Biology subjects

Ebner, D.

Publications and source records attributed to Ebner, D..

14 recordsLinked to original sources

bioRxiv2026

Refined USP25/28 inhibitors with improved selectivity towards c-Myc driven squamous lung cancer cells

The ubiquitin specific protease 28 (USP28) is implicated in tumorigenesis by controlling the turnover of substrates including the oncogene c-MYC and the ubiquitin ligase FBW7. Here, we describe small molecule inhibitors of USP25 and USP28, leading to cancer cell cycle arrest and death. However, genetic deletion of USP25/28 does not replicate this effect. An integrated -omics approach revealed off-target effects for thienopyridine and thienopyrazine carboxamide compounds in protein translation. Chemoproteomics and biochemical analyses suggested binding of such compounds to a region near the ribosome complex polypeptide exit tunnel. Structural analysis of a USP28-inhibitor complex enabled the design of modified USP25/28 inhibitor molecules which minimized translation-related off-target effects. In distinction to earlier compounds, the optimized inhibitors were non-toxic to breast cancer cells yet retained potent anti-proliferative activity in squamous lung carcinoma cells, where USP28 is associated with disease progression. Together, our results demonstrate that refined USP25/28 inhibitors can selectively suppress tumor growth by targeting c-MYC driven pathways, offering a more precise therapeutic strategy for treating squamous lung cancers whilst minimizing undesired cytotoxicity.

cancer biology↗

bioRxiv2026

SoftHybrid: A Hybrid Imputation Algorithm Optimised for Single-Cell Proteomics Data

Missing values (MVs) remain a significant barrier to reliable proteomics analysis, particularly in single-cell proteomics, where small amounts of starting material and limits in detection drive Missing-Not-At-Random (MNAR) sparsity. Existing imputation methods typically target either Missing-At-Random (MAR) or MNAR mechanisms, resulting in a trade-off between replicate consistency and preservation of biological variation, and are largely designed for bulk data. Here, we introduce SoftHybrid, a data-driven imputation framework that jointly models missingness and protein abundance to estimate the probability of MNAR, enabling continuous weighting between MAR- and MNAR-oriented strategies. SoftHybrid requires no external priors (cell type labels, group annotations, predefined missingness assumptions, etc.), enabling fully unsupervised applications. Across ground truth benchmarks and real single-cell proteomics datasets, SoftHybrid outperforms existing methods at low input and matches or exceeds their performance at the mini-bulk level. By preserving proteomic structure and abundance accuracy, it enhances the recovery of biologically meaningful signals. SoftHybrid is implemented as an R package and is freely available on GitHub.

bioinformatics↗

bioRxiv2024

A comprehensive pharmacological survey across heterogeneous patient-derived GBM stem cell models

Despite substantial drug discovery investments, the lack of any significant therapeutic advancement in the treatment of glioblastoma (GBM) over the past two decades calls for more innovation in the identification of effective treatments. The inter-and intra-patient heterogeneity of GBM presents significant obstacles to effective clinical progression of novel treatments by contributing to tumour plasticity and rapid drug resistance that confounds contemporary target directed drug discovery strategies. Phenotypic drug screening is ideally suited to heterogeneous diseases, where targeting specific oncogenic drivers have been broadly ineffective. Our hypothesis is that a modern phenotypic led approach using disease relevant patient-derived GBM stem cell systems will be the most productive approach to identifying new therapeutic targets, drug classes and future drug combinations that target the heterogeneity of GBM. In this study we incorporate a panel of patient-derived GBM stem cell lines into an automated and unbiased Cell Painting assay to quantify multiple GBM stem cell phenotypes. By screening several compound libraries at multiple concentrations across a panel of patient-derived GBM stem cells we provide the first comprehensive survey of distinct pharmacological classes and known druggable targets, including all clinically approved drug classes and oncology drug candidates upon multiple GBM stem cell phenotypes linked to cell proliferation, survival and differentiation. Our data set representing, 3866 compounds, 2.2million images and 64000 datapoints is the largest phenotypic screen carried out to date on a panel of patient-derived GBM stem cell models that we are aware of. We seek to identify agents and target classes which engender potent activity across heterogenous GBM genotypes and phenotypes, in this study we further characterize two validated target classes, histone deacetylase inhibitors and cyclin dependent kinases that exert broad and potent effects on the phenotypic and transcriptomic profiles of GBM stem cells. Here we present all validated hit compounds and their target assignments for the GBM community to explore.

