ALBERT

Description: Abstract 3635 DNA methylation is a key epigenetic mark affecting the configuration of chromatin and the potential for gene expression. Disorganization of DNA methylation contributes to the development of leukemia. There is a need for high resolution, quantitative and cost effective methods to investigate changes of methylome in leukemia. To achieve this goal, we have recently developed a digital restriction enzyme analysis of methylation (DREAM) for quantitative mapping of DNA methylation at approximately 50,000 CpG sites across the whole genome (Jelinek et al., ASH 2009, abstract 567). The method is based on creating distinct DNA signatures at unmethylated or methylated CCCGGG sites by sequential restriction digests of genomic DNA with the SmaI and XmaI endonucleases and on resolving these signatures by massively parallel sequencing. Using the DREAM method, we have analyzed DNA methylation in bone marrow cells from 2 patients with AML, 3 samples of white blood cells from healthy adults and 2 myeloid leukemia cell lines (K562 and HEL). The first patient (Pt#1) was a 72 year-old male with AML transformation of the myelodysplastic syndrome (MDS). He had 32% blasts in the bone marrow and a complex karyotype. He had received lenalidomide treatment only. The second AML patient (Pt#2) was a 28 year-old male suffering from a relapse of an AML FAB M1. The bone marrow showed 87% of blasts and a complex karyotype. The patient was heavily pretreated with daunorubicin, ara-C, etoposide, 6-thioguanine, dexamethasone and l-asparaginase. Neither of the patients received demethylating drugs. Using typically 2 sequencing lanes per sample and paired-end reads of 36 bases on the Illumina Gene Analyzer II platform, we acquired 20–38 (median 33) million sequence tags per sample; of these, 7–17 (median 12) million were mapped to SmaI/XmaI sites unique in the human genome. With a threshold of minimum 20-fold coverage, we obtained quantitative information on the DNA methylation level of 39,603-53,312 (median 44,490) CpG sites associated with 8,939-10,735 (median 9,517) genes. In general, methylation was largely absent within CpG islands (CGI). The CpG sites most protected from methylation were in CGI and within 1 kb from gene transcription start sites (TSS). These regions were represented by 13,474 CpG sites. Focusing our analysis on these CpG sites, methylation 〉10% was detected only in 268 sites in normal controls (1.9%). The numbers of sites with methylation 〉10% were significantly higher (P10% in CGI within 1 kb from TSS was observed at 2,331 sites (17.0%) in K562 and at 2,484 sites (18.1%) in HEL. Differential hypermethylation in AML patients affected 906 genes, including multiple genes previously shown to be methylated in cancer, such as CDKN1B, FOXO3, GATA2, GATA4, GDNF, HOXA9, IGFBP3, SALL1 and WT1. Methylated genes were significantly enriched in canonical pathways affecting embryonic stem cell signaling, Wnt-beta-catenin signaling and pluripotency suggesting an important role in AML stem cells. In contrast to CGI, it is known that CpG sites outside of CpG islands (NCGI) are generally fully methylated in normal cells. We analyzed 11,220 NCGI sites that were 〉1 kb from gene TSS. Methylation 〉90% was observed at 5,217 (46%) sites in normal controls, in 5,380 sites (48%) in Pt#1, while only in 1,873 sites (17%) in Pt#2 (P

Description: Abstract 3640 Background & Aims: The hypothesis that cancer is driven by Cancer Stem Cells (CSCs or Cancer-Initiating Cell) has recently attracted a great deal of attention. Epigenetic mechanism such as DNA methylation and histone modification play an important role in cancer cells and also in normal stem cells. However, their role remains unclear in CSCs. We sought to determine if CSCs have distinct epigenetic patterns in acute myeloid leukemia (AML). Methods: Peripheral blood samples in AML patients were separated to obtain stem cells (CD34+CD38-) and progenitor cells (CD34+CD38+) by magnetic cell sorting (MACS®, Myltenyi biotec). To study DNA methylation in CSCs in AML, we performed genome wide screening using methylated CpG island microarray (MCAM), which detects 7202 promoter CpG islands, 1348 non-promoter CpG islands, and 632 non-CpG island promoter methylation. MCAM was performed on 4 AML patient samples Next, we evaluated the methylation status of 7 genes which showed apparent higher DNA methylation in stem cells or progenitor cells in MCAM analysis, using a quantitative bisulfite-pyrosequencing for each population of stem cell, progenitor cell, and mature cells (CD34-) from peripheral blood samples in 6 AML patients. For histone modification analysis, we used Chromation immuprecipitation followed by massively parallel sequencing (ChIP-Seq) for stem cell and progenitor cell populations for H3K4me3 which is known to be a marker for activated genes. Results: By MCAM, we found minimal differences between stem cells and progenitor cells present in 2 out of 4 AML patients. Those few genes (

