Biology subjects

Lim, E.

Publications and source records attributed to Lim, E..

At least 19 recordsLinked to original sources

bioRxiv2026

The CUTIN SYNTHASE enzyme family was a key driver of cuticle emergence in land plants

The plant cuticle is a key adaptation acquired during the colonization of land. It forms a hydrophobic barrier at the interface with the environment, fulfilling essential protective and developmental functions. Despite its evolutionary significance and central role in land plant biology, the determinants that drove the emergence of the cuticle remain poorly understood. Here, we show that the CUTIN SYNTHASE (CUS) enzyme family, which synthesises the lipidic polyester that forms the structural framework of the cuticle in flowering plants, originated in a common ancestor of land plants, concomitant with terrestrialization. Using the moss Physcomitrium patens, we further demonstrate that CUS function is conserved among land plants. Inactivation of CUS genes disrupts gametophore development, the first tissue forming a cuticle during the moss life cycle, and compromises cuticle integrity. We also show that P. patens CUS enzymes localize to the apoplast, where they mediate the formation of a 10,16-dihydroxyhexadecanoic acid polyester using 2-mono-(10,16-dihydroxyhexadecanoyl)glycerol as substrate. Overall, our results reveal the conservation of CUS catalytic and physiological functions over 500 million years and support a pivotal role for this enzyme family in the emergence of the cuticle in an ancestral land plant during terrestrialization. SIGNIFICANCEThe cuticle is a hallmark of land plants that fulfills essential roles, ranging from protection to development. Elucidating the mechanisms underlying its emergence and formation therefore has the potential to reveal fundamental aspects of land plant evolution and biology. Here, we show that the CUTIN SYNTHASE (CUS) enzyme family, which catalyzes the formation of the cuticle framework in flowering plants, arose in an ancestor of land plants during terrestrialization. Using the moss Physcomitrium patens, we further demonstrate that CUS function has been conserved for 500 million years across bryophytes and tracheophytes. We propose that the emergence of the CUS family was a key event in establishing the plant cuticle during terrestrialization.

plant biology↗

bioRxiv2026

Ovarian hormone deficiency enhances wood smoke-induced immune dysfunction via transcriptomic and metabolic alterations

The increasing frequency and severity of wildfires have heightened public exposure to smoke, highlighting the importance of identifying susceptibility factors, including ovarian hormone deficiency. Here, we used single-cell RNA sequencing to profile bone marrow immune cells from ovariectomized (OVX) mice exposed to either filtered air (FA) or wood smoke (WS) followed by functional validation in macrophages from both OVX and Sham-operated mice. Single-cell analyses focused on the OVX context; interactions between surgery and exposure were confirmed at the functional level in assays that included both Sham and OVX groups. In OVX mice, WS broadly suppressed transcriptional programs involved in antigen processing, leukocyte activation, antiviral defense, and bone remodeling. This was associated with altered immune cell composition, including increased memory CD8L T cells and decreased granulocytes and interferon-responsive populations. Bone marrow-derived macrophages (BMDMs) from WS-exposed OVX mice displayed metabolic reprogramming, characterized by the reversal of OVX-induced suppression of oxidative phosphorylation and glycolytic activity, along with reduced expression of M2-associated genes, without concurrent induction of M1-associated genes. This immune-metabolic decoupling suggests that WS exposure under ovarian hormone deficiency may imprint a lasting program in the bone marrow macrophage axis. Together, these findings show that ovarian hormone deficiency increases vulnerability to WS-induced immune disruption in the bone marrow. WS triggers macrophage reprogramming only under ovarian hormone deficiency, leading to heightened metabolic activity alongside suppression of key immune pathways, identifying a novel mechanism of immunotoxicity. These findings emphasize the need to consider hormonal status in air pollution risk assessment.

pharmacology and toxicology↗

bioRxiv2026

Acute wood smoke exposure is associated with cell-specific hippocampal transcriptomic responses in an accelerated ovarian failure mouse model

