ALBERT

Description: Purpose: We sought to identify and characterize the functional polymorphism in the CCAAT/enhancer binding protein (C/EBP), epsilon (CEBPE) gene, which is known to harbor a genetic risk modifier for childhood acute lymphoblastic leukemia (ALL). Background: Association between ALL risk and polymorphic variation at the CEBPE gene, a zinc-finger hematopoietic and apoptotic regulator expressed exclusively in myeloid cells, was established in a genome-wide association study and replicated in other populations. The actual functional genetic variation that actually contributes the leukemia risk is not known. The discovery and functional characterization of the functional variant may lead to risk stratification, prevention, and/or therapeutic modalities. Methods: Utilizing California Childhood Leukemia Study genome-wide SNP data from Hispanic subjects, we refine the genetic signal to a specific single nucleotide polymorphism (SNP) within the CEBPE gene promoter (rs2239635). This SNP was identified first using genetic imputation using 1000 Genomes data as a framework, and further validated in additional subjects using Taqman genotyping. Functional work to characterize the impact of the polymorphism was performed using prediction methods (database searches), chromatin immunoprecipitation assays from normal subjects heterozygous for rs2239635, and luciferase expression vectors carrying the SNP. Results: The minor allele of rs2239635 conferred a 1.87-fold increased risk of B-cell ALL (95% CI = 1.51-2.30; P = 6.2x10-9) and a 2.94-fold increased risk of high hyperdiploid B-cell ALL (95% CI = 2.12-4.06; P = 8.2x10-11), but only a 1.51-fold increased risk of non-hyperdiploid B-cell ALL (95% CI = 1.11-2.04; P = 8.8x10-3). Adjusting for rs2239635 attenuated all SNP associations in the region, including the original GWAS allele, strongly suggest that the GWAS signal in CCLS Hispanics is attributable to rs2239635. We also demonstrate, using published functional data and our own chromatin immunoprecipitation assays, that this SNP disrupts a canonical transcription factor binding site for Ikaros (IKZF1), an essential hematopoietic transcription factor. Using expression vectors, we found the polymorphic locus to have transcriptional repressive properties that are diminished in the presence of the ALL risk allele. Additionally, a heritable risk allele within IKZF1 interacts with rs2239635 in a multiplicative fashion (P = 0.02) in case-control association analyses. Discussion: In sum, evidence for a functional CEBPE polymorphism links two hematopoietic developmental transcriptional factors and highlights feedback control between them. The minor, risk-associated variant of CEBPE may alter the ability of IKZF1 to repress CEBPE expression in precursor B-cells, causing lineage confusion and enhanced pre-B cell leukemia risk. This risk primarily impacts risk of pediatric ALL that exhibits the high hyperdiploid phenotype. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Modulation of the DNA methylation landscape during cell differentiation is a well-established phenomenon. The B-cell lineage represents a paradigmatic cellular model to study the dynamic epigenome during cell development and specification because major B-cell maturation stages are well defined and display differential phenotypic and gene expression features. Furthermore, different B-cell subpopulations show different proliferation abilities, microenvironmental influences and life spans, providing a window of opportunity to study the epigenome in the context of multiple processes. Methods: We performed whole-genome bisulfite sequencing (WGBS), high-density methylation microarrays and gene expression profiling of ten purified human B-cell subpopulations spanning the entire differentiation program, ranging from uncommitted progenitors to terminally-differentiated plasma cells. Results: The results of both WGBS and methylation microarrays indicate that B-cell ontogenesis involves an extensive and gradual reconfiguration of the DNA methylome. We uncovered that non-CpG methylation at CpApC trinucleotides is present in progenitor cells and disappears upon B-cell commitment independently of CpG demethylation. CpG methylation, in contrast, changed extensively during the entire B-cell maturation program, with one quarter of all measured CpGs showing dynamic methylation. B-cell enhancers suffered more extensive methylation changes than promoter regions, especially in the early differentiation steps up to the germinal center B-cell (gcBC) stage, and their demethylation seemed to be mediated by binding of lineage-specific transcription factors. Enhancers with dynamic methylation were related to genes involved in a large B-cell network that showed high gene expression variability throughout differentiation. In highly proliferative gcBCs, we observed a shift of dynamic methylation from regulatory towards non-functional elements; gcBCs start to undergo global demethylation of late-replicating heterochromatic regions and methylation of polycomb-repressed regions. This signature becomes particularly extensive in long-lived memory B cells and plasma cells, indicating that these changes start in highly proliferative cells and then accumulate in non-proliferative cells with extended lifespan. Conclusion: Our epigenomic analysis of the B-cell differentiation program extends our knowledge on how the DNA methylome is modulated during cell specification and maturation and offers a resource for researchers in the field, both at global and single gene levels. Disclosures No relevant conflicts of interest to declare.

