ALBERT

Description: The zinc finger-encoding transacting factor EKLF binds key regulatory elements of many erythroid-specific genes, and is essential for definitive erythropoiesis. Mice lacking this factor (EKLF−/−) die of anemia by E15.5 of gestation, failing to activate β-globin gene transcription, and demonstrating a block in the erythroid differentiation program at the primitive erythroblast stage. In contrast, megakaryocytic progenitors are amplified in EKLFnull embryos, with increased Fli-1 gene expression (a marker of early megakaryocytic differentiation), consistent with the idea that EKLF modulates the megakaryocyticerythroid (M-E) differentiation switch. We have demonstrated that an amino terminal mutant of EKLF (Δ221EKLF), is required to induce chromatin remodeling at the β-globin promoter in an EKLF-null erythroid cell line, but additional amino terminal sequences are required for initiation of β-globin gene transcription (Brown et al., 2002). To evaluate the role of this chromatin remodeling (CR) domain in erythroid and megakaryocytic differentiation in vivo, we have generated a knock-in allele of EKLFCR allele. Similar to EKLF-null embryos, mice homozygous for this mutant allele die of anemia by E15.5 of gestation. In contrast to erythroid cells lacking EKLF, EKLFCR/CR progenitors demonstrate appropriate binding of the CR encoding domain to all EKLF-regulatory sequences; a block in erythropoiesis at a more a mature stage in differentiation a chromatin architecture and histone modification pattern at erythroid-specific genes that recapitulates the events observed in EKLF+/+ erythroblasts at a similar stage of erythroid ontogeny; a failure of terminal erythroid gene transcription. Examining the role of EKLFCR in megakaryopoiesis, we observed inhibition of megakaryocytic progenitor amplification in EKLFCR/CR fetal hematopoietic cell populations when compared to EKLF-null embryos; loss of Fli-1 gene expression in EKLFCR expressing cells; binding of the EKLFCR mutant protein to the Fli-1 promoter with inhibition of gene transcription; a repressed chromatin architecture at megakaryocytic gene loci. In contrast to these results, mice homozygous for a knockin allele encoding the zinc finger DNA binding domain alone (Δ253EKLF), a region shown previously to be sufficient for chromatin remodeling in vitro, demonstrate erythroid and megakaryocytic phenotypes that resemble those observed in EKLF-null hematopoietic progenitors. Taken together, our results suggest strongly that the unique EKLFCR domain is necessary and sufficient to modulate the chromatin-specific roles of EKLF at erythroid- and megakaryocytic-specific loci in definitive hematopoietic cells in vivo.

Description: Abstract 2746 Introduction: Newly diagnosed and relapsed acute promyelocytic leukemia (APL) patients respond to therapy with arsenic trioxide (ATO) based regimens. Significantly more patients with relapsed APL have disease recurrence after ATO based therapy than newly diagnosed cases. We undertook a series of experiments to evaluate the potential mechanisms to explain this increased recurrence rate in patients with relapsed APL. Patients and methods: From April 2007 to March 2009 bone marrow samples from newly diagnosed and relapsed cases admitted at our center were utilized for these studies. If required the bone marrow blasts and promyelocyte component was enriched to above 90% using a lineage depletion cocktail and VarioMACS (Miltenyi Biotec, Germany). For in-vitro intracellular ATO concentration measurement, 2 × 107 cells were washed and suspended in RPMI media with 0.5 μM concentration of ATO and incubated for 24 hours. Cells were then washed, made into a pellet and solubilized with HNO3 and H2O2 and ATO content measured using an atomic absorption spectrophotometer. An in-vitro sensitivity assay of malignant cells as previously reported was standardized using an MTT assay system. The impact of co-culture of mesenchymal stromal cells (MSC) and malignant promyelocytes on ATO induced apoptosis was studied with 7AAD and Annexin staining using a flowcytometer. A gene expression array using 44k human microarray chip analysis (Agilent technologies) was done on 8 newly diagnosed and 8 relapsed cases. Results: Sixty five patients were included in this study. Of these 47 (72%) were newly diagnosed and 18 (28%) were relapsed cases. On immunophenotyping, CD34 was positive (〉20%) in 3.6% of newly diagnosed and 50% of relapsed cases (P=0.001). The mean MFI of the relapsed cases for expression of CD38, VLA-5 and CD13 was significantly lower in the relapsed group. The ability of both newly diagnosed and relapsed primary APL cells to concentrate ATO intracellular was not significantly different (15.2±9 nG/107 cell Vs. 16.3±9.7 nG/107 cell). Similarly the in-vitro IC-50 assay was not significantly different between the two groups (5.5±3.8 Vs. 4.7±4.5 μM). Neither of these assays correlate with clinical parameters such as relapse, event free (EFS) or overall survival (OS). Evaluation of the impact of MSC on ATO induced apoptosis demonstrates a protective effect in newly diagnosed and relapsed cases (Fig 1). This effect was mediated partly by the MSC conditioning media and could not be overcome by addition of VLA-4 or VLA-5 blocking antibodies (data not shown). Gene expression studies comparing the two cohorts revealed 1744 genes that were differentially expressed (〉2 fold) between samples at diagnosis and at relapse. Quantitative Real-time RT-PCR using SYBR- Green detection system was done to confirm the gene expression results obtained from microarray analysis. Using ΔΔCT method the fold difference was calculated for five selected genes which validated the microarray data. Conclusion: Relapsed patients have significant immuno-phenotypic differences from newly diagnosed cases. Mechanisms of resistance to ATO are probably multi-factorial but are unlikely to be related to intra-cellular ATO concentration. In-vitro IC-50 does not appear to predict clinical outcomes. Stromal interaction protects malignant promyelocytes from the apoptotic action of ATO which appears more pronounced in relapsed than in newly diagnosed cases. Further evaluation of parameters that enhance such stromal interaction and protect malignant promyelocytes from the apoptotic effect of ATO along with evaluation of dysregulated genes and pathways are required. Disclosure: No relevant conflicts of interest to declare.

