TNF-α/Fas-RIP-1–induced cell death signaling separates murine hematopoietic stem cells/progenitors into 2 distinct populations

Blood ◽  
2011 ◽  
Vol 118 (23) ◽  
pp. 6057-6067 ◽  
Author(s):  
Yechen Xiao ◽  
Hongling Li ◽  
Jun Zhang ◽  
Andrew Volk ◽  
Shubin Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Bone Marrow Failure ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Interacting Protein ◽  
Hematopoietic Stem ◽  
Complete Inactivation ◽  
Bone Marrow Failure Syndromes ◽  
Tnf Α

AbstractWe studied the effects of TNF-α and Fas-induced death signaling in hematopoietic stem and progenitor cells (HSPCs) by examining their contributions to the development of bone marrow failure syndromes in Tak1-knockout mice (Tak1−/−). We found that complete inactivation of TNF-α signaling by deleting both of its receptors, 1 and 2 (Tnfr1−/−r2−/−), can prevent the death of 30% to 40% of Tak1−/− HSPCs and partially repress the bone marrow failure phenotype of Tak1−/− mice. Fas deletion can prevent the death of 5% to 10% of Tak1−/− HSPCs but fails to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSPCs, suggesting that Fas might induce death within a subset of TNF-α-sensitive HSPCs. This TNF-α/Fas-induced cell death is a type of receptor-interacting protein-1 (RIP-1)–dependent programmed necrosis called necroptosis, which can be prevented by necrostatin-1, a specific RIP-1 inhibitor. In addition, we found that the remaining Tak1−/− HSPCs died of apoptosis mediated by the caspase-8–dependent extrinsic apoptotic pathway. This apoptosis can be converted into necroptosis by the inhibition of caspase-8 and prevented by inhibiting both caspase-8 and RIP-1 activities. We concluded that HSPCs are heterogeneous populations in response to death signaling stimulation. Tak1 mediates a critical survival signal, which protects against both TNF-α/Fas-RIP-1–dependent necroptosis and TNF-α/Fas-independent apoptosis in HSPCs.

TNF-α/Fas-RIP-1-Induced Cell Death Signaling Separates Hematopoietic Stem Cells/Progenitors Into Two Distinct Populations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 393-393
Author(s):  
Yechen Xiao ◽  
Andrew Volk ◽  
Shubin Zhang ◽  
Wei Wei ◽  
Peter Breslin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Failure ◽  
Inflammatory Factor ◽  
Hematopoietic Stem ◽  
Complete Inactivation ◽  
Tnf Α ◽  
Fas Signaling

