Abstract
Introduction: DNA methylation differences between normal and cancer tissue that result in differential expression of genes are a hallmark of acute myeloid leukemia (AML). DNA methylation mediated silencing of specific genes, especially transcription factors, can provide a growth advantage for malignant cells. Global DNA methylation analyses have not only led to a better understanding of AML subgroups and the impact of epigenetic aberrations in leukemogenesis, but also to the identification of new epigenetically regulated genes. We and others have recently identified the oligodendrocyte lineage transcription factor 2 (OLIG2) as differentially methylated in AML cell lines compared with normal bone marrow cells.
Aim of the study: With the hypothesis that OLIG2, which is not expressed in normal hematopoiesis, may play a role in cancerogenesis as shown for acute lymphoblastic leukemia (Lin et al., Cancer Res. 2005) and malignant glioma (Mehta et al., Cancer Cell 2011), we sought to further dissect the impact of OLIG2 in AML, implementing functional studies and primary samples.
Results: First, in a cohort of 93 AML patients, we could confirm previous results by Kröger et al. (Blood 2008) that OLIG2 is differentially methylated: using pyrosequencing, 37 patients (39.8%) showed methylation levels > 25% (range (r): 26-79%) in the 5 CpG containing amplicon of the OLIG2 promoter region, independent of cytogenetic subgroup. In a small subset of 13 patients where expression-data was available, an inverse correlation between OLIG2 DNA methylation and mRNA expression was significant (r2=0.55, p<0.005). This observation was further supported by a highly significant inverse DNA methylation/mRNA expression correlation in 10 leukemia cell lines (r2=0.74, p< 0.002). Moreover, we could demonstrate that this inverse correlation held also true for OLIG2 protein levels in cell lines with strong expression in THP-1 and NB-4, moderate expression in HL-60 and HEL and no expression in U937, KG-1A, PL-21, Kasumi-1, K-562 and Jurkat.
Interestingly, while CD 34+ cells from two healthy donors and 10 out of 12 AML patients where protein was available, showed no protein expression, OLIG2 was expressed in 2 patients, both bearing the translocation t(15;17). This corresponds well to OLIG2 expression of cell line NB-4, which also harbours t(15;17). Treatment of non-expressing cell lines PL-21 and U937 with 200 nM 5-aza-2'-deoxycytidine led to robust re-expression of OLIG2, both on mRNA and protein level, strongly implicating DNA methylation as a silencing mechanism in a subset of AML.
To investigate the relationship between OLIG2 expression and AML cell growth we used a siRNA transient knock-down in OLIG2 expressing cell lines THP-1 and NB-4. While OLIG2 protein expression measured via densitometry could be strongly reduced to 38% and 45% from pre-treatment levels in THP-1 and NB-4 cells, respectively, no change on cell viability or cell growth was detected. However, stable over-expression of OLIG2 using the lentiviral-vector pLeGO-iG in Kasumi-1 cells, led to a significant growth-inhibition of 32.2% (r: 27.0-37.3%) after 5 days and a 47.7% (r: 30.7-64.6%) increase of apoptotic cells (Annexin-V-staining) as compared to control-vector transfected cells. This negative effect on cell proliferation supports our presumption that OLIG2 could act as a growth-regulator in a subgroup of AML. This could be caused by a direct interaction between OLIG2 and a cell cycle regulator or a transcription factor complex.
Conclusion: We show that OLIG2 (I) is in part epigenetically regulated via DNA methylation in AML, resulting in an inverse correlation between DNA methylation and expression; (II) can be re-expressed upon demethylating treatment in cell lines, therefore making it an attractive biomarker to study in AML patients treated with demethylating agents; (III) shows antiproliferative activity in leukemia cell lines and thus should be further studied as a potential tumor suppressor in AML.
Disclosures
Lübbert: Cephalon / TEVA: Travel support Other.
Restoration of Epigenetically Silenced Sulfatase 1 Expression by 5-Aza-2′-Deoxycytidine Sensitizes Hepatocellular Carcinoma Cells to Chemotherapy-Induced Apoptosis
