Sir Peter MacCallum Department of Oncology - Theses

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    Mutant p53 in prostate cancer: novel therapeutics and a clinically relevant model
    Mejia Hernandez, Javier Octavio ( 2022)
    Mutation of the tumour suppressor p53 is a key driver of lethal prostate cancer (PrC). TP53 missense mutations may confer pro-survival functions following the accumulation of mutant p53 (mt-p53) protein in cells. Strategies to directly target mt-p53 have proven challenging, which led us to search for alternative methods. Using a shRNA knockdown system, we demonstrated that depleting MDM4 (a key negative regulator of wild-type p53) inhibited the growth of PrC cells harbouring mt-p53 and also those lacking p53. Our studies revealed insights into the mechanisms of response that underly growth inhibition upon MDM4 targeting in different PrC cell lines. Our preclinical data showed that MDM4 targeting was potentiated in mt-p53 PrC by the clinically relevant drug eprenetapopt (APR-246), while at equivalent doses it did not enhance the response in p53 null PrC. A rational approach to identifying the value of new treatment strategies, such as MDM4 inhibition, is to test them in animal models of PrC that reflect human disease and are clinically relevant. To this end, we developed an immune-competent model of spontaneous PrC, harbouring mt-p53 and Pten loss, which are genetic alterations common in PrC in men. Studying the role of mt-p53 in tumour initiation and progression in an immunocompetent context is important, as p53 has decisive roles in inflammation and immunosuppression in PrC. In this transgenic murine system, missense Trp53 missense mutations are knocked-in and Pten knocked out, through the prostate-specific Probascin promoter. This model is shedding light on the contribution of p53 genetic makeup to PrC onset and progression in men in the context of PTEN loss. Our data from this model showed that the influence of mt-p53 on the prostate immune landscape is clinically relevant.
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    Exploration of the clinical utility of circulating tumour DNA and immune-related biomarkers in oesophageal cancer
    Cabalag, Carlos Suhady ( 2022)
    Biomarkers, which are quantifiable biological processes, analytes, or structures that are predictive of treatment response or survival, are utilised in the care of cancer patients to facilitate treatment selection. This thesis examines the clinical utility of novel biomarkers to improve management of patients with oesophageal cancer (OC). The standard treatment for patients with locally advanced OC is a combination of chemotherapy with or without radiation followed by surgical resection. A major factor in determining the treatment of OC patients is the tumour stage, defined as the extent by which the tumour spreads to lymph nodes and other organs. Investigations used routinely for the staging of OC include computerised tomography (CT), positron emission tomography (PET) and diagnostic laparoscopy with peritoneal cytology. Limitations in current staging modalities are their inability to detect micro-metastatic disease and their poor sensitivity and specificity related to inter-observer variability. Compared to CT and PET, results from this thesis demonstrate that a patient’s circulating tumour DNA (ctDNA) status at diagnosis more accurately reflects their risk of recurrent cancer. A positive ctDNA status in node-negative patients (based on imaging) identifies a subset of patients with inferior prognosis, suggesting the presence of micro-metastatic disease not identified by imaging alone. The need for oesophagectomy in patients with a pathological complete response (pCR) to neoadjuvant treatment is currently being investigated. The diagnostic modalities used to assess tumour response following neoadjuvant therapy have limited accuracy in predicting pCR. This thesis shows that an absence of ctDNA after neoadjuvant treatment was better correlated to pCR when compared to metabolic tumour response on PET. In addition, this thesis also explores the changes in the immune landscape of OC patients following neoadjuvant treatment, with the aim of identifying immune-related prognostic biomarkers. The analysis of transcriptomic changes with immunohistochemical correlation has identified that high CD15+ tumour infiltrating neutrophils and high neutrophil to lymphocyte ratio (NLR) are prognostic for early recurrent disease within 2 years following treatment. These results were utilised to create a clinical risk prediction tool in the form of a nomogram, which was validated using an independent cohort of patients. Furthermore, these findings reveal a possible mechanistic explanation implicating neutrophils in the dissemination of metastatic disease, thus opening up a potential new therapeutic avenue. Lastly, with the advent of novel adjuvant systemic treatments, such as immune checkpoint inhibitors, that significantly prolong overall survival, the early detection of recurrent disease following curative intent treatment is a current area of focus. This thesis prospectively followed a series of patients post curative-intent treatment with ctDNA assays. Importantly, patients with positive ctDNA status have inferior progression-free survival compared to those with negative ctDNA status. For a number of patients, the detection of positive ctDNA status preceded clinical symptoms and radiological confirmation of metastatic disease. Collectively, these results demonstrate the potential of utilising ctDNA as a biomarker for early detection of recurrent disease in OC, and thus may guide patient selection for adjuvant treatment. In summary, the discovery of novel biomarkers in this thesis has the potential to enhance tumour staging accuracy and prognostication from the early diagnostic phase to the post-treatment phase of the patient journey with OC. The use of these biomarkers can facilitate individualised patient treatment selection in order to minimise harm and maximise therapeutic efficacy.