Abstract Abstract 393 Tumor necrosis factor-α (TNF-α) and Fas ligand (FasL) have been found to induce a negative regulatory effects on hematopoiesis and have been implicated in the pathogenesis of human bone marrow failure (BMF) syndromes. However, the molecular mechanism by which these factors inhibit hematopoiesis is still not completely known. We previously reported that Tak1-knockout mice (Tak1−/−) develop BMF due to the mass apoptosis of hematopoietic cells, including hematopoietic stem cells and progenitors (HSC/Ps). Taking advantage of this mouse model, we studied the effects of TNF-α and Fas-induced death signaling on HSC/Ps by examining their contributions to the development of BMF syndromes in Tak1−/− mice. To do so, TNF-α and Fas-induced signaling were genetically inactivated in Tak1−/− HSC/Ps in order to examine to what degree both the apoptosis of HSC/Ps and BMF in vivo can be prevented. We found that complete inactivation of TNF-α signaling by the deletion of both Tnfr1 and Tnfr2 (TNF receptors 1 and 2) is able to protect up to 30–40% of Tak1−/− HSC/Ps from apoptosis. In vitro studies suggested that Fas signaling also contributes to less than 10% of Tak−/− HSC/P death. However, since Fas works on the same population of cells as TNF-α, and because TNF-α signaling is dominant in vivo, inactivation of Fas signaling failed to inhibit the apoptosis of HSC/Ps and BM damage in Tak1−/− mice. In addition, inhibition of RIP-1 (Receptor-Interacting Protein-1) activity by the specific inhibitor Nec-1 (Necrostatin-1) but not inhibition of FADD/caspase-8 signaling was able to protect the same percentage of the Tak−/− HSC/Ps from death as complete inactivation of TNF-α signaling did, but was unable to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSC/Ps (Tak1, Tnfr1 and r2 compound mutant). This suggests that TNF-α, acting through RIP-1, induces death in 30 to 40% of HSC/Ps. To investigate the causes of apoptosis in the remainder of cells, we looked for factors which either protect Tak1−/−Tnfr1−/−r2−/− HSC/Ps from death or further induce such death. We found that the expression of major pro-survival genes is significantly down-regulated in Tak1−/− HSC/Ps. The survival of the Tak1−/−Tnfr1−/−r2−/− HSC/Ps can be further improved by transducing the over-expression of dominant negative (DN)-caspase-9, as well as by Bcl-xl. Our studies suggest that there is heterogeneity in BM HSC/Ps. Only a portion of HSC/Ps is responsive to TNFα/Fas-RIP-1-induced cell death, whereas the death of the remaining HSC/Ps is induced by an intrinsic apoptotic mechanism. Tak1 is involved in mediating hematopoietic cytokine- and pro-inflammatory factor-induced survival signaling, protecting against both the TNF-α/Fas-RIP-1-dependent and independent death of HSC/Ps. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ripk-Mediated Necroptosis Induces Inflammation and Bone Marrow Failure in Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1599-1599
Author(s):  
Justine E. Roderick ◽  
Nicole Hermance ◽  
Matija Zelic ◽  
Matthew Simmons ◽  
Apostolos Polykratis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Type I ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Ifn Γ

Abstract TNF-α and IFN-γ overproduction are features associated with human bone marrow failure syndromes such as Fanconi Anemia (FA) and Aplastic Anemia (AA). Cells from these patients are known to be hypersensitive to TNF-α and IFN-γ-induced cell death. The serine threonine kinases RIPK1 and RIPK3 interact to mediate necroptosis induced by TNF-α, type I or II interferons. We demonstrate that a hematopoietic RIPK1 deficiency results in hematopoietic stem and progenitor cell loss and induction of bone marrow failure. The cell death reflects cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, as Vav-iCre Ripk1fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. Hematopoietic failure in these mice is accompanied by increases in serum pro-inflammatory cytokines/chemokines and reduced hematopoietic colony formation in the presence of TNF-α, type I or II interferon. We provide genetic evidence that a RIPK3 deficiency rescues the bone marrow failure and significantly reduces serum cytokine and chemokine levels in Vav-iCre Ripk1fl/fl mice. These data reveal that in the hematopoietic lineage RIPK1 prevents inflammation by suppressing RIPK3 activity and raise the possibility that human bone marrow failure patients may benefit from selective RIPK inhibitors. Disclosures No relevant conflicts of interest to declare.

TNFα-Induced Apoptosis of Hematopoietic Progenitors Is the Major Cause of Bone Marrow Failure Observed In TAK-1 Knockout Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1557-1557
Author(s):  
Yechen Xiao ◽  
Hongling Li ◽  
Peter Breslin ◽  
Shubin Zhang ◽  
Wei Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Knockout Mice ◽  
Bone Marrow Failure ◽  
In Vitro Studies ◽  
Mutant Mice ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Induced Apoptosis

