Simon Vyse

UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

UBE2L3 is associated with increased susceptibility to numerous autoimmune diseases, but the underlying mechanism is unexplained. By using data from a genome-wide association study of systemic lupus erythematosus (SLE), we observed a single risk haplotype spanning UBE2L3, consistently aligned across multiple autoimmune diseases, associated with increased UBE2L3 expression in B cells and monocytes. rs140490 in the UBE2L3 promoter region showed the strongest association. UBE2L3 is an E2 ubiquitin-conjugating enzyme, specially adapted to function with HECT and RING-in-between-RING (RBR) E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC). Our data demonstrate that UBE2L3 is the preferred E2 conjugating enzyme for LUBAC in vivo, and UBE2L3 is essential for LUBAC-mediated activation of NF-κB. By accurately quantifying NF-κB translocation in primary human cells from healthy individuals stratified by rs140490 genotype, we observed that the autoimmune disease risk UBE2L3 genotype was correlated with basal NF-κB activation in unstimulated B cells and monocytes and regulated the sensitivity of NF-κB to CD40 stimulation in B cells and TNF stimulation in monocytes. The UBE2L3 risk allele correlated with increased circulating plasmablast and plasma cell numbers in SLE individuals, consistent with substantially elevated UBE2L3 protein levels in plasmablasts and plasma cells. These results identify key immunological consequences of the UBE2L3 autoimmune risk haplotype and highlight an important role for UBE2L3 in plasmablast and plasma cell development.

Dual Targeting of PDGFRα and FGFR1 Displays Synergistic Efficacy in Malignant Rhabdoid Tumors

Summary Subunits of the SWI/SNF chromatin remodeling complex are mutated in a significant proportion of human cancers. Malignant rhabdoid tumors (MRTs) are lethal pediatric cancers characterized by a deficiency in the SWI/SNF subunit SMARCB1. Here, we employ an integrated molecular profiling and chemical biology approach to demonstrate that the receptor tyrosine kinases (RTKs) PDGFRα and FGFR1 are coactivated in MRT cells and that dual blockade of these receptors has synergistic efficacy. Inhibitor combinations targeting both receptors and the dual inhibitor ponatinib suppress the AKT and ERK1/2 pathways leading to apoptosis. MRT cells that have acquired resistance to the PDGFRα inhibitor pazopanib are susceptible to FGFR inhibitors. We show that PDGFRα levels are regulated by SMARCB1 expression, and assessment of clinical specimens documents the expression of both PDGFRα and FGFR1 in rhabdoid tumor patients. Our findings support a therapeutic approach in cancers with SWI/SNF deficiencies by exploiting RTK coactivation dependencies.

Improved monitoring of clinical response in Systemic Lupus Erythematosus by longitudinal trend in soluble vascular cell adhesion molecule-1

BackgroundTo determine whether optimal use of serial measurements of serum levels of soluble cell adhesion molecules (CAM) can improve monitoring of disease activity in SLE.MethodsSerum levels of soluble CAM and conventional SLE biomarkers were measured in serial samples (n = 80) from 21 SLE patients during and after flare and correlated in longitudinal analysis with disease activity determined by ECLAM score. Blood samples from a second cohort of 34 SLE patients were subject to flow cytometry to correlate serum biomarkers with B cell subsets.ResultsBy adjusting for the baseline level (at the first visit), delta soluble vascular cell adhesion molecule-1 (sVCAM-1) showed stronger correlation with changes in ECLAM score and improved sensitivity and specificity for identifying SLE responders versus non-responders compared to conventional SLE biomarkers including anti-dsDNA antibody titre and complement C3. Multiple regression analysis identified delta sVCAM-1 as the best marker of SLE clinical response. sVCAM-1 levels were significantly correlated with CD95+CD27+ activated memory B cells, CD95+ plasmablasts and circulating plasma cell numbers in SLE patients.ConclusionSubtracting a baseline level of sVCAM-1 for each individual substantially improved its utility as a biomarker. Delta sVCAM-1 was superior to conventional SLE biomarkers for monitoring changes in disease activity. This suggests that serial monitoring of serum sVCAM-1 trends should be considered in SLE patients to document responses to treatment. We hypothesise that the correlation between activated B cell subsets and circulating plasma cell numbers with soluble VCAM-1 serum levels in SLE may relate to the important role of VCAM-1 in B lymphocyte survival and maturation in bone marrow and secondary lymphoid tissues.

Effect of UBE2L3 genotype on regulation of the linear ubiquitin chain assembly complex in systemic lupus erythematosus.