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    Understanding Genomic Mechanisms of Venetoclax Resistance in Haematological Malignancy
    Blombery, Piers Alexander ( 2022)
    Resistance to targeted therapies is an important cause of treatment failure in haematological malignancy. Venetoclax is one of the most effective targeted agents in chronic lymphocytic leukaemia (CLL) and lymphoid malignancy developed to date. However despite its efficacy, both primary (intrinsic) and secondary (acquired) resistance still occurs and is an important cause of treatment failure. This thesis presents data describing the first mechanism of secondary resistance identified in patients with CLL – the acquired Gly101Val mutation in BCL2. This mutation is shown to result in an approximately 180-fold reduction in venetoclax affinity for BCL2 as well as imparting resistance in cellular models. In addition, further mechanisms of secondary resistance to venetoclax are also revealed including multiple other BCL2 mutations, BCL-xL overexpression and MCL1 amplification. An exploration of the relationship between these resistance mechanisms is also presented including data at a single cell level demonstrating that oligoclonality is a dominant theme on progression on venetoclax. Given the importance of BCL2 mutations in resistance in CLL, follicular lymphoma was then studied given the characteristic presence of BCL2 mutations as a result of aberrant somatic hypermutation (aSHM) resulting from the canonical IGH-BCL2 fusion. The data presented establish that functionally relevant BCL2 mutations may exist at baseline before treatment and that overt venetoclax resistance mutations in BCL2 may emerge with venetoclax treatment. In addition these data show that clonal complexity involving BCL2 may exist outside the biopsied site and is identifiable on analysis of circulating tumour DNA. These data contribute to our understanding of the relatively high primary resistance of follicular lymphoma to venetoclax and identify causes of secondary treatment failure. Finally haematological adaptation is explored in the off-target myeloid compartment in patients with CLL treated with venetoclax. The data presented provide evidence of adaptation in the myeloid compartment through the acquisition of deleterious mutations in BAX as well as the phenomenon of lineage-specific mechanism of venetoclax adaptation through the observation of co-existing BCL2 mutations in the relapsing CLL compartment. Together the data in this thesis provide significant insights into the adaptation of CLL, follicular lymphoma and normal myeloid cells to selective pressure from venetoclax therapy and have important implications for our use of this highly effective targeted agent in the clinic.
