Medical Biology - Theses
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ItemInterrogating the cells-of-origin of BRCA mutant cancers to identify therapeutic targets for cancer prevention( 2022)It is currently estimated that approximately one woman dies every minute of breast cancer across the globe. While the greatest risk factor for developing breast or ovarian cancer is merely having female reproductive organs, the cumulative life-time risk of developing breast and ovarian cancer for women who carry pathogenic mutations in their BRCA genes is significantly higher than non-carriers. Men who harbour mutations in their BRCA genes are also at increased risk of developing breast cancer within their lifetime. Currently there are no clinically approved strategies for breast or ovarian cancer prevention in BRCA mutation carriers beyond highly invasive and irreversible surgical procedures such as prophylactic mastectomy and bilateral salpingo-oophorectomy. Targeted therapeutic strategies for cancer prevention in BRCA mutation carriers are thus a sought-after alternative. Significant headway has been made by our research group and others in identifying RANK-ligand inhibition as a putative chemoprevention strategy for the onset of breast cancer in female BRCA1 mutation carriers; subsequently, a phase 3 international clinical trial BRCA-P (ClinicalTrials.gov Identifier: NCT04711109) is currently recruiting female BRCA1 mutation carriers to assess the efficacy of RANK-ligand inhibition in preventing breast cancer development using the FDA-approved drug denosumab. A portion of this thesis describes the functional and biological consequences of denosumab treatment on the putative cell-of-origin of BRCA1 mutant breast cancer, the RANK+ luminal progenitor, from patients enrolled in the Melbourne Health BRCA-D pre-operative window study; these patients received denosumab treatments prior to undergoing prophylactic mastectomies. This work indicated that BRCA1 mutation carriers who received 1 denosumab injection per month for 3 months had significantly reduced numbers of RANK+ luminal progenitors in their breast epithelium, and these cells also displayed decreased colony forming activity ex vivo, compared to cells from untreated BRCA1 mutation carriers. This thesis also seeks to shed light on the biological mechanisms driving ovarian cancer development in BRCA1 mutation carriers, and describes novel subsets of BRCA1 mutant fallopian tube secretory cells that are putative cancer cells-of-origin. To date, there have been no prospective studies or chemoprevention trials for breast cancer development in BRCA2 mutation carriers. As such, there is a pressing need for the identification of novel therapeutic pathways for breast cancer prevention in these patients; this thesis makes several promising developments in this effort. Using preneoplastic breast tissue samples from BRCA2 mutation carriers and wildtype patients, luminal cells, including a subset of ERBB3lo luminal progenitors and mature luminal cells, were found to be expanded in breast tissue epithelium of BRCA2 mutation carriers. ERBB3lo luminal progenitors from preneoplastic BRCA2mut/+ patients were found to have increased colony forming activity ex vivo, and exhibited upregulation of genes involved in mTORC1 signalling, protein synthesis and proteostasis. Indeed, a functional protein synthesis assay revealed increased protein translation in preneoplastic luminal cells from BRCA2 mutation carriers compared to wildtype patients ex vivo. A genetically engineered mouse model of BRCA2 mutant breast cancer was used to faithfully recapitulate the preneoplastic phenotype of luminal epithelium identified in BRCA2 mutation carriers, and showed a significant delay of BRCA2mut/+ mammary tumourigenesis upon short-term treatment with an mTORC1 inhibitor in vivo. In summary, the findings detailed in this thesis describe several developments in our understanding of the mechanisms of breast and ovarian cancer development in BRCA mutation carriers, and uncover mTORC1 inhibition as a putative strategy to delay or prevent the onset of breast cancer in BRCA2 mutation carriers. Cumulatively this work provides important insights of clinical significance for women harbouring mutations in their BRCA genes.
