ALBERT

Description: Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects and cancer, particularly leukemia and squamous cell carcinomas involving the cervix or head and neck. Although the FANC proteins have been shown to be involved in DNA repair, the pathogenesis of bone marrow failure and other developmental abnormalities is still unclear, but presumably, reflects abnormalities in senescence and apoptosis. The canonical ATM-p53 pathway is a pivotal mediator of the DNA damage response, which may act as a barrier to cancer through cell cycle and apoptosis regulation but has not been fully studied in FA. To address this, we first analyzed the integrity of the ATM-p53 axis in primary fibroblasts derived from FA patients. By immunoblot assays, we found that expression and phosphorylation of ATM and p53 were all up-regulated in two different FANCA-mutant fibroblasts following irradiation (IR) or mitomycin C (MMC) treatment, when compared to identically treated isogenic mutant cells transduced with wild-type FANCA cDNA. We reasoned that these results might be explained by up-regulation of the basal levels of ATM protein in the mutant fibroblasts. By real-time PCR, we confirmed that ATM gene expression was increased in the mutant line in comparison to the gene-corrected control. We also confirmed the up-regulation of both ATM protein and transcription in a FANCA-mutant EBV-immortalized lymphoblastoid cell line in comparison to its gene-corrected control. In order to determine if these changes in ATM gene expression were directly caused by FANCA depletion, we transfected HCT116 cells (known to be p53 wild-type) with two different siRNAs against FANCA. For these experiments, we used a plasmid expression vector (OriGene Technologies, Rockville MD) that drives 29-mer short hairpin RNAs targeting FANCA. In both transient and stable transfection experiments with either siRNA, we found that knockdown of FANCA expression was associated with down-regulation of ATM expression, as assessed by real-time PCR, when compared to cells transfected with an empty vector control. These results have led us to hypothesize that knockdown of FANCA leads to down-regulation of ATM, whereas cells with mutations in FANCA up-regulate ATM, perhaps as a result of chronic genotoxic stress not seen in the knockdown cells. Up-regulation of the ATM-p53 axis, in turn, may contribute to the hypo-proliferative characteristics seen in both primary fibroblasts and hematopoietic stem cells from FA patients.

Description: Abstract 3186 Identification of new key players in erythropoiesis can lead to a better understanding of the etiology of anemia of unknown origin. Mouse models have significantly contributed to our understanding of normal erythropoiesis and the pathogenesis of erythroid disorders. Recently, we identified in the scat (severe combined anemia and thrombocytopenia) mouse model a missense mutation (G125V) in the Rasa3 gene, encoding a Ras GTPase activating protein (GAP). Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. Crisis episodes are characterized by severe anemia and thrombocytopenia while, remarkably, the phenotype reverts to normal in the remission phase. Remissions are transient, however, and 94% of scat/scat mice die by P30 during a second crisis episode. We recently demonstrated a mechanism contributing to crisis episodes. The G125V mutation in Rasa3 is a loss of function mutation causing the protein to be mislocalized to the cytosol in scat reticulocytes. This results in loss of GAP activity and, as a consequence, increased levels of active GTP-bound Ras and a severe block in erythroid differentiation. Morpholino knockdowns of rasa3 in zebrafish result in profound anemia, confirming a conserved and non-redundant role for Rasa3 in vertebrate erythropoiesis. Here, we report that the cell cycle is affected in scat erythroid progenitors and extend studies to human primary erythroid cells. Using propidium iodine and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, × ± SD p

Description: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure and leukemia predisposition, pancreatic exocrine dysfunction, and skeletal abnormalities, manifesting as skeletal dysplasia and osteoporosis. Mutations in SBDS have been shown to cause SDS, but the function of the SBDS gene product is unclear. Accelerated angiogenesis has recently been described in bone marrow cells from SDS patients. To clarify the unknown function of SBDS, we performed experiments analyzing the cellular effects of depleting SBDS by constitutive or inducible RNA interference. The growth of HeLa and NIH3T3 cells constitutively depleted of SBDS was markedly hindered when compared to cells stably transfected with siRNA against an irrelevant control gene. However, the growth abnormality was related to a significantly increased propensity to apoptosis in NIH3T3 more than in HeLa cells. We believe these differences are related to inactivation of p53 in HPV-18 positive HeLa cells, suggesting that depletion of SBDS may partially sensitize cells to p53-mediated apoptosis. As in constitutive knockdown, HeLa cells induced by doxycycline to express siRNA against SBDS exhibited growth inhibition, which was associated with modestly increased levels of apoptosis, suggesting a partial contribution of this process in inducible knockdown cells. By microarray analysis of knockdown cells, we found marked differences in expression of genes in multiple pathways, and we chose to examine a selected subset more closely using quantitative PCR arrays. In constitutive and inducible SBDS-depleted HeLa cell clones, we found 3- to 6-fold elevated mRNA levels of osteoprotegerin (OPG or TNFRSF11B) and vascular endothelial growth factor-A (VEGF-A). We confirmed significant overexpression of both secreted proteins by ELISA from supernatants of SBDS-depleted HeLa cells. Osteoprotegerin and VEGF-A are known to have diverse effects on osteoclast differentiation, angiogenesis, and monocyte/macrophage migration, all processes that may be aberrant in SDS, and we propose that overexpression of these factors may contribute to its pathology.

