ALBERT

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: Abstract 1270 Shwachman Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure syndrome mainly characterized by neutropenia, exocrine pancreatic insufficiency and an increased risk of myelodysplastic syndrome and leukemia. The phenotype in patients is variable for unclear reasons, but approximately 90% of patients have biallelic mutations in the SBDS gene. At least one action of the SBDS protein is to couple with the GTPase ELF1 to facilitate release of the eIF6 protein from the 60S ribosome subunit, thus enabling joining of the 60S and 40S ribosome subunits, a function that has prompted many to consider SDS a “ribosomopathy”. We created a cellular model of SDS using TF-1 erythroleukemia cells transduced with lentiviral vectors containing two different shRNAs against SBDS or a scrambled sequence. Clones were grown under puromycin selection and a clone from each shRNA was selected. In each clone, knockdown of SBDS was verified at the protein level by western blot, and expression levels of SBDS were less than 1%. Both clones underwent differentiation to either myeloid or erythroid colonies by culturing in GM-CSF or erythropoietin, respectively. The 2–12 clone had a significant decrease in the number and size of both myeloid and erythroid colonies (see Table) when compared with the scrambled shRNA control. In contrast, the 1–7 clone had the same number of myeloid and erythroid colonies as the control. Previous work by other investigators in SDS yeast models revealed that missense mutations in the anti-association factor, Tif6 suppress the slow growth phenotype of SDS-mutant yeast cells. In exploring the molecular basis for the difference in phenotype observed in our TF-1 cells, we therefore focused on eIF6, the human ortholog of Tif6. The 2–12 clone had similar expression of the eIF6 protein when compared to the scrambled control. However, the 1–7 clone had a significantly decreased amount of eIF6 protein compared to the control. DNA sequencing did not reveal any mutations in the eIF6 gene, and quantitative RT-PCR showed similar levels of eIF6 mRNA transcripts, suggesting that the differences in eIF6 protein levels may be due to post-translational modifications. Pressato and colleagues (Br J Haematol 157:503, 2012) have recently speculated that the relatively benign course of SDS patients with a deletion of chromosome 20q may be due to loss of the eIF6 protein (whose gene is located on 20q). Our findings add to the hypothesis that antagonizing eIF6 may modify or rescue the SDS phenotype, possibly by reducing the requirement of SBDS in giving rise to 60S subunits lacking eIF6. Scramble colonies +/− SE 2–12 colonies +/−SE 1–7 colonies +/− SE Myeloid 131+/−4.4 112+/−3.5 p

Description: Abstract 728 Background: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. The disease has been shown to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, may play a role in the pathogenesis of DBA. Previously, we described murine embryonic stem (ES) cell models of DBA and reported hematopoietic and erythroid defects common to Rps19- and Rpl5-deficient cell lines, as well as a primitive erythropoiesis defect unique to an Rpl5-deficient cell line [Blood 116(21), 877, 2010]. Methods: We studied the effects of p53 knockdown on hematopoiesis in our Rps19- and Rpl5-mutant murine ES cell lines created by gene trap technology. Small interfering RNA (siRNA) targeting p53 was transfected into mutant cell lines at the ES cell stage. A non-targeting siRNA served as a negative control. After 24 hours, cells were plated into methylcellulose medium with fetal bovine serum and stem cell factor (SCF) to generate embryoid bodies (EBs). On day 7, EBs were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 12 for total quantity and hematopoietic percentage. For secondary differentiation into primitive erythroid colonies, day 5 EBs were disrupted, and individual cells were suspended in a methylcellulose medium containing fetal bovine plasma-derived serum and epo. Primitive erythroid colonies were counted on day 7 of culture. Definitive hematopoiesis assays were performed by disruption of day 7 EBs, followed by suspension of cells in methylcellulose medium containing SCF, IL-3, IL-6 and epo. Definitive hematopoietic colonies were counted on day 10. In an independent set of experiments, we created an isogenic pair of wild-type and mutant DBA ES cells by electroporation of another Rps19- mutant line with a plasmid vector expressing wild-type Rps19 cDNA (wild-type) or an empty vector (mutant). Results: By immunoblot assays, we detected an increased amount of p53 protein in our Rps19-and Rpl5- mutant cell lines. Following p53 siRNA transfection, we confirmed 82–95% reduction