Elucidating Prostate Cancer Biology Through Integrative Computational Multi-omic Analyses

in this thesis, I aimed to elucidate the microbial landscape of castration-resistant prostate cancer (CRPC), focusing on novel biomarkers of therapeutic resistance. We employed metagenomic sequencing of over 200 CRPC patients, alongside in vitro cell Line and ex vivo patient-derived xenograft (POX), models, to investigate microbial biomarkers of progression from hormone-sensitive prostate cancer {HSPC) to CRPC. Metagenomic sequencing effectively characterised the gut microbiome ofCRPC patients. While microbiome alpha diversity between HSPC and CRPC I remained similar, species-level changes, including an increased abundance of Adlercreutzia equolifaciens, were observed in CRPC. Further, L- 1 citrulline biosynthesis by the microbiota was linked to improved AR -negative prostate cancer cell viability, highlighting its potential role in CRPC i progression. Analysis ofCRPC biopsy samples provided inconclusive evidence for intra-tumour micro biota presence, with contamination confounding microbial classifications. Single-nucleus sequencing revealed significant myeloid cell heterogeneity in CRPC, with varying degrees of oxidative stress-induced DNA damage I linked to myeloid-derived reactive oxygen species {ROS). This heterogeneity extended to tumour subclones, with genomic instability and replication I stress driving therapeutic sensitivity to ATR inhibition. Notably, the overexpression of the prognostic marker POLQ was associated with replication [ stress and ATR inhibition sensitivity, highlighting a potential therapeutic target for CRPC. These findings provide insights into the complex interplay: between the microbiome, inflammation, and cellular mechanisms driving CRPC progression and resistance and merit further functional validations

Pilot of lifestyle InterventiON to reducE brEast cancer Risk (PIONEER): the development, implementation and lessons from a randomised controlled study

This thesis describes current understanding of fixed and modifiable breast cancer risk factors. It goes on to describe the development and results of a randomised controlled pilot study to reduce breast cancer risk through lifestyle change, as well as describing the experience of running a preventative therapy clinic. It also discusses the lessons learnt through running a lifestyle change pilot study to reduce breast cancer risk, and improvements which could be made to future studies

NSD2-epigenomic reprogramming and maintenance of plasma cell phenotype in t(4;14) myeloma.

Overexpression of the H3K36 histone methyltransferase NSD2 in t(4;14) multiple myeloma (MM) is an early, oncogenic event, and understanding its impact on genomic organisation and expression is relevant to understanding MM biology. We performed epigenetic, transcriptional and phenotypic profiling of the t(4;14) KMS11 myeloma cell line and its isogenic translocation knock out (TKO) to characterise the sequelae of NSD2 overexpression. We found a marked global impact of NSD2 on gene expression and DNA organisation implicating cell identity genes; notably the early lymphocyte regulator, LAIR1 and MM cell surface markers, including CD38, a classical marker of plasma cells which was reduced in TKO cells. Plasma cell transcription factors such as PRDM1, IRF4 and XBP1 were unaffected, suggesting a downstream direct gene effect of NSD2 on cell identity. Changes in cell surface markers suggest an altered surface immunophenotype. Our findings suggest a role for NSD2 in maintaining MM cell identity, with potential implications for future therapeutic strategies based on targeting of NSD2

NXP800 Activates the Unfolded Protein Response, Altering AR and E2F Function to Impact Castration-Resistant Prostate Cancer Growth.

PURPOSE: Advanced prostate cancer is invariably fatal, with the androgen receptor (AR) being a major therapeutic target. AR signaling inhibitors have improved overall survival for men with advanced prostate cancer, but treatment resistance is inevitable and includes reactivation of AR signaling. Novel therapeutic approaches targeting these mechanisms to block tumor growth is an urgent unmet clinical need. One attractive strategy is to target heat shock proteins (HSP) critical to AR functional activity. EXPERIMENTAL DESIGN: We first did transcriptome analysis on multiple castration-resistant prostate cancer (CRPC) cohorts to correlate the association between the Gene Ontology cellular response to heat gene expression signature and overall survival. Next, we analyzed the impact of targeting the heat shock factor 1 (HSF1) pathway, with an inhibitor in clinical development, namely, NXP800 (formerly CCT361814), in models of treatment-resistant prostate cancer. Finally, we confirmed our mechanistic and phenotypic findings using an NXP800-resistant model and an in vivo model of CRPC. RESULTS: We report that in multiple CRPC transcriptome cohorts, the Gene Ontology cellular response to heat gene expression signature associates with AR signaling and worse clinical outcome. We demonstrate the effects of targeting the HSF1 pathway, central to cellular stress, with an inhibitor in clinical development, namely, NXP800, in prostate cancer. Targeting the HSF1 pathway with the inhibitor NXP800 decreases HSP72 expression, activates the unfolded protein response, and inhibits AR- and E2F-mediated activity, inhibiting the growth of treatment-resistant prostate cancer models. CONCLUSIONS: Overall, NXP800 has antitumor activity against treatment-resistant prostate cancer models, including molecular subtypes with limited treatment options, supporting its consideration for prostate cancer-specific clinical development

Unraveling complexity and leveraging opportunities in uncommon breast cancer subtypes.

