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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1181-1181 ◽  
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.

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

Blood ◽  
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.

scholarly journals Rpl22 Deficiency Predisposes Hematopoietic Stem and Progenitor Cells to Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 899-899 ◽  
Author(s):  
Bryan Harris ◽  
Jaqueline Perrigoue ◽  
Rachel M. Kessel ◽  
Shawn Fahl ◽  
Stephen Matthew Sykes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ribosomal Protein ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Mutations and deletions in ribosomal proteins are associated with a group of diseases termed ribosomopathies. Collectively, these diseases are characterized by ineffective hematopoiesis, bone marrow failure, and an increased risk of developing myelodysplastic syndrome (MDS) and subsequently acute myeloid leukemia (AML). This observation highlights the role of dysregulation of this class of proteins in the development and progression of myeloid neoplasms. Analysis of gene expression in CD34+ hematopoietic stem cells (HSC) from 183 MDS patients demonstrated that ribosomal protein L22 (Rpl22) was the most significantly reduced ribosomal protein gene in MDS. Interestingly, we observed that AML patients with lower expression of Rpl22 had a significant reduction in their survival (TCGA cohort, N=200, Log Rank P value <0.05). To assess the mechanism of reduced expression, we developed a FISH probe complementary to the RPL22 locus and assessed for deletion of this locus in an independent set of 104 MDS/AML bone marrow samples. Strikingly, we found that RPL22 deletion was enriched in high-risk MDS and secondary AML cases. We, therefore, sought to investigate whether reduced Rpl22 expression played a causal in leukemogenesis. Using Rpl22-/- mice, we found that Rpl22-deficiency resulted in a constellation of phenotypes resembling MDS. Indeed, Rpl22-deficiency causes a macrocytic reduction in red blood cells, dysplasia in the bone marrow, and an expansion of the early hematopoietic stem and progenitor compartment (HSPC). Since MDS has been described as a disease originating from the stem cell compartment, we next sought to determine if the hematopoietic defects were cell autonomous and resident in Rpl22-/- HSC. Competitive transplantation revealed that Rpl22-/- HSC exhibited pre-leukemic characteristics including effective engraftment, but a failure to give rise to downstream mature blood cell lineages. Importantly, there was a strong myeloid bias in those downstream progeny derived form Rpl22-/- HSC. Because human MDS frequently progresses to AML, we examined the potential for Rpl22-deficient HSC to be transformed upon ectopic expression of the MLL-AF9 oncogenic fusion. Indeed, Rpl22-deficient HSPC exhibited an increased predisposition to transformation both in vitro and in vivo, in MLL-AF9 knockin mice. To determine how Rpl22-deficiency increased the transformation potential of HSC, we performed whole transcriptome analysis on Rpl22-/- HSC. Interestingly, four expression signatures were observed that were consistent with the altered behavior exhibited by Rpl22-/- HSC. Rpl22-deficient HSC exhibited increased expression of: 1) genes associated with stem cell function, consistent with the basal expansion and effective engraftment of Rpl22-/- HSC upon adoptive transfer; 2) markers of the myeloid lineage, providing a potential explanation for the myeloid bias exhibited by Rpl22-/- HSC; 3) cell cycle regulators, consistent with the increased proliferation exhibited by Rpl22-/- HSC; and 4) components of the mitochondrial respiratory chain, a metabolic program on which leukemic stem cell function depends. Together, these data suggest that Rpl22 controls a program of gene expression that regulates the predisposition of HSPC to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1155-1155
Author(s):  
Stefanie Kreutmair ◽  
Rouzanna Istvanffy ◽  
Cathrin Klingeberg ◽  
Christine Dierks ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stress Response ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated. NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days). To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response. To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population. Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mir-99 Regulates Normal and Leukemic Stem Cell Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1469-1469
Author(s):  
Mona Khalaj ◽  
Carolien Woolthuis ◽  
Wenhuo Hu ◽  
Benjamin Heath Durham ◽  
Christopher Y. Park
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Gene Set Enrichment Analysis ◽  
Hematopoietic Stem