Description: Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic neoplastic disorders that are characterized by ineffective myeloid differentiation and dysplasia as well as telomere shortening and accumulated DNA damage in progenitor cells. Less understood is whether DNA damage is the instigator of impaired progenitor cell differentiation and MDS development. Telomerase deficient mice have served as a model system to demonstrate the adverse effects of wide-spread endogenous DNA damage signaling on stem cell function in vivo. In recent studies, we sought to determine whether persistent physiological DNA damage can impair the function of specific hematopoietic lineages by employing the 4-hydroxytamoxifen (OHT)-inducible telomerase reverse transcriptase-estrogen receptor (TERTER) model. For the first time, we demonstrate that late generation TERTER/ER mice with dysfunctional telomeres exhibit hallmark features of MDS, including peripheral blood cytopenias, bone marrow (BM) hyper-cellularity, and an increased myeloid-to-erythroid progenitor ratio in the absence of increased apoptosis. Severe tri-lineage myelodysplasia, and an increase of immature, morphologically abnormal myeloid blasts frequently with pronounced monocytic differentiation were consistent with refractory anemia with excess of blasts (RAEB) or chronic myelo-monocytic leukemia (CMML), a specific sub-group of MDS that is characterized by a high propensity to develop acute myeloid leukemia (AML). Accordingly, approximately 5% of aged TERTER/ER mice progressed to AML, as demonstrated by a marked increase of BM myeloid blasts, and infiltration of myeloid precursors into the splenic white-red pulp architecture, resulting in myeloid sarcoma with the complete effacement of lymphoid follicles. Compared to control mice with intact telomeres, the progenitor compartment of telomere dysfunctional mice shows a significant increase in the number of granulocyte-macrophage progenitors (GMP) with a concomitant loss of the megakaryocyte-erythroid progenitors (MEP) and slight reduction in the number of common myeloid progenitors (CMP), which is consistent with the condition of skewed myeloid differentiation occurring in MDS patients with higher risk of leukemic transformation. Transplantation experiments of long-term hematopoietic stem cells isolated from telomere dysfunctional mice into wild type congenic recipients revealed that the level of donor-derived skewed myeloid differentiation was comparable to that observed at steady state in the same telomere dysfunctional mice before transplantation, suggesting that impaired progenitor differentiation occurred as a result of cell intrinsic defects of telomere dysfunctional hematopoietic cells. In the setting of telomere dysfunction, somatic in vivo and in vitro telomerase reactivation reduced DNA damage signaling and specifically reversed defective differentiation and MDS phenotypes. Unbiased transcriptomic network analyses of CMP with telomere dysfunction revealed profound down-regulation of genes in the mRNA splicing and processing pathways which was rescued by telomerase reactivation, indicating that telomere dysfunction-induced DNA damage response can impact on the expression of genes involved in splicing regulation. RNA-seq analysis of telomere dysfunctional CMP suggested altered splicing activity at the level of exon usage and identified aberrantly spliced variants of genes implicated in chromatin remodeling, and histone modifications. The prominence of aberrantly spliced epigenetic regulators prompted us to hypothesize that there was a link between impaired myeloid differentiation and aberrant splicing patterns as a result of telomere dysfunction-induced repression of splicing components. In conclusion, our studies have revealed an unanticipated link between telomere biology, RNA splicing, and MDS pathogenesis and support the development of strategies designed to modulate the downstream targets of splicing alterations in specific hematopoietic populations. Disclosures No relevant conflicts of interest to declare.