cancer biology↗

bioRxiv2024

Drug combinations targeting FAK and MEK overcomes tumour heterogeneity in glioblastoma

Glioblastoma (GBM) is an aggressive brain tumour with limited treatment options and poor prognosis, largely due to its heterogeneity and the involvement of multiple intracellular signalling pathways that contribute to drug resistance. Standard therapies have not significantly improved patient outcomes over the past two decades. While recent advancements in targeted drug combination therapies, such as dabrafenib and trametinib, show promise for certain GBM subgroups, identifying drug combinations effective across the broader GBM population remains a challenge. Integrin-mediated signalling, particularly through Focal Adhesion Kinase (FAK), plays a pivotal role in GBM pathogenesis and invasion, making it a potential therapeutic target [1]. In our study, we utilized a chemogenomic screening approach to identify synergistic drug combinations that target FAK in glioblastoma. We initially employed a CRISPR-engineered GBM model to assess the effects of FAK depletion and discovered that combining FAK inhibitors with MEK inhibitors, particularly trametinib, demonstrated synergistic effects. This potent combination was validated through various 2D & 3D assays, including cell viability/apoptotic assessment, synergistic analysis, cellular imaging, and target engagement assays. The combination also effectively inhibited spheroid growth and invasion across a diverse panel of patient derived GBM stem cells. Molecular mechanisms underlying these effects included suppression of multiple kinase signalling pathways and enhanced apoptosis, elucidated using Reverse Phase Protein Array (RPPA) profiling and western blot validation. In vivo, the combination therapy significantly reduced tumour volume in orthotopic transplantation models. These findings suggest that combining FAK and MEK inhibitors represent a promising therapeutic strategy to overcome the challenges of GBM treatment.

cancer biology↗

bioRxiv2024

The Src family kinase inhibitor drug Dasatinib and glucocorticoids display synergistic activity against tongue squamous cell carcinoma and reduce MET kinase activity

BackgroundTongue squamous cell carcinoma (TSCC) is an aggressive cancer associated with a poor prognosis and limited treatment options, necessitating new drug targets to improve therapeutic outcomes. Our current work focuses on protein tyrosine kinases as well-known targets for successful cancer therapies. MethodsWestern blot analysis of tyrosine phosphorylation patterns in 34 TSCC lines identified Src family kinases (SFKs) as the main contributors. Inhibition of SFKs with PP2 and Dasatinib led to profound biological effects. A high-throughput screen with 1600 FDA-approved drugs was performed with three TSCC lines to discover drugs that act synergistically with Dasatinib against TSCC cell viability. Glucocorticoids emerged as potential candidates and were further investigated in 2D culture and by 3D soft agar colony formation. Dexamethasone was chosen as the major tool for our analyses. Since Dasatinib and glucocorticoids are known for their pleiotropic actions on cells, we analyzed effects on cell cycle, senescence, autophagy and cell signaling. ResultsA panel of 34 TSCC lines showed a surprisingly homogenous pTyr-protein pattern and a prominent 130 kDa pTyr-protein. Inhibition of SFK activity greatly reduced overall pTyr-protein levels and p130Cas tyrosine phosphorylation. It also impaired TSCC viability in 2D cell culture and 3D soft agar colony formation. A high-throughput drug combination screen with Dasatinib identified glucocorticoids as promising candidates for synergistic activity. Dasatinib and Dexamethasone combination treatment showed strong synergistic effects on Src and p130Cas phosphorylation and led to reduced p130Cas expression. Dexamethasone also suppressed phosphorylation of the MET kinase and its key substrate Gab1. On the cellular level, Dasatinib combination with glucocorticoids led to G1 cell cycle arrest, increased senescence and enhanced autophagy. This was also reflected by effects on cell cycle regulatory proteins, including CDKs and cyclins. ConclusionThis work is the first to show a strong synergistic activity of Dasatinib in combination with clinically used glucocorticoids in solid tumors. Furthermore, the tyrosine kinase MET and its effector protein Gab1 are newly identified glucocorticoid targets. Given the extensive research on MET as a drug target in various cancers, our findings have the potential to advance future cancer treatments.

