Medical Biology - Theses
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ItemControl of the Intrinsic Pathway of Apoptosis( 2019)Apoptosis is a cellular process of programmed cell death. The intrinsic pathway of apoptosis is triggered by mitochondrial outer membrane permeabilization, a point of no return that coincides with the release of cytochrome c into the cytosol where it activates the main effectors of cellular destruction: the caspases. The mitochondrial pathway that is centered on MOMP is tightly regulated by BCL2 family proteins, which includes some members that promote apoptosis and others that inhibit it. The interplay between these proteins with opposing roles determines whether a cell will die or survive. In a healthy cell, pro-survival BCL2 proteins inhibit the effector proteins BAX and BAK. BH3-only proteins are activated in response to cellular stress and promote apoptosis by neutralizing pro-survival proteins. Targeting BCL2 proteins to provoke apoptotic cell death has proven to be a successful strategy for cancer therapy with the BCL2-selective drug venetoclax exhibiting remarkable efficacy in treating cancers that rely on BCL2 for their survival. MCL1, a protein related to BCL2, is likewise critical for the survival of many cancer cells, making it another attractive anti-cancer drug target. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials to establish their safety and efficacy. Safety is a particular concern for MCL1 inhibitors because MCL1 is also essential for the survival of many cells in critical organs and tissues throughout the body. It remains to be seen if a sufficient therapeutic window will exist when MCL1 is targeted systemically. An alternative and potentially safer strategy to modulate MCL1 survival function would be to target pathways that regulate its activity in particular contexts. In Chapter 3 and 4, I focus on one such mechanism of MCL1 regulation: its turnover by the ubiquitin proteasome system. My work in Chapter 3 elucidated details of how MCL1 protein turnover is regulated by BH3-only protein NOXA. Using CRISPR-Cas9 screen, I discovered that the mitochondrial E3 ligase MARCH5, the E2 conjugating enzyme UBE2K and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome. I also demonstrated that this pathway is constitutively active in cells where NOXA is abundantly expressed and showed that manipulating NOXA expression in those cells impacts on MCL1 survival function. Having successfully demonstrated the power of CRISPR-Cas9 screen in Chapter 3, I undertook further screens in Chapter 4 to identify proteins, such as deubiquininating enzymes (DUBs), that might serve to enhance MCL1 protein stability. I did not identify any strong hits from these screens, possibly because multiple DUBs act redundantly on MCL1. Consistent with this hypothesis, only mild impacts on MCL1 protein stability were observed upon deleting DUBs previously reported to act on MCL1. Finally, in Chapter 5, I investigated how BH3 mimetics mimic the activity of BH3-only proteins to induce apoptosis. I studied how selective BH3 mimetic compounds perturb interactions throughout the BCL2 protein network beyond their direct protein targets. I showed that these second order impacts are crucial for effective killing. Apoptosis induced by the BCL2 selective inhibitor venetoclax, for example, typically also involves inhibition of MCL1. The impact on MCL1 in this context occurs as a consequence of displacing BH3-only proteins normally bound to BCL2.