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ItemNo Preview AvailableCharacterisation of the receptor tyrosine pseudokinases, EphB6 and EphA10( 2022)Erythropoietin-producing human hepatocellular (Eph) receptors are the largest receptor tyrosine kinase family, comprising 14 members. Like other receptor tyrosine kinases, Eph receptors consist of extracellular domains capable of ligand binding, a single transmembrane domain, and intracellular components including a tyrosine kinase domain. The cognate ligands of Eph receptors, called ephrins, are also membrane-tethered. Upon cell-cell contact, ligation of ephrins in trans results in dimerisation, oligomerisation and clustering of Eph receptors, a mechanism by which the intracellular tyrosine kinase domain autophosphorylates. The autophosphorylated Eph receptors then transmit signals to downstream effector proteins via their recruitment and phosphorylation. Typical signal outputs arising from clustered Eph receptors include cell adhesion or repulsion, depending on the cellular context. Therefore, Eph receptor/ephrin signalling is critical in embryonic development. Pathologically, deregulated Eph receptors resulting from point mutations and aberrant expression have been linked to many types of malignancies in adults. However, owing to the complexity of the activation mechanisms of Eph receptors, no therapeutics targeting Eph receptors are clinically available to date. Interestingly, two Eph receptor members, EphA10 and EphB6, are categorised as pseudokinases, as they harbour a kinase-like domain devoid of essential residues for kinase activity. Studies have suggested an oncogenic role for EphA10, and EphB6 has been proposed as a potential metastasis suppressor. Nonetheless, how these two receptor tyrosine pseudokinases exert their functions at a protein level remained largely unknown. The specific outstanding questions include: (1) How do the pseudokinase domains of EphA10 and EphB6 function? (2) Do EphA10 and EphB6 have cognate ephrin ligands, and can EphA10 and EphB6 oligomerise at the plasma membrane upon ligating to ephrins? (3) What are the signalling outputs and consequences upon binding to ephrins? This thesis aims to address these outstanding questions, with a primary focus on characterising EphB6. By applying biophysical, biochemical, structural and mass spectrometry approaches, I characterised the intracellular regions of EphB6 and EphA10, and present these studies in this thesis. The intracellular regions of EphB6 and EphA10 exhibited high conformational plasticity in solution. While the pseudokinase domains of EphB6 and EphA10 lack kinase activity, they both retained ATP binding ability, raising the possibility that they can be modulated by conventional small molecule kinase inhibitors. Furthermore, upon phosphorylation by its kinase-active cousin, EphB4, the phosphorylated EphB6 intracellular region was able to bind various Src homology 2 (SH2) domains. This suggests that, once phosphorylated, the EphB6 pseudokinase can act as a signalling hub, by recruiting adaptor and signalling proteins. To elucidate the functions of full-length EphB6, a co-culture system containing EphB6-expressing and ephrinB1-expressing cells was first established. I then employed live cell imaging, which revealed that ephrinB1 is a cognate ligand of EphB6, and is able to induce EphB6 clustering at the plasma membrane. By applying proximity-labelling techniques coupled with mass spectrometry, unique proteins enriched within clustered EphB6 were identified, implying clustered EphB6 is signalling competent and can drive cytoplasmic signal transduction. Phenotypically, clustering of EphB6 appeared to promote formation of tubules interconnecting EphB6 expressing and ephrinB1 expressing cells. By Cryo-electron tomography, our preliminary data suggested that these tubular structures consist of an unprecedented double membrane morphology, raising the prospect that clustered EphB6 may mediate a novel mode of cell-cell communication. Collectively, this thesis presents functional characterisation of EphB6 and EphA10, laying the foundation for future exploration of these two receptor tyrosine pseudokinases.
