ALBERT

Description: Erythroid cells and megakaryocytes are derived from a common precursor, the megakaryocyte-erythroid progenitor. Although these 2 closely related hematopoietic cell types share many transcription factors, there are several key differences in their regulatory networks that lead to differential gene expression downstream of the megakaryocyte-erythroid progenitor. With the advent of next-generation sequencing and our ability to precisely define transcription factor chromatin occupancy in vivo on a global scale, we are much closer to understanding how these 2 lineages are specified and in general how transcription factor complexes govern hematopoiesis.

Description: Key Points DNA methylation changes during the development of DS-AMKL occur in sequential waves of opposing losses and gains of methylation. Each wave of DNA methylation abnormalities targets specific gene networks that contribute to distinct biological features of the disease.

Description: Abstract 534 Introduction: There is increasing information about reduced intensity conditioning regimen AlloHSCT (Allogeneic Hematopoietic Stem Cell Transplantation) in children. The safety of this approach is now well established but data regarding efficacy are limited and the role in pediatric cancer has yet to be defined. Materiels and methods: We report results of a French pediatric AlloHSCT protocol with ATG-fludarabine (180 mg/m2) - Busilvex (3.2 at 4.8 mg/kg/d for 2 days) conditioning regimen. Related, unrelated bone marrow (BM) and Peripheral blood stem cell (PBSC) donors and Cord blood units (CB) were allowed. In case of CB a TBI 2 grays, Cyclophosphamide 50 mg/kg, Fludarabine 100 mg/m2 conditioning regimen was recommended. GVH prophylaxis consists in cyclosporine alone. A rapid discontinuation of systemic immunosuppression and re-injecting donor lymphocytes to initiate graft-versus-tumor effect are based on tumor assessment and blood chimerism. Inclusion criteria are children with malignancies that can be potentially cure by allograft but a conventional conditioning regimen being impossible due to toxicity and children with solid tumor or hematological malignancy remaining unresponsive to the reference strategies according to French best practices in pediatrics. Results: From April 2007 to April 2010, 40 RIC AlloHSCT were performed in 10 different French pediatric graft centers: 13 Hodgkin Lymphoma, 7 acute myeloblastic leukaemia, 2 acute lymphoblastic leukaemia, 6 neuroblastoma, 8 rhabdomyosarcoma, 3 desmoplastic tumor and 1 Ewing sarcoma. Median age at transplantation was 15 years and median time from diagnosis to transplant was 18 months. Before transplant, 15 patients are in complete response and 25 patients (14/18 solid tumors) have active disease (11 progressive, 14 partial response). 21 had already received a myeloablative therapy (18 autograft and 3 allograft). Graft source was PBSC in 17 cases (7 related and 10 unrelated), BM in 18 (10 related and 8 unrelated), and 5 CB. The RIC Bu-flu conditioning regimen permits rapid engraftment without major toxicity contrary to the Cy-TBI in CB. 1 patient had primary graft failure: 1 CB and 5 patients experienced secondary graft failure: 3 CB, 1 PBSC and 1 BM. Median time to reach an ANC of 0.5 109/l was 16 days. Median time to reach a platelet count of 20 × 109/l was 2 days. Platelet count did not decrease below 20 109/l in 10 allografts. At day 30 post-transplant, chimerism is mainly donor for 30 and partial for 6 children. At day 100 post transplant, 4 out of 6 with initial mixed chimerism were converted into full donor chimerism. 8 patients received DLI and 17 patients experienced acute graft versus host disease (GvH) (2 grade IV and 15 grade ≤ II). A low day 365 TRM of 5% is reported in these heavily pre-treated patients. With a median follow-up of 15 months, the estimated 2 yr overall survival (OS) was respectively 57 % (71% for hematological malignancies and 42% solid tumors) (fig 1) and event free survival (EFS) 36% (50% for hematological malignancies and 19% solid tumors). Univariate analysis of EFS and OS showed no effect of related versus unrelated stem cell sources and BM versus PBSC. Our analysis identified a group of patients, who had no measurable disease at transplant, with a 2 yr OS and EFS of 86%. In term of efficacy, we observe a graft versus lymphoma effect in patients with advanced active Hodgkin lymphoma. Concerning solid tumors, all children included had a very bad prognosis and detectable disease before transplant. Our results may