Milani, M
(2019)
Exploring the therapeutic potential and cellular mechanisms of BH3 mimetic-mediated apoptosis in cancer.
PhD thesis, University of Liverpool.
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Abstract
Evasion of apoptosis is one of the main hallmarks of cancer. The BCL-2 family of proteins are the main mediators of the intrinsic pathway of apoptosis and the interactions between the pro- and anti-apoptotic members of this family of proteins control the fate of a cell towards life or death. Anti-apoptotic members of the BCL-2 family, such as BCL-2, BCL-XL and MCL-1, are frequently overexpressed in a plethora of cancers promoting abnormal cell survival. During the last ten years, several small molecule inhibitors called BH3 mimetics were developed to inhibit these anti-apoptotic proteins. The potency and specificity of the BH3 mimetics make them valuable tools for targeting the pro-survival pathways in different cancers. The first known bona fide BH3 mimetic, ABT-737, has now been replaced by more selective inhibitors, such as ABT-199 (BCL-2-specific inhibitor), A-1331852 (BCL-XL-specific inhibitor), A-1210477 and S63845 (both MCL-1-specific inhibitors), all of which have been employed in the different experiments detailed in this thesis. ABT-199 has been successfully used in the treatment of haematological malignancies, such as chronic lymphocytic leukaemia. Experiments are now in progress to reproduce this success using other BH3 mimetics in a wide variety of cancers. This study is aimed at exploring (1) whether BH3 mimetic therapy can help improve on current therapy in certain haematological malignancies, (2) whether BH3 mimetics can be used as single agents or in combination with existing therapy to target certain solid tumours, and (3) the underlying cellular and molecular mechanisms by which BH3 mimetics induce apoptosis. In order to explore the therapeutic potential of BH3 mimetics in haematological malignancies, the cancer types chosen for this work were chronic and acute myeloid leukaemia (CML and AML). Tyrosine kinase inhibitors (TKIs) are the preferred drugs for treating CML, whereas AML is treated with anthracyclines such as daunorubicin. Using a series of genetic knockdown experiments, it was identified that CML is a BCL-XL dependent malignancy and that targeting BCL-XL with A-1331852 resulted in rapid and extensive apoptosis not only in the established CML cell lines but also in primary patient samples. In marked contrast, using a panel of AML cell lines, it was shown that AML cell lines and patient samples demonstrated a dual dependency on BCL-2 and MCL-1 for cell survival, which was overcome by a combination of ABT-199 and S63845. These observations were extended to solid tumours, with a focus on head and neck squamous cell carcinoma (HNSCC) and pancreatic ductal adenocarcinoma (PDAC). Unlike the dependency on BCL-XL for CML and BCL-2 and MCL-1 for AML, these solid tumours demonstrated a dual dependency on BCL-XL and MCL-1 for survival. In agreement, tissue microarray (TMA) analysis revealed high expression levels of both BCL-XL and MCL-1 in the tumour cores of these cancer patients. Furthermore, dual inhibition of these BCL-2 family proteins by a combination of A-1331852 and S63845 induced cell death and reduced clonogenic potential of several cell lines derived from these solid tumours, indicating that a combination of these inhibitors could be valuable in treating certain solid tumours. A closer analysis of the intracellular effects caused by exposure to BH3 mimetics indicated that BH3 mimetic-mediated apoptosis was accompanied by mitochondrial ultrastructural changes. BH3 mimetics induced extensive mitochondrial fragmentation, which was dependent on the mitochondrial fission machinery, of which DRP-1 is the major player. It was shown that BH3 mimetic-mediated induction of key steps of the intrinsic pathway of apoptosis, such as cytochrome c release and BAK activation, were dependent on DRP-1. This strongly suggested a linkage between the regulation of mitochondrial membrane dynamics with the mechanisms that control cell death. Assessment of the involvement of the endoplasmic reticulum (ER) in BH3 mimetic-mediated mitochondrial membrane dynamics and cell death uncovered new roles for ER shaping proteins, such as RTN-4 and CLIMP-63. Genetic silencing of these proteins indicated that, similar to DRP-1, ER shaping proteins, play an important role both in mitochondrial fission and also in the effective induction of BH3 mimetic-mediated apoptosis. Overall, this study highlights the potential of BH3 mimetics as therapeutic intervention for haematological malignancies, such as CML and AML, as well as for hard-to-treat solid tumours, such as HNSCC and PDAC. In addition, this study underscores the importance of assessing unanticipated effects of BH3 mimetics, such as mitochondrial fission. Finally, this study sheds light on how ER and mitochondrial membrane dynamics regulate key steps of the intrinsic pathway of apoptosis, by showing for the first time that ER shaping proteins can regulate mitochondrial fission and cell death.
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences > School of Medicine |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 13 Dec 2019 14:56 |
| Last Modified: | 19 Jan 2023 00:28 |
| DOI: | 10.17638/03052911 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3052911 |
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