The finely tuned regulation of haematopoietic stem and progenitor cells (HSPCs) is crucial to sustain normal haematopoiesis. The disruption of the balance between the quiescence state of haematopoietic stem cells (HSCs) and the proliferation/differentiation programs that are necessary to meet daily haematopoietic demands and respond to external insults, can lead to malignant transformation, such as acute myeloid leukaemia (AML). Therefore, it is essential to investigate the players that are responsible to maintain haematopoietic homeostasis, so that novel therapeutic targets can be identified. HSCs reside in a hypoxic environment that is crucial for their maintenance, as it protects them from over-proliferation and exhaustion. The response to a limited availability of oxygen is critically mediated by a transcription factor - hypoxia inducible factor (HIF). HIF is predominantly regulated by prolyl hydroxylases (PHDs) that are 2-oxoglutarate (2OG) dependent oxygenases. This superfamily of oxygen-sensing enzymes has been assigned important roles ranging from hypoxia signaling, DNA repair, chromatin modifications and oncogenesis Following the data published by our group attesting that HIF is dispensable for HSC survival and maintenance, we focused our investigation on HIF-independent pathways. This manuscript describes the study of the role of an oxygen-sensor enzyme, member of the 2OG oxygenases and HIF negative regulator, jumonji domain-containing protein 6 (Jmjd6), in normal and malignant haematopoiesis. Our knockout studies deleting Jmjd6 specifically within the haematopoietic system (Jmjd6fl/fl;Vav-iCre) demonstrate that the homeostasis of HSPC pool was compromised and lymphopoiesis was attenuated in Jmjd6-deficient cohorts. Upon transplantation, HSCs lacking Jmjd6 exhibited a defective chimerism and impaired capacity to fully reconstitute haematopoiesis of recipient mice. Thus, Jmjd6 is essential for HSC self-renewal and maintenance. Our assessment of the impact of Jmjd6 deletion in the context of inflammatory response and recovery from treatment with a myelotoxic agent treatment revealed that Jmjd6 is a positive regulator of HSC homeostasis and recovery from cytotoxic stress. There are accumulating data on the importance of epigenetics in the development of haematological malignancies. Being an epigenetic regulator, clearly involved in RNA splicing, we investigated Jmjd6 as possible player in leukaemogenesis. The results from our leukaemic studies unravelled a new biological function for Jmjd6 as a tumour suppressor in Meis1/Hoxa9 murine model. Altogether, our findings offer important novel insights into the biological functions of Jmjd6 and pave the way for further studies to discover on the mechanism of action of this complex enzyme. Our observations add value to the idea that Jmjd6 might constitute a good candidate for cancer diagnosis, that can be use to ameliorate patient’s prognosis and that it can be used to help patient prognosis in the future.

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.

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 myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts in circulation and/or bone marrow. Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene occurs in about 30% of AML and is associated with an inferior prognosis. Tyrosine kinase domain (TKD) mutations occur in about 5% with uncertain prognostic significance. Intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However these approaches have reached a deadlock with a cure rate of 30-40%. Targeting FLT3 in AML with multi-tyrosine-kinase inhibitors has been evaluated in Phase II/III clinical trials. Despite an initial clearance of myeloblasts, the leukemia invariably progresses despite continuous treatment. The mechanisms of drug resistance and leukemia progression, hence the effective therapeutic strategies are currently unknown, limiting its clinical application. These issues were addressed in the present study. In the first part, 13 patients with chemo-refractory or relapsed FLT3-ITD+ AML received sorafenib 200-400 mg twice daily of whom 12 patients achieved clearance or near clearance of bone marrow blasts after a median of 27 days (range 21-84 days). There was evidence of myeloid differentiation of the leukemia blasts at remission. Leukemia progression occurred in 9 patients after a median of 72 days (range 54-287 days) and in 4 out of 6 patients it was dominated by clones carrying double FLT3-ITD and -TKD mutations. Microarray studies comparing myeloblasts before sorafenib treatment (sorafenib naïve) and at subsequent progression (sorafenib resistant) demonstrated up-regulation of 64 genes including ALDH1A1, JAK3 and TESC whose functions were unknown in AML. Transplantation of sorafenib naïve and resistant myeloblasts into NOD/SCID mice recapitulated their clinical behavior when the animals were treated with sorafenib. Both ITD and TKD mutations at D835 were identified in leukemia initiating cells (LICs) from sorafenib naïve samples. These results suggested that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations. In the second part, the gene encoding tescalcin (TESC), that was up-regulated at sorafenib resistance and was known to activate a sodium/hydrogen exchange (NHE1), was evaluated to examine its link with TKI resistance. TESC was highly expressed in FLT3-ITD+ AML cell lines MOLM-13 and MV4-11 and its knock-down by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene)amiloride (HMA). Induction of sorafenib resistance in MOLM-13 cell line (MOLM-13-RE) significantly increased its sensitivity to HMA. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD+ AML cells significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. Normal CD34+ cells engraftment was not affected. HMA treatment significantly enhanced suppression of FLT3 signaling by sorafenib even in sorafenib resistant cell lines. These observations provided novel information about the pathogenetic role of TESC-NHE1-pHi in sorafenib resistance in AML. In conclusion, the information derived from the present study has provided mechanistic insights to the emergence of drug resistance during sorafenib treatment and important guide for future therapeutic strategies targeting FLT3-ITD+ AML. / published_or_final_version / Medicine / Doctoral / Doctor 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

