but transfusion dependent and one pt died from CNS bleeding. All patients developed variable degree of virilization but it was more evident with oxymetholone therapy. Older age at starting therapy was related to less virilization. Conclusion: This study shows the largest number of FA pts treated with androgen up till now. Androgen is an effective and well-tolerated treatment option for FA pts who develop BMF with 79% of them showing transfusion free after 3-5 months. This response may give us time to search for better donors. Disclosures No relevant conflicts of interest to declare.

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Activation of Non-Canonical Immune Signalling Pathways Drives Marrow Failure in a Murine Model of MDS

Blood ◽

10.1182/blood.v124.21.3246.3246 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3246-3246

Author(s):

Rawa Ibrahim ◽

Joanna Wegrzyn ◽

Linda Ya-Ting Chang ◽

Patricia Umlandt ◽

Jeff Lam ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Signalling Pathway ◽

Gene Set Enrichment Analysis ◽

Mouse Bone Marrow ◽

Bone Marrow Niche ◽

Hematopoietic Stem

Abstract The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

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Total Body Irradiation Causes Residual Bone Marrow Injury by Induction of Persistent Oxidative Stress in Murine Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.3209.3209 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3209-3209

Author(s):

Yong Wang ◽

Lingbo Liu ◽

Senthil Kumar Pazhanisamy ◽

Aimin Meng ◽

Daohong Zhou

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Total Body Irradiation ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Tissue Injury ◽

Hematopoietic Stem ◽

Chronic Oxidative Stress ◽

Total Body

Abstract Abstract 3209 Poster Board III-146 Ionizing radiation (IR) and/or chemotherapy cause not only acute tissue injury but also have late effects including long-term bone marrow (BM) suppression. The induction of residual BM injury is primarily attributable to induction of hematopoietic stem cell (HSC) senescence. However, neither the molecular mechanisms by which IR and/or chemotherapy induce HSC senescence have been clearly defined, nor has an effective treatment been developed to ameliorate the injury, which were investigated in the present study using a total body irradiation (TBI) mouse model. The results showed that exposure of mice to 6.5 Gy TBI induced a persistent increase in reactive oxygen species (ROS) production in HSCs only for up to 8 weeks, primarily via up-regulation of NADPH oxidase 4 (NOX4). This finding provides the foremost direct evidence demonstrating that in vivo exposure to IR causes persistent oxidative stress selectively in a specific population of BM hematopoietic cells (HSCs). The induction of chronic oxidative stress in HSCs was associated with sustained increases in oxidative DNA damage, DNA double strand breaks, inhibition of HSC clonogenic function, and induction of HSC senescence but not apoptosis. Treatment of the irradiated mice with N-acetyl-cysteine (NAC) after TBI significantly attenuated IR-induced inhibition of HSC clonogenic function and reduction of HSC long-term engraftment after transplantation. These findings suggest that selective induction of chronic oxidative stress in HSCs by TBI leads to induction of HSC senescence and residual BM injury and that antioxidant therapy may be used as an effective strategy to mitigate IR- and chemotherapy-induced residual BM injury. Disclosures No relevant conflicts of interest to declare.

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