BackgroundWildfire events are increasing in frequency and intensity, and it is well-known that aging individuals are more susceptible to air pollution exposures, and that air pollution exposures result in neurological sequelae. Despite this, it is unclear how declining levels of ovarian hormones that naturally occur in aging females influence brain vulnerability to air pollution. Menopause and the menopausal transition represent a period of profound physiological change that affects cardiovascular, neurological, and immune health. MethodsWe tested whether perimenopausal-like hormonal status amplifies hippocampal responses to acute wood smoke (WS) using an ovary-intact, 4-vinylcyclohexene diepoxide (VCD) model of moderate accelerated ovarian failure (AOF) in female C57BL/6 mice. Animals were exposed to HEPA-filtered air (FA) or WS for 4 h/day over 2 consecutive days ([~]0.5 mg/m3). Exposure characterization confirmed a complex mixture of combustion products with significant levels of both trace metals and gas release during WS exposure. ResultsSpatial transcriptomics (10x Visium; (n=4 sections/group) with automated cell-type annotation identified astrocytes, GABAergic and glutamatergic neurons, oligodendrocytes, revealed cell type-specific transcriptional alterations following WS exposure. Distinct transcriptional patterns were observed across all identified neuronal and glial cell populations. ConclusionTogether, these findings define a cell type-resolved transcriptional framework linking WS exposure and ovarian hormone decline and identify potential cellular pathways relevant to hippocampal vulnerability.

pharmacology and toxicology↗

bioRxiv2025

Functional Activity of HIV-1 bNAbs Across Diverse Strains is Driven by Binding Site and Can be Enhanced Through Fc Engineering

Broadly neutralizing antibodies (bNAbs) are promising tools for HIV-1 treatment and prevention, due to their ability to mediate both Fab-dependent neutralization and Fc-dependent effector functions. While antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) have been implicated in antiviral activity, the extent to which these functions vary across bNAb epitopes, viral strains, and Fc region remains unclear. Here, we systematically evaluated twenty bNAbs targeting five distinct Env epitopes against a diverse panel of nine HIV-1 strains. We found that epitope specificity and Env sequence both critically influenced bNAb effector functions. CD4 binding site (CD4bs)- and V3 glycan-targeting bNAbs mediated the broadest and most potent ADCC and ADCP, while V1/V2 apex-directed bNAbs preferentially induced ADCP. In contrast, MPER-targeting bNAbs triggered ADCC more selectively, and gp120/gp41 interface-targeting bNAbs showed limited activity. Irrespective of binding epitope, different strains exhibited a broad range of effector function sensitivities. To investigate the impact of Fc modifications on this variability, we subclass-switched and introduced previously identified Fc mutations known to change Fc{gamma} receptor affinity. Some of these mutations significantly boosted ADCC, while IgG3 subclass switching dramatically enhanced ADCP, even against highly resistant strains. Collectively, these results demonstrate that effector function is shaped by both antibody specificity and viral Env context and that rational Fc modification has the potential to improve antibody-based therapeutics against HIV. One Sentence SummaryThis study systematically evaluates the impact of bNAb epitope specificity, Env sequence, and Fc engineering on antibody-mediated effector functions.

immunology↗

bioRxiv2025

Stromal subsets modulate T-cell infiltration in early breast cancer

Recent studies of the tumour microenvironment have elucidated the heterogeneity of stromal cells, with increasing evidence suggesting that stromal subsets play an important role in regulating anti-tumour immunity in breast cancer. However, the functional diversity of these cells within the tumour immune microenvironment and how they interact with immune cells in a spatial and clinical context remain poorly understood. We performed multiplex immunofluorescence on tumour microarrays from two cohorts consisting of 591 breast cancer patients to assess the abundance and spatial co-localisation of stromal and immune subsets and their correlation with clinicopathological features and patient outcomes. We found that stromal cells were spatially distinct. A perivascular-like subset that was disseminated throughout the stroma rather than restricted to vessel-adjacent regions was enriched in an immune cold environment and associated with T-cell exclusion. An inflammatory-like cancer-associated fibroblast subset was associated with segregation of T cells from cancer cells. Our findings highlight the differential impact of stromal subsets on immune infiltration and activation within the breast cancer tumour microenvironment with potential implications for patient outcomes. SignificanceThis study characterised spatially-defined interactions between stromal and immune subsets in large clinical cohorts of early breast cancer. Findings from this study will contribute to gaps in current knowledge in how diverse stromal cell subsets, particularly novel subsets, interact with immune cells in a clinically-relevant context.

cancer biology↗

bioRxiv2025

ROCK2 inhibition has a dual role in reducing ECM remodelling and cell growth, while impairing migration and invasion

