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TP53-mutated acute myeloid leukaemia: molecular basis and identification of therapeutic targets
No full textTP53 mutations are associated with adverse risk and poor response to standard therapies in
acute myeloid leukaemia (AML) and are frequently observed in complex karyotype, therapy-related or
secondary AML. Previous studies have investigated the function of mutant p53, but the mechanism by
which mutant p53 contributes to the development of AML is incompletely understood. This thesis set
out to investigate the role of mutant p53 and loss of p53 in leukaemogenesis and identify novel
therapeutic targets for p53-mutant AML.
To this end, I generated p53-mutant AML mouse models harbouring one of three common p53
mutants or p53 loss. Characterisation of the AML models revealed that they recapitulate important
characteristics of TP53-mutated AML, including the aberrant proliferation and extramedullary infiltration
of differentiation-arrested myeloid blasts. Strikingly, the generated p53-mutant AML models displayed
an erythroid differentiation bias with expansion of megakaryocyte-erythroid progenitors. This is in line
with previous reports suggesting a link between p53 alterations and erythroid differentiation. Ex vivo
assays confirmed the erythroid phenotype. Transcriptomic analysis of the p53-mutant AML models
revealed the aberrant expression of multiple erythroid transcription factors, consistent with the erythroid
lineage bias. Further mechanistic studies are required to unravel the exact mechanistic link between
p53 alterations and the dysregulation of erythroid regulators.
To identify novel genetic vulnerabilities in p53-mutant AML, I performed genome-wide
CRISPR/Cas9 knockout screens in two cell lines derived from AML models with mutant p53 or p53
loss. From the hits, I selected the nuclear export factor NXT1 for further validation and confirmed the
dependency of murine and human AML cells on NXT1.
Taken together, this thesis explores the role of mutant p53 or p53 loss in leukaemogenesis and
erythroid differentiation and proposes NXT1 as a potential new therapy target in p53-mutant AM
Establishment of a Multidimensional Screening Platform for the Functionalisation of Putative Drivers of Castration Resistant Prostate Cancer
No full textAdvanced prostate cancer is a complex disease with significant heterogeneity, characterised by intricate interactions with the tumour
microenvironment and limited responsiveness to standard immunotherapies. This thesis focuses on developing a multimodal
platform to functionally characterise a class of transcriptional regulators that undergo genetic alterations in advanced disease. While
individually rare in primary tumours, these aberrations are significantly more frequent as a group in later stages, suggesting a crucial
role in cancer progression, castration resistance, and metastasis. Despite their clinical importance, their precise function remains
largely unknown.
These genetic alterations are thought to drive disease progression through various mechanisms, including phenotypic plasticity,
transcriptional heterogeneity, phenotypic inertia, and microenvironmental modulation. However, it remains unclear which of these
aberrations are key drivers, whether they act independently or collectively, and whether their effects are direct or mediated through
secondary pathways. Addressing these uncertainties requires a robust multimodal platform capable of integrating phenotypic and
molecular data across in vitro and in vivo models to capture their full impact on tumour biology.
This thesis begins by examining the genetic landscape and clinical outcomes of advanced castration-resistant prostate cancer
(CRPC) before narrowing its focus to a specific class of transcriptional regulators implicated in disease progression. The central
objective is to establish and validate a CRISPR-based screening system that enables systematic functional analysis of these
aberrations. This platform utilises guide RNA (gRNA) linked to a protein barcode (pro-code), enabling precise, multi-platform tracking
of genetic perturbations.
The methodology involves a thorough literature review to identify key genes, the design and optimisation of CRISPR gRNA libraries,
refinement of cloning and library balancing procedures, and the development of antibody panels for CyTOF and Phenocycler
technologies. The platform’s effectiveness is demonstrated through large-scale functional screens integrating genetic and phenotypic
data. Ultimately, this work establishes a platform designed to enhance our understanding of disease progression, supporting the
development of more precise and effective therapeutic strategies
Whole exome analyses for identification of rare variant germline susceptibility in testicular germ cell tumours
No full textTesticular germ cell tumour (TGCT) is the most common cancer in young men aged between 15 and 44, with the highest reported
incidence in populations of European ancestry. Heritability estimates of TGCT are among the highest of all cancers, half of which is
unexplained and likely embedded in rare variation.