cancer biology↗

bioRxiv2022

Small-molecule Polθ inhibitors provide safe and effective tumor radiosensitization in preclinical models

DNA polymerase theta (Pol{theta}) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Pol{theta} has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Pol{theta} DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, normal fibroblasts are not radiosensitized by Pol{theta} inhibition. Mechanistically, we show that the radiosensitization caused by Pol{theta} inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Pol{theta} inhibitor with improved metabolic stability. In vivo, the combination of Pol{theta} inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared to radiation alone. These results pave the way for future clinical trials of Pol{theta} inhibitors in combination with radiotherapy.

cancer biology↗

bioRxiv2021

Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency is a life-threatening condition caused by inheritance of the SERPINA1 gene Z variant. This single base pair mutation leads to protein misfolding, ER entrapment and gain of toxic function. Despite the significant unmet medical need presented by this disorder, there remain no approved medicines and the only curative option is liver transplantation. We hypothesized that an unbiased screen of human hepatocytes harbouring the Z mutation (ATZ) using small molecules targeted against protein degradation pathways would uncover novel biological insights of therapeutic relevance. Here we report the results of that screen performed in a patient-derived iPSC model of ATZ. Starting from 1,041 compounds we identified 14 targets capable of reducing polymer burden, including Leucine-rich repeat kinase-2 (LRRK2), a well-studied target in Parkinsons. Genetic deletion of LRRK2 in ATZ mice reduced polymers and associated fibrotic liver disease leading us to test a library of commercially available LRRK2 kinase inhibitors in both patient iPSC and CHO cell models. One of the molecules tested, CZC-25146, reduced polymer load, increased normal AAT secretion and reduced inflammatory cytokines with pharmacokinetic properties supporting its potential use for treating liver diseases. We therefore tested CZC-25146 in the ATZ mouse model and confirmed its efficacy for polymer reduction without signs of toxicity. Mechanistically, in both human and mouse models, our data show CZC-25146 inhibits LRRK2 kinase activity and induces autophagy. Cumulatively, these findings support the use of CZC-25146 and LRRK2 inhibitors in general in hepatic proteopathy disease research and as potential new treatment approaches for patients. One Sentence SummaryA small molecule screen in patient iPSCs with in vivo validation in mice identifies LRRK2 as a new therapeutic target for Alpha-1 Antitrypsin Deficiency.

cell biology↗

bioRxiv2021

Therapeutic targeting of SETD2-deficient cancer cells with the small-molecule compound RITA

The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in certain cancer types, identifying SETD2 as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to the compound RITA, resulting in significant p53 induction and apoptosis. This is further associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. RITA sensitivity is linked to the phenol sulphotransferase SULT1A1, which we find to be highly upregulated in cells that lack SETD2. Depletion of SULT1A1 or addition of the phenol sulphotransferase inhibitor DCNP abolishes these phenotypes and suppresses the sensitivity of SETD2-deficient cancer cells, identifying SULT1A1 activity to be critical in mediating the potent cytotoxicity of RITA against SETD2-deficient cells. These findings define a novel therapeutic strategy for targeting the loss of SETD2 in cancer. Significance StatementThe histone-modifying enzyme SETD2 has emerged as an important tumour suppressor in a number of different cancer types, identifying it as a promising therapeutic target. The concept of synthetic lethality, a genetic interaction in which the simultaneous loss of two genes or pathways that regulate a common essential process renders the cell nonviable, is a valuable tool for killing cancer cells that have known mutations. In this study, we conducted a synthetic lethality screen for compounds that specifically target SETD2-deficient cancer cells. The top hit, a compound called RITA, reduces cell viability and induces cell death only in the context of SETD2 loss, thereby highlighting a potential novel therapeutic strategy for treating SETD2-deficient cancers.

cell biology↗

bioRxiv2021

A genome-wide CRISPR screen identifies regulators of beta cell function involved in type 2 diabetes risk