Description: 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.

Description: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. In addition, short stature and poor skeletal growth are found in a subset of DBA patients, suggesting similar developmental abnormalities in erythropoiesis and osteogenesis in that subset. Furthermore it has been shown recently that osteoblasts secrete erythropoietin, linking the marrow niche to the modulation of erythropoiesis. DBA has been shown in the majority of cases to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, contributes to the erythroid failure of DBA. We studied two DBA genotypes in vitro using murine embryonic stem (ES) cell lines harboring gene trap mutations in ribosomal proteins RPS19 and RPL5, respectively. Both mutants had decreased embryoid body (EB) formation, decreased definitive erythroid colony formation and similar p53-dependent primitive erythroid differentiation defects (see Figure A). Cell cycle analyses were normal in the Rps19 mutant ES cells, but there was a significant G2/M arrest in the Rpl5 mutant ES cells, which was unaffected by p53 knockdown. In addition, the Rpl5 mutant cells had a more pronounced growth defect in culture compared to the Rps19 mutant cells (Figure B). ES cells were differentiated, in vitro, to osteoblasts using established culture conditions, and confirmed both by morphology and molecular characterization (e.g. RUNX2 and Osteopontin). Following 14 days of osteogenic differentiation, bone mineralization was confirmed via Alizarin Red staining. A marked reduction in Alizarin Red staining was seen in the Rpl5 mutant cells while there was only a slight diminution of staining in the Rps19 mutant ES cultures (see Figure C). Therefore the erythroid differentiation defect appears similar in both the Rps19 and Rpl5 mutant ES cells. However the Rpl5 mutant appears to have a more severe phenotype at the ES stage, as evidenced by a pronounced p53-independent G2/M arrest and slower growth rate and subsequently during osteogenic differentiation. These data suggest an explanation for the more severe non-erythroid phenotype seen in a subset of DBA patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2242 Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm. Methods and Results: To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1). Conclusions: Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2237 Background: Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes (IBMFS), characterized by erythroid hypoplasia, congenital anomalies and a cancer predisposition. DBA is caused by ribosomal protein haploinsufficiency, which somehow triggers apoptosis of erythroid precursors, possibly through activation of p53. Some DBA patients show a response to steroid treatment, while others remain transfusion-dependent. While the mechanism of action of steroids in DBA is unclear, recent work has suggested that p53 may antagonize glucocorticoid-induced proliferation of normal erythroid progenitors1. Objective: Our goal was to create a murine embryonic stem (ES) cell model of DBA with a mutation in Rps19 to study the pathophysiology of DBA and to test glucocorticoid responsiveness. Methods: The Rps19-mutated murine embryonic stem cell line, S17-10H1 was created using a gene trap strategy. The ES cells were induced to undergo primary differentiation into embryoid bodies (EBs). Day 9–11 EBs, representing definitive hematopoiesis, were re-plated with hematopoietic cytokines (stem cell factor, interleukin (IL)-3, IL-6 and erythropoietin) in methylcellulose, and secondary differentiated colonies were scored on day 10. S17-10H1 cells were transfected by electroporation with a plasmid vector expressing either wild-type Rps19 cDNA or an empty vector control. Protein and mRNA levels of the tumor suppressor p53 were measured at the ES cell stage. Secondary differentiated hematopoietic colonies were grown with and without glucocorticoids (either dexamethasone at 1nM, 10nM, 100nM, 1μM or hydrocortisone at 50nM, 2.5μM concentration). Results: Western blot analyses confirmed S17-10H1 Rps19-haploinsufficiency. The mutant cell line had reduced EB formation (hematopoietic and non-hematopoietic) following primary differentiation. Significant defects in both erythroid (BFU-E) and myeloid (CFU-GM) formation were found following secondary hematopoietic differentiation of day 9–11 EBs. These defects were specific to Rps19 haploinsufficiency since all defects were rescued by stable transfection of the mutant cell lines with an Rps19-expressing correction vector. However, glucocorticoid treatment was unable to rescue defects in secondary differentiation (hematopoietic colony formation). Finally, we also examined baseline levels of the tumor suppressor p53, which may be induced following abortive ribosome assembly resulting from ribosomal protein haploinsufficiency. Although there was no difference in transcription levels of p53 mRNA between mutant and control cells as determined by quantitative PCR, baseline levels of p53 protein were significantly increased in the mutant ES cells as compared to controls. Conclusions: (1) In our model system, Rps19 mutation was associated with a relatively early defect in hematopoietic progenitors, since both erythroid and myeloid (or mixed) colony formation was diminished. (2) Hematopoietic defects in our murine ES cell model of DBA were rescued by Rps19 gene transfer but not by glucocorticoid therapy. (3) We found significant accumulation of baseline p53 protein (but not mRNA) levels in mutant ES cells, suggesting that Rps19 haploinsufficiency leads to inhibition of the p53-degradation pathway. (4) Taken together, these results suggest that p53 may antagonize glucocorticoid-induced proliferation in our Rps19-mutant ES cell model. Experiments to test this hypothesis are in progress. Reference: Ganguli G, Back J, Sengupta S, Wasylyk B. The p53 tumour suppressor inhibits glucocorticoid-induced proliferation of erythroid progenitors. EMBO Rep. 2002;3:569-574. Disclosures: No relevant conflicts of interest to declare.