Choroidal neovascularisation (CNV) is a hallmark of wet/neovascular age-related macular degeneration (wAMD), characterized by aberrant blood vessel growth from the choroid into the retina. Both pathological angiogenesis and inflammation contribute to disease progression. Here we show that analsysis of single-cell RNA sequencing of experimental CNV lesions revealed upregulation of Zeb1 in angiogenic endothelial cells (ECs). We generated an endothelial-specific Zeb1 knockout (Zeb1iECKO) mouse model to assess its functional role. CNV was induced via laser photocoagulation, and vascular leakage and inflammation were evaluated using fluorescein angiography, immunohistochemistry, and transcriptomic analyses. Zeb1iECKO mice exhibited increased fluorescein leakage and enhanced vascular invasion during CNV, indicating destabilized neovascular structures. However, leukocyte infiltration within CNV lesions was not elevated. In vitro, ZEB1 knockdown in human ECs led to downregulation of inflammatory signalling pathways and reduced expression of adhesion molecules in response to TNF-α stimulation yet retained angiogenic capacity. ZEB1 coordinates angiogenic and inflammatory responses in CNV. Its loss enhances neovascularisation without promoting inflammation, suggesting a potential therapeutic target for modulating pathological angiogenesis in wAMD while minimizing inflammatory damage.

Cancer cells dynamically reprogram their metabolism to adapt to changing microenvironmental conditions during tumor growth and metastatic dissemination. Metastasis of solid tumors-the principal cause of cancer-related mortality-is often driven through activation of epithelial-mesenchymal transition (EMT), regulated by the transcription factor ZEB1, which is frequently upregulated during tumor progression. To investigate the role of metabolic plasticity in metastasis, we employed murine pancreatic ductal adenocarcinoma (PDAC) cell lines with distinct EMT states, ZEB1 expression and lung colonization capacities. Highly plastic epithelial-type cancer cells (KPCepi) efficiently colonize the lung, whereas Zeb1-deficient cancer cells (KPCZ) with compromised metabolic plasticity show markedly reduced colonization, correlated with absent glycolytic reserve, mitochondrial dysfunction, and reduced anti-oxidant metabolite levels. Interestingly, mesenchymal-type cancer cells (KPCmes) also exhibit poor lung colonization despite retaining normal glycolytic capacity and a high proportion of functional mitochondria; however, similar to KPCZ cells, they display diminished levels of detoxifying metabolites. Low metastatic capacity correlates with increased susceptibility to ferroptosis even in epithelial-type KPCZ cells, indicating a limited ability to counteract reactive oxygen species under stress. Together, these findings demonstrate that metabolic plasticity and redox homeostasis are essential prerequisites for efficient lung colonization. Thus, concurrent targeting of metabolic adaptability and redox buffering may represent a promising strategy to prevent metastasis in aggressive PDAC tumors.

PURPOSE:To investigate the role of Zeb1 in regulating fibrosis in endothelial-mesenchymal transition (MT) in the cornea. siRNA knockdown of Zeb1 inhibits FGF2-induced MT in the corneal endothelium. Endothelial cells that undergo MT show increased expression of endothelial-MT-associated genes, including COL1, fibronectin, and vimentin, and decreased expression of E-cadherin, leading to fibrous retrocorneal membrane formation. METHODS:To address potential off-target effects of siRNA knockdown, Zeb1flox/flox:UBC-CreERT2 mouse was generated to allow for spatiotemporal control of Zeb1 targeting and studying the role of Zeb1 in the corneal endothelium in vivo. RESULTS:Intracameral injection of 4-hydroxytamoxifen inhibited Fgf2-induced expression of Zeb1 mRNA and protein in the corneal endothelium of Zeb1flox/flox:UBC-CreERT2 mouse. Fgf2 and surgical injury-dependent expression of MT-related genes and suppression of E-cadherin were inhibited by conditional targeting of Zeb1 in the mouse corneal endothelium in vivo. Surgical injury led to corneal edema with a central corneal thickness of 189.0 ± 14.6 µm (injury) vs 92.3 ± 2.8 µm (control), and injury-induced edema was significantly attenuated by Zeb1 targeting in the corneal endothelium with a central corneal thickness of 182.2 + 15.5 µm (injury) vs 106.8 ± 11.1 µm (injury + 4-hydroxytamoxifen), F = 120.9, P < .00001. Moreover, conditional targeting of Zeb1 also inhibited injury-dependent retrocorneal membrane formation in the mouse corneal endothelium in vivo. CONCLUSIONS:These results suggest that ZEB1 signaling could be targeted for inhibiting retrocorneal membrane formation and anterior segment fibrosis and could be leveraged to treat certain forms of corneal blindness without relying on transplantation.

