ALBERT

Description: Although recent data suggests that osteoblasts play a key role within the hematopoietic stem cell (HSC) niche, the mechanisms underpinning this remain to be fully defined. The studies described herein examine the role in hematopoiesis of Osteopontin (Opn), a multidomain, phosphorylated glycoprotein, synthesized by osteoblasts, with well-described roles in cell adhesion, inflammatory responses, angiogenesis, and tumor metastasis. We demonstrate a previously unrecognized critical role for Opn in regulation of the physical location and proliferation of HSCs. Within marrow, Opn expression is restricted to the endosteal bone surface and contributes to HSC transmarrow migration toward the endosteal region, as demonstrated by the markedly aberrant distribution of HSCs in Opn–/– mice after transplantation. Primitive hematopoietic cells demonstrate specific adhesion to Opn in vitro via β1 integrin. Furthermore, exogenous Opn potently suppresses the proliferation of primitive HPCs in vitro, the physiologic relevance of which is demonstrated by the markedly enhanced cycling of HSC in Opn–/– mice. These data therefore provide strong evidence that Opn is an important component of the HSC niche which participates in HSC location and as a physiologic-negative regulator of HSC proliferation.

Description: Abstract 1279 Poster Board I-301 The MLL (mixed-lineage leukemia) gene at chromosome band 11q23 is rearranged frequently in AML and ALL, and associated with poor prognosis. The consequence of these translocations is the formation of a chimeric oncogenic transcription factor that specifies a unique expression signature distinct from other subtypes of acute leukemia. However, it is poorly understood, which changes in gene expression in leukemic cells are under the direct control of MLL fusion proteins (fusion), nor is it clear what is the potential overlap between MLL wild type (WT) and fusion target genes. In the present study, we used genome-wide location analysis to determine the genomic loci that are specifically bound by MLL fusion proteins. Combining the binding analysis with expression profiling, we further defined the subset of MLL fusion-bound genes whose expression is regulated by the presence of MLL fusion proteins. Using ChIP-chip (Chromatin Immunoprecipitation coupled with micro-array), we determined the MLL-bound regions in 5 myeloid leukemic cell lines using a custom array containing the entire genomic region of 200 genes previously found to have altered expression in MLL-rearranged leukemias. Examination of these 200 genomic loci revealed a largely overlapping set of genes bound by MLL (wild type and/or fusion proteins) in WT/WT (U937: 110 genes, HL60: 79 genes) and WT/Fusion cells (MV4;11: 62 genes, THP-1: 89 genes). Surprisingly, the MLL-bound genes in fusion/fusion (ML-2) cells (25 genes) are a small subset of that found in each of the other 4 cell lines, despite comparable levels of detected MLL binding signal across all lines examined. These data suggest that the MLL fusion protein is likely only localized to a limited portion of genomic loci occupied by the MLL wild type protein. To test this hypothesis in a more systematic way, we examined an inducible MLL-ENL–ER transformed cell line (Slany et al, MCB 2004), which grow as myeloblastic cells in the presence of MLL-ENL, and differentiate into neutrophils upon inactivation of the fusion protein. As MLL-ENL promotes histone H3 lysine 79 (H3K79) methylation, we determined both MLL binding and H3K79 methylation using a genome-wide location analysis. We anticipated that MLL-fusion bound genomic regions would exhibit a significant drop in either MLL and/or K79 signal upon inactivation of MLL-ENL. Unexpectedly, among thousands of genes that are bound by MLL, only 10% of them (222 genes) showed a pattern of binding increase between MLL-ENL induced and un-induced conditions. To explore the impact of MLL fusion protein on gene activation, we performed whole genome expression profiling in the presence or absence of MLL-ENL. Increased levels of either MLL binding or H3K79 methylation are significantly associated with differential gene expression. Among 222 MLL fusion target genes, 12 of them are differentially up-regulated in the presence of MLL-ENL, indicating that a large fraction of MLL fusion bound genes do not exhibit significant changes in mRNA expression. The identified 12 genes include key regulators in cellular differentiation and cell cycle regulation, as well as Meis1, Hoxa9 which are known to be essential for the development of MLL leukemia. To explore the apparent discrepancy between the massive expression changes in MLL rearranged leukemia and the small number of direct fusion target genes we identified, we tested the hypothesis that a significant portion of the MLL fusion protein expression signature was derived from its direct fusion target genes Meis1 and Hoxa9. Using publicly available data, we compared the MLL leukemia associated expression profile with the set of genes that were down-regulated upon knock-down of Meis1 and Hoxa9. We found significant enrichment of Hoxa9/Meis1 downstream targets in the expression profile defined by MLL fusion proteins. Altogether, our data suggest that MLL fusion proteins are likely to contribute to the development of acute leukemia through direct activation of a very small set of genes. The results have important implications in understanding the mechanisms of target gene specificity involving oncogenic transcription factors. Disclosures No relevant conflicts of interest to declare.