The Rho-associated coiled-coil containing protein kinases 1/2 (ROCK1/2) are key signalling proteins involved in the regulation of the actin cytoskeleton and control a variety of cellular processes. This includes cell proliferation, stemness, cell migration and invasion as well as actomyosin contraction and extracellular matrix (ECM) remodelling. Due to a lack of ROCK2 specific inhibitors, previous pharmacological studies of ROCK function have relied on pan-ROCK1/2 inhibition. Here, we use the ROCK2 specific inhibitor, GV101, in the context of breast cancer (BC) models to uncouple the effects of ROCK2 pharmacological inhibition on both epithelial and stromal fibroblast cell populations. We demonstrate that ROCK2 inhibition with GV101 reduces fibroblast-mediated remodelling of pre-existing and de novo synthesised ECM resulting in a reduction in biomechanical stiffness, while also limiting epithelial cell growth in 2D and 3D settings. We also demonstrate that ROCK2 inhibition exposes epithelial cell vulnerability to fluid flow-induced shear stress. Furthermore, using 3D co-cultures we reveal that GV101 reduces single-cell migratory capacity, while the disruption of stromal ECM architecture is sufficient to impede collective cell migration and invasion. Finally, we assessed ROCK2 expression in human BC patient tumours revealing that ROCK2 expression is up-regulated during BC progression and that high ROCK2 expression correlates with poor patient outcome in the triple-negative subtype of BC. Together, these results demonstrate that ROCK2 specific inhibition disrupts key functions in both epithelial cell and stromal fibroblast compartments warranting further assessment of ROCK2 in highly proliferative, fibrotic or metastatic diseases, such as BC. HighlightsO_LIFibroblast-mediated remodelling of pre-existing and de novo synthesised extracellular matrix is reduced upon ROCK2 specific targeting. C_LIO_LIROCK2 inhibition impairs epithelial cancer cell growth and colony formation in 2D and 3D contexts, while also limiting single-cell migration and cell survival upon fluid flow-induced shear stress. C_LIO_LIROCK2 targeting during fibroblast-mediated ECM remodelling is sufficient to disrupt collective cell migration as well as 3D invasion into organotypic fibroblast-contracted collagen matrices. C_LIO_LIROCK2 expression is up-regulated during breast cancer progression and high expression correlates with poor patient outcome in triple-negative breast cancer. C_LI

cell biology↗

bioRxiv2025

Genetic barcoding uncovers the clonal makeup of solid and liquid biopsies

Intra-tumoral heterogeneity (ITH) is fueling tumor progression in breast cancer, as specific clones present within a tumor may have a selective advantage to colonize distant organs and escape therapy. Accurate sampling of ITH is therefore a pressing challenge in clinical oncology, to adequately predict recurrence and inform rational and personalized therapies. Here we used cellular barcoding to track the spatio-temporal composition of human breast cancer clones in six preclinical models - across two cell lines and four patient derived xenografts (PDXs). This allowed direct side-by-side quantitative comparison not only of intra-tumor clonal composition, but also of how that composition was reflected in needle biopsies and cell-free DNA (cfDNA). These analyses highlighted several clinically relevant findings. First, the use of orthogonal genetic and optical barcoding revealed that clonal diversity in the center of non-necrotic primary tumors was significantly higher compared to their periphery. Second, cfDNA barcode analysis suggested that DNA shedding in the vasculature varied largely, not only depending on necrosis and tumor burden, but also between models. Third, combining information captured in both solid and liquid biopsies can provide a more robust assessment of tumor clonal composition. Taken together, these results showcases the utility of these barcoded models to optimize the use of solid and liquid biopsies as surrogate markers of ITH.

cancer biology↗

bioRxiv2025

Interplay between T3SS effectors, ExoY activation, and cGMP signaling in Pseudomonas aeruginosa infection

ExoY is a nucleotidyl cyclase, secreted by the Pseudomonas aeruginosa type 3 secretion system (T3SS), which generates various cyclic nucleotides within host cells, predominantly cGMP. How ExoY-catalyzed cNMPs production contributes to the infection process remains unclear. Using recombinant P. aeruginosa strains expressing ExoY variants with different substrate specificities, we identified that ExoY-derived cGMP modulates the cytotoxicity of ExoT, a co-injected T3SS exotoxin. Our results demonstrated that dephosphorylation of the host CrkII adaptor protein, a downstream effect of the ADP-ribosyltransferase activity of ExoT, is limited by ExoY-derived cGMP, therefore antagonizing ExoT-induced cell retraction. We also revealed a reciprocal interaction between T3SS effectors, showing that ExoT and ExoS, can in turn, suppress ExoY activity and its production of cGMP in certain cell types, thereby limiting their regulation by ExoY-synthesized cGMP. These findings highlight the intricate interplay between T3SS effectors and uncover a novel host cell-dependent regulation within P. aeruginosa infections.

