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Synthetic oligonucleotides as DHX8 inhibitors: An oligomimetic approach
No full textThis thesis presents the development of selective inhibitors and tool compounds targeting DEAH-Box Helicase 8 (DHX8), guided by an X-ray crystal structure of poly(A)6 RNA bound within the protein’s RNA-binding tunnel.1 To achieve this, chemically modified oligonucleotides (ONs) were designed and screened according to an oligomimetic approach—a novel approach intended to transform RNA-derived scaffolds into drug-like compounds with enhanced therapeutic potential. Compared to conventional small-molecule approaches, the oligomimetic framework has the potential to expand binding site coverage, overcome potency plateaus, potentially minimise toxicity, and improve selectivity. The development of oligomimetic DHX8 inhibitors in this thesis serves as a proof of concept for extending this approach to other disease relevant RNA-binding proteins (RBPs), which are notoriously difficult to drug due to their shallow, polar, and diffuse binding interfaces.
This strategy is a marriage between recent advances in ON therapeutics and a traditional medicinal chemistry approach to early-stage drug discovery. It involved systematic testing of poly(A)6 analogues incorporating single-point changes to the nucleobases, phosphates and ribose rings, followed by the evaluation of combined modifications to determine synergistic effects. Substitutions were based on ON modifications known to enhance cell permeability, bioavailability and nuclease stability.
This led to the identification of a poly(A)4-based minimum pharmacophore and several modified poly(A)4 derivatives, or oligomimetics, exhibiting improvements in potency of up to more than 1,000-fold in a biochemical competitive binding assay. Of these, oligomimetics 47, 56 and 57 were selected for further testing and demonstrated inhibitor-like functional activity in an ATPase assay, selectivity against DHX15, and cellular activity in an MCL1S/L alternative splicing assay. X-ray crystal structures of 47 and 56 were obtained, revealing a new intermediate conformational state of DHX8.
Overall, an oligomimetic approach was successfully employed in the discovery of selective DHX8 inhibitors from an RNA starting point. Compounds were initially screened in competitive binding assays based on Fluorescence Polarisation (FP) and/or Homogenous Time-Resolved Fluorescence (HTRF) and subsequently validated by Surface Plasmon Resonance (SPR) and, in some cases, Ligand-Observed NMR (LO-NMR). Results and hypotheses were supported by X-ray crystallography and in silico studies
A novel model of glioblastoma recurrence to identify therapeutic vulnerabilities.
No full textGlioblastoma remains incurable and recurs in all patients. Here we design and characterize a novel induced-recurrence model in which mice xenografted with primary patient-derived glioma initiating/stem cells (GIC) are treated with a therapeutic regimen closely recapitulating patient standard of care, followed by monitoring until tumours recur (induced recurrence patient-derived xenografts, IR-PDX). By tracking in vivo tumour growth, we confirm the patient specificity and initial efficacy of treatment prior to recurrence. Availability of longitudinally matched pairs of primary and recurrent GIC enabled patient-specific evaluation of the fidelity with which the model recapitulated phenotypes associated with the true recurrence. Through comprehensive multi-omic analyses, we show that the IR-PDX model recapitulates aspects of genomic, epigenetic, and transcriptional state heterogeneity upon recurrence in a patient-specific manner. The accuracy of the IR-PDX enabled both novel biological insights, including the positive association between glioblastoma recurrence and levels of ciliated neural stem cell-like tumour cells, and the identification of druggable patient-specific therapeutic vulnerabilities. This proof-of-concept study opens the possibility for prospective precision medicine approaches to identify target-drug candidates for treatment at glioblastoma recurrence
Towards the Rational Design of Monovalent Degraders: Lessons Learnt from Cyclin K Degraders.