Description: Thrombopoietin (TPO) and TPO mimetics have been shown to be beneficial in the treatment of thrombocytopenia. Low molecular weight, orally available compounds offer the advantage of convenience and lack of immunogenicity when compared to protein-based drugs. STS-T4 is a novel low-molecular weight (1 mM. These data suggest that further evaluation of safety and efficacy of STS-T4 for the treatment of thrombocytopenia is warranted. In addition, the results imply that CD34+ progenitor cells can differentiate into megakaryocytes expressing high levels of CD41 and CD42b in the absence of a full rhTPO-like proliferative response, which may support further investigation of new therapies for the treatment of thrombocytopenia that provide the benefit of megakaryocyte maturation while avoiding excessive expansion of hematopoietic stem cells.

Description: Abstract 718 Introduction: The presence of donor white blood cells (WBC) in transfused blood products can induce alloimmunization, and reducing or eliminating this response may prove to be of clinical benefit. The use of a pathogen reduction method based on UV light illumination in the presence of riboflavin has been shown to induce changes in WBCs that result in a failure to bind to, or induce proliferation of allogeneic PBMCs in vitro. In addition, a study in rats has shown a reduction in alloimmunization in vivo using this treatment. Transfusion of cells illuminated with UV light at other doses without riboflavin has been shown to induce some degree of tolerance with a reduced antibody response to subsequent allogeneic transfusions. We sought to assess both the degree of alloimmunization in mice given pathogen reduced versus untreated allogeneic platelets, as well as determine if cells from mice given pathogen reduced platelets exhibited signs of tolerance ex vivo. Methods: Peripheral blood was collected from C57Bl/6 and Balb/cJ mice into CPDA-1, and platelet rich plasma (PRP) was prepared by gentle centrifugation. WBCs were isolated from the remainder of the blood and were added back to a portion of the PRP to generate either WBC-enriched or WBC-poor PRP. These products were either left untreated or pathogen reduced using the Mirasol pathogen reduction technology system, which uses a combination of riboflavin and UV illumination. These products were transfused via tail vein injection into Balb/cJ mice. Two weeks after transfusion the treated mice were sacrificed, and peripheral blood and spleens were collected. Serum levels of circulating alloantibodies were measured by flow cytometry. Splenocytes were cultured for 48 hours in the presence or absence of C57Bl/6 splenocytes, and levels of secreted cytokines were measured in culture supernatants using multiplexing techniques. Groups were compared using one-way ANOVA with Tukey's multiple comparison post-test, α=0.05. Results: Mice given allogeneic PRP transfusions had significantly elevated levels of alloantibodies compared with non-transfused control mice, whereas mice given syngeneic PRP or pathogen reduced PRP did not. Mice given either the WBC-enriched PRP or WBC-poor PRP generated alloantibodies, though higher levels of antibodies were observed with WBC-enriched PRP. Levels of IFN-γ, TNF-α, IL-10 and GM-CSF were significantly higher following secondary allogeneic challenge of cells from mice given untreated allogeneic PRP compared with those given no transfusion or syngeneic PRP, but not with those given pathogen reduced PRP. Levels of IL-1β, IL-4, IL-5, IL-6, IL-12(p70), and IL-13 were significantly reduced following secondary allogeneic challenge of cells from mice given pathogen reduced allogeneic PRP compared with those given no transfusion or syngeneic PRP. Conclusions: Treatment of allogeneic PRP with riboflavin and UV light prior to transfusion blocks alloimmunization in mice. Furthermore, secondary cytokine responses to allogeneic cells ex vivo are reduced, in some cases bellow the levels observed in cells from mice without prior exposure, suggesting induction of tolerance. Disclosures: Marschner: CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment. Norris:CaridianBCT Biotechnologies: Consultancy, Research Funding.