This thesis presents two whole-exome sequencing case-control studies (Study 1 and Study 2), the largest of their kind worldwide to
date, with the goal of investigating the rare germline architecture of TGCT. The first meta-analysis comprises 1,435 men with TGCT
from three datasets and 18,284 cancer-free individuals. The second meta-analysis from two datasets includes 7,627 TGCT cases
and 35,565 non-TGCT controls. For the latter study, I prepared >7,000 case DNA samples for sequencing. I performed rigorous
quality control to limit false positives and address confounding factors. I conducted various gene-level association analyses under a
dominant (including X/Y-linked genes) and recessive model. I initially analysed 120 cancer susceptibility genes to investigate
pleiotropic effects. Then, I conducted association testing of protein-truncating and nonsynonymous disruptive variants (i) across
biologically related candidate genes (655 genes), and (ii) exome-wide (19,355 genes). I also undertook detailed examinations of
gene-set enrichment and power.
In Study 1, no individual gene-disease association was identified following multiple testing correction. In Study 2, the CEP290 ciliary
gene, was associated with TGCT with a protective effect (p=1.60x10-15). Both studies were well-powered to detect rare variation of
moderate/high effect sizes (OR≥5), but power diminished for modest effect sizes (OR<5). In both studies, functional gene-set
analyses identified excess associations with genes involved in microtubular/ciliary pathways (Study 1: p=1.69x10-8; Study 2:
p=0.013).
Uncovering the sources of missing heritability in TGCT remains a major challenge. By highlighting microtubular/ciliary pathways, this
work provides a foundation for future functional studies that could inform risk prediction and therapeutic approaches
A phase 1, first-in-human, dose escalation study of JNJ-80038114, a PSMAxCD3 bispecific antibody, in participants with metastatic castration-resistant prostate cancer.
No full textPURPOSE: Prostate-specific membrane antigen (PSMA) has been identified as a therapeutic target for metastatic castration-resistant prostate cancer (mCRPC). The recent success of radioligands targeting PSMA spurred development of new PSMA-targeting agents including immunotherapy. JNJ-80038114 is a bispecific antibody that binds PSMA on tumor cells and CD3 on T cells to induce anti-tumor activity. METHODS: This was a phase 1, open-label, multicenter study of JNJ-80038114 in participants with mCRPC and ≥ 1 prior systemic therapy. JNJ-80038114 was administered subcutaneously every 3 weeks (Q3W), starting at 0.1 mg. The primary endpoint was safety. Secondary endpoints included pharmacokinetics (PK), immunogenicity, and prostate-specific antigen (PSA). RESULTS: At final analysis, 39 participants received 0.1-180 mg JNJ-80038114 across 11 dose-escalation cohorts for a median of 9.3 weeks (range, 0.1-31.1). The most common treatment-related adverse events (TRAEs; ≥20%) included cytokine release syndrome (CRS, 51.3%, all Grade 1-2), injection-site reactions (46.2%), and fatigue (30.8%). Related Grade ≥ 3 TRAEs occurred in 51.3% of participants; dose-limiting toxicities occurred in 3 (7.7%). Four participants (10.3%) developed clinically significant neuropathies. In 37 PK-evaluable participants, mean exposure (Cmax, AUC) increased with increasing doses. Anti-drug antibodies (ADA) were reported in 56.8% (21/37) participants. One participant had confirmed PSA decrease ≥ 50%. Three participants had radiological responses in the context of rapidly rising PSA. Following a review of data, the study was terminated. CONCLUSIONS: This first-in-human study of JNJ-80038114 was discontinued early due to its lack of preliminary clinical activity, neurologic toxicities, high rates of CRS, and the development of ADAs impacting PK. GOV INFORMATION: NCT05441501, Registered July 1, 2022
Outcomes of pelvic and para-aortic stereotactic reirradiation for gynaecological cancer recurrence
No full textInvestigating the mechanisms of DNA damage-dependent cGAS activation
No full textcGAS is a major sensor of double-stranded DNA. Upon DNA binding, cGAS produces the second
messenger cGAMP, which in turn activates innate immune responses such as inflammatory gene
expression, cell death, and senescence. Under unperturbed conditions, cGAS is prevented from
self-sensing through its inhibition on chromatin. Despite this, cGAS can be activated by genotoxic
stress, but the mechanism and regulation of this process remain unknown. Although most cGAS
activation models involve cytoplasmic DNA, a substantial pool of nuclear cGAS exists. Here, we
hypothesise that cGAS inhibition is relieved upon genotoxic stress, potentially activating nuclear
cGAS and leading to subsequent immune responses.