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ItemStudies of the role of Mcl-1 in haemopoiesis and leukaemia( 2015)Cell death by apoptosis plays a critical role during embryonic development and in maintaining tissue homeostasis. Consequently, defective apoptosis can lead to degenerative diseases, autoimmunity and tumour development. In mammals, there are two converging apoptosis pathways: the ‘extrinsic’ pathway, which is triggered by engagement of cell surface ‘death receptors’ such as Fas; and the ‘intrinsic’ pathway, which is triggered by diverse cellular stresses, and is regulated by pro- and anti-apoptotic members of the Bcl-2 family of proteins. The principal focus of my studies is Mcl-1, an inhibitor of the intrinsic apoptosis pathway. Mcl-1 is overexpressed in a variety of cancers, including acute myeloid leukaemia (AML) where high levels of Mcl-1 are associated with poor prognosis and drug resistance. Using mouse genetic models, I have investigated the consequences of overexpression of Mcl-1 for haemopoiesis and autoimmunity (Part I) and for the development and treatment of AML (Part II). I. To determine the impact of simultaneously inhibiting the intrinsic apoptosis pathway via overexpression of Mcl-1 and the extrinsic apoptosis pathway via a non-functional Fas receptor, mcl-1 transgenic mice were crossed with faslpr/lpr mice. The combined mutations had little impact on myelopoiesis apart from an increase in macrophages, mainly in the spleen. All major lymphoid subsets were elevated, however, including the “unusual” T cells characteristic of faslpr/lpr mice. Furthermore, the onset of autoimmune disease was markedly accelerated. Thus, consistent with other genetic studies, the intrinsic and extrinsic apoptosis pathways synergise to control autoimmunity. II. To determine the impact of Mcl-1 in AML, I used a mouse model induced by retroviral expression of MLL-AF9, the fusion oncoprotein created by the t(9;11) translocation often found in childhood and treatment-induced adult AML. Overexpression of Mcl-1 or its pro-survival relative, BCL-2, increased the leukaemic burden in the spleen and blood of sick mice although it did not accelerate morbidity. AMLs overexpressing Mcl-1 or BCL-2 tended to have a higher proportion of mature cells compared to ‘wild type’ MLL-AF9 leukaemias. Unlike ‘wild type’ MLL-AF9 leukaemias, which were readily transplantable in non-irradiated recipients, most MLL-AF9 leukaemias overexpressing Mcl-1 and many overexpressing BCL-2 would only transplant if injected into lightly-irradiated recipients. Possible reasons for this unexpected result are discussed. In vitro experiments using short-term lines derived from primary tumours demonstrated that overexpression of Mcl-1 or BCL-2 in MLL-AF9 tumours increased resistance to standard drugs used to treat AML in the clinic. However, even those overexpressing Mcl-1 or BCL-2 were sensitive to the proteasome inhibitor, bortezomib, and to various CDK inhibitors as single agents. The addition of the BH3-mimetic ABT-737 enhanced the response of MLL-AF9 AMLs of all genotypes to standard therapeutics. In contrast, when added to bortezomib or CDK inhibitors, ABT-737 only enhanced the sensitivity of the AMLs that overexpressed BCL-2. Future studies will compare the efficacy of these drug regimens in vivo in transplanted syngeneic immuno-competent mice.
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ItemBIM is critical for DNA damage-induced apoptosis and enforces tumour suppression( 2013)Apoptosis is a highly regulated, ordered form of cell death that is critical for a wide variety of physiological processes. Loss of key apoptotic regulators or other defects in apoptosis can result in either excessive or insufficient removal of cells leading to severe consequences, such as embryonic lethality, autoimmunity or cancer. The BCL-2 family of proteins are key regulators of apoptosis that control the ‘point of no return’ integrating diverse upstream signalling pathways to determine whether a cell will live or die under conditions of stress. Due to their ability to regulate cell survival, abnormalities in the expression of the BCL-2 family members are frequently observed in human cancer. Furthermore due to their ability to initiate apoptosis signalling, the BH3-only subfamily of the BCL-2 protein family are required for mediating tumour cell killing following treatment with many chemotherapeutic agents. Accordingly, mutation or loss of the BH3-only proteins, or their upstream regulators, is associated with chemoresistance and poor treatment response. The tumour suppressor p53 is a critical direct transcriptional activator of the genes encoding the BH3-only proteins PUMA and NOXA. Mutations in p53, with