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ItemIdentification of synthetic lethal interactions with the KRAS oncogene for targeted cancer treatment( 2021)Cancer is a major public health issue globally, ranking as the second most common cause of death. Molecularly targeted therapies, focused on exploiting tumour cell dependency on certain oncogenic driver mutations for growth and survival, have greatly improved patient outcomes. However, despite these advances, some of the most frequent oncogenic mutations in cancer, such as those found in KRAS, are extremely challenging to target directly. One promising strategy to expand the range of actionable targets for cancer drug development is the exploitation of synthetic lethal interactions. Synthetic lethality is the term used to describe the death of cells in response to the co-existing disruption of two genes, neither of which is lethal alone. In this setting, targeting a gene that is synthetic lethal with a cancer-relevant mutation has the potential to induce the death of vulnerable cancer cells while leaving healthy cells unaffected. With this background in mind, my lab participated in a focused ENU mutagenesis screen in zebrafish with the aim of identifying genes that are essential for high rates of cell proliferation during endodermal organ development but not required by quiescent tissues. This yielded mutants that exhibited either ‘cell death’ or ‘growth arrest’ phenotypes in the liver, intestine and pancreas. I investigated two of the underlying mutant genes, ahctf1 and rnpc3, for their capacity to engage in synthetic lethal interactions with the kras oncogene. In Chapter 3, I investigated the impact of ahctf1 heterozygosity on the growth and survival of KrasG12V-expressing hepatocytes in a zebrafish model of hepatocellular carcinoma (HCC), TO(krasG12V). ahctf1 encodes Elys, a multifunctional nucleoporin with essential roles in nuclear pore assembly and mitosis. I found that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of Tp53-dependent and Tp53-independent cell death pathways which reduced tumour burden. Importantly, ahctf1 heterozygosity did not impact normal liver development, advancing ELYS as an attractive target for cancer therapy with a viable therapeutic window. In Chapter 4, I examined if rnpc3 heterozygosity also reduced tumour burden in the TO(krasG12V) model. rnpc3 encodes 65K, a unique protein component of the U12-dependent spliceosome, a specialised splicing machinery required for the correct splicing of a very small percentage (3.7%) of genes. In hepatocytes expressing krasG12V, rnpc3 heterozygosity reduced the number of cells in S phase of the cell cycle and increased cell death, together reducing tumour burden, without affecting normal tissue. In Chapter 5, I demonstrated that the zebrafish model of HCC is a powerful platform for testing novel therapeutics. I evaluated the efficacy of PRMT5 and KAT6A/B inhibitors early in their development, and showed that they were effective in reducing tumour growth and worthy of future investigation. In conclusion, my studies revealed two promising new targets for cancer treatment. I also demonstrated that the zebrafish HCC model is highly amenable to pharmacological inhibition and provides a valuable system for the pre-clinical examination of drug treatments in vivo.
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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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ItemTargeting regulators of natural killer cell homeostasis in cancer immunotherapy( 2018)The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory receptors and from cytokines such as IL-15. The ability to recognise tumour cells in the absence of antigen presentation has garnered significant interest in NK cells as novel targets for immunotherapy development. However, successful developments in this area have led to limited success. This is, in part, due to the lack of understanding of the underlying mechanisms governing inhibitory and stimulatory pathways in NK cells. In this work, we aimed to identify the key regulator of NK cell proliferation in order to further our understanding of NK cell activity both in the steady-state and in the setting of inflammation. Here, we have identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signalling in NK cells. IL-15 is the main driver of NK cell proliferation, survival, differentiation and function, and thus a highly relevant checkpoint in NK cell homeostasis. We found Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFNγ production and cytotoxicity toward tumours, in vitro. This was associated with increased JAK-STAT signalling in Cish-deficient NK cells. Cish-deficient mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity, uncovering CIS as a potent intracellular checkpoint in NK cell-mediated tumour immunity. Under homeostatic conditions, phenotypic changes in NK cells lacking Cish were observed in vivo. This included an increase in terminally differentiated NK cells as well as increased expression of cell cycle markers, suggesting that under steady-state conditions, CIS also plays a role in maintaining IL-15 driven regulation of NK cells in vivo. Additionally, the changes observed in steady state Cish-deficient NK cells manifested in a lower activation threshold, evidenced by the redundancy of exogenous IL-15 to induce augmented production of inflammatory cytokines and cytotoxicity when stimulated ex vivo. These data suggest that Cish not only regulates NK cell responsiveness to IL-15, but may also play a role in maintaining an activation threshold, consequently regulating effector functions in vivo. Furthermore, inhibition of CIS was found to be conserved between human and mouse NK cells, emphasising its potential role as a novel immunotherapy target for the treatment of human cancer.