in p53 expression by quantitative PCR, whereas ES cells transfected with non-targeting siRNA did not alter p53 expression. For both Rps19- and Rpl5- mutants, previously shown to have EB formation defects in comparison to parental controls, p53 knockdown significantly improved EB formation, especially hematopoietic-type EBs, compared to mutants treated with non-targeting siRNA. In addition, p53 knockdown in both mutants reversed the definitive hematopoiesis defect by increasing the ratio of erythroid colony to myeloid colony formation. Furthermore, p53 siRNA transfection of the Rpl5- mutant rescued the primitive erythropoiesis defect previously shown by us. To further explore the mechanistic basis of our findings, we additionally tested the effects of Rpl11 knockdown in our DBA models. The presence of free RPL11 secondary to abortive ribosome assembly has been hypothesized to be responsible for increased p53 in DBA by binding to and inhibiting the p53 inhibitor HDM2 (Mdm2 in mice). Transfection of Rpl11 siRNA into both Rps19- and Rpl5-mutant cell lines at the ES cell stage led to a marked reduction in EB formation, compared to cells transfected with non-targeting siRNA. Finally, we also extended our analysis to an isogenic pair of Rps19- wild-type and mutant cells. In the mutant line, we confirmed a 5–8 fold rescue of EB formation with siRNA targeting p53 when compared to the non-targeting siRNA. In order to clarify the role of two major downstream effectors of p53, siRNA targeting either Bax or p21 was transfected into the mutant cell line. Surprisingly, neither siRNA was able to rescue the EB formation defect of the mutant cells. Conclusions: (1) Knockdown of p53 markedly improves erythroid defects of Rps19- and Rpl5-deficient murine ES cell models of DBA, while inhibition of the upstream target Rpl11 causes significant toxicity to cells already haploinsufficient for Rps19 or Rpl5. (2) Knockdown of either Bax or p21 does not recapitulate knockdown of p53, suggesting that neither plays a significant individual role in downstream signaling from p53 in this model. (3) Further exploration of the p53 pathway may provide insights into the pathogenesis of DBA and identify new targets for therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1343 Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome linked to defects in ribosome synthesis. The heterogeneous array of clinical findings associated with this disease state most commonly includes exocrine pancreas insufficiency, neutropenia, and metaphyseal chondroplasia. Patients also show a predisposition for progression to myelodysplastic syndromes and acute myelogenous leukemia. Mutations in the gene SBDS are known to be responsible for most cases of SDS. Initial studies of the yeast ortholog of SBDS, Sdo1, revealed that this family of proteins is involved in the maturation of 60S subunits. Other studies have suggested that SBDS is a multifunctional protein affecting processes other than ribosome synthesis. Most recently it has been shown that reactive oxygen species are dysregulated in TF-1 erythroleukemic cells depleted of SBDS leading to increased cell death (Pediatr Blood Cancer. 2010 Dec 1;55(6): 1138–44). In an effort to elucidate potential sources of increased reactive oxygen species we investigated mitochondrial function in yeast and human models of SDS. Yeast cells lacking Sdo1 fail to grow on media containing only respiratory carbon sources, indicative of a defect in mitochondrial energy metabolism. Related studies in human TF-1 cells revealed that cells depleted of SBDS exhibit reduced oxygen consumption relative to controls. Given that the largest producer of reactive oxygen species is the mitochondrial electron transport chain, perturbation of respiratory function in cells depleted of SBDS family members could be a potential source of elevated reactive oxygen species. To investigate the potential molecular mechanisms underlying these respiratory deficient phenotypes we carried out a proteomic analysis comparing yeast cells depleted of Sdo1 with controls. Our data reveal that cells lacking Sdo1 overexpress Por1, an ortholog of human VDAC1. VDAC1 is a voltage dependent anion channel of the mitochondrial outer membrane that is thought to be an essential component of the mitochondrial permeability pore. Both over and under expression of VDAC1 have been shown to disrupt mitochondrial function and lead to enhanced apoptosis. Current efforts are focused on possible changes in VDAC1 expression and the role they play in the respiratory deficient phenotype in human SDS models. These studies continue to shed further insight into the molecular mechanisms underlying SDS pathophysiology. Disclosures: No relevant conflicts of interest to declare.