consequent loss of its transcriptional activity, constitute the most frequent abnormality in human cancer and are associated with poor response to anti-cancer therapeutics, particularly those that cause DNA damage. Since p53 functions upstream of the BH3-only proteins in apoptosis signalling, therapies that act independently of p53 to induce the expression of BH3-only proteins are likely to be more efficacious for the treatment of patients that harbour p53-deficient tumours. In this thesis I describe the characterisation of the mechanisms by which DNA damage can induce apoptosis in the absence of p53 function, focussing on those that are relevant to the treatment of p53-deficient tumours. To do this I generated a panel of p53-deficient thymic lymphoma-derived cell lines and determined their ability to undergo apoptosis in response to DNA damaging agents that induce different types of DNA lesions, such as γ-irradiation, etoposide and cisplatin. I have examined changes in protein expression of members of the BCL-2 family following treatment with these DNA damaging agents and identified those members of the BH3-only sub-group that are up-regulated. By generating lymphoma-derived cell lines from mice that lack both p53 and select members of the BH3-only proteins, I have been able to determine which are essential for the induction of DNA damage-induced apoptosis in the absence of p53. Through this approach I have identified novel mediators of DNA damage induced-apoptosis both in lymphoma cell lines and also in primary non-transformed cells deficient for p53. I then sought to determine whether this newly identified pathway plays a critical role in the elimination of pre-leukaemic cells that have sustained physiological DNA damage in vivo (e.g. due to replication induced stress or the stress elicited by oncogene activation). To address this cohorts of mice deficient for p53 plus candidate transducers of this pathway were aged and the lymphoma incidence was compared to those lacking p53 alone. The additional loss of this novel DNA damage induced pathway provoked a pronounced acceleration in lymphoma onset in the p53-deficient mice and also resulted in a more aggressive tumour phenotype. The importance of this tumour suppressor pathway was further characterised by ageing p53-heterozygous mice deficient for pathway members and comparing their tumour incidence to that of mice lacking only a single allele of p53; revealing a profound acceleration of tumour development in this context. BIM has been shown to be a critical tumour suppressor in the Eμ-Myc mouse model of lymphoma and recent evidence has shown that the pro-survival BCL-2 family member BCL-XL is critical to promote survival of cells undergoing neoplastic transformation in this model. To gain further insight into the processes that govern cell survival during neoplastic transformation, I performed experiments to determine whether concomitant loss of BIM would be sufficient to abrogate the delay in lymphomagenesis observed in the absence of BCL-XL. Loss of a single allele of Bim was sufficient to revert this delay and loss of both alleles of Bim provoked a further acceleration in lymphoma onset. Thus through my investigations in vitro into the manner in which apoptosis can be induced in cells lacking p53 and my studies utilising various mouse models of lymphoma development I have identified a novel apoptotic pathway important for the elimination of in p53-deficient pre-leukaemic cells and malignant lymphoma cells. These data provide new mechanistic insights into DNA damage induced apoptosis in vitro and for tumour suppression in vivo.
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ItemThe role of BOK in apoptosis and development( 2013)The intrinsic (also known as the mitochondrial or BCL-2 regulated) pathway of apoptosis is regulated by members of the BCL-2 family, which can be divided into two main groups depending on whether they posses a prosurvival or pro-death function. BAX and BAK are two key players that are directly involved in the execution of apoptosis by mediating mitochondrial outer membrane permeabilisation (MOMP), which unleashes the cascade of caspases that lead to cellular demolition. The essential overlapping roles of BAX and BAK in the intrinsic apoptotic pathway is demonstrated by the fact that many cell types deficient for both proteins are highly resistant to a broad range of cytotoxic stimuli. In addition, mice lacking both proapoptotic proteins (Bax-/-Bak-/-) displayed several developmental abnormalities (such as persistence of interdigitating cells and excess neurons in the brain), and the few survivors that lived to early adulthood all developed severe lymphadenopathy and autoimmune