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ItemHow do cytokines promote gastrointestinal cancer?( 2016)Gastric cancer is one of the most common cancers in the world, and is the third most common cause of cancer related mortality. Due to the largely asymptomatic nature of this disease, patients are often diagnosed only when the cancer has undergone metastasis, for which there are limited treatment options. While our understanding of the genetic landscape of gastric cancer is still in its infancy, there is clear evidence that chronic inflammation contributes to its onset and progression. Inflammatory cells present in the tumour microenvironment are a major source of numerous cytokines, many of which have been associated with the development of colorectal cancers. For this reason, targeting pro-inflammatory cytokines, as a means to modulate cancer growth, has been of great therapeutic interest. The Th17 cell-associated cytokines IL-17A, IL-22, and IL-23 are elevated in gastric cancer patients. However, there have been limited studies exploring the contribution of these cytokines to gastric tumorigenesis. The first aim of this thesis therefore involved the analysis of Gp130F/F mice, which represent a mouse model of inflammation-associated intestinal-type gastric cancer, following ablation of Il17a, Il22, Il23a, Il22ra1, or the Th17 cell transcription factor Rorc and demonstrate stage-specific roles for each of these cytokines during gastric tumour development and progression. Loss of IL-17A promoted tumour growth, but did not affect tumour burden during late-stage disease. In contrast, IL-22RA1 deficiency exacerbated early-stage tumour development, while IL-23A and RORγT were dispensable for disease onset and progression. The second aim of this thesis investigated the role of IL-1β and IL-18, which are elevated in the serum of gastric cancer patients. Although the role of these cytokines has been well characterised in other inflammation-associated cancers including colorectal cancer, the contribution of these two cytokines has not been directly compared in gastric cancer. For this reason, Gp130F/F mice deficient for Il1r1 or Il18 were generated. Gp130F/F: Il1r1-/- compound mutant mice displayed a small reduction in tumour growth, but no change to tumour burden, during late-stage disease, while Gp130F/F: Il18-/- mice had reduced tumour burden and growth compared to Gp130F/F mice during late-stage disease. Additionally, tumours from Gp130F/F: Il18-/- mice displayed a significant reduction in macrophage number, which preceded the reduction in tumour burden. These results suggest that IL-18 may have a dominant role over IL-1 signalling in tumorigenesis in this model. The final aim of the work presented was to explore the relationship between the cytokines IL-11 and IL-22 in the colon, which have protective effects in various models of intestinal damage. The latter is associated with induction of genes that promote cellular proliferation and survival, and in the case of IL-22, the induction of anti-microbial peptides and mucus-associated genes. Given the importance of both cytokines in maintaining epithelial barrier integrity, and the fact that IL-22 can induce IL-11 expression in the gastrointestinal tract, we directly compared the effect of loss of both cytokines during intestinal disease. Following irradiation to study epithelial regeneration, we demonstrated comparable cell proliferation between Il11ra1-/-: Il22ra1-/- and WT mice. In DSS-induced colitis, we observed a significant increase in disease severity, as determined through endoscopy monitoring and by a reduction in colon length on autopsy, in Il22-/- and Il11ra1-/-: Il22ra1-/- mice compared to WT mice, although no significant differences were observed between Il22ra1-/- and Il11ra1-/-: Il22ra1-/- mice. These results suggest that the loss of both IL-11 and IL-22 signalling does not result in catastrophic intestinal damage. Taken together, this study expands our knowledge about the role of cytokine signalling in gastric cancer, and suggests that they have stage specific roles in either promoting or protecting against tumour development and progression. These findings will have implications for the therapeutic utility of cytokine inhibition. This study has also begun to explore the importance of loss of multiple cytokine signalling pathways to intestinal regeneration during injury, and suggests that loss of both IL-11 and IL-22 signalling does not result in fatal intestinal damage. Together, these results suggest that cytokines have evolved different roles that are dependent on both the timing and location of cytokine expression, receptor expression, and subsequent activation of downstream signalling in the gastrointestinal tract.