Abstract Abstract 1557 In bone marrow (BM) hematopoietic stem cells/progenitors (HSC/Ps), the apoptotic machinery is tightly controlled by a complex interplay between intrinsic signals and stimuli from the surrounding microenvironment, inducing a dynamically balanced network between pro-apoptotic and anti-apoptotic influences. Disruption of this balance can result in hematopoietic disorders such as various BM failure syndromes. Studies have suggested that tumor necrosis factor-α (TNF-α) and Fas-ligand induce programmed cell death and/or differentiation of HSC/Ps, thus exercising negative regulation over hematopoiesis. However, whether the apoptotic cell death induced by these factors plays a role in BM failure syndromes remains ambiguous. We have reported that transforming growth factor beta-activated kinase-1 (TAK-1) plays an essential role in the survival of HSC/Ps. Mice with TAK-1 deletion in BM hematopoietic cells develop BM failure due to the apoptotic death of mutant HSC/Ps. We have taken advantage of the dramatic phenotypes of this mutant mouse line to closely examine TNF-α and Fas signaling in order to understand which of these is related to the induction of apoptosis of HSC/Ps, and to what degree. To do so, we generated TAK-1/TNFR1a, TAK-1/TNFR1b and TAK-1/Fas compound-mutant mice in order to evaluate which signaling system, when inactivated, permitted the rescue the BM failure defects observed in TAK-1 knockout mice, and the degree to which it did so, using both in vivo and in vitro studies. We found that, as was the case with TAK-1 knockout mice, TAK-1/TNFR1b and TAK-1/Fas compound-mutant mice died within 8 to 10 days after induction of TAK-1 deletion with exactly the same type of BM failure observed in TAK-1 knockout mice. In vitro studies indicated that neither TNFR1b nor Fas deletion was able to protect cells from apoptosis, nor could they rescue the colony-forming ability of TAK-1 mutant HSC/Ps. However, TAK-1/TNFR1a-mutant mice appeared to be healthy one month after induction of TAK1 deletion. By careful analysis, we found that TNFR1a deletion partially rescued the BM failure phenotype of TAK-1 knockout mice. The total numbers of nucleated BM and splenic cells in TAK-1/TNFR1a- mutant mice are approximately 54.7% and 83.8% (respectively) of those of their wild-type littermate controls. These percentages represent significant increases comparing to their littermates with TAK-1 deletion only (7.5% and 17% of WT control). In vitro studies demonstrated that TNFR1a deletion restored colony-forming ability in 20–30% of TAK1-knockout HSC/Ps. Currently, we are in the process of analyzing the hematopoietic phenotypes of TAK-1/TNFR1a/1b triple-mutant mice in order to determine whether the complete inactivation of TNF-α signaling further reverses the hematopoietic defects seen in TAK1-knockout mice. Our study demonstrated that TNF-α, via its receptor 1a-induced apoptosis, contributes substantially to the loss of HSC/Ps in TAK-1 knockout mice. Our results also suggest that pro-apoptotic signaling other than TNF-α is also involved in the BM failure syndrome observed in TAK-1 mutant mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Secondary CNL after SAA reveals insights in leukemic transformation of bone marrow failure syndromes

2020 ◽  
Vol 4 (21) ◽  
pp. 5540-5546
Author(s):  
Laurent Schmied ◽  
Patricia A. Olofsen ◽  
Pontus Lundberg ◽  
Alexandar Tzankov ◽  
Martina Kleber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Failure ◽  
Driver Mutations ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Proinflammatory Responses ◽  
Stem And Progenitor Cells

Abstract Acquired aplastic anemia and severe congenital neutropenia (SCN) are bone marrow (BM) failure syndromes of different origin, however, they share a common risk for secondary leukemic transformation. Here, we present a patient with severe aplastic anemia (SAA) evolving to secondary chronic neutrophilic leukemia (CNL; SAA-CNL). We show that SAA-CNL shares multiple somatic driver mutations in CSF3R, RUNX1, and EZH2/SUZ12 with cases of SCN that transformed to myelodysplastic syndrome or acute myeloid leukemia (AML). This molecular connection between SAA-CNL and SCN progressing to AML (SCN-AML) prompted us to perform a comparative transcriptome analysis on nonleukemic CD34high hematopoietic stem and progenitor cells, which showed transcriptional profiles that resemble indicative of interferon-driven proinflammatory responses. These findings provide further insights in the mechanisms underlying leukemic transformation in BM failure syndromes.