Special histologic subtypes of breast cancer (BC) exhibit unique phenotypes and molecular profiles with diagnostic and therapeutic implications, often differing in behavior and clinical trajectory from common BC forms. Novel methodologies, such as artificial intelligence may improve classification. Genetic predisposition plays roles in a subset of cases. Uncommon BC presentations like male, inflammatory and pregnancy-related BC pose challenges. Emerging therapeutic strategies targeting genetic alterations or immune microenvironment are being explored

Open-label, phase Ia study of STING agonist BI 1703880 plus ezabenlimab for patients with advanced solid tumors.

BI 1703880, a novel STimulator of INterferon Genes (STING) agonist, has demonstrated preclinical antitumor activity. As STING activation can upregulate programmed death ligand 1 and human leukocyte antigen in tumor cells, a combination of BI 1703880 and an anti-programmed cell death protein 1-antibody, such as ezabenlimab, may improve efficacy. This first-in-human phase Ia study (NCT05471856) is evaluating BI 1703880 plus ezabenlimab in patients with advanced solid tumors. The study utilizes an innovative lead-in design; all patients receive BI 1703880 monotherapy in Cycle 1 and combination therapy from Cycle 2. The primary endpoint is dose-limiting toxicities during the maximum tolerated dose evaluation period. Results will inform the future development of BI 1703880 for treatment of metastatic or recurrent malignancies.Clinical Trial number: NCT05471856

UK guidelines for the management of soft tissue sarcomas.

Soft tissue sarcomas (STS) are rare tumours arising in mesenchymal tissues and can occur almost anywhere in the body. Their rarity, and the heterogeneity of subtype and location, means that developing evidence-based guidelines is complicated by the limitations of the data available. This makes it more important that STS are managed by expert multidisciplinary teams, to ensure consistent and optimal treatment, recruitment to clinical trials, and the ongoing accumulation of further data and knowledge. The development of appropriate guidance, by an experienced panel referring to the evidence available, is therefore a useful foundation on which to build progress in the field. These guidelines are an update of the previous versions published in 2010 and 2016 [1, 2]. The original guidelines were drawn up by a panel of UK sarcoma specialists convened under the auspices of the British Sarcoma Group (BSG) and were intended to provide a framework for the multidisciplinary care of patients with soft tissue sarcomas. This iteration of the guidance, as well as updating the general multidisciplinary management of soft tissue sarcoma, includes specific sections relating to the management of sarcomas at defined anatomical sites: gynaecological sarcomas, retroperitoneal sarcomas, breast sarcomas, and skin sarcomas. These are generally managed collaboratively by site specific multidisciplinary teams linked to the regional sarcoma specialist team, as stipulated in the recently published sarcoma service specification [3]. In the UK, any patient with a suspected soft tissue sarcoma should be referred to a specialist regional soft tissues sarcoma service, to be managed by a specialist sarcoma multidisciplinary team. Once the diagnosis has been confirmed using appropriate imaging and a tissue biopsy, the main modality of management is usually surgical excision performed by a specialist surgeon, combined with pre- or post-operative radiotherapy for tumours at higher risk for local recurrence. Systemic anti-cancer therapy (SACT) may be utilised in cases where the histological subtype is considered more sensitive to systemic treatment. Regular follow-up is recommended to assess local control, development of metastatic disease, and any late effects of treatment

Contribution of Prediagnostic Host Factors to Shaping the Stromal Microenvironment of Breast Cancer among Sub-Saharan African Women.