Abstract Background Progression and metastasis of solid cancers are orchestrated by activation of epithelial-mesenchymal transition (EMT) in the primary tumor. This process is typically restricted to a limited number of cells that acquire partial or hybrid EMT states to unleash cellular plasticity. Capturing such dynamic and often reversible events in vivo on the single cell level is hampered by the lack of proper labeling tools that yet often induce permanent staining persisting beyond transient EMT activation. Methods To enable live-tracking of EMT in vivo, we utilized CRISPaint and homologous recombination to endogenously tag ZEB1, one key transcription factor to activate EMT during tumorigenesis. Using the bright fluorescent protein mNeonGreen, we generated ZEB1-Neon fusion knock-in alleles in MDA-MB-231 and MCF10A cells, as well as in mice. Results We demonstrate that mNeonGreen fluorescence is suitable to faithfully report on ZEB1 expression in vitro over time, becomes properly upregulated by TGFβ, and allows separation of ZEB1hi and ZEB1lo cells to capture different cellular properties, e.g., handling of DNA damage. The fusion does not affect ZEB1 function as evident by proper EMT induction, embryogenesis, and tissue homeostasis when present homozygously. Moreover, introducing Zeb1-Neon into the KPC mouse model of pancreatic cancer permits tracking of ZEB1+ cells in precision-cut slices and time-lapse imaging of isolated tumor cells. Conclusions In summary, we provide a versatile tool that allows precise detection and live cell imaging of EMT, which will help to more accurately decipher the role of EMT in tumor progression and to identify therapeutic agents that can specifically manipulate EMT for novel combination therapies. Graphical Abstract

Tumor-associated macrophages (TAMs) dynamically influence anti-tumor immunity. Understanding TAM function is therefore critical to design immunotherapies. By combining syngeneic models of colorectal and pancreatic cancer with cell type-specific deletion of the epithelial-to-mesenchymal transition driver Zeb1, which is expressed in subsets of TAMs, we discovered that ZEB1 is an intrinsic regulator of TAM-controlled T cell trafficking and anti-tumor immune responses. ZEB1 supports secretion of a subset of chemokines via the constitutive pathway, including CXCL10, CCL2 and CCL22, by regulating their biosynthesis, vesicular transport and release. This elevates cytotoxic T cell (CTL) recruitment in vitro and fosters immunosurveillance by CTLs in tumors and metastases as well in an organotypic model for therapeutic CD8 + T cell addition. Our study identifies ZEB1 in TAMs as a facilitator of anti-tumor immunity, suggests a window of opportunity for cytokine-guided CTL tropism and reinforces the importance of onco-immunological context, particularly in the design of macrophage- and/or cytokine-depleting strategies.

Background The structure and function of cardiomyocytes rely on tightly regulated gene networks that control development, maturation, and homeostasis. Disruption of these networks is associated with cardiomyopathies such as hypertrophic and dilated cardiomyopathy, leading to heart failure. Our previous work identified the transcription factor “Zinc Finger E-Box Binding Homeobox 1” (ZEB1) as a downstream effector of Cytoplasmic Polyadenylation Element Binding Protein 4 (CPEB4), an RNA-binding protein responsive to cardiac stress. While ZEB1 is known for its role in cancer metastasis and epithelial-to-mesenchymal transition (EMT), its function in cardiomyocytes is not well understood. Based on previous findings, we hypothesize that ZEB1 is essential for maintaining the structural integrity and contractile function of cardiomyocytes. Hypothesis and objectives We hypothesized that ZEB1 plays a critical role in maintaining cardiomyocyte structure and function. Deletion of Zeb1 may result in sarcomeric disorganization and mitochondrial dysfunction, whereas its overexpression appears to promote maladaptive hypertrophy. Our objectives are to: 1. Investigate if ZEB1 overexpression induces pathological cardiac remodeling. 2. Examine the effects of ZEB1 deletion on cardiomyocyte structure and function. 3. Identify ZEB1-regulated transcriptional networks. Methods AAV9-Zeb1 was used for the overexpression of Zeb1. Using a myosin heavy chain alpha (αMHC) Cre system, we created a Zeb1 conditional knockout mouse. To evaluate the cardiac function, we used methods like echocardiography, electron microscopy, immunohistochemistry. We identified differential expressed genes upon deletion of Zeb1 using RNA-seq and integrated the data set with ChIP-seq. Results ZEB1 deletion leads to sarcomere damage, mitochondrial dysfunction, and dedifferentiation, with more pronounced effects in females. Overexpression promotes hypertrophic remodeling. Echocardiographic analysis showed progressive systolic dysfunction, and histology revealed sarcomeric disarray, again especially in females. A tamoxifen-inducible ZEB1 knockout mouse model confirmed ZEB1’s crucial role in fully differentiated cardiomyocytes. Conclusions ZEB1 is critical for cardiomyocyte homeostasis, and maintaining its function is necessary for normal cardiac performance and structure. Condensed Abstract We identified Zeb1 as a downstream target of Cpeb4 in cardiac hypertrophy. While ZEB1’s role in cancer and EMT is known, its impact on cardiomyocytes remains largely underexplored. We hypothesize that ZEB1 regulates cardiomyocyte maturation and function, which is crucial for structural integrity and contractility. Zeb1 deletion disrupts sarcomeric organization and induces cardiac dysfunction, notably more severe in females. RNA sequencing confirms that ZEB1 loss in females increases the expression of epithelial and extracellular matrix (ECM) genes. ZEB1’s overexpression induces pathological hypertrophy and progressive cardiac dysfunction. These findings underscore ZEB1’s critical role in cardiac structure and function, further increasing our understanding of gene regulatory networks in cardiomyocytes. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. * Abbreviation list : 1. ZEB1 : Zinc finger E-box-binding homeobox 1 2. TAC : Transverse Aortic Constriction 3. DCM : Dilated Cardiomyopathy 4. HCM : Hypertrophic Cardiomyopathy 5. NRCMs : Neonatal Rat Cardiomyocytes 6. PE : Phenylephrine 7. AAV9 : Adeno-Associated Virus serotype 9 8. cKO : Conditional Knockout 9. icKO : Inducible Conditional Knockout 10. EF : Ejection Fraction Deutsche Forschungsgemeinschaft, 552055378 CRC1550 [1]: pending:yes