Investigation of TCR-Vβ CDR3 Spectrotypes in CD4 and CD8 Lymphocytes from Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2827-2827
Author(s):  
Akiko Nakamura ◽  
Tsutomu Shichishima ◽  
Hideyoshi Noji ◽  
Kazuhiko Ikeda ◽  
Yukio Maruyama
Keyword(s):  
Bone Marrow ◽  
Healthy Volunteer ◽  
Bone Marrow Failure ◽  
Interferon Γ ◽  
Relative Increase ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Abnormal Cells ◽  
Ifn Γ ◽  
Cd4 Lymphocytes

Abstract PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome. It is considered that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) and interferon-γ (IFN-γ) contribute to hypoplastic bone marrow of these disorders. In addition, PNH is an acquired clonal disorder of the hematopoietic stem cell. Recently, it has been reported that analysis of T cell-antigen receptor (TCR)-Vβ repertoires, especially TCR-Vβ CDR3 (complementarity- determining region 3) spectrotypes, is an effective tool to study immunologic mechanisms by CTLs in pathophysiology of PNH (Karadimitris et al, Blood, 2000; Kook et al, Blood, 2002; Risitano et al, Blood, 2002). In the present study, we investigated 21 kinds of TCR-Vβ repertoires by flow cytometry in CD4 and CD8 lymphocytes from 5 PNH patients and a healthy volunteer and the TCR-Vβ CDR3 spectrotypes using polymerase chain reaction assay in CD4 and CD8 lymphocytes from 3 of 5 PNH patients and the control. We also quantitated intracellular IFN-γ in CD4 and CD8 lymphocytes from 5 PNH patients and the control according to the method by Sloand et al (Blood, 2002). We found no specific TCR-Vβ repertoires in CD4 and CD8 lymphocytes from PNH patients compared with the control. The TCR-Vβ repertoires with relative increase of CD4 or CD8 lymphocytes (over 10 of ratio of the proportion of each TCR-Vβ repertoire in a PNH patient/the proportion of the same TCR-Vβ repertoire in a healthy volunteer) were 13.6 or 4 and 22 in Case 1, 3 and 11 or 1 in Case 2, 3 and 13.6 or 3 in Case 3, 5.3 and 7.2 or 2, 3, 7, and 18 in Case 4, and 4, 5.2, 13.6, 16, and 23 or 1 and 14 in Case 5, respectively. TCR-Vβ CDR3 spectrotyping showed that in CD4 lymphocytes most CDR3 patterns were chiefly polyclonal, except for one oligoclonal (Case 1) and one monoclonal (Case 3) patterns of TCR-Vβ25; in CD8 lymphocytes most CDR3 consisted of polyclonal, oligoclonal, and/or monoclonal patterns, suggesting the possibility that CD8 lymphocytes recognize much more antigens of abnormal cells, probably including PNH clones, than CD4 lymphocytes. Unfortunately, we found the same patterns as described above in CD8 lymphocytes from the control, although CD4 lymphocytes from the control presented only polyclonal pattern of CDR3. Quantitative analyses of IFN-γ showed that index values of IFN-γ in CD4 and CD8 lymphocytes from PNH patients were higher than those from the control. However, we did not find any significant correlations between the spectrotypes of TCR-Vβ CDR3 and the index values of IFN-γ in PNH patients, suggesting that TCR-Vβ repertoires with monoclonal and oligoclonal CDR3 patterns do not necessarily produce much IFN-γ. In conclusion, our findings suggest that TCR-Vβ CDR3 spectrotyping is more effective tool to resolve some immune mechanisms of pathophysiology in PNH, especially by auto-reactive CTLs.