BACKGROUND A single risk haplotype across UBE2L3 is strongly associated with systemic lupus erythematosus (SLE) and many other autoimmune diseases. UBE2L3 is an E2 ubiquitin-conjugating enzyme with specificity for RING-in-between-RING E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC), which has a pivotal role in inflammation, through crucial regulation of NF-κB. We aimed to determine whether UBE2L3 regulates LUBAC-mediated activation of NF-κB, and determine the effect of UBE2L3 genotype on NF-κB activation and B-cell differentiation. METHODS UBE2L3 genotype data from SLE genome-wide association studies was imputed by use of 1000 Genomes data. UBE2L3 function was studied in a HEK293-NF-κB reporter cell line with standard molecular biology techniques. p65 NF-κB translocation in ex-vivo B cells and monocytes from genotyped healthy individuals was quantified by imaging flow cytometry. B-cell subsets from healthy individuals and patients with SLE, stratified by UBE2L3 genotype, were determined by multicolour flow cytometry. FINDINGS rs140490, located at -270 base pairs of the UBE2L3 promoter, was identified as the most strongly associated single nucleotide polymorphism (p=8·6 × 10(-14), odds ratio 1·30, 95% CI 1·21-1·39). The rs140490 risk allele increased UBE2L3 expression in B cells and monocytes. Marked upregulation of NF-κB was observed with combined overexpression of UBE2L3 and LUBAC, but abolished by dominant-negative mutant UBE2L3 (C86S), or UBE2L3 silencing. The rs140490 genotype correlated with basal NF-κB activation in ex-vivo human B cells and monocytes, as well as NF-κB sensitivity to CD40 or tumour necrosis factor (TNF) stimulation. UBE2L3 expression was 3-4 times higher in circulating plasmablasts and plasma cells than in other B-cell subsets, with higher levels in patients with SLE than in controls. The rs140490 genotype correlated with increasing plasmablast and plasma cell differentiation in patients with SLE. INTERPRETATION This study shows that NF-κB activation mediated by LUBAC is exquisitely sensitive to the expression level of UBE2L3. The UBE2L3 risk haplotype is correlated with TNF and CD40 induced NF-κB activation in primary human cells, and with plasmablast and plasma cell expansion in SLE, consistent with the dependence of these cells on NF-κB as a survival factor. Since UBE2L3 is highly expressed in plasma cells, UBE2L3 could be a novel therapeutic target in SLE. FUNDING Arthritis Research UK, Wellcome Trust, George Koukis Foundation, European Communitys Seventh Framework Programme.

Advances in mass spectrometry based strategies to study receptor tyrosine kinases

This review discusses advances in mass spectrometry-based strategies to investigate receptor tyrosine kinase signalling networks and activation dynamics.

Exploiting receptor tyrosine kinase co-activation for cancer therapy

Studies over the past decade have shown that many cancers have evolved receptor tyrosine kinase (RTK) co-activation as a mechanism to drive tumour progression and limit the lethal effects of therapy. This review summarises the general principles of RTK co-activation and discusses approaches to exploit this phenomenon in cancer therapy and drug discovery. Computational strategies to predict kinase co-dependencies by integrating drug screening data and kinase inhibitor selectivity profiles will also be described. We offer a perspective on the implications of RTK co-activation on tumour heterogeneity and cancer evolution and conclude by surveying emerging computational and experimental approaches that will provide insights into RTK co-activation biology and deliver new developments in effective cancer therapies.

Exploiting Synthetic Lethality and Network Biology to Overcome EGFR Inhibitor Resistance in Lung Cancer

Despite the recent approval of third-generation therapies, overcoming resistance to epidermal growth factor receptor (EGFR) inhibitors remains a major challenge in non-small cell lung cancer. Conceptually, synthetic lethality holds the promise of identifying non-intuitive targets for tackling both acquired and intrinsic resistance in this setting. However, translating these laboratory findings into effective clinical strategies continues to be elusive. Here, we provide an overview of the synthetic lethal approaches that have been employed to study EGFR inhibitor resistance and review the oncogene and non-oncogene signalling mechanisms that have thus far been unveiled by synthetic lethality screens. We highlight the potential challenges associated with progressing these discoveries into the clinic including context dependency, signalling plasticity, and tumour heterogeneity, and we offer a perspective on emerging network biology and computational solutions to exploit these phenomena for cancer therapy and biomarker discovery. We conclude by presenting a number of tangible steps to bolster our understanding of fundamental synthetic lethality mechanisms and advance these findings beyond the confines of the laboratory.

Primary Cilia Mediate Diverse Kinase Inhibitor Resistance Mechanisms in Cancer

Summary Primary cilia are microtubule-based organelles that detect mechanical and chemical stimuli. Although cilia house a number of oncogenic molecules (including Smoothened, KRAS, EGFR, and PDGFR), their precise role in cancer remains unclear. We have interrogated the role of cilia in acquired and de novo resistance to a variety of kinase inhibitors, and found that, in several examples, resistant cells are distinctly characterized by an increase in the number and/or length of cilia with altered structural features. Changes in ciliation seem to be linked to differences in the molecular composition of cilia and result in enhanced Hedgehog pathway activation. Notably, manipulating cilia length via Kif7 knockdown is sufficient to confer drug resistance in drug-sensitive cells. Conversely, targeting of cilia length or integrity through genetic and pharmacological approaches overcomes kinase inhibitor resistance. Our work establishes a role for ciliogenesis and cilia length in promoting cancer drug resistance and has significant translational implications.

Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model

The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three‐dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non‐malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1–HRAS and UBA2–PDCD2L expressed in‐frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer‐relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer.

Systematic analysis of tumour cell-extracellular matrix adhesion identifies independent prognostic factors in breast cancer

Tumour cell-extracellular matrix (ECM) interactions are fundamental for discrete steps in breast cancer progression. In particular, cancer cell adhesion to ECM proteins present in the microenvironment is critical for accelerating tumour growth and facilitating metastatic spread. To assess the utility of tumour cell-ECM adhesion as a means for discovering prognostic factors in breast cancer survival, here we perform a systematic phenotypic screen and characterise the adhesion properties of a panel of human HER2 amplified breast cancer cell lines across six ECM proteins commonly deregulated in breast cancer. We determine a gene expression signature that defines a subset of cell lines displaying impaired adhesion to laminin. Cells with impaired laminin adhesion showed an enrichment in genes associated with cell motility and molecular pathways linked to cytokine signalling and inflammation. Evaluation of this gene set in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort of 1,964 patients identifies the F12 and STC2 genes as independent prognostic factors for overall survival in breast cancer. Our study demonstrates the potential of in vitro cell adhesion screens as a novel approach for identifying prognostic factors for disease outcome.