Description: Purpose: We sought to define the drivers and functional consequences of DNA methylation changes in childhood pre-B cell leukemia. A comprehensive analysis of such DNA methylation changes will reveal aspects of the etiology and pathogenesis of leukemias, and suggest potential therapeutic modalities. Background: The epigenome is extensively altered in carcinogenesis, but the proximate causes, functional consequences, and overall patterning of DNA methylation changes in the pediatric leukemias is not well defined. Methods: We bisulfite sequenced at single base pair resolution two common pre-B cell leukemia case DNAs and a pre-B cell control (CD19+/CD34+ pre-B cells from normal marrow), and analyzed DNA methylation by high definition microarray in another 227 subjects. RNA expression was available for 82 samples, including the sequenced pre-B cells and leukemias. Results: Epigenetic alteration of B-ALLs occurred in two tracks: de novo methylation of small functional compartments and demethylation of large inter-compartmental backbones. The de novo methylation occurred preferentially at polycomb targets and binding sites for the transcriptional co-repressor CtBP2. DNA methylation of ETV6-AML1 leukemia was overall equivalent to the control, with high hyperdiploid methylation levels being 4.4% lower. Hierarchical clustering revealed four groups, with ETV6-AML1 and hyperdiploid leukemias enriched within two separate groups. DNA methylation deviations were exaggerated in lamina-associated domains, with differences corresponding to these methylation clusters and/or cytogenetic groups. DNA methylation changes were correlated with gene expression changes, and a key polycomb expression signature was reversed when DNA methylation marks were pharmacologically removed in leukemia cell lines. Discussion: While leukemia cells retain the majority of their developmentally determined DNA methylation patterns, key modifications with leukemogenesis revealed both broad patterns and functional targets with some dependence on known characteristics of pre-B cell leukemias. Our data suggested a pivotal role of polycomb and CTBP2 in de novo methylation, which may be traced back to bivalency status of embryonic stem cells. Driven by these potent epigenetic modulations, suppression of polycomb target genes was observed along with disruption of developmental fate and cell cycle and mismatch repair pathways and altered activities of key upstream regulators. An appreciation and validation of the observed changes will lead to new insights into etiology, pathogenesis, and treatments of childhood ALL. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3476 Childhood B-cell acute lymphoblastic leukemias (B-ALL) arise from diverse genetic events. Gene dysregulation through abnormal DNA methylation is also believed to play an important role in the tumorigenesis of B-ALL but has not been thoroughly investigated in a large set yet. We performed a genome-wide methylation analysis for 238 childhood B-ALL patients, including 77 hyperdiploid and 64 t(12;21)(p13;q22) [ETV6/RUNX1–fusion] patients, using the Infinium HD450 Methylation array (Illumina). The methylation profile was compared with that of 24 normal precursor B-cell samples, including pro-B, pre-B-I, pre-B-II and immature B cells, which were obtained from 8 normal fetuses and separated by flow cytometry using a panel of antibodies targeting the CD antigens 3/13/19/33/34/56, and IgM antibodies. A total of 3,641 CpGs in 1,953 genes, including 1,007 polycomb target genes, were differently methylated in B-ALL compared to normal precursor B-cells (at a P value below 1.0×10−4; beta difference〉0.5). The Ingenuity Pathway Analysis System demonstrated