This study aimed to establish assays to measure cGAS activation to define the mechanisms by
which cGAS activation occurs following genotoxic stress. Since studying cGAS activation in cells
can prove difficult due to feedback control and cellular heterogeneity, I have established cell-free
Xenopus egg extracts to study cGAS activation. This model can now be used to assess the effects
of DNA damage and defined DNA damage structures on cGAS activity. However, irradiation and
aphidicolin treatment did not appear to cause cGAS activation in extract. This potentially correlates
with findings from our group obtained in cells, which indicated that DNA damage does not
generate a strong cGAS stimulus, and only activates cGAS in a minor fraction of cells. To begin to
understand the nature of this heterogeneity, I have set up and performed a whole kinome siRNA
microscopy based-screen using a novel cell-based reporter for cGAS activation developed in our
group and have identified some potential regulators of cGAS activation following DNA damage.
Collectively, these results provide insight into the mechanisms underlying cGAS activation and its
regulation and may provide a basis for future co-treatment strategies to boost cGAS activation
during anti-cancer therapy
Unravelling the molecular basis of fidelity in mRNA splicing through DEAH-box helicases DHX38 and DHX8
No full textThe spliceosome is a large and dynamic complex splicing together exons in pre-mRNA to generate mature mRNA, which is then translated into a protein. RNA helicases drive the transitions between different stages of the spliceosome assembly and regulate the selection of a splice junction. This is a normal phenomenon, but when deregulated is a hallmark of cancer. The DEAH-box RNA helicases DHX38 and DHX8 remodel the spliceosome between the C and C* complex and between the C* and P complex at the later stages of spliceosome assembly. They play crucial roles in regulating splicing fidelity by rejecting sub-optimal splice sites. Previous experiments have identified a complex comprising DHX38, DHX8 and other spliceosomal proteins. Using double co-immunoprecipitation, I confirmed that DHX38 and DHX8 co-elute together. Using mass spectrometry, I identified several other spliceosome related proteins involved which I term the ‘fidelity complex’.
DHX38 is a potential therapeutic cancer drug target, as it is upregulated in cancer cells, but the full-length structure is unknown. Therefore, a second aim of my research involved its structure determination using cryo-electron microscopy, because crystallisation of full length DHX38 proved unsuccessful. Here I report a low-resolution anisotropic map of the closed conformation of the full length DHX38. The truncated helicase domain of DHX38 was co-expressed with the novel chaperone NudCD1. NudCD1 is a tumour associated antigen with a conserved p23 domain which allows for chaperone activity. NudCD1 has been found to bind to multiple DEAH-box helicases however its function is still poorly understood. Here I report the low-resolution density maps of NudCD1 on its own, and the NudCD1-DHX38 complex, which shows the WD40 domain of NudCD1 packing against the C-terminal domain of DHX38, with the helicase core in a possible open conformation. Furthermore, the beta sheet of NudCD1 interacting with the RecA domain
Towards the Rational Design of Monovalent Degraders
No full textSmall molecules that induce protein degradation hold the potential to overcome several limitations of currently available inhibitors. Monovalent or molecular glue degraders, in particular, enable the benefits of protein degradation without the disadvantages of high molecular weight and the resulting challenge in drug development that are associated with bivalent molecules like Proteolysis Targeting Chimeras. One key challenge in designing monovalent degraders is how to build in the degrader activity – how can an inhibitor be converted into a degrader? If degradation activity requires very specific molecular features, it will be difficult to find new degraders and challenging to optimize those degraders toward drugs. Herein, an unexpectedly wide range of modifications to the degradation-inducing group of the cyclin K degrader CR8 are shown to be tolerated, including both aromatic and non-aromatic groups. These findings were subsequently utilised to convert the pan-CDK inhibitors dinaciclib and AT-7519 to Cyclin K degraders, leading to a novel dinaciclib-based compound with improved degradation activity compared to CR8 and which confirm the mechanism of degradation. These compounds were not capable of degrading related targets such as other cyclins or CDKs, suggesting that the degradation event may be highly specific to the interface formed. Having identified a range of cyclin K degraders, the next aim was to explore the feasibility of identifying degraders of a new target. A key challenge in the TPD field is the lack of targeted screening methods to identify novel degraders, with low hit rates and reliance on high-throughput screening rendering degrader discovery a slow process that is inaccessible to many. To tackle this, a new screening approach was devised and run as a pilot screen to understand if degraders can be identified through a less resource-intensive strategy. Here, multi-kinase inhibitors were modified at the solvent-exposed region with the hypothesis that increasing the number of potential binding targets may significantly reduce the library size required to identify a degrader. A library of 77 modified multi-kinase inhibitors was designed, synthesized and tested against a panel of fourteen NLuc-tagged kinases. The screen did not identify any confirmed selective kinase degraders, with multiple compounds causing non-specific effects, such as translation inhibition and cell death, that decreased Nluc signal. This identified several key areas for improvement of the screen to improve its utility, such as pre-screening compounds for cell toxicity and increasing the library size by ten-fold. Whilst rational design of monovalent degraders is not yet possible, the field is seeing an increasing number of discoveries due to a heightened understanding of features capable of triggering degradation, parallel synthesis and improved screening platforms. As such, the guidelines for monovalent degrader design are slowly being elucidated
Decoding the mechanotransduction role of Piezo2 in Pancreatic Cancer
No full textPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a low and stagnant survival rate of
12.5%, largely due to limited treatment options, therapeutic resistance, and relapse driven by cancer stem cells
(CSCs). These cells play a pivotal role in tumour growth, heterogeneity, metastasis, and recurrence due to their
unique ability to self-renew and differentiate into various cell types. Identifying CSC regulators that can be
therapeutically exploited is essential to improve current treatment outcomes by reducing the risk of relapse and
metastasis.
