ClassifieR 2.0: expanding interactive gene expression-based stratification to prostate and high-grade serous ovarian cancer.

Background: Advances in transcriptional profiling methods have enabled the discovery of molecular subtypes within and across traditional tissue-based cancer classifications. Such molecular subgroups hold potential for improving patient outcomes by guiding treatment decisions and revealing physiological distinctions and targetable pathways. Computational methods for stratifying transcriptomic data into molecular subgroups are increasingly abundant.

Ribosome Quality Control mitigates the cytotoxicity of ribosome collisions induced by 5-Fluorouracil.

Ribosome quality control (RQC) resolves collided ribosomes, thus preventing their cytotoxic effects. The chemotherapeutic agent 5-Fluorouracil (5FU) is best known for its misincorporation into DNA and inhibition of thymidylate synthase. However, while a major determinant of 5FU's anticancer activity is its misincorporation into RNAs, the mechanisms by which cancer cells overcome the RNA-dependent 5FU toxicity remain ill-defined.

Nanoencapsulation of MDM2 Inhibitor RG7388 and Class-I HDAC Inhibitor Entinostat Enhances their Therapeutic Potential Through Synergistic Antitumor Effects and Reduction of Systemic Toxicity.

Inhibitors of the p53-MDM2 interaction such as RG7388 have been developed to exploit latent tumor suppressive properties in p53 in 50% of tumors in which p53 is wild-type. However, these agents for the most part activate cell cycle arrest rather than death, and high doses in patients elicit on-target dose-limiting neutropenia. Recent work from our group indicates that combination of p53-MDM2 inhibitors with the class-I HDAC inhibitor Entinostat (which itself has dose-limiting toxicity issues) has the potential to significantly augment cell death in p53 wild-type colorectal cancer cells.

Dual-Hit Strategy for Therapeutic Targeting of Pancreatic Cancer in Patient-Derived Xenograft Tumors.

Purpose: Paracrine activation of pro-fibrotic hedgehog (HH) signaling in pancreatic ductal adenocarcinoma (PDAC) results in stromal amplification that compromises tumor drug delivery, efficacy, and patient survival. Interdiction of HH-mediated tumor-stroma crosstalk with smoothened (SMO) inhibitors (SHHi) "primes" PDAC patient-derived xenograft (PDX) tumors for increased drug delivery by transiently increasing vascular patency/permeability, and thereby macromolecule delivery. However, patient tumor isolates vary in their responsiveness, and responders show co-induction of epithelial-mesenchymal transition (EMT).

surviveR: a flexible shiny application for patient survival analysis.

Kaplan-Meier (KM) survival analyses based on complex patient categorization due to the burgeoning volumes of genomic, molecular and phenotypic data, are an increasingly important aspect of the biomedical researcher's toolkit. Commercial statistics and graphing packages for such analyses are functionally limited, whereas open-source tools have a high barrier-to-entry in terms of understanding of methodologies and computational expertise. We developed surviveR to address this unmet need for a survival analysis tool that can enable users with limited computational expertise to conduct routine but complex analyses.

Circulating tumour DNA kinetics in recurrent/metastatic head and neck squamous cell cancer patients.

Purpose: Immune checkpoint blockade (ICB) has become a standard of care in the treatment of recurrent/metastatic head and neck squamous cell cancer (R/M HNSCC). However, only a subset of patients benefit from treatment. Quantification of plasma circulating tumour DNA (ctDNA) levels and on-treatment kinetics may permit real-time assessment of disease burden under selective pressures of treatment.

Autocrine activation of MAPK signaling mediates intrinsic tolerance to androgen deprivation in LY6D prostate cancer cells.

The emergence of castration-resistant prostate cancer remains an area of unmet clinical need. We recently identified a subpopulation of normal prostate progenitor cells, characterized by an intrinsic resistance to androgen deprivation and expression of LY6D. We here demonstrate that conditional deletion of PTEN in the murine prostate epithelium causes an expansion of transformed LY6D progenitor cells without impairing stem cell properties.

Investigating the suitability of cell lines as models for the major subtypes of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most fatal gynaecological malignancy, accounting for over 200,000 deaths worldwide per year. EOC is a highly heterogeneous disease, classified into five major histological subtypes-high-grade serous (HGSOC), clear cell (CCOC), endometrioid (ENOC), mucinous (MOC) and low-grade serous (LGSOC) ovarian carcinomas. Classification of EOCs is clinically beneficial, as the various subtypes respond differently to chemotherapy and have distinct prognoses.

Bcl-xL Is a Key Mediator of Apoptosis Following KRASG12C Inhibition in KRASG12C-mutant Colorectal Cancer.

Novel covalent inhibitors of KRASG12C have shown limited response rates in patients with KRASG12C-mutant (MT) colorectal cancer. Thus, novel KRASG12C inhibitor combination strategies that can achieve deep and durable responses are needed. Small-molecule KRASG12C inhibitors AZ'1569 and AZ'8037 were used.

Activation of innate-adaptive immune machinery by poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer.

Objective: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy.

Design: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of and stroma-rich models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours.

classifieR a flexible interactive cloud-application for functional annotation of cancer transcriptomes.