Identifying the most biologically meaningful microRNA (miRNA) targets remains challenging, as predictive and biochemical methods yield many weak or non-productive interactions. Transcription factors (TFs) are enriched among miRNA targets and amplify miRNA effects through their broad regulatory influence. Frequently, these same TFs also regulate the miRNA, forming double negative feedback loops that enforce bistable gene expression and cell-fate decisions. We investigated this regulatory motif by focusing on reciprocal repression between the miR-200 family and ZEB1/2, which governs epithelial–mesenchymal plasticity. Employing a system isolating ZEB-dependent effects of miR-200c and combining Weighted Gene Co-expression Network Analysis (WGCNA) with Exon-Intron Split Analysis (EISA), as well as functional cell biology assays, we show this circuit reinforces mutually exclusive epithelial and mesenchymal states through complex networks of intertwined direct and indirect, transcriptional and post-transcriptional, ZEB-dependent and independent mechanisms. Our findings highlight how miRNA-TF feedback loops can act as bistable switches to lock cell identity and emphasize the pivotal role of strongly regulated TFs within miRNA target networks.

Cell plasticity is a crucial trait for cancer progression towards metastasis and treatment resistance. Research efforts from the past 20–30 years have revealed that the dynamic flux of the epithelial–mesenchymal transition (EMT) programme is one of the major underlying processes enabling cancer cell plasticity and greatly facilitates these major causes of cancer mortality. The spectrum of evidence ranges from extensive data from cell line and animal model studies across multiple cancer types through a rapidly expanding body of work demonstrating associations between EMT biomarkers and disease progression and mortality in patients. EMT is also implicated in resistance to most of the major treatment modalities, yet our efforts to harness this knowledge to improve therapeutic outcomes are currently in their early stages. In this Review, we describe clinical evidence supporting a role of EMT and the associated epithelial–mesenchymal plasticity in various stages of cancer in patients and discuss the subsequent clinical opportunities and challenges associated with attempts to implement this knowledge as novel therapies or clinical management approaches. Despite several decades of research that has revealed roles in the development and progression of many solid tumours, clinical translation of research targeting epithelial–mesenchymal transition (EMT) has thus far been limited. In this Review, the authors provide a summary of the role of EMT in cancer development and progression in the context of this lack of clinical translation, summarize the current status of direct or indirect EMT-modulating agents in clinical development, and highlight the major barriers to the development of EMT-related clinical interventions.

