published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

Acute myeloid leukemia (AML) is a heterogeneous disease both clinically and biologically. Approximately 55% of AML harbour karyotypic changes, and one of the most common chromosomal aberrations is the t(8;21)(q22;q22), which leads to the AML1-ETO fusion protein. Previous studies have found that this fusion protein recruits the N-CoR/mSin3A/HDAC complex, thereby acts as a transcriptional repressor. Recently, DNA methylation array studies have shown that DNA methylation patterns can stratify AML cases into different subgroups, and some of these correspond to certain chromosomal abnormalities, such as the t(8;21). These findings suggest a possible link between the fusion transcript AML1-ETO and epigenetic modifications. Additionally, c-kit mutations have emerged as an important disease modifier in the t(8;21) AML and are correlated with poor overall survival and event free survival in patients with t(8;21) AML. We therefore sought to investigate whether there are different DNA methylation patterns in t(8;21) AML with or without c-kit mutations. In our series, 52.2% of the t(8;21) AMLs harbored c-kit mutations, which were correlated with poor event free survival. We next performed pyrosequencing on a selected panel of genes and pinpointed the THBS4 and PAWR genes as hypermethylated in their promoter CpG islands in 86.4% and 59.1% of the t(8;21) AML patients, respectively. These data suggest that THBS4 and PAWR may be important in the pathogenesis of t(8;21) AML. / published_or_final_version / Pathology / Master / Master of Philosophy

Acute promyelocytic leukemia (APL) is one of the subtypes of acute myeloid leukemia carrying t(15;17), and constitutes 10 to 15% of adult AMLs. One of the mechanisms of gene inactivation is hypermethylation of promoter-associated CpG islands. Cancers are characterized by global hypomethylation with locus-specific hypermethylation and hence silencing of tumor suppressor genes. Apart from tumor suppressor genes, microRNA, a class of non-coding RNA measuring 19-25 nucleotides, with tumor suppressive function is also found to be inactivated by DNA methylation in hematological malignancies. microRNAs repress target gene translation and hence expression by binding to 3'-untranslated region of corresponding mRNA. Because TP53 mutation is frequently involved in solid cancer carcinogenesis but is rarely found in APL, TP53 network may be dysregulated through epigenetic inactivation of tumor suppressor gene/miRNAs engaged in TP53 tumor suppressor network. This thesis aimed to study DNA methylation of tumor suppressor genes and miRNAs engaged in TP53 tumor suppressor network in APL. Overall survival (OS) and event free survival (EFS) of patients with or without candidate gene/miRNA hypermethylation were compared to examine their prognostic significances. Promoter methylation of DAPK1, p14ARF, miR-34a, miR-34b/c and miR-605 were studied in 10 normal bone marrow samples, NB4 cell line and 60 APL primary samples at diagnosis by methylation-specific PCR (MSP). DAPK1, miR-34a, miR-34b/c and miR-605 were completely unmethylated in normal bone marrow samples but completely methylated in NB4. Treatment of NB4 by 5'-Aza-2'-deoxyctidine (5-azadC) resulted in promoter demethylation together with re-expression of DAPK1, miR-34a, miR-34b/c and miR-605. Promoter methylation of DAPK1, p14ARF, miR-34a were absent while miR-34b/c and miR-605 methylation were detected in 43% and 10% APL samples respectively. However, methylation of miR-34b/c and miR-605 bore no prognostic significance. Overexpression of miR-34b in NB4 resulted in inhibition of proliferation. In short, methylation of DAPK1, miR-34a, miR-34b/c and miR-605 is associated with gene/miRNAs silencing. miR-34b/c is frequently methylated whereas miR-605 is methylated in small number of APL patients. miR-34b/c is a tumor suppressive miRNA in APL. Methylation of miR-34b/c may contribute to APL leukemogenesis. / published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

