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Item Understanding the homeostatic response to PARP inhibitors in breast cancer(Institute of Cancer Research (University Of London), 2025-05-13)PARPi show significant antitumor activity in BRCA1/2 mutant tumours, but many patients develop resistance, and the underlying causes are often unknown. Predominantly our understanding of resistance to PARPi has originated from studies utilising genetic approaches, and relatively little is understood about the protein expression changes induced by PARPi, and how these changes could give rise to PARPi resistance. In this thesis, I used mass spectrometry based proteomic profiling of both tumour cell lines and patient derived tumour organoids to define the proteomic response to PARPi exposure. I integrated the information gained from this proteomic profiling with genetic perturbation screens, particularly CRISPR activation screens, to identify candidate gain of function mechanisms of PARPi resistance. I identified upregulation of the cohesin loader NIPBL as a candidate driver of PARP inhibitor resistance. Examination of tumour RNA-seq data from advanced breast cancer patients with PARPi resistance indicated that NIPBL might be upregulated in the PARPi resistant state. By conducting functional studies in in vitro models, I confirmed that upregulation of NIPBL confers PARPi resistance, and likely does so by restoring the localisation of RAD51 to the site of DNA damage in BRCA1/2 mutant cells that otherwise have a RAD51 defect. Together, the data presented in this thesis offers a foundation for understanding the homeostatic responses to PARPi exposure which could drive PARPi resistance in BRCA1/2 mutant tumours.Item FIRRM cooperates with FIGNL1 to promote RAD51 disassembly during DNA repair.(AMER ASSOC ADVANCEMENT SCIENCE, 2023-08-09)Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.Item The dynamic process of covalent and non-covalent PARylation in the maintenance of genome integrity: a focus on PARP inhibitors(Oxford University Press (OUP), 2023-06-09)Abstract Poly(ADP-ribosylation) (PARylation) by poly(ADP-ribose) polymerases (PARPs) is a highly regulated process that consists of the covalent addition of polymers of ADP-ribose (PAR) through post-translational modifications of substrate proteins or non-covalent interactions with PAR via PAR binding domains and motifs, thereby reprogramming their functions. This modification is particularly known for its central role in the maintenance of genomic stability. However, how genomic integrity is controlled by an intricate interplay of covalent PARylation and non-covalent PAR binding remains largely unknown. Of importance, PARylation has caught recent attention for providing a mechanistic basis of synthetic lethality involving PARP inhibitors (PARPi), most notably in homologous recombination (HR)-deficient breast and ovarian tumors. The molecular mechanisms responsible for the anti-cancer effect of PARPi are thought to implicate both catalytic inhibition and trapping of PARP enzymes on DNA. However, the relative contribution of each on tumor-specific cytotoxicity is still unclear. It is paramount to understand these PAR-dependent mechanisms, given that resistance to PARPi is a challenge in the clinic. Deciphering the complex interplay between covalent PARylation and non-covalent PAR binding and defining how PARP trapping and non-trapping events contribute to PARPi anti-tumour activity is essential for developing improved therapeutic strategies. With this perspective, we review the current understanding of PARylation biology in the context of the DNA damage response (DDR) and the mechanisms underlying PARPi activity and resistance.Item ZNF432 stimulates PARylation and inhibits DNA resection to balance PARPi sensitivity and resistance(Oxford University Press (OUP), 2023-11-10)Abstract Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.Item Development and implementation of efficient adaptive designs in early phase oncology trials for targeted agents(Institute of Cancer Research (University Of London), 2025-05-06)Recent advancements in molecular biology and genomic research have led to the development of a new class of cancer treatments known as targeted agents. Unlike cytotoxic therapies, which work by directly killing cancer cells, targeted agents focus on specific molecular markers that drive tumour growth. This distinct mechanism presents significant challenges to the traditional early-phase clinical trial design, which was originally tailored for cytotoxic treatments. This thesis focuses on three pivotal challenges posed by targeted therapies to the conventional design paradigm. Key approaches have been proposed to address these challenges. The occurrence of late-onset toxicities and rapid patient recruitment in early-phase trials highlights the challenge of incomplete toxicity information. We conducted a methodological review of existing designs that accommodate incomplete toxicity and evaluated the implementation of these designs in published trials in Chapter 2. Our findings underscore the importance of reporting sufficient information to ensure trial replicability while addressing the issue of incomplete toxicity data. To support more robust decision-making under such conditions, we propose the incorporation of the Dose Transition Pathways (DTP) look ahead strategy tool with two time-to-event toxicity designs in Chapter 3. This approach aims to balance the trade-off between accelerated dose escalation and recruitment suspension when toxicity outcomes are still pending. Non-monotonic dose-efficacy relationships and delayed responses further complicate the use of traditional designs. In Chapter 4, we introduce the DTP-TITE-CFO-ET design, which accounts for delayed toxicities, delayed efficacy, as well as non-monotonic dose-efficacy relationship. We also extend the DTP look-ahead strategy to the joint outcomes setting with incomplete information. This method allows for more informed and timely dose decisions, enhancing the flexibility and applicability of advanced early phase trial designs for targeted therapies. In Chapter 5, we propose a novel two-stage single-arm design that uses progression-free survival (PFS) as an intermediate endpoint in the interim analysis and overall survival (OS) in the final analysis. By employing a multi-state survival model to link PFS and OS, we improve the statistical power of trials while providing a framework for early stopping when appropriate. Collectively, the designs presented in this thesis offer a comprehensive approach to overcoming the limitations of traditional early-phase clinical trials when applied to targeted therapies. These advancements not only address the design challenges introduced by targeted agents but also enhance decision-making through real-time data integration and advanced statistical methods
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