Description: The pathogenesis of MDS is multifactorial including both cell intrinsic alterations, such as mutations, and cell extrinsic stimuli such as immune deregulation. The prognosis of patients that lose response to hypomethylating agents (HMAs) is very poor and the mechanisms are not understood. Leukemia cells develop multiple resistance mechanisms to escape host immune response. Programmed death-1 (PD-1) is a negative costimulatory receptor on activated T lymphocytes. Expression of PD-1 ligands on tumor cells can induce immunosuppressive T-cells. Recent studies have indicated that demethylation of PD-1 leads to exhausted CD8+ T cells after chronic virus infection. We hypothesized that dysregulated PD-1 immune checkpoint signaling may be involved in pathogenesis of MDS and resistance to HMAs. We first evaluated the mRNA expression levels of PD-L1, PD-L2, PD-1 and CTLA4 by Q-PCR in bone marrow CD34+ cells from 124 patients which include 69 with MDS, 46 with CMML and 9 with AML. Seventy two (58%) patients were previously untreated. We observed aberrant up-regulated mRNA expression of PD-L1 in 39 patients (34% with fold 0-843.3), PD-L2 in 17 patients (14% with fold 0-22.5), PD-1 in 18 patients (15% with fold 0-76.7) and CTLA4 in 10 patients (8% with fold 0-25.1). No significant differences in gene expression were observed when comparing patients that had and had not received prior therapy. Statistically significant relationships were identified between elevated PD-1 expression and increased age (p=0.008), while elevated PD-L2 expression correlated significantly with female gender (p=0.005). Both elevated PD-L1 and CTLA4 expression correlated with MDS subtype, (p=0.034) and (p=0.012), respectively. Additionally, elevated CTLA4 expression correlated with higher white blood cell count (p=0.021), lack of prior therapy (p=0.02) and lower MDS IPSS score (p=0.027). We then performed an analysis of the impact on survival of the 4 gene expression in patients that had not received prior therapy. Patients with lower expression of PD-L1 had a non-significant trend towards better survival (31.5 months versus 16.2, p=0.24). Forty six of 124 patients analyzed received HMAs, and within these 46 patients lower PD-L1 expression correlated with a significantly improved overall response rate (67% vs 25%, p=0.038). Aberrant up-regulation of these 4 genes was also observed in peripheral blood mononuclear cell (PBMNC) from 61 MDS, CMML and AML patients. The relative expression of PD-L1 was significantly higher in MDS (p=0.018) and CMML (p=0.0128) compared to AML. Of interest, mRNA expression of these 4 genes was significantly higher in PBMNC than in CD34+ cells except PD-L1. By immunohistochemical (IHC) analysis, a strong correlation was observed between protein and mRNA expression. By IHC, we observed that leukemia blasts were positive for PD-L1 whereas stroma/non-blast cellular compartment was positive for PD-1 in bone marrow biopsies from MDS, CMML and AML patients. We then analyzed effect of HMAs on these four gene expression in cohort of 61 patients treated with different trials of epigenetic therapy. Treatment resulted in up regulated expression of PD-L1 in 57% of the patients (with maximum induction fold of 4.8), PD-L2 in 57% (maximum fold of 15.77), PD-1 in 58% (maximum fold of 50.26) and CTLA4 in 66% (maximum fold of 29.9). Of importance, patients resistant to therapy had increased gene expression compared to patients that achieved response (fold 5.3 vs 0.4 for PD-L1, 6.2 vs 0.4 for PD-L2, 3.0 vs 0.4 for PD-1 and 5.4 vs 0.7 for CTLA4). Comparing patients without and with expression induction, the median survival was 11.7 and 6.6 months (p=0.122) for PD-L1, 12.5 and 4.7 months (p=0.029) for PD-L2, indicating a better prognosis in patients without PD-L1 and PD-L2 induction treated with HMAs. To model this observation, we treated KG-1 and THP1 cells with different concentrations of decitabine (0 to 10 uM) and observed a dose dependent up-regulation of PD-L1 and PD-L2 in THP1 cells, and CTLA4, PD-1, PD-L1 in KG1 cells. Exposure to decitabine resulted in demethylation of PD-1 in these cell lines, and the demethylation effect was also observed in HMAs treated MDS and AML patients. This study suggests immune checkpoint PD-1/PD-1 ligands signaling may be involved in MDS pathogenesis and resistance mechanisms to HMAs. Blockade of this pathway can be a potential therapy in MDS and AML. Fig.1 PD-L1 expression in MDS CD34+ cells by IHC. Fig.1. PD-L1 expression in MDS CD34+ cells by IHC. Disclosures: No relevant conflicts of interest to declare.