Background: Transcriptionally informed predictions are increasingly important for sub-typing cancer patients, understanding underlying biology and to inform novel treatment strategies. For instance, colorectal cancers (CRCs) can be classified into four CRC consensus molecular subgroups (CMS) or five intrinsic (CRIS) sub-types that have prognostic and predictive value. Breast cancer (BRCA) has five PAM50 molecular subgroups with similar value, and the OncotypeDX test provides transcriptomic based clinically actionable treatment-risk stratification.

VPRBP Functions Downstream of the Androgen Receptor and OGT to Restrict p53 Activation in Prostate Cancer.

Unlabelled: Androgen receptor (AR) is a major driver of prostate cancer initiation and progression. O-GlcNAc transferase (OGT), the enzyme that catalyzes the covalent addition of UDP-N-acetylglucosamine (UDP-GlcNAc) to serine and threonine residues of proteins, is often highly expressed in prostate cancer with its expression correlated with high Gleason score. In this study, we have identified an AR and OGT coregulated factor, Vpr (HIV-1) binding protein (VPRBP) also known as DDB1 and CUL4 Associated Factor 1 (DCAF1).

Molecular Subtyping Resource: a user-friendly tool for rapid biological discovery from transcriptional data.

Generation of transcriptional data has dramatically increased in the past decade, driving the development of analytical algorithms that enable interrogation of the biology underpinning the profiled samples. However, these resources require users to have expertise in data wrangling and analytics, reducing opportunities for biological discovery by 'wet-lab' users with a limited programming skillset. Although commercial solutions exist, costs for software access can be prohibitive for academic research groups.

Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists.

Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models, whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to suppress MEDI3039-induced cell death.

TNF-α synergises with IFN-γ to induce caspase-8-JAK1/2-STAT1-dependent death of intestinal epithelial cells.

Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn's disease (CD). Th1-type cytokines-IFN-γ and TNF-α-occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms.

Clinical Positioning of the IAP Antagonist Tolinapant (ASTX660) in Colorectal Cancer.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance.

Dissecting the DNA binding landscape and gene regulatory network of p63 and p53.

The transcription factor p53 is the best-known tumor suppressor, but its sibling p63 is a master regulator of epidermis development and a key oncogenic driver in squamous cell carcinomas (SCC). Despite multiple gene expression studies becoming available, the limited overlap of reported p63-dependent genes has made it difficult to decipher the p63 gene regulatory network. Particularly, analyses of p63 response elements differed substantially among the studies.

Investigating Radiotherapy Response in a Novel Syngeneic Model of Prostate Cancer.

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa.

Chronic loss of STAG2 leads to altered chromatin structure contributing to de-regulated transcription in AML.

Background: The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member.

Methods: Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss.

Tumor suppressor p53: from engaging DNA to target gene regulation.

The p53 transcription factor confers its potent tumor suppressor functions primarily through the regulation of a large network of target genes. The recent explosion of next generation sequencing protocols has enabled the study of the p53 gene regulatory network (GRN) and underlying mechanisms at an unprecedented depth and scale, helping us to understand precisely how p53 controls gene regulation. Here, we discuss our current understanding of where and how p53 binds to DNA and chromatin, its pioneer-like role, and how this affects gene regulation.

Pevonedistat (MLN4924): mechanism of cell death induction and therapeutic potential in colorectal cancer.

Pevonedistat (MLN4924), a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit (NAE1), has demonstrated significant therapeutic potential in several malignancies. Although multiple mechanisms-of-action have been identified, how MLN4924 induces cell death and its potential as a combinatorial agent with standard-of-care (SoC) chemotherapy in colorectal cancer (CRC) remains largely undefined. In an effort to understand MLN4924-induced cell death in CRC, we identified p53 as an important mediator of the apoptotic response to MLN4924.

The pseudo-caspase FLIP(L) regulates cell fate following p53 activation.

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53.

The SCF ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L).

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCF). We find that SCF can interact with both TRAIL-R2's pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC).

A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis.

The long FLIP splice form FLIP(L) can act as both an inhibitor and promoter of caspase-8 at death-inducing signalling complexes (DISCs) formed by death receptors such as TRAIL-R2 and related intracellular complexes such as the ripoptosome. Herein, we describe a revised DISC assembly model that explains how FLIP(L) can have these opposite effects by defining the stoichiometry (with respect to caspase-8) at which it converts from being anti- to pro-apoptotic at the DISC. We also show that in the complete absence of FLIP(L), procaspase-8 activation at the TRAIL-R2 DISC has significantly slower kinetics, although ultimately the extent of apoptosis is significantly greater.

Comparison of Molecular Assays for HPV Testing in Oropharyngeal Squamous Cell Carcinomas: A Population-Based Study in Northern Ireland.

Background: Determination of human papillomavirus (HPV) status has become clinically relevant for patient stratification under UICC TNM8 staging. Within the United Kingdom, a combination of p16 IHC and HPV DNA-ISH is recommended for classifying HPV status. This study will assess a series of clinically applicable second-line molecular tests to run in combination with p16 IHC to optimally determine HPV status.