No full textMonovalent degraders can enhance pre-existing surface complementarity between a target protein and a ligase to induce target degradation via the proteasome. For the most part, degraders have been discovered serendipitously and structure-activity relationship (SAR) studies have been limited, making it difficult to rationally design new compounds. Here we discuss how work on the SAR of cyclin K degraders demonstrates that a broad range of compounds can stabilise protein-protein interactions to induce degradation and how it lays the foundation for further monovalent degrader discovery
Combining (chemo)radiotherapy with immunomodulatory agents in head and neck cancer
No full textDespite advances in immuno-oncology, attempts to improve outcomes for patients with locally
advanced, unresectable squamous cell carcinoma of the head and neck (SCCHN) using immune
checkpoint inhibitors alongside standard radiotherapy (RT) and chemoradiotherapy (CRT) have largely
failed. Motivated by these translational setbacks, this thesis employed a reverse translational approach
to investigate the immunological effects of RT and CRT using an immunocompetent murine model of
SCCHN, with particular focus on CD8⁺ T-cell dynamics, antigen engagement, and T-cell receptor (TCR)
clonality.RT alone enhanced local tumour control and increased intratumoural CD8⁺ T-cell infiltration
but also triggered immunosuppressive responses. Nr4a3-Tocky reporter analysis revealed a timedependent
rise in CD8⁺ T-cell antigen engagement, with peak expansion of PD-1hi “persistent-arrested”
subsets at days 7-10 post-RT. Despite their activation, these antigen-engaged T-cells were not
functionally required for tumour control, which was shown to be CD8⁺ T-cell-independent. In contrast,
CRT displayed CD8⁺ T-cell-dependent tumour control, suggesting addition of cisplatin sensitises
tumours to T-cell-mediated killing. Single-cell analysis revealed that CRT, more so than RT, enhanced
CD8⁺ T-cell clonal expansion and enriched for effector-like and progenitor-exhausted phenotypes
versus terminally exhausted. However, these shifts were insufficient to synergise with anti-PD-1
therapy, highlighting persistent immunosuppressive barriers. Addition of the inhibitor of apoptosis
protein (IAP) antagonist xevinapant to CRT, while increasing local tumour cell death, paradoxically
impaired the CRT-induced immunological benefits by diminishing CD8⁺ T-cell cell function and
disrupting key innate and adaptive immune pathways.These findings underscore the necessity of
thoroughly characterising the immunomodulatory effects of standard-of-care treatments such RT and
CRT prior to the integration of immunotherapeutic agents. They further highlight the complexity of
combination treatments and the critical need for mechanistic validation to inform rational and effective
combination strategie
Risk adapted therapy for newly diagnosed multiple myeloma delivered through local cytogenetic laboratories in a National Clinical Trial: UKMRA RADAR study.
No full textThe UKMRA RADAR study is a phase II/III clinical trial for newly diagnosed multiple myeloma patients eligible for autologous stem cell transplant. It offers a risk-adapted approach with the addition of isatuximab for genetically high-risk patients, evaluated using local cytogenetics laboratories rather than centralised testing. We have observed excellent overall success rates, with > 90% patients assigned to a risk-adapted pathway following cytogenetic testing in 25 local laboratories nationwide, with clinically-relevant turnaround times allowing > 70% patients to commence isatuximab at the earliest opportunity if indicated. This paves the way for providing standard-of-care risk-adapted treatment for multiple myeloma patients in the UK
Immunological characterisation of muscle invasive bladder cancer: insights from multidimensional profiling of urine, blood and tumour samples from clinical trials patients
No full textDespite recent advances, treatment for muscle-invasive bladder cancer (MIBC) remains a major clinical and research challenge. Bladder cancer immunology likely impacts variable responses to chemo-, radio-, and immunotherapy, but is incompletely understood. Research into immune involvement could identify biomarkers of response, optimize immunotherapy strategies, and improve longitudinal disease monitoring. Liquid biopsies, such as blood and urine, offer promising non-invasive research and clinical opportunities, whilst profiling tumour tissue provides valuable insights into the immune tumour microenvironment.
This thesis integrated genomic, transcriptomic, and immunological studies of blood, tumour, and urine samples from MIBC clinical trial patients with relevant clinical outcomes. The aim was to construct an integrated, in-depth translational profile of immune involvement in bladder cancer. Chapter One is a general introduction to bladder cancer, immunology (particularly of T-cells), outstanding research needs and overarching thesis aims. Chapter Two outlines clinical trials investigated and general sample processing methodologies.