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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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ItemTumour suppression by p53 and therapeutic targeting of the p53-MDM2 interaction( 2014)The transcription factor p53 plays an essential role in tumour suppression and in the way that cancer cells respond to DNA damage-inducing anti-cancer therapies. Activated in response to a diverse range of cellular stresses p53 can coordinate the expression of ~600 targets genes. Through this p53 is able to regulate activity in a broad range of downstream effector pathways including; cell cycle arrest, cellular senescence, apoptotic cell death, coordination of DNA repair and coordination of metabolism. Many conventional anti-cancer strategies act through induction of extensive DNA damage in tumour cells. Such therapeutics limit cancer cell growth and promote tumour regression, in a large part, through DNA damage-induced activation of p53 and its downstream effector pathways e.g. apoptosis (via PUMA and NOXA), cell cycle arrest and cellular senescence (via p21). Concerns that genotoxic strategies for anti-cancer treatment may lead to further mutations within cancer cells (leading to treatment resistance) or in non-cancerous tissues (leading to secondary cancers) has led to the development of novel anti-cancer therapeutics that can activate p53 via non-genotoxic mechanisms. Small molecule inhibitors of MDM2, such as Nutlin3a, are the first of such compounds to enter clinical trials. Utilising gene-targeted mice lacking p21, PUMA and/or NOXA, I have shown that PUMA- (and to a lesser extent NOXA-) mediated apoptosis and not p21-mediated cell cycle arrest and/or senescence is essential to drive treatment responses to Nutlin3a in both non-transformed lymphoid and malignant Eμ-Myc lymphoma cells. In addition to its fundamental role in cellular responses to DNA damage, p53 has been shown to play an essential role in the suppression of cancer development, with its loss frequently observed in human cancers (e.g. Burkitt’s lymphoma) and in animal models of MYC-driven lymphomagenesis (e.g. Eμ-Myc mice). Apoptosis induced downstream of p53 was originally thought to be the sole effector process required to limit MYC-driven lymphomagenesis. However loss of PUMA fails to completely remove the requirement for mutation/deletion of p53 in Eμ-Myc lymphoma or accelerate lymphoma onset in mice to the same extent as loss of a single allele of p53. Together these data suggest that processes in addition to PUMA- (and NOXA-) mediated apoptosis are required for suppression of MYC-driven cancers. The CDK inhibitor, p21, has been suggested to be critical effector of tumour suppression downstream of p53. To determine if p21 and PUMA cooperate in p53-mediated suppression of MYC-driven lymphomagenesis I generated Eμ-Myc mice lacking p21 (or both p21 and PUMA). Surprisingly, lymphoma onset in Eμ-Myc;p21-/- mice was profoundly delayed. This suggests that within this context p21 acts to promote, rather than suppress, lymphomagenesis. The delay in lymphoma onset observed in Eμ-Myc;p21-/- mice was largely overcome with the concomitant loss of PUMA, suggesting that induction apoptosis is required for the survival advantage observed in these mice. Cell cycle arrest mediated by p21 may promote lymphoma development by allowing nascent cancer cells to arrest and repair damage to DNA. This process if impaired, e.g. by deletion of p21, may allow continued replication of cells bearing aberrant changes to their DNA, and this may in turn trigger clearance of these cells by PUMA-mediated apoptosis. Loss of p53 protein activity, either directly, e.g. through deletion or mutation of p53, or indirectly, e.g. through over-expression of MDM2, is observed in almost all human cancers. Interestingly, the majority of mutations to p53 observed in human cancer occur within the p53’s DNA binding domain and thus impair its transcriptional activity. This suggests that p53’s ability to block early “cell-transforming” events is critically linked to its ability to modulate transcription of its ~600 target genes. Given p53-mediated transcriptional induction of PUMA, NOXA and p21 (and hence induction of apoptosis, cell cycle arrest and/or cellular senescence) is critical for p53-mediated responses to DNA damage (and enforced oncogene expression), it was widely believed that these same processes would also be essential for p53’s ability to suppress spontaneously forming cancers. In order to gain concrete insight into whether apoptosis (via PUMA and NOXA) and/or cell cycle arrest and/or senescence (via p21) are required for p53-mediated suppression of spontaneous tumour development, I generated mice completely lacking all three of these critical effectors. In stark contrast to p53-deficient mice, which all succumb to spontaneous thymic lymphoma and/or sarcoma development by 250 days of age, p21-/-Puma-/-Noxa-/- mice remain tumour free for up to 500 days. These results demonstrate beyond doubt that suppression of spontaneous tumorigenesis by p53 does not require PUMA/NOXA-mediated apoptosis or p21-mediated cell cycle arrest and cellular senescence. The discovery that processes are not essential for p53-mediated tumour suppression (despite their critical importance for cellular responses to acute DNA damage and enforced oncogene expression) re-launches the search for the p53 target genes and effector processes that are critical to prevent cancer development. Gaining insight into the mechanisms by which p53 suppresses early “cell-transforming” events will have important ramifications for the development of novel strategies for cancer therapy and possibly even cancer prevention.
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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.