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    Mechanisms of FOLFOX Resistance in Metastatic Colorectal Cancer
    Behrenbruch, Corina Camille ( 2022)
    Over half of patients diagnosed with colorectal cancer develop liver metastases during the course of their disease. In patients with metastases isolated to the liver, surgical resection offers the only chance of cure, improving 5-year survival rates from 13% with chemotherapy alone, to 33-55% if all sites of disease can be resected. A substantial proportion of patients receive neoadjuvant FOLFOX (5-Fluorouracil/oxaliplatin/leucovorin) prior to surgery, either to downstage to enable surgical resection or reduce the risk of recurrence. Unfortunately, the majority of patients progress during treatment or recur following liver resection. Despite its widespread usage and toxicity, the molecular mechanisms that contribute to FOLFOX resistance remain poorly understood. Improved understanding of FOLFOX resistance could enable better patient selection and/or the identification of combination regimens that may improve response rates and survival outcomes. In this thesis, a biobank of patient-derived tumour organoids (PDTOs) from patients with metastatic colorectal cancer (mCRC) was generated to study resistance mechanisms to FOLFOX. Using a combination of phenotypic, transcriptomic and genomic analyses of both tumour samples derived from patients with mCRC and matching PDTOs, a novel FOLFOX resistance signature was identified. Resistant PDTOs, identified after in vitro exposure to FOLFOX, manifested elevated expression of genes involved in the E2F pathway as well as activated TP53-independent S phase, G2/M and spindle assembly checkpoints (SAC). Colorectal liver metastases collected from patients with progressive disease during neoadjuvant FOLFOX demonstrated similar molecular features, highlighting the relevance of this finding. In parallel, results of a large (n=423 compounds), PDTO kinase inhibitor screen indicated that drugs targeting regulators of the DNA damage response, G2M checkpoint and SAC had cytotoxic effects on PDTOs generated from patients whose disease progressed during FOLFOX treatment. The combined results of this thesis indicate that targeting DNA damage induced and TP53-independent S-phase or G2M cell cycle checkpoints may significantly improve response rates to FOLFOX in patients with mCRC.
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    Discovery and validation of novel ovarian carcinoma predisposition genes
    Subramanian, Deepak Naga ( 2021)
    Epithelial ovarian carcinoma (EOC) has a significant hereditary component, over half of which cannot be explained by known hereditary breast and ovarian cancer (HBOC) genes (e.g. BRCA1 and BRCA2). Gene discovery studies to date have generally been limited to a small number of candidate genes in relatively few families and have failed to consistently identify any compelling new genes that may account for this missing heritability. The underlying hypothesis of this thesis is that the remaining unexplained EOC families are due to individually rare deleterious variants in numerous genes, each explaining a small proportion of families. To overcome the limitations of earlier targeted panel sequencing efforts, germline whole exome sequencing (WES) was performed on 516 likely familial high-grade serous ovarian cancer (HGSOC) patients with no pathogenic variants in BRCA1 or BRCA2 to discover novel predisposition genes. Forty-three candidate genes enriched for rare loss-of-function (LoF) variants were identified, along with LoF variants in several proposed EOC predisposition genes (e.g. ATM, PALB2). A high degree of genetic heterogeneity was observed, with no single gene harbouring LoF variants in more than 1% of cases. These candidate genes represent diverse functional pathways, with relatively few involved in DNA repair and only a small enrichment for genes involved in homologous recombination (HR) repair. This suggests that many of the remaining HGSOC families are explained by genes in pathways that have been previously under-explored. Since candidate gene variants were individually very rare, orthogonal approaches of tumour sequencing and segregation analysis were undertaken to validate these genes. WES and/or Sanger sequencing was performed on tumour DNA from 105 germline variant carriers, along with bisulphite sequencing of promoter CpG islands for selected genes, to identify evidence of biallelic inactivation and mutational signatures that might support a causative role for that gene. Two genes previously implicated as HGSOC predisposition genes, PALB2 and ATM, displayed biallelic inactivation in nearly every germline variant carrier tumour, associated with characteristic mutational signatures defined principally by the presence or absence of HR repair deficiency, respectively. Of the candidate genes, 19 out of 38 demonstrated biallelic inactivation in at least one tumour from affected carriers, but only three- LLGL2, SCYL3 and MIPOL1- displayed this result consistently in multiple samples, with the others showing loss of the variant allele or returning inconclusive results. Distinctive mutational signatures were found in the LLGL2 and SCYL3 tumours, similar to those for ATM and PALB2, respectively. In the limited number of segregation studies performed amongst six families, none of the tested germline variants consistently segregated with disease. In conclusion, these studies provided data supporting PALB2 and ATM as likely moderate-risk HGSOC predisposition genes, demonstrating the utility of this approach for validating novel familial cancer genes. Several candidate genes showed evidence to indicate a potential predisposing role, but the extreme genetic heterogeneity of unexplained familial HGSOC will necessitate larger studies to confirm these findings.