Tumor-associated macrophages (TAMs) shape the tumor microenvironment (TME) and exert a decisive impact on anti-tumor immunity. Understanding TAM function is therefore critical to understand anti-tumor immune responses and to design immunotherapies. Here, we describe the transcription factor ZEB1, a well-known driver of epithelial-to-mesenchymal transition, as an intrinsic regulator of TAM function in adaptive anti-tumor immunity. By combining cell type-specific deletion of Zeb1 with syngeneic models of colorectal and pancreatic cancer, we discovered an unexpected function of ZEB1 in the TAM-mediated control of T cell trafficking. ZEB1 supports secretion of a subset of chemokines including CCL2 and CCL22 by promoting their transcription and translation as well as by safeguarding protein processing. ZEB1 thereby elevates cytotoxic T cell (CTL) recruitment in vitro and in vivo and fosters immunosurveillance during tumor as well as lung metastatic outgrowth. Our study spotlights ZEB1 as a crucial facilitator of adaptive anti-tumor immunity and uncovers a potential therapeutic window of opportunity for cytokine-guided enhancement of CTL infiltration into tumors and metastases.### Competing Interest StatementThe authors have declared no competing interest.

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.

Emerging evidence implicates the epithelial-mesenchymal transition transcription factor Zeb1 as a critical regulator of hematopoietic stem cell (HSC) differentiation. Whether Zeb1 regulates long-term maintenance of HSC function remains an open question. Using an inducible Mx-1-Cre mouse model that deletes conditional Zeb1 alleles in the adult hematopoietic system, we found that mice engineered to be deficient in Zeb1 for 32 weeks displayed expanded immunophenotypically defined adult HSCs and multipotent progenitors associated with increased abundance of lineage-biased/balanced HSC subsets and augmented cell survival characteristics. During hematopoietic differentiation, persistent Zeb1 loss increased B cells in the bone marrow and spleen and decreased monocyte generation in the peripheral blood. In competitive transplantation experiments, we found that HSCs from adult mice with long-term Zeb1 deletion displayed a cell autonomous defect in multilineage differentiation capacity. Long-term Zeb1 loss perturbed extramedullary hematopoiesis characterized by increased splenic weight and a paradoxical reduction in splenic cellularity that was accompanied by HSC exhaustion, lineage-specific defects, and an accumulation of aberrant, preleukemic like c-kit+CD16/32+ progenitors. Loss of Zeb1 for up to 42 weeks can lead to progressive splenomegaly and an accumulation of Gr-1+Mac-1+ cells, further supporting the notion that long-term expression of Zeb1 suppresses preleukemic activity. Thus, sustained Zeb1 deletion disrupts HSC functionality in vivo and impairs regulation of extramedullary hematopoiesis with potential implications for tumor suppressor functions of Zeb1 in myeloid neoplasms. (c) 2024 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGF beta stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1. Schwab, Rao et al. report that Zeb1 mediates enhanced ferroptosis sensitivity in cancer cells after EMT activation, associated with altered expression of selected lipogenic enzymes and an subsequent increase in the PUFA:MUFA ratio.

Luteinizing hormone (LH), a heterodimeric glycoprotein produced by pituitary gonadotrope cells, regulates gonadal function. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates LH synthesis and secretion. GnRH induces LHβ subunit (Lhb) expression via the transcription factor, early growth response 1 (EGR1), acting on the Lhb promoter. In contrast, overexpression of zinc finger E-box binding homeobox 1 (ZEB1) represses LH production in mice, but the underlying mechanism was not previously elucidated. Here, we observed that ZEB1 inhibited GnRH-stimulated but not basal Lhb mRNA expression in homologous murine LβT2 cells. Moreover, ZEB1 blocked GnRH and/or EGR1 induction of murine Lhb but not human LHB promoter-reporter activity in these cells. Using chimeric reporters, we mapped the species-specific ZEB1 sensitivity to sequence differences, including in Z- and E-boxes, in the proximal Lhb/LHB promoters, immediately upstream of the transcription start sites. ZEB1 bound to the murine Lhb promoter with higher affinity than to the human LHB promoter in this region. To examine ZEB1's physiological role in LH synthesis, we characterized gonadotrope-specific Zeb1 knockout mice. Loss of ZEB1 in gonadotropes did not affect LH production or secretion. Collectively, the data suggest that ZEB1, when overexpressed, can inhibit GnRH/EGR1 induction of murine Lhb transcription but does not play a necessary role in LH synthesis in mice.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

