Biology subjects

Villunger, A.

Publications and source records attributed to Villunger, A..

16 recordsLinked to original sources

bioRxiv2026

TET2 loss promotes premalignant survival and clonal selection in MYC-driven B cell lymphoma

The DNA demethylase ten-eleven translocation enzyme 2 (TET2) is frequently inactivated in hematologic malignancies, yet how its loss shapes oncogene-driven transformation remains unclear. Using a mouse model in which MYC is overexpressed in the B cell lineage, driving aggressive B cell lymphoma, we show that Tet2 loss increases lymphoma penetrance and biases disease toward an IgM immunophenotype. Established lymphomas are broadly similar across genotypes, suggesting that Tet2 loss exerts much of its effects before lymphoma onset. Accordingly, Tet2 loss expands a premalignant IgM B cell subset with reduced apoptotic sensitivity and an increased frequency of BCL2BIMhi cells. Consistently, Tet2 deficient IgM B cells persist better in in vitro cultures, show increased clonogenic survival, and exhibit clonal skewing. These findings support a model in which Tet2 loss heightens MYC-driven lymphoma penetrance by promoting the survival and selection of premalignant B cells under apoptotic stress.

cancer biology↗

bioRxiv2026

Ex Vivo Assay for Organ-Specific Cancer Cell Invasion

BackgroundMetastasis is the leading cause of cancer-related mortality, yet experimental models often fail to recapitulate the tissue-specific microenvironments that shape metastatic dissemination. While in vivo systems provide physiological relevance, they are poorly suited for mechanistic studies. Conversely, conventional in vitro assays lack the organ-specific extracellular matrix (ECM) context that critically regulates invasive behavior. There is therefore a need for accessible models that balance biological relevance with experimental feasibility. ResultsIn this study, we developed an ex vivo invasion platform based on mild detergent decellularization of mouse organs followed by vibratome slicing. This approach generates optically transparent lung, liver, and intestine ECM scaffolds that preserve native matrix architecture, mechanical properties, and retain biochemical hallmarks of their tissues of origin. Organ-derived matrices were integrated into standard microfluidic channels and analyzed using conventional fluorescence microscopy to enable quantitative assessment of cancer cell invasion. Benchmarking with breast cancer cell lines of defined invasive capacity, we could demonstrate the robustness and biological relevance of the system. Non-invasive MCF7 cells failed to infiltrate any organ scaffold, whereas highly invasive MDA-MB-231 cells exhibited pronounced organ-specific invasion. These cells preferentially invaded lung and liver ECM while showing minimal invasion of intestinal scaffolds, recapitulating clinically observed metastatic tropism. Quantitative invasion rates closely matched values previously reported in vivo using intravital microscopy. ConclusionsThis ex vivo organ-derived ECM platform provides a scalable, cost-effective, and experimentally accessible system to study ECM-driven determinants of metastatic invasion. By preserving tissue-specific matrix cues while reducing reliance on animal models, this approach provides a powerful tool to interrogate ECM-driven determinants of metastasis and to evaluate potential therapeutic interventions.

cancer biology↗

bioRxiv2025

tRNA thiolation defects disrupt cellular proteostasis and tissue homeostasis in mammals

Sulfur modification of tRNA wobble uridines is an evolutionarily conserved mechanism that ensures efficient protein synthesis. In humans, loss of this anticodon modification due to mutations in CTU2 (cytosolic thiouridylase 2) causes DREAM-PL syndrome, a severe congenital disorder often leading to early postnatal death. However, the mechanisms by which loss of tRNA thiolation drives pathology remain unclear. Here, we show that loss of CTU2 triggers significant cellular proteostasis defects in patient cells and model cell lines. Structural and biochemical analyses reveal that the pathogenic CTU2L63P mutation destabilizes the CTU1/CTU2 complex and abolishes tRNA binding and thiolation. Acute loss of CTU2 caused codon-specific ribosome pausing at A-ending codons decoded by thiolated tRNAs, and decreased ribosome occupancy of A-rich transcripts in a dosage-dependent manner. Codon-biased mRNAs transcribed from genes critical for ciliogenesis are predicted to be most affected, linking their reduced translation to DREAM-PL etiology in humans. Surprisingly, Ctu2L63P mice display severe thiolation defects, but develop normally, are viable and fertile. Our findings highlight the importance of functional tRNA thiolation for organismal health in humans and identify species-specific vulnerabilities during embryonic development in mammals.