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    Developing novel methods of infection surveillance in haematology-oncology patients and implications for health policy
    Valentine, Jake Christopher ( 2021)
    Healthcare-associated and opportunistic infections are a leading cause of mortality, morbidity, and increased healthcare costs in haematology-oncology patients. Surveillance is recognised as the cornerstone of infection prevention to guide clinical decision making and to monitor quality improvement. The utility of current case ascertainment methods is poorly delineated in patients with underlying malignancy. Administrative data comprise a standardised ontology to classify disease with the potential to support large scale infection surveillance programmes; however, it is unclear if administrative data can support surveillance activities in high-risk settings. The overall aims of this thesis were to: (i) present and argue the case for novel case ascertainment methods; (ii) develop and apply a methodology using administrative data to identify infection in haematology-oncology patients, and determine the classification performance and healthcare funding implications of these data in line with current health policy; and (iii) establish and evaluate a hospital-wide linked dataset integrating multiple data sources, together with administrative data, to achieve maximal performance for automated surveillance in patients with haematological malignancies. Methods include a systematic review to describe the scope of existing surveillance methods among haematology-oncology units, analysis of continuous statewide surveillance datasets and hospital-level administrative data extracts, performance evaluation of administrative data to classify infection and simulation of pay-for-performance funding methodology in a cancer casemix, and development of a hospital-wide linked dataset to identify discrete data combination yielding highest performance for automated infection surveillance. The thesis findings demonstrate significant heterogeneity in existing infection monitoring methods among haematology-oncology patients. Estimates of the burden of disease in a predefined haematology-oncology population relative to a statewide cohort were determined, together with longitudinal trends in incidence over time. Administrative data show to be a feasible alternative to current surveillance data to enable standardised comparison of intra- and interhospital infection epidemiology in patients with underlying malignancy, however, at the expense of poor-to-moderate classification performance associated with significant shortfalls in hospital remuneration. Linkage of administrative data with microbiology, histopathology, and antimicrobial-dispensing data according to specific data combinations demonstrated improvements in classification performance for discrete opportunistic and healthcare-associated infections in patients with haematological malignancy. It was demonstrated that although administrative data enable standardised comparison of infection epidemiology, these data are an unreliable proxy for infection surveillance in Australian haematology-oncology units. Refinements to current pay-for-performance funding specifications are necessary before administrative data can reliably be used as quality improvement measures in a cancer casemix. This thesis posits data linkage as an efficient means to optimise the utility of administrative data, together with hospital-level datasets, to support an automated surveillance strategy in haematology-oncology patients. Future research agenda are outlined regarding the evaluation of electronic medical record data and other codified nomenclature to support electronic surveillance and quality improvement monitoring.