Description: DNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P 〈 .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance.

Description: Abstract 1716 Cytosine methylation is an epigenetic mark affecting accessibility of DNA to transcription. Cancer is associated with hypermethylation in CpG islands (dense clusters of CpG sites frequently present around gene transcription starts) and hypomethylation of sparse CpG sites outside CpG islands. Complex changes of DNA methylation in leukemia permanently disturb epigenetic regulation and participate in leukemogenesis. To characterize epigenetic aberrations in myeloid neoplasms, we analyzed DNA methylation in 16 patients with myelodysplastic syndrome (MDS), 7 patients with acute myeloid leukemia (AML) and 5 healthy controls. Using Digital Restriction Enzyme Analysis of Methylation, we quantified DNA methylation at CpG dinucleotides within approximately 40,000 CCCGGG restriction sites across the genome. We analyzed methylation differences between healthy controls and patients with MDS and AML. CpG sites within CpG islands (CGI sites) are typically not methylated in normal tissues. We found 18,738 CGI sites with methylation 20% in these sites, ranging from 5 to 2720 (median 186) hypermethylated sites in individual patients. The median number of hypermethylated CGI sites was 146 in MDS and 1234 in AML patients. Altogether, we found 5069 CGI sites corresponding to 2183 genes differentially hypermethylated in MDS or AML. GpG sites outside CpG islands (NCGI sites) are generally methylated. We found only 3262 NCGI sites unmethylated (80% in normal controls. Methylation levels

Description: The RUNX family of transcription factors forms the DNA binding α-chain partners of the heterodimeric core binding factor (CBF) complex. Each of the RUNX proteins, RUNX1, RUNX2, and RUNX3, can form heterodimers with CBFβ. In the M4Eo subtype of human acute leukemia, the chromosomal translocation resulting in inversion 16 encodes a chimeric protein in which CBFβ is fused to smooth muscle myosin heavy chain (SMMHC). Although the exact mechanism of leukemogenesis by this chimera is unknown, it is thought that CBFβ-SMMHC sequesters RUNX1 in the cytoplasm and antagonizes its normal function. Although the role of RUNX1 in hematopoiesis has been previously well-established, recent data have indicated that the RUNX3 gene may also play a key role in the development of human acute leukemias. To clarify the role of RUNX3 in acute myeloid leukemia (AML), we investigated its expression and promoter DNA methylation in leukemia cell lines and patient samples. Eleven human leukemia cell lines of myeloid origin and twelve of lymphoid origin were used in this study. Cell suspensions from bone marrow aspirate specimens from patients with AML (69 cases), MDS (19 cases) and ALL (6 cases) were obtained prior to therapy from established tissue blocks. Peripheral blood samples were obtained from four healthy volunteers, and CD34+ cells were obtained from another four individuals. Methylation status of the gene promoters of RUNX1, RUNX2 and RUNX3 were evaluated using the Pyrosequencing Methylation Assay (PMA) method, and expression of RUNX3 was analyzed by quantitative real-time PCR and immunohistochemical staining. Hypermethylation of RUNX1 and RUNX2 was rare in cell lines; RUNX1 was not hypermethylated in any of the studied samples, and RUNX2 was hypermethylated in only two cell lines. In contrast, we found that the RUNX3 promoter was hypermethylated in 17 of the cell lines (74%). Interestingly, we observed a trend toward