cell biology↗

bioRxiv2025

Integrated Metabolic Complex Genetic Interaction 1 Network of Chromosome 4p Loss in Basal Breast Cancer

Basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and exhibits a recurrent large chromosomal deletion in chromosome 4p (chr4p). Chr4p loss is associated with poor survival, evolves early in tumorigenesis and confers on cells a proliferative state. Here, we map the integrated metabolic complex genetic interaction network of chr4p in basal breast cancer to identify targetable vulnerabilities. Differential gene expression analysis of patient derived xenografts and cancer cell models revealed that chr4p loss is associated with changes in cellular energetics and reduction/oxidation balance. Analysis of DepMap pooled genome-wide CRISPR-Cas9 screens identified complex genetic interactions specific to chr4p deletion in basal breast cancer cell models. Functional assays revealed that chr4p loss is associated with disrupted mitochondrial respiratory function and reduced glycolytic capacity, suggesting metabolic rewiring. Increased reactive oxygen species and lipid peroxidation compromised antioxidant defense mechanisms. Ultimately, this study sheds light on targeted therapies for basal breast cancer harboring large chromosomal deletions.

genomics↗

bioRxiv2025

Dual function of ERH in primary miRNA biogenesis

MicroRNAs are small non-coding RNAs that mediate post-transcriptional silencing of most mammalian genes. They are generated in a multi-step process initiated by the Microprocessor, a protein complex composed of DROSHA and DGCR8. Recent studies have described the phenomenon of "cluster assistance", in which a prototypic primary miRNA hairpin can license the Microprocessor-mediated processing of a clustered suboptimal hairpin in cis. Genetic screening and mechanistic analyses led to the identification of two critical factors for this process, SAFB2 (scaffold attachment factor B2) and ERH (enhancer of rudimentary homolog), which have been shown to associate with the N-termini of DROSHA and DGCR8, respectively, but also form a complex with each other. However, it remains unclear how SAFB2 and ERH can alter the Microprocessor substrate specificity, and whether the described protein-protein interactions are required for cluster assistance. In this study, we focused on the role of ERH and show that its loss largely phenocopies the effect of SAFB1/2 deletion on the miRNA transcriptome, suggesting that both factors are involved in the same processes of primary miRNA biogenesis. In this context, our data demonstrate that both SAFB1/2 and ERH are required for efficient Microprocessor feedback regulation via processing of pri-miR-1306, uncovering a clear physiological function of cluster assistance. Mechanistically, our data show that ERH-mediated cluster assistance depends neither on its direct association with SAFB2 nor on its described interaction with DGCR8. In contrast, disrupting the ERH binding site within DGCR8 drives the processing of a subset of cluster assistance-unrelated pri-miRNAs. Thus, this study reveals dual roles of ERH in primary miRNA biogenesis, a largely suppressive one driven by its direct binding to DGCR8, and the other in cluster assistance that does not require DGCR8 binding. HighlightsO_LILoss of SAFB1/2 and ERH, respectively, induces overlapping, but not identical defects in primary miRNA biogenesis. C_LIO_LIBoth SAFB2 and ERH are involved in Microprocessor feedback regulation. C_LIO_LIERH-mediated cluster assistance functions independent of its binding to the DGCR8 N-terminus. C_LIO_LIBinding of ERH to the DGCR8 N-terminus confers a largely inhibitory function for primary miRNA biogenesis C_LI

molecular biology↗

bioRxiv2025

Sequential PIDD1 auto-processing is essential for ploidy control in liver and heart