microbiology↗

bioRxiv2025

National Scale Genomic Engine for Precision Medicine: Singapore PRECISE-SG100K Experience

The whole-of-nation National Precision Medicine (NPM) programme in Singapore aims to integrate a genomics backbone into standard healthcare. Phase II recently completed the sequencing of 102,202 genomes from the diverse Asian population of Singapore, generating the PRECISE-SG100K dataset. The scaling from the pilot SG10K_Health phase to national production scale demanded a central shift: from earlier generation workflows to an integrated, industrial grade "Genomic Engine". The initiative was co-developed under a public-private partnership model. Our Genomic Engine was designed with an automation-first approach, as a modular platform linking a purpose-built, centralized biospecimen processing and DNA extraction facility (EXTRACT Lab), a high-throughput sequencing operation (Illumina Data Generation Infrastructure, IDGI), with the cloud-native analytics (GRIDS) running on a fully integrated PRECISE Sample Tracking System (STS). Here we describe the key design features of our Genomic Engine. Highlighting the Stress Test phase where the automated DNA extraction yields failed to meet expectations. We share the operational adjustments which were implemented to stabilize production and resulted in a three-fold reduction in sample triage due to improved DNA recovery. Without establishing the operational guardrails during the Stress Test, the Engine would not have reached a steady-state production evidenced by >3,000 genomes per month, median turnaround time 11 days, 99.6% operational success, within in six months. This framework offers a practical blueprint for other population-scale initiatives seeking to bridge the gap between scientific piloting and industrial delivery.

genomics↗

bioRxiv2025

JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

PurposeEndocrine therapy in combination with CDK4/6 inhibition doubles the progression-free survival of patients with advanced ER+ breast cancer, but resistance is inevitable, leaving patients with limited treatment options. Experimental DesignWe performed unbiased genome-wide CRISPR/Cas9 knockout screens using ER+ breast cancer cells to identify novel drivers of resistance to combination endocrine therapy (tamoxifen) and CDK4/6 inhibitor (palbociclib) treatment. Screen hits were validated by CRISPR/Cas9 knockout models, mechanistic analyses and evaluation of patient samples. ResultsOur screens identified the inactivation of JNK signalling, including loss of the kinase MAP2K7, as a key driver of combination resistance. We developed multiple CRISPR/Cas9 knockout ER+ breast cancer cell lines (MCF-7 and T-47D) to investigate the effects of MAP2K7, MAPK8 and MAPK9 loss. MAP2K7 knockout increased metastatic burden in vivo and led to impaired JNK-mediated stress responses, as well as promoting cell survival and reducing senescence entry following endocrine therapy and CDK4/6 inhibitor treatment. Mechanistically, this occurred via loss of the AP-1 transcription factor c-JUN, leading to an attenuated response to combination endocrine therapy plus CDK4/6 inhibition. Furthermore, we analysed ER+ advanced breast cancer patient cohorts and found that inactivation of the JNK pathway was associated with increased metastatic burden, and low pJNKT183/Y185 activity correlated with a poorer response to systemic endocrine and CDK4/6 inhibitor therapies. ConclusionsOverall, we demonstrate that suppression of JNK signalling enables persistent growth during combined endocrine therapy and CDK4/6 inhibition. Furthermore, our data provide a pre-clinical rationale to screen patients tumours for JNK signalling deficiency prior to receiving combined endocrine therapy and CDK4/6 inhibition. STATEMENT OF TRANSLATIONAL RELEVANCEResistance to CDK4/6 inhibitors in the context of endocrine therapy resistance presents an urgent clinical challenge for the management of estrogen receptor positive (ER+) breast cancer. However, the mechanisms driving resistance to this therapeutic combination remain poorly understood. Here, we have identified inactivation of JNK signalling, specifically loss of the JNK kinase MAP2K7, as a major determinant of resistance to combined endocrine therapy and CDK4/6 inhibition in ER+ breast cancer. We uncovered that MAP2K7 loss augments tumour survival in vitro and in vivo. This occurs via disruption of activator protein-1 (AP-1) transcription factors, and thus prevents the induction of therapy-induced senescence and JNK-induced stress response. These findings reveal a critical tumour suppressor role for the JNK pathway in ER+ breast cancer, highlighting the importance of identifying patients with deficient JNK signalling and cautioning against the development of JNK inhibitors for this setting.