suggest that an immune-mediated effect cannot be excluded in some refractory solid pediatric tumors particularly in neuroblastoma. The main cause of failure of this approach is disease progression. Immunologic approaches after transplantation may help cure more of these very-high-risk patients. Conclusion: Even if further follow up is needed, this prospective study suggest that RIC regimen provides promising outcome in children previously not eligible for myeloablative AlloHSCT. This study “RICE” was registered at www.clinicaltrials.gov as NCT 007 50 126 Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2605 GATA family transcription factors play critical roles in various mammalian developmental processes, including hematopoiesis. In particular, GATA-1 expression is necessary for proper terminal differentiation of mast cells, red blood cells, eosinophils, and megakaryocytes. GATA-2 is required for proliferation and survival of hematopoietic stem and progenitor cells, and is also expressed in erythroid precursors, mast cells, and early megakaryocytes. In developing erythrocytes, GATA-2 and GATA-1 are responsible for temporal control of a multi-factor transcriptional regulatory network that involves (a) GATA-2 positively regulating its own gene transcription, (b) GATA-2 positively regulating the expression of the Gata1 gene, (c) GATA-1 positively regulating its own gene transcription, and (d) GATA-1 negatively regulating Gata2 gene transcription. During this sequence of events, a “GATA switch” occurs, wherein GATA-1 replaces GATA-2 at canonical GATA binding sites within the regulatory regions of the Gata2 and Gata1 genes, as well as at many other genomic loci that encode genes responsible for proliferation or differentiation of erythroid progenitors. Similarly, in early megakaryocytic progenitors, GATA-2 promotes proliferation and suppresses expression of alternative-lineage genes; subsequent activation of GATA-1 precipitates terminal differentiation with concomitant downregulation of proliferative genes and activation of megakaryocyte-specific genes. The presence or role of a GATA switch in megakaryocytes has not yet been formally investigated. To address the role of the GATA switch in megakaryocytic differentiation, we performed massively parallel sequencing of chromatin immunoprecipitation (ChIP-Seq) material for GATA-2 and GATA-1 before or after GATA-1 restoration in the GATA1-null megakaryocytic progenitor cell line, G1ME. We obtained 22 million unique GATA-2 tags and 10 million unique GATA-1 tags and identified 14985 and 5102 high-confidence GATA-2 and GATA-1 binding sites, respectively. Additionally, we used 13 million tags from ChIP for H3K4me3 to identify 24909 genomic sites enriched for the presence of trimethylated lysine-4 on histone H3. Trimethylated H3K4 marks nearly half of all GATA-1 bound sites and one-third of GATA-2 bound sites. Over 40% of the sites bound by GATA-1 in differentiating G1ME cells were also bound by GATA-2 in proliferating G1ME cells, indicating that a GATA switch does indeed occur during megakaryocyte development. Coordinated analyses of these occupancy data with previously published gene expression datasets show that the lists of bound genes are significantly enriched for differentially expressed genes and the data depict a generally antagonistic relationship between GATA-2 and GATA-1. Interestingly, we find that even among genes that don't contain GATA switch sites, greater than 40% of those bound by GATA-1 were also occupied by GATA-2 at distinct sites. To further characterize the occupied loci, we surveyed the genomic regions bound by GATA-1 and GATA-2 to detect motifs enriched in the sequences surrounding the peak calls. As expected, we found that over 80% contained the canonical WGATAR binding motif. In contrast to reports of motifs enriched in GATA-1 ChIP studies in erythroid cells, we failed to observe significant enrichment of LRF binding motifs. Rather, the GATA-1 and GATA-2 bound regions in megakaryocytes are strongly enriched for motifs that match the binding sites for Ets family transcription factors. Finally, we have found that these genomic regions are indeed occupied by one or more Ets factors in proliferating G1ME cells. Together, these data establish the presence of a GATA switch in megakaryocyte development and provide novel insights into coordinated gene regulation by GATA factors and the differences between the closely related erythroid and megakaryocyte lineages. Disclosures: No relevant conflicts of interest to declare.