higher frequency of hypermethylation of RUNX3 in cell lines of myeloid (90%) compared to lymphoid (57%) origin. In patient samples, RUNX3 promoter methylation was below 15% in normal samples, and hypermethylation was found in 32/69 AML samples (46%), 4/19 MDS samples (21%), and 6/6 ALL samples (100%). Of the 69 AML samples, 19 were classified as AML M4Eo, and 50 were other types of AML. 84% of the human AML M4Eo samples were hypermethylated at the RUNX3 promoter region, whereas only 34% of the other AML subtypes were hypermethylated. We also evaluated DNA methylation of RUNX1 and RUNX2 in a subgroup of these samples (66 samples for RUNX1 and 72 for RUNX2) and found that, as in cell lines, these genes are almost universally unmethylated; with the exception of a single AML case, all studied samples presented no promoter methylation. As support of functional outcome, hypermethylation of RUNX3 was correlated with both lower levels of mRNA and protein, as confirmed by qRT-PCR and immunohistochemistry analysis in cell lines and patient samples, and treatment with the DNA demethylating agent Decitabine resulted in mRNA re-expression of RUNX3 concomitantly with decreased promoter methylation. Finally, we compared clinicopathological features of patients with and without RUNX3 methylation. In this analysis, only non-M4Eo AML cases were compared because of the small number of non-methylated patients in the M4Eo group. Differences were found neither for blood counts nor for overall survival probability. However, relapse-free survival was significantly better for the unmethylated group (p=0.016). In summary, we showed that promoter methylation of the RUNX3 gene and down regulation of RUNX3 expression occurs almost universally in M4Eo/inversion 16 AMLs, and that in cell lines, RUNX3 repression can be reversed by treatment with the hypomethylating agent decitabine. These results suggest that silencing of RUNX3 is likely an important target in CBF leukemia and that future studies should be dedicated to further characterize the role of RUNX3 in inversion 16 AML and its predictive value of relapse-free survival in AML. Disclosures No relevant conflicts of interest to declare.

Description: In myotonic dystrophy type 1 (DM1), somatic mosaicism of the (CTG)n repeat expansion is age-dependent, tissue-specific and expansion-biased. These features contribute toward variation in disease severity and confound genotype-to-phenotype analyses. To investigate how the (CTG)n repeat expansion changes over time, we collected three longitudinal blood DNA samples separated by 8–15 years and used small pool and single-molecule PCR in 43 DM1 patients. We used the lower boundary of the allele length distribution as the best estimate for the inherited progenitor allele length (ePAL), which is itself the best predictor of disease severity. Although in most patients the lower boundary of the allele length distribution was conserved over time, in many this estimate also increased with age, suggesting samples for research studies and clinical trials should be obtained as early as possible. As expected, the modal allele length increased over time, driven primarily by ePAL, age-at-sampling and the time interval. As expected, small expansions