Background: Chronic myeloid leukaemia (CML) is a haematological malignant disease involving haematopoietic stem cells. It is caused by a known reciprocal chromosomal t(9;22)(q34;q11) translocation, or known as Philadelphia chromosome. The translocation results in the formation of a chimeric BCR-ABL fusion gene. In the most recent guidelines published by NCCN and European LeukemiaNet in 2013, tyrosine kinase inhibitors (TKI) specifically inhibiting the Bcr-Abl tyrosine kinase, are the first-line therapy for patients with chronic phase CML. Imatinib is the oldest among the 3 TKI, and is the most commonly prescribed. Despite its proven therapeutic role in CML, imatinib is a drug of extreme high cost. Estimated annual drug cost is HKD$223,380for a standard 400mg adult daily dose. Therefore, this study aims to survey on the prescribing pattern of imatinib in CML patients, its funding status, response; and estimate its economic burden on the Hong Kong population. Methodology: This is a retrospective patient chart review study. All patients who were diagnosed with CML from 2003 to 2012 and were managed in QMH or QEH were reviewed. Electronic records were retrieved to see whether imatinib was started as first-line treatment within 6 months of diagnosis. The reasons for not initiating imatinib were also investigated. Patients’ response to imatinib, and funding source for the drug, were documented. Annual drug cost of imatinib was estimated from all CML patients who attended all Hospital Authority institutions in 2012 who were prescribed with the drug. Results: Total 153 patients from the 2 institutions were reviewed. One hundred twenty four (81%) of them started imatinib as first-line therapy within 6 months of diagnosis. Nine patients started second generation TKI as first-line. Among those who did not start TKI, the most common reasons are patient preference (3.9%) and financial difficulties (3.3%). Twelve paediatric patients are identified, and all but one of them started imatinib. Seventy one% patients on imatinib experienced side effects. Most frequently reported adverse reactions are thrombocytopenia, oedema and neutropenia. Twenty eight% switched to second generation TKI due to suboptimal response or intoleranceto imatinib. During their course of treatment, 46.3% patients on imatinib require social subsidy from Samaritan Fund. From the dispensing records, the average drug cost per patient per year is HK$113,902. The estimated annual cost burden on the whole Hong Kong population is HK$43,425,878. Conclusion: The prescribing rate of imatinib in chronic phase CML patients in Hong Kong is comparable to overseas prescribing rate. The drug has become a significant financial burden to patients’ family and the society as a whole. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences

Thesis (Ph. D.)--University of Adelaide, Dept. of Medicine, 1998. / Includes bibliographical references (leaves 91-126).

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine; and, Institute of Medical and Veterinary Science, Division of Haematology, 2005. / Title from title page of source document; viewed 19 July 2005. Bibliography: p. 157-184 of source document. Also available in print.