Abstract Acute myeloid leukemia (AML) is composed of functionally heterogeneous cells including leukemic stem cells (LSCs), which exhibit the ability to self-renew and propagate disease. It is thought that failure of common chemotherapy regimens is due to insufficient eradication of LSCs. However, the mechanisms that maintain stem cell function in the hematopoietic system are not well understood. MicroRNAs play an important role in the regulation of normal and malignant hematopoietic stem cells. Our studies showed that miR-99, a miRNA highly expressed in AML patient cell populations enriched for LSC activity, is among the most highly expressed miRNAs in hematopoietic stem cells (HSCs), suggesting that miR-99 plays a role in regulating normal HSCs as well as LSCs. To test the role of miR-99 in normal hematopoiesis, we knocked down (KD) miR-99 in mouse HSCs (Lin-cKit+Sca1+CD34-SLAM+), which resulted in ~3 fold reduced methylcellulose colony formation upon secondary plating (P=0.01), as well as accelerated granulopoiesis as demonstrated by increased Gr1+Mac1+ cells 7 days after culture initiation (P<0.01), suggesting that miR-99 functions to suppresses differentiation. Consistent with this model, transplantation assays demonstrated >10-fold reduction in long-term engraftment capacity of miR-99 KD compared to scrambled controls (P=0.0004). In addition, Ki-67/DAPI staining of stably engrafted miR-99 KD hematopoietic stem and progenitor cells (HSPCs) showed increased cell cycling, demonstrating that miR-99 also maintains HSPC quiescence. Gene set enrichment analysis (GSEA) of RNA-sequencing data generated from stably engrafted Lin-Sca-1+c-Kit+ cells revealed that miR-99 KD induces significant depletion of LT-HSC gene signatures (P<0.001) and induction of a late progenitor signature (P<0.001), providing further evidence that miR-99 normally functions to maintain HSPCs in the undifferentiated state. To test whether miR-99 maintains LSCs, we performed miR-99 KD experiments using the MLL-AF9 retroviral mouse model. miR-99 KD resulted in a significant extension in survival in secondary transplants compared to scrambled controls (median 92 days vs. 48 days, P<0.001). Evaluation of the bone marrow at the time of death revealed ~2.5 fold decrease in the frequency of LSCs (P<0.01) and ~2 fold increase in the percentage of cycling LSCs (in SG2M) (P<0.001). Analysis of RNA-seq data from miR-99 KD LSCs revealed induction of a differentiated normal progenitor signature (P<0.001) and depletion of a shared HSC/LSC gene signature (P=0.05). Giemsa staining of peripheral blood showed miR-99 KD also induced a significant increase in the number of differentiated myeloid precursors in the peripheral blood (P<0.001), reminiscent of AML differentiation-inducing agents used in the clinic such as ATRA. Consistent with a role in regulating leukemic blast differentiation, microRNA-Seq data from the 153 AML patients in the TCGA database revealed that miR-99 expression inversely correlated with their French-American-British classifications, with low expression levels associated with M4 and M5 subtypes. Compatible with a role in maintaining LSCs, miR-99 KD in a primary AML sample reduced long-term engraftment upon xenotransplantation into NSG mice, and the engrafting cells displayed increased CD14 expression. Together, these data demonstrate that similar to normal HSPCs, miR-99 maintains LSCs function. As miR-99 functions to maintain both LSCs and HSCs, we asked which miR-99 target genes mediate miR-99 KD phenotypes. To address this question, we performed a shRNA library-based forward genetic screen designed to rescue the reduced HSC function following miR-99 KD. We designed 180 shRNAs against 45 predicted miR-99 targets that we identified as upregulated upon acute miR-99 KD in mouse HSPCs. Among the conserved miR-99 targets, Hoxa1, a member of the Hox family of transcription factors, was among the top hits, with all 4 shRNAs being enriched compared to controls. Ectopic expression of Hoxa1 in MonoMac6 AML cells was sufficient to induce differentiation, a phenotype similar to miR-99 KD. These data indicate that Hoxa1 is an important downstream mediator of miR-99 function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Aberrant rRNA 2'-O-Methylation Causes Bone Marrow Failure and Defective Immune Function

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Daphna Nachmani ◽  
Lourdes M. Mendez ◽  
Pier Paolo Pandolfi
Keyword(s):  
Bone Marrow ◽  
Rna Binding ◽  
Cell Function ◽  
Immune Cell ◽  
Germ Line ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Hematopoietic Stem ◽  
Npm1 Mutation