Immunogenetic Analysis Reveals the Association of INF-γ (+874 A/T) Hypersecretor Genotype in AA and a Low Frequency of KIR-2DL3/C1 Mismatch in Responders to Immunosuppression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1700-1700
Author(s):  
Bianca Serio ◽  
Giridharan Ramsingh ◽  
Ramon Tiu ◽  
Antonio M. Risitano ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Cytotoxic T Cells ◽  
Bone Marrow Failure ◽  
Receptor Gene ◽  
Low Frequency ◽  
Hematopoietic Stem ◽  
Hla Ligand ◽  
Bone Marrow Failure Syndromes ◽  
Immune Mediated ◽  
Self Tolerance ◽  
Tnf Α

Abstract Clinical and laboratory evidence support an immune pathogenesis in most cases of idiopathic aplastic anemia (AA) and closely related disorders such as paroxysmal nocturnal hemoglobinuria (PNH). While external triggers are likely necessary, a complex constellation of immunogenetic factors may determine disease susceptibility. Many immunogenetic factors can influence the quality of immune response and affect the propensity to immune-mediated attack on hematopoietic stem cells in AA. Here we investigated whether KIR and KIR-L (HLA-A) genotype and cytokine/receptor gene variants are over-represented in AA and PNH. We studied a cohort of 77 patients with AA (23 AA, 20 AA/PNH and 34 PNH), 10 with hypocellular MDS and 175 healthy controls. The following SNPs in immunoregulatory genes were analyzed: IL-1α (−889 T/C), IL-2 (−330 T/G +166 G/T), IL-4 (−1098 T/G −590 T/C −33 T/C), IL-1R (−1970 C/T), IL-1Rα (mspa111100 T/C), IL-4RA (+ 190 G/A), IL-1β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, −819 C/T, −592 C/A), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), INF-γ (+874 A/T), TNF-α (−308 G/A, −238 G/A) and immunomodulatory receptor genes including CTLA-4 exon 6 (+49 G/A), FcRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of NK and cytotoxic T cells, we examined the combined impact of KIR/KIR-L genotypes on the risk of AA and PNH syndrome. In AA we found a decreased frequency of inhibitory KIR-2DL3 genes (68% vs. 89%, p=.0002); analysis of the KIR genotype in correlation with the corresponding KIR-L profile, revealed a decreased frequency of stimulatory 2DS1/C2 mismatch resulting in a potentially enhanced cytotoxic activity (14% vs.44%, p=.003). No association was found for most of the SNPs tested. However, when we examined the frequency TGF-β genotypes, increased frequency of GG variant in codon 25 (61% vs. 35% in controls, p=.03), associated with the “high secretor” phenotype, was found in AA. This relationship was also present in hypocellular MDS (82% vs. 32%, p=.007). Additionally, we found a lower incidence of TT genotypes for the IL-1Rα gene (33% vs. 62% p=.02). We confirm that the hypersecretor genotype T/T of INF-γ was over-represented in AA (28% vs. 10% in controls, p=.02). Subgroup analysis revealed that the T/T genotype of IFN-γ (35% vs. 14% p=.01) correlated with presence of a PNH clone. Previously, we have shown the association of HLA-DR15 with responsiveness to immunosuppression. When AA patients were subgrouped according to response to ATG/CsA, therapy refractoriness correlated with the presence of the C2/C2 haplotype (30% vs. 0% p=.02) and inhibitory KIR-2DL3/C1 mismatch (70% vs. 0%, p=.01) which may result in a greater propensity to breach of self-tolerance. In comparison, in the total AA group, C2/C2 haplotype and KIR-2DL3/C1 mismatch were present in 17% vs. 24% and 8% vs. 16% of controls, respectively. An increase in the frequency of 2DL3 and a decrease in 2DS1 mismatch may result in imbalance between cytotoxicity and KIR inhibition. In sum, our findings demonstrate that complex inherited traits involving immunogenetic factors may genetically determine propensity to bone marrow failure syndromes.