cancer biology↗

bioRxiv2024

Gut Microbiome Signatures During Acute Infection Predict Long COVID

Long COVID (LC), manifests in 10-30% of non-hospitalized individuals post-SARS-CoV-2 infection leading to significant morbidity. The predictive role of gut microbiome composition during acute infection in the development of LC is not well understood, partly due to the heterogeneous nature of disease. We conducted a longitudinal study of 799 outpatients tested for SARS-CoV-2 (380 positive, 419 negative) and found that individuals who later developed LC harbored distinct gut microbiome compositions during acute infection, compared with both SARS-CoV-2-positive individuals who did not develop LC and negative controls with similar symptomatology. However, the temporal changes in gut microbiome composition between the infectious (0-1 month) and post-infectious (1-2 months) phases was not different between study groups. Using machine learning, we showed that microbiome composition alone more accurately predicted LC than clinical variables. Including clinical data only marginally enhanced this prediction, suggesting that microbiome profiles during acute infection may reflect underlying health status and immune responses thus, help predicting individuals at risk for LC. Finally, we identified four LC symptom clusters, with gastrointestinal and fatigue-only groups most strongly linked to gut microbiome alterations.

genomics↗

bioRxiv2024

Federated deep learning enables cancer subtyping by proteomics

Artificial intelligence applications in biomedicine face major challenges from data privacy requirements. To address this issue for clinically annotated tissue proteomic data, we developed a Federated Deep Learning (FDL) approach (ProCanFDL), training local models on simulated sites containing data from a pan-cancer cohort (n=1,260) and 29 cohorts held behind private firewalls (n=6,265), representing 19,930 replicate data-independent acquisition mass spectrometry (DIA-MS) runs. Local parameter updates were aggregated to build the global model, achieving a 43% performance gain on the hold-out test set (n=625) in 14 cancer subtyping tasks compared to local models, and matching centralized model performance. The approachs generalizability was demonstrated by retraining the global model with data from two external DIA-MS cohorts (n=55) and eight acquired by tandem mass tag (TMT) proteomics (n=832). ProCanFDL presents a solution for internationally collaborative machine learning initiatives using proteomic data, e.g., for discovering predictive biomarkers or treatment targets, while maintaining data privacy. Statement of SignificanceA federated deep learning approach applied to human proteomic data, acquired using two distinct proteomic technologies from 40 tumor cohorts from eight countries, enabled accurate cancer histopathological subtyping while preserving data privacy. This approach will enable privacy-compliant development of large-scale proteomic AI models, including foundation models, across institutions globally.

cancer biology↗

bioRxiv2023

An experimental comparison of the Digital Spatial Profiling and Visium spatial transcriptomics technologies for cancer research

BackgroundSpatial transcriptomic technologies are powerful tools for resolving the spatial heterogeneity of gene expression in tissue samples. However, little evidence exists on relative strengths and weaknesses of the various available technologies for profiling human tumour tissue. In this study, we aimed to provide an objective assessment of two common spatial transcriptomics platforms, 10X Genomics Visium and Nanostrings GeoMx DSP. MethodThe abilities of the DSP and Visium platforms to profile transcriptomic features were compared using matching cell line and primary breast cancer tissue samples. A head-to-head comparison was conducted using data generated from matching samples and synthetic tissue references. Platform specific features were also assessed according to manufacturers recommendations to evaluate the optimal usage of the two technologies. ResultsWe identified substantial variations in assay design between the DSP and Visium assays such as transcriptomic coverage and composition of the transcripts detected. When the data was standardised according to manufacturers recommendations, the DSP platform was more sensitive in gene expression detection. However, its specificity was diminished by the presence of non-specific detection. Our results also confirmed the strength and weakness of each platform in characterising spatial transcriptomic features of tissue samples, in particular their application to hypothesis generation versus hypothesis testing. ConclusionIn this study, we share our experience on both DSP and Visium technologies as end users. We hope this can guide future users to choose the most suitable platform for their research. In addition, this dataset can be used as an important resource for the development of new analysis tools.