Identification of the genes and processes mediating genetic association signals for complex disease represents a major challenge. Since many of the genetic signals for type 2 diabetes exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of- function screen in human pancreatic beta cells. We focused on the regulation of insulin content as a disease-relevant readout of beta cell function. We identified 580 genes influencing this phenotype: integration with genetic and genomic data provided experimental support for 20 candidate type 2 diabetes effector transcripts including the autophagy receptor CALCOCO2. Our study highlights how cellular screens can augment existing multi-omic efforts to accelerate biological and translational inference at GWAS loci.

genomics↗

bioRxiv2020

Optimized chemogenomic library design strategies for precision oncology

Designing a focused compound screening library of bioactive small molecules is a challenging task since many compounds modulate their effects through multiple protein targets with various degrees of potency and selectivity. We describe here several analytic procedures with adjustable cut-off parameters that enable one to design anticancer target-focused compound libraries optimized for library size, cellular activity, biological and chemical diversity and target selectivity. Even though our focus was on designing compound libraries to enable a comprehensive investigation of the target biology of glioblastoma (GBM), the compound collections cover a wide range of protein targets and biological pathways implicated in various types of cancers, making the libraries widely applicable in precision oncology studies. We published the final screening set library, called the Comprehensive anti-Cancer small-Compound Library, or C3L.We hope these general library design principles and the current, widely annotated small molecule libraries will prove useful for the community in various phenotypic screening experiments in GBM and other cancers.

cancer biology↗

bioRxiv2020

IGF targeting perturbs global replication through ribonucleotide reductase dysfunction

IGF receptor (IGF-1R) inhibition delays repair of radiation-induced DNA double-strand breaks (DSBs), prompting us to investigate whether IGF-1R influences endogenous DNA damage. We demonstrate that IGF-1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. We detect delayed replication fork progression in IGF-inhibited or IGF-1R depleted cancer cells, with activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflects unanticipated regulation of global replication by IGF-1, mediated via AKT, MEK/ERK and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. IGF-1R inhibition or depletion downregulate RRM2, compromising RNR function and dNTP supply. The resulting delay in fork progression and hallmarks of replication stress are rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF-1 regulates replication. Following targeted compound screens, we identify synergy between IGF inhibition and ATM loss, with evidence that IGF inhibition compromises growth of ATM null cells and xenografts. This synthetic lethal effect reflects conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. These data implicate IGF-1R in alleviating replication stress, and the identified reciprocal IGF:ATM co-dependence provides an approach to exploit this effect in ATM-deficient cancers.

cancer biology↗

bioRxiv2019

The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

cell biology↗

bioRxiv2019

Germline and somatic genetic variants in the p53 pathway interact to affect cancer risk, progression and drug response

Insights into oncogenesis derived from cancer susceptibility loci could facilitate better cancer management and treatment through precision oncology. However, therapeutic applications have thus far been limited by our current lack of understanding regarding both their interactions with somatic cancer driver mutations and their influence on tumorigenesis. Here, by integrating germline datasets relating to cancer susceptibility with tumour data capturing somatically-acquired genetic variation, we provide evidence that single nucleotide polymorphism (SNPs) and somatic mutations in the p53 tumor suppressor pathway can interact to influence cancer development, progression and treatment response. We go on to provide human genetic evidence of a tumor-promoting role for the pro-survival activities of p53, which supports the development of more effective therapy combinations through their inhibition in cancers retaining wild-type p53. SignificanceWe describe significant interactions between heritable and somatic genetic variants in the p53 pathway that affect cancer susceptibility, progression and treatment response. Our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel therapeutic targets.

cancer biology↗

bioRxiv2019

Cyclin F-Chk1 synthetic lethality mediated by E2F1 degradation

Cyclins are central engines of cell cycle progression when partnered with Cyclin Dependent Kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but forms an E3 ubiquitin ligase, assembling through the F-box domain, an Skp1-Cul1-F-box (SCF) module. Although multiple substrates of cyclin F have been identified the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging cells with more than 200 kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. The DNA replication catastrophe depends on the accumulation of E2F1 in cyclin F depleted cells. We observe that SCFcyclin F promotes E2F1 degradation after Chk1 inhibitors in a CDK dependent manner. Thus, Cyclin F restricts E2F1 activity during cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.

cancer biology↗