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    Novel approaches to harness the anti-tumour activity of natural killer cells
    Freeman, Andrew John ( 2022)
    Despite revolutionary advances in cancer treatment with immunotherapy, durable clinical benefit is limited to a subset of patients. Current forms of immunotherapy, including checkpoint inhibition and chimeric antigen receptor T cells, are primarily restricted to targeting cytotoxic CD8+ T cells of the adaptive immune system. Natural killer (NK) cells are the cytotoxic effector cells of the innate immune response and are emerging as a promising and alternative target for cancer immunotherapy, however, comprehensive functional genetic studies examining anti-tumour NK cell activity are limited. Using genome-scale clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 screening technology, we sought to comprehensively identify and validate tumour genes that potently influence sensitivity to primary NK cells. We first demonstrate that B16-F10 mouse melanoma cells lacking genes encoding proteins involved in interferon-gamma (IFN-gamma) signaling and antigen processing/presentation are highly sensitive to killing by NK cells, a process dependent on the absence of major histocompatibility complex class I expression. As checkpoint inhibition-resistant melanoma patients present with loss-of-function mutations within these pathways, our findings highlight intratumoural NK cells as a potent strategy to limit and/or overcome resistance to CD8+ T cell attack during conventional checkpoint inhibition immunotherapy. We additionally identify Rnf31 as a novel negative regulator of tumour cell sensitivity to NK cell killing. Rnf31 encodes for HOIP, which has an established role in driving tumour necrosis factor (TNF)-mediated gene induction and inhibition of TNF-induced cell death in certain cell types. HOIP-deficient melanoma cells not only exhibited increased sensitivity to NK cells, but also to antigen-specific CD8+ T cells. We surprisingly demonstrate that HOIP protects tumour cells from apoptosis induced by combined IFN-gamma and TNF, rather than TNF alone. We provide valuable mechanistic insight into the transcription-dependent form of apoptosis induced by combined IFN-gamma and TNF limited by HOIP, pharmacological validation using a HOIP inhibitor, and in vivo validation using HOIP-deficient B16-F10 ovalbumin-expressing tumours that exhibit enhanced CD8+ T cell-mediated control. Inhibition of tumour HOIP activity may therefore enhance NK and CD8+ T cell anti-tumour responses through unlocking the apoptotic potential of combined IFN-gamma and TNF secreted by these immune cells, representing a potential new combinatorial target for current and emerging immunotherapies. Collectively, we impartially identify tumour-specific genes that powerfully modulate tumour cell sensitivity to primary NK cells. We provide validation of established yet clinically relevant tumour genes associated with evasion from CD8+ T cells that mediate resistance to checkpoint inhibition immunotherapy, and additionally validate Rnf31 as a novel tumour regulator of not only sensitivity to NK cells, but also CD8+ T cells. We lastly present appropriate methodology and molecular tools that may facilitate analogous screening within NK cell lines to identify targetable regulators of tumour cell-induced IFN-gamma production by NK cells. Taken together, we advocate that NK cells may play an important role in combating resistance to checkpoint inhibition therapy in melanoma and identify HOIP as a potential combinatorial tumour target that may enhance future NK cell-based immunotherapy endeavours through secreted IFN-gamma and TNF, which may form the basis of a future immunotherapeutic strategy.
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    Overcoming resistance to PI3K inhibitors in colorectal cancer
    de las Heras Vila, Francesc d'Assis ( 2021)
    Colorectal cancer (CRC) is responsible for the second highest number of cancer deaths worldwide, with stage IV patients having a 5-year survival rate of only 14%. One of the treatments in development involves a targeted therapy directed at PI3K alpha, a protein mutated in 18% of CRC patients. Particularly, PI3K alpha is involved in several cancer hallmarks including survival, metabolism and migration. The introduction of PI3K inhibitors as targeted therapy in clinical trials caused an increase in overall survival and a decrease in disease progression. However, ultimately, treatment failure and tumour progression still occur due to development of drug resistance. Therefore, the aim of this project was to identify mechanisms of resistance to PI3K targeted therapy that can be exploited to overcome treatment failure. Specifically, the focus in this study is on BYL719, a PI3K alpha specific inhibitor. Firstly, a novel subcutaneous syngeneic mouse model of CRC was generated by the syngeneic transplant of Pik3ca mutated and Apc deleted gastrointestinal tumours from a mouse model of CRC. Tumour growth in this model was reduced upon treatment with BYL719. Mice were then chronically treated with BYL719 to induce resistance. Although the development of resistance was not confirmed within the time-frame of this thesis, this model proved to be a useful tool for pharmacological studies. Secondly, in silico analyses were performed, correlating the sensitivity of solid cancer cell lines to PI3K inhibition with CRISPR KO/mRNA/protein/metabolite data from online pharmacogenomic datasets. These analyses identified multiple pathways potentially involved with sensitivity to PI3K inhibition, including proteins involved in the regulation of the cytoskeleton, the PI3K/MAPK pathway, the endomembrane system and lipid and glutamine metabolism. Finally, an in vitro approach was performed using genome-wide CRISPR KO screen techniques, to identify additional functional pathways involved with sensitivity to PI3K inhibition. The results identified potential involvement of PI3K/Akt/MAPK signaling, mTOR/protein synthesis pathways, the Wnt pathway, TGF-beta pathway and metabolism, including lipids, glycolysis and the mitochondrial respiratory chain. Ultimately, these findings should enable the rational design of novel combination treatments with PI3K inhibitors to prevent or overcome resistance. Moreover, the findings might also be used to screen patients to predict their response to PI3K inhibitors.