Description: Abstract 461 Krüppel-like factor 1 (KLF1) is essential for erythroid gene expression. Key molecular mechanisms modulated by this transacting factor have been elucidated at the b-globin locus. KLF1 has been associated with recruitment of SWI/SNF and RNA polymerase (PolII) complexes necessary for chromatin remodeling and gene transcription respectively, and for facilitating the apposition of the promoter with the far-upstream locus control region. More recently, KLF1 has been implicated in the regulation of an erythroid-specific gene program unlinked to the b-globin locus. Coordinated expression of these genes, including Alpha Hemoglobin-Stabilizing Protein (AHSP), a factor required for globin tetramer stability, and the red cell membrane protein Dematin, are critical for erythroid ontogeny. To compare the role(s) of KLF1 at these loci, we have used a unique 4-OH-Tamoxifen (4-OHT) inducible erythroid cell line, which facilitates the characterization of the temporal kinetics of KLF1-dependent erythroid gene activation. In preliminary experiments, we observed that KLF1 binding was maximal at the three loci within 60 minutes of 4-OHT induction. AHSP and dematin primary RNA transcripts followed similar kinetics, being maximal at 60-90 minutes post-induction. In contrast, b-globin gene transcription reached a plateau 4-6 hours post-induction. From these observations, we hypothesized that transcriptional activation at AHSP and dematin differs from that observed at the b-globin cluster. Consistent with this hypothesis, we observed significant differences in chromatin remodeling at the three loci. At the b-globin promoter, we observed a small but statistically significant increase in DNaseI sensitivity, a measure of chromatin remodeling, with KLF1 binding. In contrast, we observed a complete loss of DNaseI resistance after KLF1 binding at the AHSP and dematin promoters. Consistent with these findings, we observed a five-fold reduction in histone H3 occupancy at the AHSP and dematin promoters, contrasting with no significant change in occupancy at the b-promoter. Importantly, these differences were not observed in regions 1-5 kb upstream of the promoters. These observations, coupled with similar differences in DNaseI hypersensitivity and histone occupancy in fetal liver erythroblasts from wild type and KLF1-null mice, suggest a profound difference in the mechanisms of chromatin remodeling at KLF1-dependent erythroid gene loci. To explore the potential mechanisms underlying these differences in chromatin accessibility, we examined the kinetics of recruitment of other transacting factors and co-activators to the three loci. We observed similar increases in binding of serine-5 phosphorylated PolII, GATA-1, and p45NF-E2 at the promoters. In contrast, binding of BRG1, the core ATPase component of the SWI/SNF complex differed between the b–promoter and the other erythroid genes. Although BRG1 binding was co-incident with KLF1 binding to the b-gene, we observed significant albeit weak binding of this complex to the AHSP and Dematin promoters only after maximal gene transcription had occurred. Our results suggest that different KLF1 multiprotein complexes are recruited to remodel target gene promoters in vivo. Furthermore, we propose that KLF1's chromatin remodeling capabilities are not limited to the recruitment of the SWI/SNF complexes Disclosures: No relevant conflicts of interest to declare.

Description: Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.