Description: Key steps in hematopoiesis and the expression of genes encoding hemoglobin subunits are critically dependent upon specific members of the GATA factor family of transcription factors. Our recent efforts have focused on elucidating how GATA factors select functional sites in chromatin and how they function combinatorially with additional regulatory factors. GATA motifs are often arranged in close proximity to E-boxes, and such composite elements commonly mediate GATA factor- and Scl/TAL1-dependent transcriptional responses. Only a small fraction of these composite elements in chromatin are occupied by GATA factors and Scl/TAL1, and a specific epigenetic signature distinguishes occupied versus unoccupied elements genome-wide. In the context of hemoglobin synthesis, we are using genetic and molecular approaches to dissect the multistep mechanism underlying the control of β-globin transcription. GATA-1-containing complexes assemble at the β-globin Locus Control Region (LCR) prior to the murine adult βmajor promoter. Though the LCR physically interacts with the βmajor promoter, this interaction is not required for the binding of several trans-acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems. promoter. Though the LCR physically interacts with the β promoter, this interaction is not required for the binding of several -acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems.

Description: Abstract 3808 Poster Board III-744 DNA methylation is a common epigenetic mechanism of gene silencing in patients with the Myelodysplastic Syndrome (MDS) and Acute Myelogenous Leukemia (AML). Epigenetic therapy with drugs which inhibit DNA methylation such as 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) have proven to be clinically potent in MDS and AML. In addition to DNA methylation inhibitors, histone deacetylase inhibitors (HDACi) have activity in leukemias, and at low doses show epigenetic synergy with DNA methylation inhibitors. To test this synergy in the clinic, we designed a phase II randomized study comparing decitabine alone (20 mg/m2 IV daily x 5 every 4 weeks) to decitabine (same dose) plus valproic acid (50 mg/kg PO daily for 7 days started at the same time as decitabine). We have previously reported interim results from this study, showing an overall response rate of 64% in MDS/CMML (CR in 39%) and 46% in AML (CR in 25%) with no significant differences in response or survival between the two arms. We now report on molecular analyses in this trial. We have studied DNA methylation of ALOX12, LINE1, MapK15, miR124a-1 and 3 and P15 using bisulfite-pyrosequencing, and expression of ATM, mi124a, p15 and p21 by qPCR at baseline and at days 5, 12 and 30 after initiation of therapy in 60 (32 for expression) patients treated on the study (33 received decitabine, 27 received decitabine + valproic acid, overall there were 31CRs or HI's and 28 NRs, 1 patient was inevaluable for response). Global methylation (measured by LINE1) decreased at day 5 by an average of 6.8 ±1.8% in the DAC arm and 3.5 ± 1.2% in the DAC/VPA arm (p=0.20). At day 12, the decrease (from baseline) was by 10.2 ± 2.2% in the DAC arm and 7.0 ± 1.5% in the DAC/VPA arm (p=0.32). At day 30 we observed a decrease of 6.4 ± 1.4% in the DAC arm and 4.8 ±2.2% in the DAC/VPA arm. We found no statistical differences between the two arms in any of the other genes studied for hypomethylation. By qPCR, expression of p15 at day 5 increased by 1.2±0.6 fold in the DAC arm and by 2.5±0.7 fold in the DAC/VPA arm (p=0.01). We found no differences in the other 3 genes studied between the two arms. We next asked about correlations between epigenetic modulation and response. There was no association between LINE1 methylation change at days 5, 12 or 30 and response. By contrast, sustained hypomethylation of miR124a1 correlated with response; at day 5, miR124a methylation had changed by -17.9 ± 3.7% in responders vs. -15.2 ± 5.8% in non-responders, while at day 30, methylation decreased further to -24±6.5%% in responders, but had already partially recovered to -5.3 ±5.8% of baseline in non-responders (p=0.029 for a comparison between responders and non-responders). Additionally we found that responders hypomethylated miR124a-3 faster by a change in methylation of -34.1 ± 6.3% at day 5 compared to non-responders who had -14.8 ± 6.3% at day 5 (p= 0.039). However there was no difference at day 30. By qPCR we studied the same genes as previously listed. We found that responders had a larger induction of p15 gene expression at day 5, 2.3 ± 0.75 fold compared to non-responders who had a 0.91 ± 0.66 fold increase (p=0.018). We also found a similar pattern in expression induction in the ATM gene, where responders at day 5 had a 1.92 ± 0.51 fold increase as compared to non-responders who had a 0.3 ± 0.64 fold increase (p=0.034). Similarly, for the mature miR124a locus, responders