diseases. However, perhaps surprisingly, some organs that are thought to require apoptosis for proper morphogenesis still developed normally in these Bax-/-Bak-/- animals. This indicates the existence of BAX/BAK-independent apoptotic or other cell death processes for the removal of superfluous cells. BOK, a BAX/BAK-related protein, may play a role in cell death signaling during embryogenesis either on its own, or in a manner overlapping with BAX or/and BAK. This thesis describes the first functional analysis of BOK in embryonic development and apoptosis in a physiological context through the study of BOK knockout mice. Since many BCL-2 family members exert their effects in the hematopoietic system, the Bok-/- animals were examined with particular emphasis on their lymphoid organs. Western blot and quantitative PCR analysis demonstrated that BOK is most prominently expressed in the brain and reproductive tissues. However, BOK-deficient animals remain fertile, displayed no obvious abnormalities, and failed to develop reproducible age-related diseases up to at least one year of age. In response to diverse cytotoxic insults, leukocytes from Bok-/- mice underwent apoptosis at a normal rate. These findings demonstrate that BOK is dispensable for embryonic development, fertility, and diseases-free survival of mice. To investigate whether BOK functions in a manner overlapping with BAX or BAK, Bok-/-Bak-/- and Bok-/-Bax-/- doubly deficient mice were generated. These animals also appeared largely normal, and the additional loss of BOK on top of BAX did not exacerbate or rescue infertility in BAX-deficient males. Surprisingly, aged Bok-/-Bax-/- females were found to accumulate abnormally increased follicles at various developmental stages in their ovaries, suggesting that BOK may function together with BAX to regulate follicle atresia. In the final part of this study, Bok-/-Bax-/-Bak-/- triple knockout mice were generated to investigate whether functional overlap exists between all three proteins. This also tested for the presence of alternative cell death mechanisms in the absence of all proapoptotic multi BH-domain BCL-2 family members (i.e. BOK, BAX and BAK). Although the majority of Bok-/-Bax-/-Bak-/- mice died perinatally, a very small number still managed to survive to weaning, indicating the existence of other (perhaps still uncharacterized) processes that regulate tissue morphogenesis through cell killing or cell loss (e.g. shedding of cells into luminal structures) during certain aspects of murine development. In addition, BOK/BAX/BAK triple knockout embryos, unlike those doubly deficient for BAX and BAK (Bax-/-Bak-/-), were already present at a significantly lower than expected Mendelian frequency just before birth (embryonic day E19), implying that BOK may be required, along with BAX and BAK for normal embryogenesis. Finally, the consequences of the combined loss of all three proteins in the hematopoietic system were examined with the use of chimeric mice. Such analyses demonstrated that BAX and BAK alone are sufficient for the development and homeostasis of hematopoietic cells, whereas BOK is dispensable in these processes.
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ItemFunctional characterisation of Bcl-G( 2011)Apoptosis is a controlled and precise form of cell death necessary for maintaining tissue homeostasis and normal development. Perturbations of this cell death process contribute to a wide range of diseases such as cancer and autoimmunity. The Bcl-2 family regulates the intrinsic pathway of apoptosis and members share homology at one or more of the four different Bcl-2 Homology (BH) domains. They can be subdivided into the pro-survival proteins, the multi-domain pro-apoptotic proteins and the BH3-only proteins. The physiological functions and mechanism of action of the main players of the Bcl-2-regulated apoptotic pathway have been studied extensively using mouse models. However, the roles played by less prominent Bcl-2 family members are not as well characterised. In this thesis, we described the characterisation of Bcl-G, an evolutionarily conserved novel Bcl-2 family member implicated in cancer. Human BCL-G produces two major isoforms, BCL-GL and BCL-GS. BCL-GS only contains a BH3 domain while BCL-GL also contains a BH2 domain. While BCL-GL exhibited little killing ability, in vitro over-expression studies suggested that BCL-GS kills cells by binding BCL-XL. The mouse Bcl-G gene only produces one isoform, which contains both the BH2 and BH3 domains. Little is known about its functions and roles in apoptosis. To characterise the tissue distribution and subcellular localisation of mBcl-G, we produced monoclonal antibodies specific for this protein. Bcl-G was