cancer biology↗

bioRxiv2022

Treatment with anti-inflammatory viral serpin modulates immuno-thrombotic responses and improves outcomes in SARS-CoV-2 infected mice

1.Severe acute respiratory distress syndrome (ARDS) during SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infection, manifests as uncontrolled lung inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies can reduce COVID-19 severity if administered in the early viremic phase, but treatments for later stage immuno-thrombotic syndrome and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases during thrombotic, thrombolytic and immune responses. The myxoma poxvirus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated coagulation and complement protease pathways as part of a self-defense strategy to combat viral clearance by the innate immune system. When purified and utilized as an anti-immune therapeutic, Serp-1 is effective as an anti-inflammatory drug in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 (PEGSerp-1) as a therapy for immuno-thrombotic complications during ARDS. Treatment with PEGSerp-1 in two distinct mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved clinical outcomes. PEGSerp-1 significantly reduced M1 macrophage invasion in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR) and complement membrane attack complex (MAC). Sequential changes in urokinase-type plasminogen activator receptor (uPAR) and serpin gene expression were observed in lung and heart with PEGSerp-1 treatment. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for treatment of severe viral ARDS with additional potential to reduce late SARS-CoV-2 complications related to immune-thrombotic events that persist during long COVID. SignificanceSevere acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infection manifests as uncontrolled tissue inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies reduce COVID-19 severity if administered early, but treatments for later stage immuno-thrombosis are limited. Serine protease inhibitors (SERPINS) regulate thrombotic, thrombolytic and complement pathways. We investigate here systemic treatment with purified poxvirus-derived PEGSerp-1 as a therapeutic for immuno-thrombotic complications in viral ARDS. PEGSerp-1 treatment in two mouse-adapted SARS-CoV-2 models (C57Bl/6 and BALB/c) significantly reduced lung and heart inflammation and improved clinical outcomes, with sequential changes in thrombolytic (uPAR) and complement expression. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for immune-thrombotic complications in severe viral ARDS and has potential benefit for long COVID.

microbiology↗

bioRxiv2021

Pre-clinical validation of an RNA-based precision oncology platform for patient-therapy alignment in a diverse set of human malignancies resistant to standard treatments

Predicting tumor sensitivity to antineoplastics remains an elusive challenge, with no methods demonstrating predictive power. Joint analysis of tumors--from patients with distinct malignancies who had progressed on multiple lines of therapy--and drug perturbation transcriptional profiles predicted sensitivity to 28 of 350 drugs, 26 of which (93%) were confirmed in low-passage, patient-derived xenograft (PDX) models. Drugs were prioritized based on their ability to either invert the activity of individual Master Regulator proteins, with available high-affinity inhibitors, or of the modules they comprise (Tumor-Checkpoints), based on de novo mechanism of action analysis. Of 138 PDX mice enrolled in 16 single and 18 multi-protein treatment arms, a disease control rate (DCR) of 68% and 91 %, and an objective response rate (ORR) of 12% and 17%, were achieved respectively, compared to 6% and 0% in the negative controls arm, with multi-protein drugs achieving significantly more durable responses. Thus, these approaches may effectively complement and expand current precision oncology approaches, as also illustrated by a case study.

systems biology↗

bioRxiv2021

Epigenetic therapy suppresses endocrine-resistant breast tumour growth by re-wiring ER-mediated 3D chromatin interactions

Three-dimensional (3D) epigenome remodelling is an important mechanism of gene deregulation in cancer. However, its potential as a target to overcome therapy resistance remains largely unaddressed. Here we show that FDA-approved epigenetic therapy Decitabine (5-Aza-mC) suppresses tumour growth in preclinical metastatic ER+ breast tumour xenograft models. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher order chromatin structure and loss of topologically associated domain boundary insulation. Significant DNA hypomethylation at ER-enhancer elements was associated with gain in ER binding, creation of ectopic 3D enhancer-promoter interactions and concordant activation of ER-mediated transcription pathways. Importantly long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates how epigenetic therapy has potential to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions associated with suppression of tumour growth.