Chapter Three presents optimization of sample processing. Protocols developed were tailored to planned downstream analyses; in particular, improved yield of selected biological materials from urine was achieved. Chapter Four presents whole exome sequencing (WES) data and RNA sequencing data acquired from diagnostic tissue from MIBC patients. Chapter Five utilized TCR sequencing from blood, tissue and urine to compare and track T-cell metrics and dynamics at baseline and throughout treatment. Chapter Six presents flow cytometry data, including from urinary lymphocytes, for longitudinal tracking of immune responses throughout treatment. Chapter Seven employed tissue staining and imaging techniques including multiplex immunofluorescence to visualise the immune tumour microenvironment. Final chapters present integration of data from the aforementioned techniques, and detail final discussions and conclusions arising from the work presented, including clinical implications, and future research directions. In summary, this thesis used multimethodology to provide new insights into immune involvement in bladder cancer, with potential further research and clinical implications
Pembrolizumab plus Abiraterone Acetate and Prednisone in Patients with Chemotherapy-naïve Metastatic Castration-resistant Prostate Cancer: Results from KEYNOTE-365 Cohort D.
No full textBACKGROUND AND OBJECTIVE: Abiraterone acetate (abiraterone) plus prednisone is approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Our aim was to evaluate the efficacy and safety of pembrolizumab plus abiraterone in mCRPC. METHODS: In cohort D of the phase 1b/2 KEYNOTE-365 study (NCT02861573), patients were chemotherapy-naïve, had disease progression ≤6 mo before screening, and had either not received prior next-generation hormonal agents for mCRPC or had received prior enzalutamide for mCRPC and had disease progression or became intolerant to enzalutamide. Patients received pembrolizumab 200 mg intravenously every 3 wk plus abiraterone 1000 mg orally once daily and prednisone 5 mg orally twice daily. The primary endpoints were safety, prostate-specific antigen (PSA) response rate, and objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) by blinded independent central review (BICR). Secondary endpoints included radiographic progression-free survival (rPFS) according to Prostate Cancer Clinical Trials Working Group 3-modified RECIST v1.1 by BICR and overall survival (OS). KEY FINDINGS AND LIMITATIONS: For the 103 patients who were treated, median follow-up was 28 mo (interquartile range 26-31). The confirmed PSA response rate was 56% (58/103 patients). The ORR for patients with RECIST v1.1-measurable disease was 16% (6/37 patients). Median rPFS was 15 mo (95% confidence interval 9.2-22) and median OS was 30 mo (95% confidence interval 23-not reached); the estimated 24-mo OS rate was 58%. In total, 91% of patients experienced treatment-related adverse events, and 39% experienced grade 3-5 events. Grade 3/4 elevation of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) was observed in 12% and 6.8% of patients, respectively. One patient died due to treatment-related myasthenic syndrome. Study limitations include the single-arm design. CONCLUSIONS: Pembrolizumab plus abiraterone and prednisone demonstrated antitumor activity and acceptable safety in patients with chemotherapy-naïve mCRPC. Higher incidence of grade 3/4 elevated ALT/AST occurred than was reported for the individual agents. PATIENT SUMMARY: For patients with metastatic castratation-resistant prostate cancer, the drug combination of pembrolizumab plus abiraterone and prednisone showed antitumor activity and acceptable safety
Blood tumor load: combining biomarkers to increase the proportion of informative patients.
No full textBACKGROUND: The load of circulating tumor cells (CTC) and tumor-derived extracellular vesicles (tdEV) strongly correlates with poor clinical outcomes and can be used to evaluate treatment response and the presence of treatment targets. However, the frequency of CTC is low, making an accurate assessment impossible in most patients. Here, we introduce blood tumor load (BTL), in which CTC and tdEV are combined into one value ranging from 0 (low) to 1 (high) to simplify result interpretation and increase the percentage of patients from which a reliable assessment can be made. PATIENTS AND METHODS: The CTC and tdEV counts were obtained from the ACCEPT analysis of the CellSearch image datasets of 98 metastatic breast cancer patients (mBC) and 157 castration-resistant prostate cancer patients (CRPC). The BTL generated using these counts was used in human epidermal growth factor receptor 2 (HER2) expression assessment in mBC patients. The BTL scores of CRPC patients at baseline and first follow-up time points were evaluated, and a change in BTL, indicating response to therapy, was measured in the patients. RESULTS: Using 10 CTCs as a threshold, the HER2 positivity could be determined in 34/98 (35%) breast cancer patients, whereas with BTL, the positivity increased to 76/98 (78%). The BTL showed an improved Cox hazard ratio for overall survival in 157 CRPC patients before and at first follow-up points compared with CTC and tdEV alone. A decrease in BTL indicating response to therapy was seen in 45% of CRPC patients, and an increase in BTL was seen in 9%, indicating progression on treatment. The remaining 46% of patients showed no change. CONCLUSIONS: In this study, we demonstrated the applications of BTL in improving the reliability of measuring response to therapy and increasing the proportion of patients from which the presence of a treatment target can be assessed
Challenging the concept of functional high-risk myeloma through transcriptional and genetic profiling.