This thesis investigates two complementary strategies to improve the understanding and treatment of PDAC. In the
first part, an organoid-based drug screen identified Thapsigargin and Halofuginone as promising candidates,
targeting endoplasmic reticulum (ER) stress and stromal remodelling, respectively.
The second part, and main focus, of this thesis centres on Piezo2, a mechanosensitive ion channel, and its role in
PDAC progression. A rare PIEZO2-expressing subpopulation exhibits stem-like properties that are essential for
organoid initiation and tumour formation. Using gain- and loss-of-function approaches in organoid and mouse
models, I demonstrate that Piezo2 acts as a cell-state-dependent regulator of epithelial-to-mesenchymal transition
(EMT), with a more pronounced impact on cells in mesenchymal-like states. Transcriptomic and functional analysis
suggest that Piezo2 modulates the YAP/TAZ-Hippo and AKT-mTOR signalling pathways. Furthermore, in vivo
experiments using the KPCY mouse model reveal that loss of Piezo2 reduces metastatic tumour burden in the liver,
highlighting its role in the metastatic cascade.
These findings position PIEZO2 as a potential therapeutic target in PDAC, supporting a mechanobiology-based
strategy to reduce metastasis and improve treatment outcomes
The Prognostic and Predictive Impact of ctDNA Levels in Patients with Advanced Breast Cancer Enrolled on the plasmaMATCH Trial.
No full textPURPOSE: ctDNA dynamic levels may identify patients who will respond to therapy. We assessed ctDNA baseline levels and on-treatment dynamics in patients with advanced breast cancer on the plasmaMATCH trial with mutation-targeted therapies (cohorts A-D) and triple-negative breast cancer on olaparib and ceralasertib combination (cohort E). EXPERIMENTAL DESIGN: Blood samples were collected at baseline [cycle 1 day 1 (C1D1)] and before treatment on cycle 2 day 1 (C2D1). Samples were sequenced using error-corrected targeted panels (Guardant360/GuardantOMNI). Circulating DNA ratio was calculated as the ratio of C2D1/C1D1 circulating DNA ratio, and baseline ctDNA levels were associated with progression-free survival (PFS) and confirmed objective response rates (ORR). RESULTS: A total of 167 patients had assessable C1D1-C2D1 ctDNA results. There was a strong association between baseline ctDNA levels and response in cohort E; low baseline levels were associated with longer PFS (HR, 0.33; P = 0.001) and higher ORR (40% vs. 9.7%; P = 0.02). In cohorts A to D, there was a weaker association with PFS (HR, 0.60; P = 0.03) and ORR (15.2% vs. 5.7%; P = 0.17). Associations of baseline ctDNA level and ORR were validated in the independent PEARL study. For on-treatment dynamics, suppression of ctDNA below median was predictive in cohorts A to D (HR, 0.47; P = 0.001) but not in cohort E (HR, 1.02; P = 0.94). Undetectable ctDNA levels at C2D1 were associated with good outcomes in both cohorts: in cohort E with improved PFS (HR, 0.25; P = 0.01) and improved ORR (86% vs. 11%; P = 0.01). Six of seven patients with undetectable on-treatment ctDNA were BRCA1/BRCA2/PALB2 wild type. CONCLUSIONS: Baseline low ctDNA levels predict response to targeted therapy, potentially suggesting shared mechanisms between high ctDNA release and resistance to therapy. Both baseline ctDNA levels and on-treatment dynamics are a promising surrogate endpoint for drug development, with clearance of ctDNA being a robust cross-therapy surrogate for outcomes