Description: Inflammation and innate immunity have an essential role in the pathogenesis of myelodysplastic syndromes (MDS). One of the key inflammatory cytokines that is upregulated in MDS and acute myeloid leukemia (AML) is IL-8. [Schinke C et al, 2015]. The expression of IL-8 and other inflammatory cytokines is controlled by phosphodiesterase 4 (PDE4), which has yet to be studied in MDS and AML. Furthermore PDE4 inhibitors may be useful therapeutic targets for MDS. Based on this, we evaluated the expression of PDE4 and examined its potential impact on clinical outcomes in MDS. Transcriptomic profiling was obtained from the Illumina Genome Analyzer IIx platform, using the NuGEN Ovation RNA sequencing (RNA-seq) system. Total RNA was extracted from CD34+ bone marrow hematopoietic cells from healthy individuals (n=10) and patients with MDS (n=24) or CMML (n=19). We interrogated the RNA-seq dataset to analyze expression levels of each isoform of the PDE4 (A, B, C and D) using Z-score to normalize the results. Median overall survival (OS) was estimated using Kaplan-Meier methods with the log-rank test. Survival rates for each isoform of the PDE4 were stratified by negative or positive Z-scores (low or high expression groups, respectively). The study cohort had a median follow-up of 21.2 months (range: 0.2-68). Median age at diagnosis was 69 years (range: 43-87) with 67.5 % of diagnosed patients being ≥65 years. Most of patients (72%) were male, and 21% had a prior chemotherapy and/or radiotherapy. The median count (x 109/L) for white blood cells and platelets was 12.2 (range: 0.7-67) and 97.2 (range: 11-331), respectively. IPSS risk was low and int-1 in 51.2% of patients, int-2 in 37.2%, high in 6.9%, and not available in 4.7%. The cytogenetic groups of risk were: very good and good in 53.5% of patients, intermediate in 30.2%, poor in 14% and not available in 2.3%. The overall response rate (ORR) to hypomethylation agents (HMA) was 51.1%, with 37.2% having complete response (CR), 6.9% having bone marrow CR, and 6.9% having hematologic improvement. Median OS for the whole cohort was 17.6 months (95% confidence interval [CI]: 9.6-25.6). Detailed PDE4 isoform-specific analyses are presented in Table1. Importantly, there appears to be an impact on OS depending on PDE4 isoform expression levels, which should be confirmed in a larger patient cohort. PDE4 expression may be useful as both a prognostic factor and a potential therapeutic target for patients with MDS. The effects of PDE4 inhibitors should be investigated in vitro against MDS cell lines and in preclinical mouse models of MDS. One potential application for this study is to exploit the blockade of the PDE4 pathway on the IL8 expression, which may be a good therapeutic strategy against MDS and AML stem cells [Schinke C et al, 2015]. PDE4 inhibitors could also be included in our therapeutic arsenal in the context of the HMA failure in selected patients with MDS. Table 1. PDE4 isoform expression stratification by expression levels and by disease state DISEASE STATE STRATIFICATION Mean expression (range) p value EXPRESSION LEVEL STRATIFICATION Number of patients (%) Mean expression (range) Median OS, months [95% CI] p value PDE4A 0.26 PDE4A 0.24 MDS 0.26 (0-5.2) Low 27 (62.7) 0.26 (0-0.08) 15.8 [11.1-37.1] Normal 0.108 (0-0.5) High 16 (37.3) 0.65 (0.1-5.2) 29.2 [7.60-36.4] PDE4B 0.90 PDE4B 0.87 MDS 4.90 (0.06-23.95) Low 28 (65.1) 2.25 (0.06-4.87) 24.1 [11.7-19.8] Normal 4.92 (0.06-23.95) High 15 (34.9) 9.85 (5.21-23.95) 22 [26.7-31.8] PDE4C 0.03 PDE4C 0.25 MDS 0.004 (0-0.045) Low 29 (67.4) 0.0003 (0-0.002) 26.7 [18.6-34.8] Normal 0.0009 (0-0.004) High 14 (32.6) 0.01 (0.004-0.045) 12.3 [3.40-21.1] PDE4D 0.89 PDE4D 0.95 MDS 1.55 (0.007-5.19) Low 25 (58.1) 0.92 (0.007-1.49) 26.7 [4.60-48.80] Normal 1.50 (0-3) High 18 (41.9) 2.42 (1.51-5.10) 16.8 [13.3-20.25] Disclosures No relevant conflicts of interest to declare.