Proapoptotic Bid Preserves HSC Function through Restraint of Necroptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 251-251
Author(s):  
Patrice N. Wagner ◽  
Qiong Shi ◽  
Yuri D. Fedoriw ◽  
Sandra S. Zinkel
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Mouse Model ◽  
Caspase 3 ◽  
Bone Marrow Failure ◽  
Marrow Cell ◽  
Caspase 8 ◽  
Complex Ii ◽  
A Cell

Abstract Multicellular organisms remove damaged or superfluous cells through a highly regulated cellular process known as programmed cell death. There are two main forms of programmed cell death, apoptosis and necrosis. Necrosis (necroptosis) previously thought to be an unregulated death pathway was recently found to be highly regulated. The manner by which a cell dies has important implications. In apoptotic cell death, caspases digest the cell to cause implosion in an immunologically silent process. In necroptotic cell death, increased Rip kinase signaling effects rupture of the plasma membrane, cellular explosion, and the activation of an inflammatory response. Death receptors, such as the TNFα receptor, can activate either apoptotic or necroptotic death. The upstream activators and transducers including Caspase-8, Rip1, and Fadd, are common to both forms of cell death. Interestingly, Caspase-8 and c-FlipL, a caspase homolog, were recently shown to inhibit the necrotic pathway during embryonic development through the formation of a catalytically active complex. The BH3-only Bcl-2 family member, Bid is one of the strongest substrates of Caspase-8, placing it at the interface of the apoptotic and necroptotic pathways, and in position to mediate cell death fate. The role of apoptosis in hematopoietic homeostasis has been well characterized. We developed a mouse model of unrestrained necroptosis in order to determine how unrestrained necroptosis impacts hematopoietic homeostasis and bone marrow function. To do this we generated a mouse model in which apoptosis is prevented by the deletion of the pro-apoptotic effectors Bax and Bak. We further deleted the upstream activator Bid (VavBaxBakBid TKO mice). Surprisingly, these mice die of bone marrow failure due to unrestrained necroptotic cell death. TKO bone marrow displays necroptotic cells by electron microscopy, and markedly increased Rip1 expression by immunofluorescence. TKO mice die of bone marrow failure with marked myeloid dysplasia between the age of 3 and 12 months, and a small number develop leukemia, a phenotype that closely resembles MDS. Further analysis revealed expansion and increased BrdU incorporation of the SLAM-HSC population, consistent with increased HSC proliferation in response to death of more mature cells. To assess function of these HSCs, we performed competitive reconstitution assays. TKO bone marrow initially outcompetes WT bone marrow, but the mice eventually succumb to bone marrow failure beginning at 5 months post–transplantation, despite the presence of ~10-15% wild type bone marrow. These results demonstrate that increased necroptotic signaling results in a cell autonomous stem cell defect. In addition, the presence of necroptotic bone marrow also kills normal HSCs in a non cell-autonomous manner, due to a feed-forward inflammatory process. To further characterize how necroptotic cell death is regulated, we developed myeloid progenitor cell lines (MPCs) from the bone marrow of WT, Bid KO, BaxBak DKO, and BaxBakBid TKO mice to facilitate biochemical and mechanistic studies. Our studies demonstrated increased activation (phosphorylation) and markedly increased levels of Rip1 in the pronecrotic complex (Complex II) with Rip3, Caspase-8, and Fadd in our TKO MPCs following LPS treatment. This association of Rip1 with Complex II is abrogated by reintroduction of Bid by retrovirus into TKO MPCs, demonstrating that Bid inhibits Rip1 association with complex II, suggesting that Bid is a key factor that determines cell death fate. Increased bone marrow cell death is well documented in MDS. To determine if necroptosis plays a role in this bone marrow cell death, we evaluated RIP1 and Caspase 3 expression in 17 human MDS samples. Remarkably, we found increased RIP1 expression, but not activated caspase 3 in bone marrow samples from patients with the RCMD, RAEB-1, and RAEB-2 subtype of MDS, but not in 4 control bone marrow samples (normal lymphoma staging marrows). Our study thus demonstrates that increased necroptosis signaling can result in bone marrow failure with dysplasia, and that necroptotic cell death signaling is increased in bone marrow from MDS patients, highlighting the potential importance of this targetable signaling pathway in bone marrow failure disorders such as MDS. Disclosures No relevant conflicts of interest to declare.