RUNX3是RUNX轉錄因子家族的其中一位成員。RUNX轉錄因子家族是負責調控細胞的增殖和分化。最近研究表明RUNX3可能在造血過程中扮演其中一個角色。可是，它在髓系細胞中的調節角色依然未明。此前，我們發現在核心結合因子急性骨髓性白血病中的融合蛋白RUNX1-ETO和CBFB-MYH11會抑制RUNX3基因表達，並且RUNX3表達水平對兒童急性骨髓性白血病的預後有顯著影響。本研究的目的是要調查RUNX3在成人急性骨髓性白血病的預後價值，並透過闡明RUNX3的轉錄調節去了解其在髓系細胞分化扮演的角色。 / 首先，我們透過實時定量聚合鏈反應去量化在174個成人急性骨髓性白血病的患者骨髓中的RUNX3表達，從而調查RUNX3表達與成人急性骨髓性白血病預後的關係。我們發現低RUNX3表達與較好預後的核型(P=0.045)，NPM1基因突變(P=0.014) 和較年青患者(P=0.084) 有關聯。在存活分析中，我們把有完整生存數據的非急性前骨髓性白血病病人分成高RUNX3表達和低RUNX3表達兩組。在成人急性骨髓性白血病中，高RUNX3表達和較差整體存活率(OS) (P=0.011)和無事件存活率(EFS) (P=0.003)有顯著的關聯，這和我們在兒童急性骨髓性白血病所觀察的一致。高RUNX3表達和較差存活率的關係在有野生型FLT3基因的病人中更為明顯(OS, P=0.004; EFS, P=0.001)。由於低RUNX3表達和較好預後核型有關聯，我們進一步只對擁有較差預後核型的病人作將存活分析，發現RUNX3表達仍是影響EFS的一個顯著因素(P=0.017)。在多元分析中，高RUNX3表達在所有病人(EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356)，野生v 型FLT3基因的病人(OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372)和較差預後核型的病人(EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896) 中都是一個獨立的不利預後因素。 / 接著，我們研究RUNX3基因的表達調控。我們鑒定出一個最小啟動子區對於在髓系細胞的基因表達有關鍵作用。透過預測啟動子區和轉錄因子結合位點的分析，顯示這個活性區域含有PU.1，AP-1和Sp1轉錄因子結合位點。我們透過報告基因系統研究，染色質免疫沈澱技術及電泳遷移率改變分析去闡明PU.1，c-Jun及Sp1和相對的轉錄因子結合位點參與RUNX3基因的表達調控。我們進一步透過PU.1基因剔除去證實RUNX3是PU.1的直接下遊靶基因並發現PU.1與RUNX3表達在急性骨髓性白血病人中呈正相關性。 / 由於RUNX3基因表達受到PU.1, c-Jun及Sp1的控制，我們繼續研究RUNX3在髓系細胞分化的功用。我們透過實時定量聚合鏈反應及流式細胞儀檢測發現RUNX3過度表達誘導K562細胞株作單核細胞及粒細胞分化。RUNX3能激活髓系基因的啟動子。它在成熟髓系細胞的表達水平明顯比血幹細胞為高。根據以上結果，RUNX3也許在單核細胞及粒細胞分化中有一定功能。但是，有別於其他癌細胞，RUNNX3不能在髓系細胞誘導細胞凋亡和周期阻滯。 / 總括而言，RUNX3表達在成人急性骨髓性白血病中是一個獨立的預後因素。除此之外，本研究表明RUNX3受到PU.1，c-Jun及Sp1的表達調控並在單核細胞及粒細胞分化中有一定功能。 / RUNX3 is a member of Runt-related domain (RUNX) transcription factor family, which regulates cell proliferation and differentiation. Recent studies have suggested a role of RUNX3 in hematopoiesis. However, its regulatory function in myeloid cells remains unclear. Our group previously showed that RUNX3 expression was repressed by the fusion proteins RUNX1-ETO and CBFB-MYH11 in core-binding factor acute myeloid leukemia (CBF-AML) and had prognostic implication in childhood AML patients. The aim of this study is to investigate the prognostic value of RUNX3 in adult AML patients and its role in myeloid differentiation by elucidating its transcriptional control. / To investigate the relationship between RUNX3 expression and prognosis of adult AML, RUNX3 expression in the diagnostic bone marrow samples from 174 adult AML patients were quantified by real time quantitative PCR (RQ-PCR). Low RUNX3 expression was found to be associated with favorable cytogenetic group (P=0.045), NPM1 mutations (P=0.014) and younger age (P=0.084). For the survival analysis, 110 non-acute promyelocytic leukemia (non-APL) patients with complete survival data were dichotomized into high and low expression groups. Concordant with our previous observation in childhood AML, a significant association between high RUNX3 expression and poorer overall survival (OS) (P=0.011) and event-free survival (EFS) (P=0.003) was observed. The association between high RUNX3 expression and poorer survival was further strengthened in patients with wild-type FLT3 (P=0.004 and 0.001 for OS and EFS respectively). Since low RUNX3 expression was associated with favorable cytogenetics, the analysis was next restricted to patients with non-favorable cytogenetics and RUNX3 expression remained as a significant factor for EFS (P=0.017). In multivariate analysis, high RUNX3 expression was an independent adverse prognostic factor in the whole cohort (EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356), patients with wild-type FLT3 (OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372) and patients with non-favorable genetics (EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896). / Next, the transcriptional regulation of RUNX3 in myeloid cells was investigated. A minimal promoter region was identified to be critical for myeloid-specific promoter activity. Sequence analysis of the fragment revealed potential transcription factor binding sites for PU.1, AP-1 and Sp1.The involvement of these putative binding sites and corresponding transcription factors in transcriptional regulation of RUNX3 was demonstrated by promoter reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA).Furthermore, PU.1 knockdown in U937 cells confirmed RUNX3 was a direct downstream target of PU.1 and a positive correlation between PU.1 and RUNX3 expression was observed in AML patient samples. / As RUNX3 was shown to be transcriptionally regulated by PU.1, c-Jun and Sp1, a role of RUNX3 in myeloid differentiation was postulated. Overexpression of RUNX3 induced both monocytic and granulocytic markers in K562 myeloid cells as detected by flow cytometry and RQ-PCR. RUNX3 was also found to activate myeloid-specific gene promoters and its expression was significantly higher in mature myeloid cells than in hematopoietic stem cells. This suggested a role of RUNX3 in both monocytic and granulocytic differentiation. However, unlike in other solid tumors, RUNX3 did not induce apoptosis and cell cycle arrest in myeloid cells. / In conclusion, RUNX3 expression was an independent prognostic factor in adult AML. Furthermore, our findings showed that RUNX3 was transcriptionally regulated by the master myeloid regulator PU.1 along with c-Jun and Sp1 and implicated a role in monocytic and granulocytic differentiation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Kwan, Tsz Ki. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 171-202). / Abstracts also in Chinese.