Polyploidization refers to the balanced increase in gene copy number and is a feature of specialized cells in different mammalian tissues, including the liver and the heart. During organogenesis, hepatocytes and cardiomyocytes undergo scheduled polyploidization events to increase their cellular or nuclear DNA content. This is thought to improve cellular output and enable for rapid genetic adaptation in response to stress. Yet, excessive increases in ploidy can also be disadvantageous and increase the risk of genome instability. Hence, a dedicated machinery, the PIDDosome multi-protein complex, has evolved to prevent exacerbated increases in DNA content. Using targeted mutagenesis in mice, we show that the PIDDosome controls hepatocyte ploidy in a cell-autonomous manner and that sequential and quantitative auto-processing of PIDD1 is key for accurate control of ploidy in postnatal development of liver and heart. Stoichiometric imbalances in bioactive PIDD1-fragments impair p53-dependent and independent cell cycle arrest responses during organogenesis, as well as caspase-2-dependent apoptosis caused by centrosome amplification. Strikingly, targeted mutagenesis of the caspase cleavage motif in the critical E3-ligase controlling p53 protein levels, Mdm2, impairs ploidy control in hepatocytes, but not in cardiomyocytes, indicative of the existence of alternative caspase-2 substrates that help to restrict ploidy in the heart.

cell biology↗

bioRxiv2025

WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2

Inhibitors of the protein kinase WEE1 have emerged as promising agents for cancer therapy. In this study, we uncover synergistic interactions between WEE1 small- molecule inhibitors and defects in mRNA translation, mediated by activation of the integrated stress response (ISR) through the kinase GCN2. Using a pooled CRISPRi screen, we identify GSPT1 and ALKBH8 as factors whose depletion confer hypersensitivity to the WEE1 inhibitor, AZD1775. We demonstrate that this synergy depends on ISR activation, which is induced by the off-target activity of WEE1 inhibitors. Furthermore, PROTAC-based WEE1 inhibitors and molecular glues show reduced or no ISR activation, suggesting potential strategies to minimise off-target toxicity. Our findings reveal that certain WEE1 inhibitors elicit dual toxicity via ISR activation and genotoxic stress, with ISR activation being independent of WEE1 itself or cell-cycle status. This dual mechanism highlights opportunities for combination therapies, such as pairing WEE1 inhibitors with agents targeting the mRNA translation machinery. This study also underscores the need for more precise WEE1 targeting strategies to mitigate off-target effects, with implications for optimising the therapeutic potential of WEE1 inhibitors.

cancer biology↗

bioRxiv2024

The PIDDosome controls cardiomyocyte polyploidization during postnatal heart development

The adult mammalian heart is characterized by post-mitotic polyploid cardiomyocytes (CMs). Understanding how CMs regulate cell cycle exit and ploidy can help developing new heart regenerative therapies. Here, we uncover that the PIDDosome, a multi-protein complex activating the endopeptidase Caspase-2, helps to implement a CM-specific differentiation program that limits ploidy during postnatal heart development. DNA content analyses show that PIDDosome-loss causes a cell-autonomous increase in nuclear and cellular CM ploidy. Remarkably, increased ploidy does not affect cardiac structure nor function. PIDDosome-imposed ploidy restriction commences at postnatal day 7 (P7), reaching a plateau on P14. PIDDosome activation requires ANKRD26, targeting PIDD1 to mother centrioles. Opposite to prior observations in liver development, the PIDDosome limits CM polyploidization in a p53-independent manner but reliant on p21/Cdkn1a, a notion supported by nuclear RNA sequencing and genetic deletion experiments. Our results provide new insights how proliferation of polyploid CMs is restricted during postnatal heart development.

developmental biology↗

bioRxiv2024

Exact centriole counts are critical for B cell development but not function

Centrioles define centrosome structure and function. Deregulation of centriole numbers can cause developmental defects and foster malignant disease. The p53 tumor suppressor limits the growth of cells lacking or harboring additional centrioles and can be engaged by the "mitotic surveillance" or the "PIDDosome pathway", respectively. Here, we show that early B cell progenitors frequently present extra centrioles that are rapidly lost during maturation. Increasing centriole counts beyond physiological levels by Polo-like kinase 4 (PLK4) overexpression induces apoptosis, suggesting clearance of such cells during development. Remarkably, this apoptotic response is independent of PIDD1 or p53, but can be blocked by excess BCL2. In contrast, loss of centrosomes upon Plk4 deletion arrests B cell development at the pro B cell stage. This defect can be rescued by co-deletion of Usp28, a critical component of the mitotic surveillance pathway that restores cell number and function in the absence of centrioles. In both scenarios, too many and too few centrosomes, mitochondrial apoptosis is engaged to kill B cells with abnormal centriole counts during their development with progenitor B cells being intolerant to centriole loss but permissive to centriole amplification. Unexpectedly, our findings show that centrioles are dispensable for mounting an effective humoral immune response.