had a 2.91 ± 0.88 fold increase in expression at day 30 compared to non-responders who had 1.1 ± 0.24 fold change in gene expression (p=0.03). In conclusion, we found that adding Valproic acid to decitabine enhances activation of P15, but also shows trends for reducing hypomethylation induction, which is consistent with in-vitro studies. These opposing trends may explain why the response rate is not dramatically different in the two arms. We also found that sustained gene specific hypomethylation correlates with response, as does induction of expression of P15, miR124 and ATM, which confirms and extends our prior studies. Thus, modulation of DNA methylation and gene expression appears to be associated with response to decitabine, and testing whether histone deacetylase inhibitors enhance this response will require non-overlapping dosing regimens and, likely, more potent HDAC inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: MicroRNAs (miRs) are small RNA molecules of approximately 1–722 bps in length. They have been shown to control gene expression at the mRNA and protein levels by binding to the 3′ UTR of genes within a RNA Induced Silencing Complex (RISC). Recently miRs have been shown to have globally altered expression in several types of hematological malignances. However there is little data on how these miRs may affect the biology of the disease or their effects on response to chemotherapy. Some miRs have also been shown to be regulated at an epigenetic level, and we speculated that their expression may be involved in response to epigenetic therapy. Here, we focused on miR124 in patients with Acute Mylogenous Leukemia (AML) and Myelodysplastic Syndrome (MDS) who were treated with epigenetic chemotherapy, decitabine or decitabine and valproic acid. This miR is transcribed in three different regions of the genome hsa-miR124a-1 chr.8p, hsa-miR124a-2 chr.8q and hsa-miR-124a-3 chr.20q, however only two have CpG islands: a-1 and a-3. MiR124a-1 and a-3 have recently been shown to be frequently methylated in patients with AML which may lead to altered expression of the miR as well as the miR targets. We began our study by looking at baseline (pretreatment) methylation, by bisulfite pyrosequencing, in 56 patients (36=MDS,20=AML) and found that miR124a-1 was hypermethylated in 78% of MDS and 95% of AML patients compared to the 95% CI of normal controls. We also looked at methylation of miR124a-3 at baseline and found that 47% MDS patients and 65% of AML patients were hypermethylated above the 95% CI of normal controls. In a separate set of 60 MDS patients, we found that methylation of miR124a-1 at the time of diagnosis could significantly predict shorter overall survival in patients who were methylated (p=0.04). Next we studied how the DNA methylation inhibitor decitabine (DAC) may affect the methylation of miR124a-1 and a-3. To do this we looked at miR124a-1 and miR124a-3 methylation levels at day 5, 12 and 30 post DAC treatment in patients who showed pretreatment methylation. We found that patients who responded to DAC (Complete Response CR or Hematolgic Imporvement HI) had a significant decrease in methylation at day 5 (a-1 p=0.020 and a-3 p=0.014) and at day 12 (a-1 p=0.043 and a-3 p=0.029) whereas patients who did not respond to therapy had no significant de-methylation during these times. Additionally we found no significant differences in the patients treated with DAC(n=40) vs. the patients treated with DAC + valproic acid(n=16). Since promoter methylation has been well correlated with gene silencing we looked for altered expression levels of the mature miR by real time PCR. We first found that miR124 was expressed in CD34+ cells from cord blood and normal bone marrow CD34+ cells. Additionally we found that out of 30 (15 CR/HI, 15 Non Responders) patients with available RNA; patients who responded to DAC had a significant increase in expression at day 5 (p=0.03) whereas patients who did not respond to therapy had no significant change in expression. Since miR124 has been shown to down regulate CDK6, a proto-oncogene, we proceeded to see how its expression was effected in these patients at the mRNA and protein level. We found that again patients who responded to therapy had a significant decrease in CDK6 mRNA expression at day 5 (p=0.002) whereas patients with no response had no significant change in expression. These data were confirmed by western blot where we found that 7/9 responders had a decrease in expression and 4/4 non-responders had no changes in expression. Also we found that miR124a-1 methylation was positively correlated with CDK6 expression (p=0.007), while there was no correlation with miR124a-3. In summary we have found that miR124 hypomethylation and subsequent re-expression is a good marker of response to decitabine, possibly also mediating these responses by reducing the levels of CDK6.