Premature Senescence in Hematopoietic Stem and Progenitor Cells in Ribosomopathy-Associated Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 298-298
Author(s):  
Hengjun Chao ◽  
Johnson M. Liu
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Premature Senescence ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Bone Marrow Failure Syndromes ◽  
P53 Activation ◽  
Adult Bone

Abstract Introduction: Aged hematopoietic stem cells (HSCs) are known to functionally decline and are prone to development of myeloid malignancies. Recent work has highlighted the twin roles of replication stress and decreased ribosome biogenesis as drivers for the accumulation of DNA damage and senescence. Certain bone marrow failure syndromes, including Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), and the acquired 5q- syndrome, are characterized by defects in ribosome biogenesis. Furthermore, recent work has suggested a role for p53 activation, through the 5S ribonucleoprotein particle (RNP), in driving cells to senescence following perturbation of ribosome biogenesis. Methods and Results: Here, we have used multiplexing flow cytometry protocols to define, enumerate, and characterize hematopoietic cells of distinct differentiation stages and lineages in 2 DBA cord bloods and 4 adult bone marrows (2 SDS, 1 DBA, and 1 patient with a diminutive somatic deletion of 5q: ages 27, 32, 40, and 30, respectively), as compared with 4 normal cord bloods and 6 normal adult bone marrows. We included a patient with bona fide MDS (diminutive somatic deletion of 5q including RPS14 in a young adult) to compare with the SDS and DBA patients, who do not meet criteria for MDS. Our preliminary results revealed significant defects in the primitive HSC and multipotent progenitor (MPP) compartments in both DBA and SDS. Specifically, we found in DBA and SDS bone marrow and cord blood samples (compared to normal controls): significantly decreased numbers of primitive HSCs (Lin-CD34+CD133+CD38-CD45RA-CD49f+CD90+) and MPPs (Lin-CD34+CD133+CD38-CD45RA-CD49f-CD90-); increased levels of apoptosis and dysregulated proliferation; and G0-1/S cell cycle arrest. We also found significant increases in senescence-associated β-galactosidase staining and G0-1/S cell cycle arrest in Lin-CD34+ and Lin-CD34+CD38-CD133+ subpopulations in all 4 adult patient bone marrows, as compared with normal adult bone marrows processed in identical fashion [see Fig. 1 for representative data from Lin-CD34+CD133+ hematopoietic progenitor cells (HPCs) from one SDS patient]. Foci of the phosphorylated form of the variant histone H2AX (γH2AX) mark DNA damage, and γH2AX staining was similarly increased in comparison to controls (Fig. 1). The mechanism whereby disturbed ribosome biogenesis induces senescence has been suggested as involving 5S RNP-mediated p53 activation. However, our experiments did not demonstrate increased levels of p53 in the SDS patient marrows, as assessed by intracellular staining. Levels of p16, a well known marker of senescence, were markedly increased in the SDS patient samples, when compared to controls. Finally, in the 2 DBA cord bloods analyzed, there was increased senescence-associated β-galactosidase staining but to a lesser degree than in the adult bone marrow samples (as might be expected with temporal progression). Discussion: Taken together, our data suggest that ribosomopathies (which often present in childhood) are disorders of premature senescence. Consequent DNA damage accumulation and decreased repair and compensation may account for the development of MDS and acute myeloid leukemia, disorders seen in young ribosomopathy patients that ordinarily are rare in the general pediatric and young adult population. Disclosures No relevant conflicts of interest to declare.

Utility of Telomere in Diagnosis of Bone Marrow Failure Syndromes

Blood ◽  
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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