found to be predominantly cytoplasmic when over-expressed in HeLa cells and is present in a wide range of mouse tissues including spleen, thymus, lung, intestine and testis. Anti-Bcl-G immunohistochemistry revealed that it is expressed highly by some dendritic cell (DC) subtypes and certain epithelial cell types including those lining the gastrointestinal tract. To study the functions of mBcl-G and gain clues on its human orthologue, I generated and characterised Bcl-G-deficient mice. Bcl-G-1- mice were born viable, developed normally and had similar numbers of DCs and other immune cell subtypes when compared to their wild-type counterparts. Notably, Bcl-G-deficient cells were normally sensitive to the range of apoptotic stimuli tested. I also analysed the gastrointestinal tract of the Bcl-G-1- mice but did not observe any abnormalities in the gross morphology of the stomach, small intestine and colon. However, Bcl-G loss attenuated the colitis response to acute dextran sodium sulphate (DSS) administration, resulting in reduced immune infiltration and destruction of crypt architecture. The role mBcl-G plays in DSS-induced colitis is unclear and currently under investigation. To gain clues on the processes in which Bcl-G is involved in, I conducted a search for binding partners using two different approaches: co-immunoprecipitation coupled with mass spectrometric analysis (IP-MS) and yeast two-hybrid screening. Importantly, no Bcl-2 family member, whether it is pro-survival or pro-apoptotic, was pulled-down in either screen. In fact, in vitro over-expression studies revealed that Bcl-G’s BH3 domain was incapable of binding Bcl-XL and activating apoptosis when placed in the context of BH3-only protein Bim. These results raise doubts that mBcl-G interacts with the pro-survival proteins and is involved in the Bcl-2-regulated apoptotic pathway. Instead, Trappc6b, a subunit of the Trafficking protein particle (Trapp) complex, was identified as a potential Bcl-G binding partner. Mammalian Trapp is a multi-subunit protein complex involved in intracellular membrane traffic. This finding proposes a novel role of this Bcl-2 family member in protein trafficking and vesicle transport. In a second project, I have studied how BH3-only members activate the essential effectors Bax and Bak. Two models have been proposed, but the issue remains controversial. The indirect activation model suggests that BH3-only proteins simply neutralize all of the pro-survival proteins, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak by direct binding. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of pro-survival relatives but lost interaction with Bax. Analysis of these mice showed that Bim's pro-apoptotic activity is not solely determined by its ability to engage its pro-survival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.
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ItemThe impact of BH3-only proteins on the response of murine lymphoma to anti-cancer therapy( 2011)Apoptosis, a genetically programmed process for cell killing, is critical for the elimination of damaged or redundant cells during development, homeostasis and defence against pathogens. Defective apoptosis can promote tumour development and impair the response of tumour cells anti-cancer therapy. Apoptosis in response to developmental cues as well as a diverse range of stress stimuli is mediated by the interplay between the pro- and anti-apoptotic members of the Bcl-2 protein family. The BH3-only proteins represent one subset of pro-apoptotic proteins within the Bcl-2 family. The eight mammalian BH3-only proteins that exist are essential for initiation of apoptosis following a death stimulus, and display death stimulus-specific as well as cell type-restricted activity. Some BH3-only proteins, including Puma, Bim and Bid, can bind avidly to all pro-survival Bcl-2-like proteins and therefore have potent apoptotic activity, whilst others, such as Noxa, Bik, Bad and Bmf display more limited binding capabilities and thus more limited killing activity. Conventional cytotoxic chemotherapy drugs, such as cyclophosphamide and etoposide, that are used to treat a variety of cancers, including many haematological malignancies, act by inducing DNA damage in cells, thereby activating the tumour suppressor p53 with consequent triggering of the Bcl-2-regulated apoptotic pathway. The BH3-only protein family members puma and noxa are direct transcriptional targets of p53. Recently, Puma has been shown to play a major, and Noxa a more restricted role in DNA damage-induced p53-mediated apoptosis of normal (non-transformed) cells. Most strikingly, in thymocytes, the combined loss of Puma and Noxa was capable of conferring as much