cell biology↗

bioRxiv2024

Caspase-2 kills cells with extra centrosomes

Centrosomes are membrane-less organelles that orchestrate a wide array of biological functions by acting as microtubule organizing centers. Here, we report that caspase-2-driven apoptosis is elicited in blood cells failing cytokinesis and that extra centrosomes are necessary to trigger this cell death. Activation of caspase-2 depends on the PIDDosome multi-protein complex and priming of PIDD1 at extra centrosomes is necessary for this pathway. Accordingly, loss of its centrosomal adapter, ANKRD26, allows for cell survival and unrestricted polyploidization in response to cytokinesis failure. Mechanistically, cell death is initiated upstream of mitochondria via caspase-2-mediated processing of the BCL2 family protein BID, driving BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Remarkably, BID-deficient cells enforce apoptosis by engaging p53-dependent pro-apoptotic transcriptional responses initiated by caspase-2. Consistently, BID and MDM2 act as shared caspase-2 substrates, with BID being kinetically favored. Our findings document that the centrosome limits its own unscheduled duplication by the induction of PIDDosome-driven mitochondrial apoptosis to avoid potentially pathogenic polyploidization events.

cell biology↗

bioRxiv2023

Chronic deregulation of the spindle assembly checkpoint triggers myelosuppression and gastrointestinal atrophy

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralises the critical APC cofactor, CDC20, preventing mitotic exit. In cancer cell lines, this can provoke apoptosis involving pro-apoptotic BCL2 family members BIM and NOXA while BCL2 overexpression blocks mitotic cell death, facilitating SAC adaptation. However, the consequences of apoptosis after SAC perturbation in vivo are unclear. By conditional MAD2 overexpression across tissues in mice, we observed that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy. While myelosuppression was tolerated, gastrointestinal atrophy was detrimental. Remarkably, co-deletion of Bim/Bcl2l11, but not Bid, Puma/Bbc3 or Noxa/Pmaip, prevented developing gastrointestinal syndrome caused by chronic SAC activity, identifying BIM as rate-limiting for mitotic cell death in the gastrointestinal epithelium. In contrast, only BCL2 overexpression but none of the BH3-only protein deficiencies tested could mitigate myelosuppression, highlighting tissue and cell type-specific survival dependencies in response to SAC perturbation in vivo.

cell biology↗

bioRxiv2022

The miR-15a/16-1 and miR-15b/16-2 clusters regulate early B cell development by limiting IL-7 receptor expression

Pleiotropic functions of miRNAs as transcriptional repressors have been reported for multiple biological processes. One prominent miRNA family is the miR-15 family, which is a well-established tumor-suppressor in B-cell chronic lymphocytic leukemia (CLL). The miR-15 family consists of three bicistronic clusters, miR-15a/16-1, miR-15b/16-2 and miR-497/195, all sharing the same seed sequence suggesting that loss one cluster can be functionally compensated by the remaining miR-15 family members. Thus, a combined deletion may be necessary to reveal its physiological function in vivo. A combined knockout of the most prominent miR-15 clusters, miR-15a/16-1 and miR-15b/16-2 in the hematopoietic system reveals a novel role of the miR-15 family in early B cell development highlighted by an increase of the pro-B cell compartment. Mechanistically, this effect is mediated by enhanced IL-7 receptor expression, which we identified as direct miR-15 target gene. Notably, elevated IL-7 receptor levels were sufficient to trigger increased activation of the STAT5 and PI3K/AKT pathways. Moreover, derepression of directly targeted cell cycle regulators such as Ccne1, Chek1 and Wee1 further facilitates G-to-S transition. Thus, by deregulating a target gene network of cell cycle and signaling mediators, loss of the miR-15 family establishes a pro-proliferative milieu manifesting in an enlarged pro-B cell pool.

immunology↗

bioRxiv2021

The BH3-only protein NOXA serves as an independent predictor of breast cancer patient survival and defines susceptibility to microtubule targeting agents

