DNA去甲基化藥物於血癌細胞亞群的表觀遺傳效應整合分析

Integrative analysis of epigenomic responses to DNA demethylating agents in leukemic cell subpopulations

陳彥瑋(Yen-Wei Chen)

指導教授 ： 蔡幸真

國立臺灣大學/醫學院/毒理學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703534

摘要

表觀遺傳學是一門研究不改變DNA序列卻能夠影響基因表現，並將基因表現之變化在細胞分裂中遺傳至子細胞的學問。該門學問與胚胎幹細胞及血液幹細胞的分化息息相關。而對於具有腫瘤生成能力的癌症幹細胞，表觀遺傳學的研究仍有進步空間。因此本研究使用急性骨髓性白血病的Kasumi-1細胞株，先透過細胞表面的CD34蛋白標記，分出了可以在老鼠中引發白血病的CD34陽性細胞以及無法在老鼠中引發白血病的CD34陰性細胞亞群，接著使用地西他濱(decitabine)分別處理各細胞亞群，抑制細胞內之DNA甲基轉移脢、以促使CD34陽性血癌細胞分化、並降低其在老鼠體內形成血癌之能力, 再配合高通量分析技術，分析這些細胞全基因之轉錄體、甲基體以及組蛋白修飾情形。以期能找出表觀遺傳機轉於CD34陽性幹細胞致癌性及分化能力所扮演的角色，以及低劑量地西他濱如何透過去甲基化以外的機轉影響形成血癌的能力。我的研究發現，甲基化以及組蛋白修飾對於幹細胞特性相關基因調控扮演重要的角色，幹細胞特性基因之啟動子於CD34陽性血癌細胞中甲基化程度低及H3K27me3組蛋白修飾低，導致幹細胞特性基因高度表達。而以低劑量去甲基化藥物地西他濱處理後，除了發現基因甲基化程度整體下降以及分化相關基因表達上升之外，亦發現特定之組蛋白修飾如H3K4me3以及H3K27me3於基因啟動子處都明顯下降的現象。。除此之外，也發現decitabine處理過後再表現以及沒有再表現的基因，即使和處理前的甲基化還有組蛋白修飾相比都是明顯下降。處理前的H3K27me3仍可以提供線索告訴我們他會不會再表現。透過這個特性進一步發現被decitabine所促進的分化相關基因，都在處理前具有較高的H3K27me3以導致其在處理後的再表現。除此之外也注意到了decitabine的處理可以降低CD34陽性細胞還有陰性細胞的分化基因表現差距，以及調控這些基因的甲基化和組蛋白修飾H3K27me3差距都有下降的現象，說明了decitabine有可能降低癌症中次族群中的差異性。整體而言這份研究說明了癌症幹細胞致癌特性會受到甲基化以及組蛋白修飾影響，而經過地西他濱處理後會經過複雜的表觀遺傳學機制降低其幹細胞致癌特性。

關鍵字

表觀遺傳學 ； 去甲基化藥物 ； 急性骨髓性白血病

並列摘要

Epigenetics refers to study of heritable gene expression changes without changing DNA sequence. Epigenetic mechanisms play important roles in many biological processes, including embryonic and somatic stem cell differentiation. However, how epigenetic mechanisms regulate cancer-initiating capacity of cancer stem cells is still unclear. Thus, we used Kasumi-1 cells as model system, which is an acute myelogenous leukemia cell line containing leukemogenic CD34-positive (CD34pos) and non-leukemogenic CD34-negative (CD34neg) cell subpopulations. We conducted integrative bioinformatics analysis on genome-wide transcriptomes (gene expression arrays), methylation profiles (Infinium 450K arrays), and histone modifications (ChIP-seq) to investigate how cancer stemness is regulated by epigenetic mechanisms. Furthermore, we studied whether and how decitabine, a DNMT inhibitor, acts beyond DNA demethylation to diminish leukemogenicity through chromatin changes. The analyses revealed lower DNA methylation and H3K27me3 levels at the promoters of stemness-related genes in CD34pos cells, correlated with higher transcriptional activities of these genes. After decitabine treatment, a global decrement of H3K4me3 and H3K27me3 enrichments near transcription start site was noted, possibly due to loosening of chromatin configuration. In addition, genes with higher H3K27me3 promoter enrichments at baseline tend to be upregulated upon decitabine treatment, as opposed to genes with low H3K27me3 enrichments. This suggests pretreatment epigenetic patterns may dictate gene responses to decitabine therapy. Finally, our data indicate that decitabine may diminish epigenetic differences between two cell subpopulations by pushing all cells towards a more differentiated state. In conclusion, we have shown that leukemia initiating capacity is regulated not only by DNA methylation but also chromatin modifications. Decitabine treatment may decrease stemness features through complex interactions of epigenetic controls.

並列關鍵字

Epigenetics ； DNA demethylating agents ； Acute myeloid leukemia

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