Description: There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than one-third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression; and in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we show that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification.

Description: Abstract 1602 The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction and aims: Patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia are known to display several immunological abnormalities. Azacitidine represents a therapeutic options for these disorders and, beside the well known effects on bone marrow precursors, has been demonstrated to potentially influence T-cell polarization. The aim of this study is to monitor the kinetic of the T-cell receptor (TCR) repertoire during Azacitidine treatment in order to explore its potential ability, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these patients. Materials and methods: Our study consisted in a flow cytometric and spectratyping analysis performed on the peripheral blood of 11 patients (5 with MDS and 6 with AML with multilineage dysplasia) and 30 normal controls. Each patient was evaluated at baseline and then every 3 cycles of Azacitidine. The flow cytometry analysis was based on a panel of 24 beta variable (BV) family-specific antibodies. A BV expansion was defined as any value of BV family expression higher than the mean + 3 standard deviations calculated in controls. The profile of the third complementarity-determining-region (CDR3) in separated helper and cytotoxic T-cells was then analyzed by spectratyping. After immunomagnetic CD4+/CD8+ cell separation, RNA extraction and reverse transcriptase PCR, CDR3 fragment analysis was performed through capillary electrophoresis. Spectratyping evaluation was carried out by determining the percentage of Gaussian, skewed and oligoclonal BV subfamilies. Results: Our patients had a median of 3 assessments during their treatment with Azacitidine. On flow cytometry at baseline, in CD4+ cells 5 patients did not show any lymphocyte expansion while 5 of them showed a single BV expansion. One of these expansions disappeared after 3 cycles while 4 were stable during treatment. When reassessed during therapy, 2 of the patients showed each the appearance of 3 new BV expansions, some of which however disappeared at the following evaluations. Overall CD4+ expansions during the all period of study were 11 (5 at baseline and 6 emerged during treatment) and their size ranged from 5.1% in BV 13.6 to 23.9% in BV 11. Within the CD8+ subset, at baseline 7 out of 10 patients showed at least one T-cell expansion. In details 3 patients showed a single expansion while 4 of them displayed 2 different BV expansions. Of these 11 baseline expansions 7 were stable during treatment, while 4 of them quickly disappeared. Remarkably, one of these expansions which had disappeared in a patient in remission reappeared at disease relapse. Six patients showed the appearance of a single BV expansion during treatment, which once again was usually transient. Overall CD8+ expansions during the all period of evaluation were 17 (11 at baseline and 6 emerged later) and their size ranged from 2.5% in BV 5.3 to 50.1% in BV 3. Noteworthy, when analyzed by spectratyping during their treatment our patients showed significant changes in their CDR3 profiles, which were much more evident in helper T lymphocytes. In fact, the frequency of BV showing a skewed CDR3 profile was significantly decreased from baseline to the following evaluations in the CD4+ subset (mean 81.45% vs. 70.17%, p= 0.004). This pattern was even more pronounced in patients responding to Azacitidine (mean 89.60% vs. 61.47%, p= 0.002). Also in the CD8+ subset a trend towards a reduction in the frequency of skewed CDR3 profiles (mean 99.27% vs. 98.74%, p= 0.01) was demonstrated. In the patient with the longest follow up it was possible to observe a dramatic decrease in the degree of CD4+ CDR3 skewing from 91% at baseline to 22% at 18 months, when he was still responding to Azacitidine. Conclusions: Our findings firstly confirmed in our patients an overall derangement of the TCR repertoire. However this pattern seems to gradually improve during Azacitidine treatment, as witnessed by the disappearance of some BV expansions observed on flow cytometry but much more by the progressive restoration of the CDR3 diversity detected by spectratyping, especially within the CD4+ subset. Therefore our data suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of patients with MDS and AML with multilineage dysplasia. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3643 GATA-1 is a zinc finger transcription factor that regulates the differentiation of megakaryocytes and erythrocytes from the megakaryocyte-erythrocyte progenitor (MEP). Mutations in GATA1 are associated with hematologic malignancies of these two related lineages. Acquired mutations that lead to expression of the short isoform of GATA-1, termed GATA-1s, are associated with Acute Megakaryocytic Leukemia in children with Down syndrome (DS-AMKL). Moreover, inherited mutations in the N-finger of GATA1, such as V205M, cause a set of related diseases characterized by dyserythropoietic anemia and thrombocytopenia. These latter mutations disrupt recruitment of the essential cofactor FOG-1 and thus promote disease by interfering with the ability of the GATA-1:FOG complex to properly regulate gene expression. Despite the fact that V205 lies along the surface of the zinc finger opposite to DNA, previous studies suggest that FOG-1 may regulate the chromatin binding activity of GATA-1 at a subset of sites. In contrast to V205 mutation, the precise mechanisms by which GATA-1s contributes to disease is poorly understood. Previous studies have shown that GATA-1s uncouples megakaryocyte proliferation from differentiation, likely by an inability of GATA-1s to properly repress expression of a subset of GATA-1 target genes. We hypothesized that both inherited and acquired GATA1 mutations contribute to disease by interfering with not only target gene activation or repression, but also with GATA-1 chromatin binding. In order to define the chromatin binding activity of GATA-1 and its disease associated mutants, we performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-Seq) for wild-type GATA-1, GATA-1s and GATA-1V205G in the G1ME cell line. G1ME cells, which were derived from GATA-1 null ES cells, approximate an MEP in that they can differentiate into either erythroid cells (in the presence of EPO) or megakaryocytes (in the presence of TPO) upon reconstitution with GATA-1. We expressed GATA-1, GATA-1s, or GATA-1V205G in G1ME cells by retroviral transduction and subjected the cells to ChIP for GATA-1. The resulting DNA was sequenced on the Illumina GAII, yielding 11.8M, 10.4M, and 8.5M uniquely mapped reads. Analysis of the datasets using QuEST yielded 2367, 963, and 4130 peaks, respectively, with an FDR of