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    Investigating the role of Mucosal-Associated Invariant T (MAIT) cells in cancer
    Petley, Emma Victoria ( 2021)
    The success of immunotherapy in patients has highlighted the importance of the anti-tumour role of the immune system. The function of conventional T cells within the tumour microenvironment (TME) have been intensively studied, while the role of mucosal-associated invariant T (MAIT) cells is yet to be determined. MAIT cells are abundant in humans and enriched in mucosal tissues, such as the colon and lung, and have been found within primary and metastatic tumours. Upon activation, MAIT cells exert rapid effector functions and can secrete both the anti-tumour cytokines (IFNg and TNF) and pro-tumour cytokines (IL-17 and IL-22). MAIT cells also produce granzyme B and perforin, suggesting they are capable of killing target cells. Although direct evidence of MAIT cells precise function in cancer is limited, some studies show that increased numbers of MAIT cells within tumours are correlated with a good prognosis, whilst other studies have indicated MAIT cells are associated with a poorer prognosis. These divergent results have made it difficult to interpret whether MAIT cells have an anti-tumour or pro-tumour role. Therefore, this thesis investigated the role of MAIT cells in cancer and the potential for MAIT cells to be exploited for adoptive cellular therapy. The first results chapter of this thesis explores the anti-tumour role of MAIT cells in both murine and ex vivo human models. It was observed that at steady-state, MAIT cells negatively regulate NK cell maturation and anti-tumour activity. Conversely, activating MAIT cells through either pulsing of tumour targets or intranasal administration of free MAIT cell antigen, led to robust protection against the development of lung metastases. Upon further investigation, it was discovered that activated MAIT cells enhance NK cell maturation and anti-tumour activity in an IFNg-dependent manner. This chapter proposes the existence of a MAIT-NK cell axis that can control NK cell mediated anti-tumour efficacy. The second results chapter aims to further improve the anti-tumour efficacy of activated MAIT cells, by combining this therapy with additional immunotherapies. The additional immunotherapies tested in combination with MAIT cell activation were selected on the basis of their ability to activate MAIT cells and/or NK cells. Notably, additional therapies that increased both MAIT cell and NK cell activity were most promising. This chapter also found that intravenous administration of MAIT cell antigen led to systemic expansion of MAIT cells and an increase in MAIT cells within the tumour tissue, broadening the application of activating MAIT cells in the clinic. The third results chapter aims to investigate the potential of MAIT cells in the context of Chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapy is currently ineffective in solid tumours, due to the immunosuppressive TME, antigen heterogeneity, poor trafficking to solid tumours and decreased persistence. Furthermore, this therapy requires autologous generation of CAR cells in order to avoid graft versus host disease (GVHD). Excitingly, MAIT cells represent an allogeneic source of CAR cells as they are not restricted to conventional MHC. Chapter 5 demonstrates that MAIT cells are able to be efficiently transduced with CAR and upon target recognition CAR MAIT cells produce cytokines and directly kill tumour cells. Collectively, this data illustrates the potential anti-tumour activity of MAIT cells through a MAIT-NK cell axis. Furthermore, this thesis demonstrates the potential for MAIT cells to be used in adoptive cellular therapy, namely as CAR MAIT cells.
