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

Chemoresistance remains a major clinical obstacle to effective management of ovarian cancer. Cancer stem cells (or tumor-initiating cells, TICs) have been discovered recently, and have played a pivotal role in changing the view of cancer development; however, the molecular mechanisms by which these cells escape conventional therapies remain elusive. In this study, TICs were isolated from ovarian cancer cells as tumor spheres with specific stem properties under TIC-selective conditions. Unlike non-TICs, TICs strongly express stem cell factor (SCF) and c-Kit. Blocking SCF-c-Kit by SCF neutralizing antibodies, c-Kit small interfering RNA (siRNA) or imatinib (Gleevec), a clinical drug that inhibits c-Kit signaling, significantly inhibited TIC proliferation. Although cisplatin and paclitaxel killed the non-TICs, they did not eliminate TICs. Importantly, the combination of cisplatin/paclitaxel with c-Kit siRNA or imatinib inhibited the growth of both non-TICs and TICs. Similar results were obtained when patient-derived TICs were used. The findings also indicate that tumor-predisposing microenvironment, such as hypoxia, may promote ovarian TICs through upregulating c-Kit expression. Furthermore, I have showed that c-Kit expression induced activation of Phosphatidylinositol 3-kinases (PI3K)/Akt, -catenin, and ATP-binding cassette G2, which could be reversed by treatment with the PI3K/Akt inhibitor or -catenin siRNA. I further studied potential gene expression in TICs using cDNA and microRNA (miRNA) microarrays. The result from these microarrays provided a general profile in gene expression of TICs compared with the bulk tumor cells. In particular, let-7a, b, and c were shown to be downregulated in TICs compared to bulk tumor cells, suggesting that their loss may contribute to ovarian cancer development. Together, this study reveals a previously undescribed therapeutic effect of SCF-c-Kit signaling blockade to prevent ovarian cancer progression by eliminating TICs and the altered genes or miRNAs may represent possible molecular targets. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph.D.) - University of Glasgow, 2007. / Thesis submitted in part fulfilment of the Ph.D. to The Beatson Institute for Cancer Research, Faculty of Medicine, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Thesis (M.D.) - University of Glasgow, 2007. / M.D. thesis submitted to the Centre for Oncology and Applied Pharmacology, Cancer Research UK Beatson Laboratories, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Studies of drug resistance mostly characterize genetic mutations, and we know much less about the phenotypic mechanisms of drug resistance, especially at a quantitative level. p53 is an important mediator that regulates the cellular response to chemotherapy, but even cancer cells with wild-type p53 exhibited variable drug sensitivity for unclear reasons. In this PhD thesis, I investigated the mechanistic basis underlying differential p53 pathway activation in response to two types of chemotherapeutics, i.e., etoposide (a DNA-damaging drug) and 5-fluorouracil (5-FU, an antimetabolites), which led to distinct cell fate outcome in drug sensitive vs. resistant cancer cells. Specifically, I uncovered a new resistance mechanism to etoposide through bimodal modulation of p53 activation dynamics and characterized a four-component regulatory module, involving ATM, p53, Mdm2 and Wip1, which generates bimodal p53 dynamics through coupled feed-forward and feedback loops. Moreover, I found that the inhibitory strength between ATM and Mdm2 determined the differential modular output between drug sensitive and resistant cancer cell lines, and that combinatorial inhibition of Mdm2 and Wip1 was an effective strategy to alter p53 dynamics in resistant cancer cells and sensitize their apoptotic response, pointing to p53 pulsing as a potentially druggable mechanism that mediates resistance to DNA damaging chemotherapy. As for response to 5-FU, preliminary results illustrated that 5-FU activated p53 and differential cell fate outcome via ribosomal stress, rather than DNA damage. Different from dose response to etoposide, 5-FU-induced p53 activity was not only regulated by p53 induction level but also p53 phosphorylation by kinases, such as DNA-PK. Overall, this thesis presented original results that elucidated phenotypic mechanism of chemoresistance and provide new angles towards developing more effective combinatorial anticancer therapy.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-104). Also available in print.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 97-108). Also available in electronic version. Access restricted to campus users.