that genes involve in the G-protein coupled receptor signaling are significantly demethylated or hypermethylated (P=8.7×10−6), followed by genes in antigen presentation, B-cell development, cyclic adenosine monophosphate (cAMP)-mediated signaling, and calcium-induced T-lymphocyte apoptosis pathways (P=6.1×10−5, 6.1×10−5, 1.0×10−4, and 1.2×10−3, respectively). Gene Set Enrichment Analysis revealed that genes containing motifs for SP1, LEF1, MAZ, TCF3, MLLT7, NFAT, ETS2, MYOD1, FOXA1, and VSX1 molecules were significantly enriched (P0.5). Many of those genes were involved in the molecular mechanism of cancer (P=6.9×10−5), with several genes showing large differences in methylation status between hyperdiploid and ETV6/RUNX1. Heatmap generation and clustering analysis identified a demethlyation cluster and a hypermethylation cluster specific for hyperdiploid, and a demethylation cluster for ETV6/RUNX1. In addition, we discovered evidence of a highly variable CpG cluster, by which each cytogenetics group could be divided into two separate subgroups. Some genes in the cluster are previously shown to have an important role in hematopoiesis. An integrative analysis using the preexisting expression database for B-ALL identified that differences in methylation beta values between hyperdiploid and ETV6/RUNX1 generally have an inverse correlation with differences in expression levels. By filtering both methylation and expression data, we could identify several genes that may potentially attribute to the biological difference between hyperdiploid and ETV6/RUNX1. These data indicate that numerous genes are aberrantly hypermethylated or demethylated in B-ALL, with changes in some genes having a significant impact on the leukemogenesis as well as on the biologic differences in each B-ALL subgroup. Heterogeneity in methylation profile also appears to be present even within a well-established cytogenetics group. Disclosures: No relevant conflicts of interest to declare.

Description: Expression of the CD4 antigen was observed on human fetal liver, fetal bone marrow (BM), and umbilical cord blood progenitors expressing high levels of CD34. Using clonal and liquid-culture assays, CD4+ CD34++ Lin− (lineage = CD3, CD8, CD10, CD14, CD15, CD16, CD19, CD20, and glycophorin A) fetal liver progenitors were found to have a greater proliferative potential than CD4− CD34++ Lin− progenitors, whereas the CD4− fraction was more enriched for erythroid progenitors. Both the CD4+ and the CD4− progenitor subpopulations also gave rise to multilineage engraftment upon transplantation into human fetal bone fragments, supportive of B-lymphoid and myeloid growth, or into human fetal thymic fragments, supportive of T-cell growth, implanted in scid/scid (SCID) mice. However, in SCID-hu mice transplanted with graded doses of donor cells ranging from 2.0 × 102 to 2.0 × 104 cells, BM reconstitution by the CD4+ fraction of CD34++ Lin− cells was more frequent than by the CD4− fraction when low numbers of cells were injected. These functional data strongly suggest that stem cells reside among CD4+ CD34++ Lin− fetal liver cells. This hypothesis was further supported by the observations that CD4+ CD34++ Lin− fetal liver cells were enriched for CDw90+ (Thy-1), CD117+ (kit), CD123+, HLA-DR+, CD7−, CD38−, CD45RA−, CD71−, CD115− (fms), and rhodamine 123dull cells, a phenotypic profile believed to represent fetal stem cells. Furthermore, all CD4+ CD34++ Lin− fetal liver cells also expressed CD13 and CD33.

Description: Key Points Exosomes in blood are proinflammatory and may contribute to transfusion-related immune modulation. Exosomes act via antigen-presenting cells to potentiate T-cell survival and mitogen-induced proliferation.