Epithelial-mesenchymal transition (EMT) constitutes an important pathway in organ fibrosis seen in the lungs, liver, eye, and salivary glands. This review summarizes the EMT observed within the lacrimal gland during its development, tissue damage and repair along with possible translational implications. Existing animal and human studies have reported the increased expression of EMT regulators i.e., transcription factors like Snail, TGF-β1 within the lacrimal glands, and a possible role of reactive oxygen species, which might be initiating the cascade of EMT. In these studies, EMT is typically detected by reduced E-cadherin expression in the epithelial cells and increased Vimentin and Snail expression within the lacrimal glands' myoepithelial or ductal epithelial cells. Other than specific markers, electron microscopic evidence of disrupted basal lamina, increased collagen deposition, reorganised cytoskeleton of myoepithelial cells also indicated EMT. Very few studies have shown myoepithelial cells to be the cells transitioning into mesenchymal cells with increased extracellular matrix deposition within the lacrimal glands. EMT in animal models seemed reversible as glands got repaired after damage with IL-1α injection or duct ligation and transiently used the EMT as a means for tissue repair. The EMT cells also expressed nestin, a marker for progenitor cells in a rabbit duct ligation model. However, lacrimal glands of ocular graft versus host disease and IgG4 dacryoadenitis demonstrate irreversible acinar atrophy along with signs of EMT-fibrosis, reduced E-cadherin, and increased Vimentin and Snail expression. Future studies exploring the molecular mechanisms of EMT and thereby developing targeted therapies capable of transforming the mesenchymal cells into epithelial cells or blocking the EMT might help in the restoration of the lacrimal gland function.

Osteoclasts are bone-resorbing polykaryons responsible for skeletal remodeling during health and disease. Coincident with their differentiation from myeloid precursors, osteoclasts undergo extensive transcriptional and metabolic reprogramming in order to acquire the cellular machinery necessary to demineralize bone and digest its interwoven extracellular matrix. While attempting to identify new regulatory molecules critical to bone resorption, we discovered that murine and human osteoclast differentiation is accompanied by the expression of Zeb1, a zinc-finger transcriptional repressor whose role in normal development is most frequently linked to the control of epithelial-mesenchymal programs. However, following targeting, we find that Zeb1 serves as an unexpected regulator of osteoclast energy metabolism. In vivo, Zeb1-null osteoclasts assume a hyperactivated state, markedly decreasing bone density due to excessive resorptive activity. Mechanistically, Zeb1 acts in a rheostat-like fashion to modulate murine and human osteoclast activity by transcriptionally repressing an ATP-buffering enzyme, mitochondrial creatine kinase 1 (MtCK1), thereby controlling the phosphocreatine energy shuttle and mitochondrial respiration. Together, these studies identify a novel Zeb1/MtCK1 axis that exerts metabolic control over bone resorption in vitro and in vivo.

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers is reduced upon fibroblast-specific loss of Zeb1 . Single-cell transcriptomics, histological and in vitro characterization reveal a crucial role in CAF polarization, promoting myofibroblastic features whilst restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1 -deficient CAF repertoire sensitizes to immune checkpoint inhibition, pointing to a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.

The DNA damage response (DDR) and epithelial-to-mesenchymal transition (EMT) are two crucial cellular programs in cancer biology. While the DDR orchestrates cell-cycle progression, DNA repair, and cell death, EMT promotes invasiveness, cellular plasticity, and intratumor heterogeneity. Therapeutic targeting of EMT transcription factors, such as ZEB1, remains challenging, but tumor-promoting DDR alterations elicit specific vulnerabilities. Using multi-omics, inhibitors, and high-content microscopy, we discover a chemoresistant ZEB1-high-expressing sub-population (ZEB1hi) with co-rewired cell-cycle progression and proficient DDR across tumor entities. ZEB1 stimulates accelerated S-phase entry via CDK6, inflicting endogenous DNA repli-cation stress. However, DDR buildups involving constitutive MRE11-dependent fork resection allow homeo-static cycling and enrichment of ZEB1hi cells during transforming growth factor (3 (TGF-(3)-induced EMT and chemotherapy. Thus, ZEB1 promotes G1/S transition to launch a progressive DDR benefitting stress toler-ance, which concurrently manifests a targetable vulnerability in chemoresistant ZEB1hi cells. Our study thus highlights the translationally relevant intercept of the DDR and EMT.

Radial glia-like (RGL) stem cells persist in the adult mammalian hippocampus, where they generate new neurons and astrocytes throughout life. The process of adult neurogenesis is well documented, but cell-autonomous factors regulating neuronal and astroglial differentiation are incompletely understood. Here, we evaluate the functions of the transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) in adult hippocampal RGL cells using a conditional-inducible mouse model. We find that ZEB1 is necessary for self-renewal of active RGL cells. Genetic deletion of Zeb1 causes a shift toward symmetric cell division that consumes the RGL cell and generates pro-neuronal progenies, resulting in an increase of newborn neurons and a decrease of newly generated astrocytes. We identify ZEB1 as positive regulator of the ets-domain transcription factor ETV5 that is critical for asymmetric division.