RNA modifications are emerging as key determinants of development and disease. Understanding the mechanisms regulating their functional impact is crucial to uncovering their relevance for disease pathology. The NPM1 gene is frequently a target of genetic alteration in hematological tumors, particularly of the myeloid lineage. While extensively studied, the mechanisms by which NPM1 exert its impact on HSPCs are still not fully elucidated. Recently, we have identified NPM1 as an essential regulator of rRNA 2'-O-methylation (2'OMe). We found that NPM1, through its RNA-binding activity, binds C/D box small nucleolar RNAs (snoRNAs) in the nucleolus, to regulate rRNA 2'OMe, and thereby controls translation. Additionally, we identified NPM1 germ-line mutations in dyskeratosis congenita patients presenting with BMF. Characterization of these mutations found them as selectively deficient in snoRNA binding, and thus their expression led to reduce 2'OMe levels and aberrant translation in patient cells. To causally link reduced 2'OMe to dyskeratosis congenita manifestation, we generated mice harboring a dyskeratosis congenita germ-line NPM1 mutation. The KI mice phenocopy both hematological and non-hematological DC features, thus casually linking mutated NPM1 and reduced 2'OMe to bone marrow failure. Currently, we are taking advantage of our Npm1-mutated mouse model to explore the role of 2'OMe in mature immune cell function. We find that aberrant 2'OMe in mature macrophages (due to Npm1 mutation) does not affect their maturation, however, it leads to altered functions. Specifically, we find that Npm1-mutated macrophages, with reduced 2'OMe, display reduced production of reactive oxygen species, chemotactic properties and phagocytic capacity. These studies demonstrate a role for Npm1 and 2'OMe in adult immune cells, and reveal the importance of translation regulation in both hematopoietic stem cells and mature macrophage function. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

The Hematopoietic Stem Cell (HSC) Compartment in Tristetraprolin (TTP) Knock-out Mice Is Abnormal Phenotypically but Normal Functionally.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2541-2541
Author(s):  
Ian M Kaplan ◽  
Sebastien Morisot ◽  
Diane Heiser ◽  
Wen-Chih Cheng ◽  
Christina LaDana ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Half Life ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Regulation Of Gene Expression ◽  
Rna Degradation ◽  
Fine Tuning ◽  
Hematopoietic Stem ◽  
Marker Combination

Abstract Abstract 2541 Poster Board II-518 The RNA binding protein TTP binds to AU-rich elements (AREs) located in the 3′UTR of mRNAs and dramatically reduces their half-life through exosome-mediated RNA degradation. TTP provides a conserved mechanism for the temporal and fine-tuning regulation of gene expression. We found that the Lin−Kit+Sca+ (KSL) population, a phenotypic marker combination that enriches for HSCs, in adult (6–8 week old) C57Bl/6 marrow expressed TTP mRNA at a level 25-fold higher than whole bone marrow mononucuclear cells. Furthermore, adult TTP KO mice had twice the number of KSL cells per mouse compared to WT littermates, with no difference in bone marrow cellularity. Thus, we reasoned that TTP might represent an as yet unexplored avenue for HSC regulation. The TTP KO mouse has been described previously* and displays myeloid hyperplasia, cachexia and arthritis. Consistent with the reported phenotype there was a 2-fold increase in the number of phenotypic granulocyte-monocyte progenitors (GMPs) and normal numbers of both common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs) in KO marrow. Within the KSL population of KO marrow, we observed an increase in the Flt3−CD34− fraction, which has been shown to be highly enriched for long-term HSCs (LT-HSCs). However, when we measured the frequency of functional long-term engrafting cells by limiting dilution competitive repopulation transplantation assays, there was no difference between WT and KO marrow. We also found no difference in the in vivo responses of WT and KO HSCs in chimeric transplant recipients to bi-monthly 5-FU injections for 18 weeks. We tested the hypothesis that the increase in the number of KSL cells is due to a lack of regulation of the Sca1 mRNA in KO marrow. In an RNA half-life assay in NIH3T3 cells, TTP had no effect on the half-life of a reporter message carrying the Sca1 3′UTR; whereas the TNFa 3′UTR, a known TTP target, did cause the half-life of the reporter message to decrease in the presence of TTP in our assay. Interestingly, the Lin−CD150+CD48−Kit+Sca+ labeling scheme gave a much more accurate representation of the relative frequency of HSCs in KO vs WT marrow. In studying HSC biology, the ability to prospectively identify alterations in the HSC compartment of genetically and pharmacologically manipulated marrow is of utmost importance. These studies demonstrate that in genetically manipulated marrow, the phenotypic and functional phenotypes are not always in accordance. *Taylor GA and Blackshear PJ et al. Immunity (4) 1996 Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

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

Blood ◽  
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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