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    Therapeutically targeting RNA Polymerase II transcription in leukaemia
    Todorovski, Izabela ( 2021)
    RNA Polymerase II (RNAPII) transcription is a discontinuous and unidirectional process, control of which is necessary for normal gene expression and cellular functioning. Regulation occurs at discrete checkpoints throughout transcription and is in part mediated by Cyclin Dependent Kinases (CDKs). This includes CDK7, where it phosphorylates RNAPII among other transcriptional substrates to enable transcriptional initiation and coordination of co-transcriptional processes, such as splicing. However, the genome-wide role of CDK7 in nascent RNA synthesis and splicing, in addition to whether its function is negatively regulated, has not been robustly established. The loss of RNAPII transcription control invariably leads to disease, including cancer. Mutations in cancer can affect several proteins involved in regulating RNAPII and engenders a reliance on sustained functioning of the core RNAPII machinery for continued malignant cell survival and proliferation. This dependence is termed ‘transcription addiction’ and is the basis for targeting RNAPII core components for therapeutic benefit. Small molecule perturbation of transcription in these cancers results in selective gene expression changes attributed to the target gene’s specific chromatin context, which includes the occupancy of oncogenic transcription factors (TFs) and association with genomic elements such as super enhancers (SEs). Whether selective transcriptional changes are also influenced by RNA stability however, is largely unexplored. In this thesis, we demonstrated that RNA decay rates largely determined gene expression changes to clinically relevant transcriptional and epigenetic compounds. Transcripts that were the most down-regulated on the total RNA level had both short half-lives and high production rates and often encoded TFs, including the proto-oncogene c- MYC. Moreover, genetically engineering the three prime untranslated region (3’UTR) of c-MYC to prolong its RNA half-life rendered c-MYC transcripts resistant to transcriptional targeting on the total RNA level. Therefore, these finding highlight an under-appreciated role of RNA stability in gene expression changes to therapeutic RNAPII perturbation in cancer. Among the most promising therapeutic transcriptional targets is CDK7, and inhibitors targeting this protein are currently clinical trials and have been expedited for Food and Drug Administration (FDA) approval. Proteomic and transcriptomic characterization of the CDK7 inhibitor, YKL-5-124, in the THP-1 Acute Myeloid Leukaemia (AML) cell line revealed that it perturbed the phosphorylation of several proteins involved in cell cycling, transcription and co-transcriptional processes, namely splicing. This was paralleled with a global decrease in de novo RNA synthesis, most prominently towards the 5’ ends of genes, and total RNA levels. In addition, CDK7 inhibition increased alternative isoform expression, most strikingly related to exon skipping and intron retention. On a phenotypic level, these changes were also associated with a decrease in THP-1 cell proliferation. Taken together, these findings improve our understanding of the significance of CDK7 kinase activity in RNAPII transcription and splicing. To identify factors that functionally antagonize CDK7 and may confer resistance to sustained CDK7 inhibition, CRISPR-Cas9 knockout positive enrichment screening in THP-1 cells was performed using YKL-5-124 treatment as a selective pressure. The screen showed that knockout of histone acetyltransferase (HAT) and core structural components of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex mediated resistance to CDK7 inhibition. Further investigation into the resistance mechanism revealed that cells knocked out for the SAGA HAT subunit, TADA2B, were less sensitive to nascent RNA down-regulation with YKL-5-124. Moreover, this was concomitant with maintained H2BK120 mono-ubiquitin levels across gene bodies genome-wide. Therefore, these data highlight SAGA complex components as novel negative regulators of CDK7 and how cancers might overcome the effect CDK7 inhibitors in the clinic. Overall, the results presented herein improve our understanding of CDK7 in RNAPII transcription and the importance of RNA decay in defining the anti-cancer effects of therapeutic perturbation of the core RNAPII machinery.