molecular biology↗

bioRxiv2021

PLURIPOTENT STEM CELL-DERIVED CARDIOVASCULAR PROGENITORS DIFFFERENTIATED ON LAMININ 221 REGENERATE AND IMPROVE FUNCTION OF INFARCTED SWINE HEARTS

BackgroundIschemic heart disease is a huge global burden where patients often have irreversibly damaged heart muscle. State-of-the-art technology using stem cell-derived products for cellular therapy could potentially replace damaged heart muscle for regenerative cardiology. Methods and ResultsPluripotent human embryonic stem cells (hESCs) were differentiated on a laminin LN521+221 matrix to cardiovascular progenitors (CVPs). Global transcriptome analyses at multiple time points by single-cell RNA-sequencing demonstrated high reproducibility (R2 > 0.95) between two hESCs lines. We identified several CVP signature genes as quality batch control parameters which are highly specific to our CVPs as compared to canonical cardiac progenitor genes. A total of 200 million CVPs were injected into the infarcted region caused by permanent ligation of the coronary arteries of 10 immunosuppressed pigs and maintained for 4- and 12-weeks post transplantation. The transplanted cells engrafted and proliferated in the infarcted area as indicated by IVIS imaging, histology staining and spatial transcriptomic analysis. Spatial transcriptomic analysis at 1 week following transplantation showed that the infarcted region expressed human genes in the same area as immunohistology sections. Heart function was analyzed by magnetic resonance imaging (MRI) and computerized tomography (CT). Functional studies revealed overall improvement in left ventricular ejection fraction by 21.35 {+/-} 3.3 %, which was accompanied by significant improvements in ventricular wall thickness and wall motion, as well as a reduction in infarction size after CVP transplantation as compared to medium control pigs (P < 0.05). Immunohistology analysis revealed maturation of the CVPs to cardiomyocytes (CMs) where the human grafts aligned with host tissue forming end-to-end connections typical for heart muscle. Electrophysiology analyses revealed electric continuity between injected and host tissue CMs. Episodes of ventricular tachyarrhythmia (VT) over a period of 25 days developed in four pigs, one pig had persistent VT, while the rest remained in normal sinus rhythm. All ten pigs survived the experiment without any VT-related death. ConclusionsWe report a highly reproducible, chemically defined and fully humanized differentiation method of hESCs for the generation of potent CVPs. This method may pave the way for lasting stem cell therapy of myocardial infarction (MI) in humans. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIWe present a highly reproducible, chemically defined and fully humanized laminin-based differentiation method for generation of large amounts of cardiovascular progenitors (CVP); 20 million cells in a 10 cm2 culture dish which were used for a preclinical study in pigs. C_LIO_LITransplantation of the CVPs into the myocardial infarcted pig hearts yields maturation of the progenitor cells to cardiomyocytes (CMs) and improved cardiac function (21.35 {+/-} 3.3 % LVEF improvement) using only 200 million CVPs. C_LIO_LITemporary episodes of ventricular arrhythmia (50%) were observed after CVP transplantation. No fatal ventricular arrhythmia occurred. C_LI What are the clinical implications?O_LIOur laminin-based approach generated potent CVPs in vivo and largely restored function of the damaged heart. C_LIO_LICardiovascular progenitors may provide a new and safe therapeutic strategy for myocardial infarction. C_LIO_LIThe results may have a significant impact on regenerative cardiology. C_LI

cell biology↗

bioRxiv2020

DNA methylation and survival differences associated with the type of IDH mutation in 1p/19q non-codeleted astrocytomas

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1R132H mutations. This high prevalence is important as IDH1R132H is presumed to be relatively poor at producing D-2-hydroxyglutarate (D-2HG) whereas high concentrations of this oncometabolite are required to inhibit TET2 DNA demethylating enzymes. Indeed, patients harbouring IDH1R132H mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of site of origin. For 1p/19q non-codeleted glioma patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring non-R132H mutated tumours have better outcome (HR 0.41, 95% CI [0.24, 0.71], p=0.0013). Non-R132H mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p<0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H mutated 1p/19q non-codeleted gliomas have a more favourable prognosis than their IDH1R132H mutated counterpart is clinically relevant and should be taken into account for patient prognostication. Single sentence summaryAstrocytoma patients with tumours harbouring IDH mutations other than p.R132H have increased DNA methylation levels and longer survival

cancer biology↗