Breast cancer (BC) treatment frequently involves microtubule-targeting agents (MTAs), such as paclitaxel, that arrest cells in mitosis. Sensitivity to MTAs is defined by a subset of pro- and anti-apoptotic BCL2 family proteins controlling mitochondrial apoptosis. Here, we aimed to determine their prognostic value in primary tumour samples from 92 BC patients. Our analysis identified high NOXA/PMAIP mRNA expression levels as an independent prognostic marker for improved relapse-free survival (RFS) and overall survival (OS) in multivariate analysis in BC patients, independent of their molecular subtype. Analysis of available TCGA datasets of 1060 BC patients confirmed our results and added a clear predictive value of NOXA mRNA levels for patients who received MTA-based therapy. In this TCGA cohort, 122 patients received MTA-treatment and high NOXA mRNA levels correlated with their progression-free interval (PFI) and OS. Our follow-up analyses in a panel of BC cell lines of different molecular subtypes identified NOXA protein expression as a key determinant of paclitaxel sensitivity in triple-negative breast cancer (TNBC) cells. Moreover, we noted highest additive effects between paclitaxel and chemical inhibition of BCLX, but not BCL2 or MCL1, documenting dependence of TNBC cells on BCLX for survival and paclitaxel sensitivity, defined by NOXA expression levels.

cancer biology↗

bioRxiv2020

PIDDosome-induced p53-activation for ploidy restriction facilitates hepatocarcinogenesis

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. The PIDDosome multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome-deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome-deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome-deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts of survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and tumor cell density serves as a novel prognostic marker for recurrence free survival in HCC patients.

cancer biology↗

bioRxiv2019

SAFB2 enables the processing of suboptimal stem-loop structures in clustered primary miRNA transcripts

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally silence most protein-coding genes in mammals. They are generated from primary transcripts containing single or multiple clustered stem-loop structures that are thought to be recognized and cleaved by the DGCR8/DROSHA Microprocessor complex as independent units. Contrasting this view, we here report an unexpected mode of processing of a bicistronic cluster of the miR-15 family, miR-15a-16-1. We find that the primary miR-15a stem-loop is a poor Microprocessor substrate and is consequently not processed on its own, but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage, and describe SAFB2 as a novel accessory protein of DROSHA. Notably, SAFB2-mediated cluster assistance expands to other clustered pri-miRNAs including miR-15b, miR-92a and miR-181b, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal DGCR8/DROSHA substrates in clustered primary miRNA transcripts. HighlightsO_LIthe primary miR-15a stem-loop structure per se is a poor Microprocessor substrate C_LIO_LIcleavage of pri-miR-15a requires the processing of an additional miRNA stem-loop on the same RNA C_LIO_LIsequential pri-miRNA processing or "cluster assistance" is mediated by SAFB proteins C_LIO_LISAFB2 associates with the Microprocessor C_LI

molecular biology↗

bioRxiv2018

Checkpoint kinase 1 is essential for fetal haematopoiesis and hematopoietic stem cell survival

Checkpoint kinase 1 is critical for S-phase fidelity and preventing premature mitotic entry in the presence of DNA damage. Tumour cells have developed a strong dependence on CHK1 for survival and hence this kinase has developed into a popular drug-target. Chk1-deficiency in mice results in blastocyst death due to G2/M checkpoint-failure showing that it is an essential gene and may be difficult to target therapeutically without side-effects. Here, we show that chemical inhibition of CHK1 kills murine hematopoietic stem and progenitor cells (HSPCs) as well as human CD34+ HSPCs by the induction of BCL2-regulated but p53-independent apoptosis. Moreover, Chk1 is essential for stem cell survival and definite hematopoiesis in the mouse embryo. Remarkably though, cell death inhibition in hematopoietic stem cells (HSC) cannot restore blood cell formation in utero as Chk1 loss causes severe DNA damage that ultimately prevents HSC expansion. Our findings establish a previously unrecognized role for CHK1 in establishing hematopoiesis; they also suggest adverse effects of therapeutic CHK1-inhibtion, particularly under conditions forcing stem cells out of dormancy, such as chemotherapy-induced myelosuppression.

developmental biology↗