BACKGROUND: The stromal microenvironment (SME) is integral to breast cancer biology, impacting metastatic proclivity and treatment response. Emerging data indicate that host factors may impact the SME, but the relationship between prediagnostic host factors and SME phenotype remains poorly characterized, particularly among women of African ancestry. METHODS: We conducted a case-only analysis involving 792 patients with breast cancer (17-84 years) from the Ghana Breast Health Study. High-accuracy machine-learning algorithms were applied to standard H&E-stained images to characterize SME phenotypes [including percent tumor-associated connective tissue stroma, Ta-CTS (%); tumor-associated stromal cellular density, Ta-SCD (%)]. Associations between prediagnostic host factors and SME phenotypes were assessed in multivariable linear regression models. RESULTS: Decreasing Ta-CTS and increasing Ta-SCD were associated with aggressive, mostly high-grade tumors (P-value < 0.001). Several prediagnostic host factors were associated with Ta-SCD independently of tumor characteristics. Compared with nulliparous women, parous women had higher levels of Ta-SCD [mean (standard deviation, SD) = 31.3% (7.6%) vs. 28.9% (7.1%); P-value = 0.01]. Similarly, women with a positive family history of breast cancer had higher levels of Ta-SCD than those without family history [mean (SD) = 33.0% (7.5%)] vs. 30.9% (7.6%); P-value = 0.03]. Conversely, increasing body size was associated with decreasing Ta-SCD [mean (SD) = 31.6% (7.4%), 31.4% (7.3%), and 30.1% (8.0%) for slight, average, and large body sizes, respectively; P-value = 0.005]. CONCLUSIONS: Epidemiological risk factors were associated with varying degrees of stromal cellularity in tumors, independently of clinicopathological characteristics. IMPACT: The findings raise the possibility that epidemiological risk factors may partly influence tumor biology via the stromal microenvironment. See related In the Spotlight, p. 459

Fluorescent Probes for Imaging Reactive Cell Metabolites

This thesis reports the design and development of activity-based sensors (ABS) form vitro imaging of reactive oxygen (ROS) and nitrogen species (RNS). The ABS have been designed on established boronic pinacol ester sensing moieties and a range of reporters including: 4-nitroaniline, 4-methyl-7-amino-coumarin, and 1,8-naphthalimide. Chapter 1 includes: a thesis introduction, background to reactive intracellular metabolites such as hydrogen peroxide and peroxynitrite, current methods of ROS/RNS imaging, fluorescence imaging, and a history of activity-based sensing. Chapter 2 comprises an in-depth investigation into known and novel proof-of-concept based ABS. their synthesis, and all non-biological fluorescence testing, including the selectivity, stability. and sensitivity of such compounds. Chapters 3 and 4 include two strategies to enhance the intracellular retention time of fluorescent reporters for ABS. The first hypothesis for improving the intracellular retention time of reporters relies on lowering passive permeability, a concept termed ''the polarity switch". This chapter investigates the entrapment of positively charged reporters unmasked via ROS/RNS mediated mechanisms. The second strategy and the following chapter explore using covalent warheads for the promiscuous covalent attachment of ABS within the cell. This chapter also includes the design and development of a novel masked covalent warhead which is unreactive until activation by ROS/RNS. Lastly, chapter 5 includes all biological and imaging data of the compounds generated as part of this thesis, including a discussion on the permeability and stability of ABS under biological conditions

Addressing uncertainty in hereditary colorectal cancer: the role of a regional expert multidisciplinary team meeting.

There is frequent uncertainty in both the precise quantification of risk, and the application of clinical interventions, designed to mitigate increased heritable colorectal cancer (CRC) susceptibility. We evaluated the role of a collaborative specialist multidisciplinary team meeting (MDM) for familial and hereditary CRC, led by the St Mark's Hospital Centre for Familial Intestinal Cancer specifically in supporting the clinical management of uncertainty. A retrospective thematic analysis of meeting outcomes from inception in June 2020 until March 2023 was performed. Descriptive statistics were employed to ascertain clinicopathological data, clinical queries and whether MDM recommendations were outside the scope of current guidelines. In total 260 cases were discussed from 13 regional institutions. A prior personal history of cancer was present in 215 (82.6%), and a family history of CRC in 107(41.2%) and non-CRC 27(10.4%) cases. In thematic analysis uncertainty related to indications for genetic testing was considered in 148 (56.9%) of cases, with unexplained mismatch repair deficiency (u-dMMR) in 78 (30%) of cases, and resolution of molecular interpretation in 61 (23.5%). Surveillance related queries represented 55 (21.1%), and mainstreaming 29 (11%) of cases. Management was recommended beyond the scope of existing guidelines in 64 (24.6%) cases. This regional hereditary CRC MDM provides clinicians with support in areas of uncertainty in diagnosis and clinical management, supporting clinical decision-making where evidence and clinical guidelines may be limited. This model could be replicated to support complexity in clinical care in other geographical regions or other health conditions