No full textFunctional high-risk (FHR) multiple myeloma (MM) is defined as an unexpected, early relapse (ER) of disease in the absence of baseline molecular or clinical risk factors (RF), making FHR MM inherently dependent on which RFs were assessed at diagnosis, and what treatment patients received. To establish the true incidence of FHR, we analyzed uniformly treated, transplant-eligible patients from the Myeloma-XI (MyXI) trial that had been profiled for the International Myeloma Society and Working Group (IMS/IMWG) defined high-risk cytogenetic aberrations (HRCA), and the SKY92 gene expression HR signature (GEP-HR). A total of 135 MyXI patients were studied, with a median follow-up of 88 months; 25 (18.5%) experienced ER, defined as relapse <18 months from maintenance randomization post-autologous stem-cell transplantation. Hereof, 15 (60%) were IMS/IMWG-HR at diagnosis, of whom 8 were also GEP-HR. Another 6 patients were GEP-HR only and would have been missed by IMS/IMWG-HR. Among 4 patients with IMS/IMWG- and GEP-standard risk, 2 had isolated HR markers at diagnosis, leaving only 2 patients (8% of ER; 1.5% of all) truly meeting all FHR-criteria. Combined IMS/IMWG-HR and GEP-HR profiling identified 84% of ER, and differentiated long-term outcome across all 135 patients: co-occurring IMS/IMWG and GEP-HR was associated with very short overall survival compared to the absence of both (HR = 13.1; 95% CI, 6.5-26.1, P < .0001), followed by GEP-HR only (HR = 5.1; 95% CI, 2.4-11.1, P < .0001) and IMS/IMWG-HR only (HR = 3.2; 95% CI, 1.6-6.2, P = .0007). Our results support more comprehensive baseline diagnostic profiling to identify those at risk of ER upfront. The trials were registered at the ISRCTN Registry as ISRCTN49407852 and at clinicaltrials.gov as #NCT01554852
Effect of drain omission after mastectomy on cosmesis, patient satisfaction and interval to adjuvant therapy.
No full textINTRODUCTION: Omission of closed suction drains in women undergoing simple mastectomy has become the standard in the United Kingdom (UK) with studies demonstrating no difference in symptomatic seroma rates or complications. A theoretical concern is that a large-volume seroma distorts the skin envelope, potentially resulting in inferior long-term postoperative aesthetic appearance and patient satisfaction. Furthermore, the seroma may lead to a delay in adjuvant treatment, in particular, chest wall radiotherapy. There is currently no objective scoring system to evaluate the postoperative appearance after simple mastectomy. METHODS: Patients who had undergone a drainless unilateral simple mastectomy at the Royal Marsden Hospital attending for annual surveillance contralateral mammography between October 2016 and July 2017 were invited to complete a BREAST-Q questionnaire and attend medical photography for panel assessment of aesthetic outcome. Patient satisfaction in this cohort was compared with results from the UK National Mastectomy and Breast Reconstruction Audit (NMBRA) 2011, which was conducted at a time when surgical drains were routinely placed. RESULTS: The proportion of patients satisfied with their appearance was similar to that of NMBRA 2011. BREAST-Q results were in line with the published literature. A panel assessment scoring system for simple mastectomies was developed. There was no difference in delays to adjuvant treatment between the study and NMBRA cohort. CONCLUSIONS: Omission of drains in women undergoing simple mastectomy did not result in inferior aesthetic outcomes or lower patient satisfaction, nor did it result in delay to adjuvant treatment. BREAST-Q results were in line with the literature. A panel assessment scoring system for simple mastectomy was developed