protection from DNA damage-induced killing as loss of p53 itself. How DNA damaging drugs kill tumour cells is currently still poorly defined. To define the essential requirement of the various BH3-only proteins in the response of tumour cells to DNA damage inducing drugs, gene-targeted mice lacking BH3-only proteins were crossed with Eμ-myc transgenic mice to obtain Eµ-myc lymphomas with deficiencies in one or more BH3-only proteins. Analysis of the response of these lymphomas to DNA damaging drugs in vitro and in vivo revealed that Puma was the most critical of the BH3-only proteins in apoptosis induction. Interestingly, combined loss of both p53 activated BH3-only proteins, Noxa and Puma did not result in resistance as severe as that observed with p53 deficiency and these lymphomas displayed no greater drug resistance than lymphomas lacking Puma alone. These observations contrast those reported for primary, non-transformed lymphoid cells, and demonstrate the differences in the requirements for BH3-only proteins for apoptosis induction following DNA damaging drug treatment between Myc-driven tumour cells and non-transformed lymphocytes. As the combined loss of Puma and Noxa could not recapitulate the drug resistance caused by p53 loss, the involvement of other BH3-only proteins was additionally assessed. The induction of another BH3-only protein, Bim, although not known to be a direct p53 target, was also observed following DNA damage in Eµ-myc lymphomas but not in Eµ-myc lymphomas lacking p53. Bim deficiency did not significantly alter the sensitivity of Eµ-myc lymphomas to DNA damaging drugs, however, knockdown of bim levels markedly increased the drug resistance of Eµ-myc lymphomas lacking Noxa and Puma. Remarkably, c-MYC driven lymphoma cells generated from noxa-/-puma-/-bim-/- haematopoietic stem/progenitor cells were as resistant as those lacking p53. These findings demonstrate that the combinatorial action of Puma, Noxa and Bim is critical for the efficacious killing of lymphoma cells by DNA-damaging chemotherapeutic agents and these three BH3-only proteins therefore represent biomarkers for treatment outcome in the clinic. Several BH3-only proteins have been proven to exert tumour suppressive actions in vivo. Loss of the ‘potent’ BH3-only proteins, Bim and Puma that are both capable of binding all pro-survival Bcl-2 family members, has been shown to markedly accelerate Eµ-myc lymphomagenesis, albeit to a lesser extent than p53 deficiency. Interestingly loss of the more selective BH3-only proteins, Bmf and Bad, which are capable of binding only Bcl-2, Bcl-w and Bcl-xL but not Mcl-1 or A1, have also recently been shown to accelerate Eµ-myc-induced lymphoma development. Bik, another selective pro-apoptotic BH3-only protein with similar binding specificity to Bmf and Bad, is widely expressed and has been postulated to function as a tumour suppressor on the basis that its deficiency and loss of function have been reported in many human cancers, including lymphoid malignancies. Surprisingly however, Bik deficiency did not accelerate Eµ-myc induced lymphomagenesis, indicating that in this context, Bik does not function as a tumour suppressor, at least on its own. Moreover, although p53-mediated induction of Bik has been previously documented, loss of Bik did not cause resistance in Eµ-myc lymphomas to DNA damaging drug-induced apoptosis, demonstrating that other BH3-only proteins could adequately compensate for its loss. When ectopically over-expressed, co-operation between Bik and the BH3-only protein Noxa (capable of binding avidly to Mcl-1 and A1) has been described to potently induce apoptosis and this has been attributed to their complementary binding specificities for pro-survival Bcl-2 family proteins. In contrast to these previously published reports, the combined deficiency of Bik and Noxa did not affect onset of Eµ-myc lymphoma development or alter the sensitivity of Eµ-myc lymphomas to killing by DNA damaging drugs. Together, these results suggest that at physiological levels, Bik alone, or even in combination with Noxa, is insufficient to impose a barrier to c-Myc-driven tumorigenesis, and that these two BH3-only proteins on their own do not play a significant role in the p53-mediated DNA damaging drug-induced apoptosis of lymphoma cells. The findings in this thesis highlight the necessity in understanding the crucial requirements of specific BH3-only proteins for tumour suppression, as well as for efficient killing of tumour cells by DNA damaging chemotherapeutic drugs. This knowledge has important implications for predicting treatment success or failure, and ultimately, aid the development of novel anti-cancer agents with improved therapeutic efficacy.