This review explores the dual roles of lipid-laden macrophages (LL-Macs) in inflammation, atherosclerosis and cancer, emphasizing both their shared and divergent functions across physiological and pathological conditions. Lipid metabolism regulates the polarization of macrophages in homeostasis and inflammation. In atherosclerosis, LL-Macs contribute to plaque formation and inflammation, while in cancer, LL-Macs play crucial roles in immune suppression and tumor progression. The article outlines the molecular mechanisms driving macrophage lipid accumulation in each of these scenarios and how the process is influenced by the distinct local microenvironments in inflammation, atherosclerosis and cancer. Single-cell RNA sequencing (scRNA-seq) studies have identified both common and unique gene signatures between LL-Macs in atherosclerosis and cancer, reflecting the varying microenvironmental cues that shape macrophage function and disease outcomes. Finally, we examine lipid-modulating strategies in atherosclerosis, assess their potential in cancer treatment, and highlight research gaps for developing new LL-Macs targeted therapies in cancer.

A significant proportion of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) cases harbor a gain-of-function, heterozygous somatic mutation of the methyltransferase gene EZH2. While this factor is known to cooperate with the proto-oncogene MYC during malignant B cell development, the effect of interfering with both factors remains underexplored. Here we undertook the simultaneous evaluation of two epigenetic drugs targeting EZH2 methyltransferase activity and BRD4-mediated control of MYC transcription, CPI169 and CPI203, using preclinical models of DLBCL and FL with distinct EZH2 mutational status. We observed a specific and synergistic antiproliferative effect of these compounds in EZH2-mutated cells and mouse xenograft models, that was related to the abrogation of MYC transcriptional program and to tumor cell proliferation blockade at the G1 cell cycle phase. Gene expression profile, exploratory data analysis, and siRNA screening identified the PI3K/AKT-regulated gene and mitosis regulator, YPEL2, as a crucial factor involved in the efficacy of MYC/EZH2 dual targeting both in vitro and in vivo. Altogether, our results provide first pre-clinical evidence that simultaneous targeting of MYC and EZH2 is a safe and efficient approach that can be monitored by specific biomarkers, in aggressive lymphoid tumors of germinal center origin.

Vaccinia virus, known for its clinical safety, has a tropism for primary and metastatic tumors as well as ovarian tissue. Consequently, oncolytic approaches with recombinant vaccinia viruses have emerged as attractive agents against ovarian cancer. Unfortunately, oncolytic vaccinia monotherapies are yet to live up to their potential promise. Given this, there is a need to identify combination agents that improve the effectiveness of vaccinia in ovarian cancer treatment. We screened 9,000 compounds to identify drugs that enhance the ability of a recombinant vaccinia virus lacking VGF and F1 (ΔVF) to induce death of ID8 Trp53 -/- murine ovarian cancer cells. We identified a class of tubulin polymerization inhibitors including vinorelbine. The combination of vinorelbine and vaccinia induces ID8 Trp53 -/- cell death via apoptosis. In a syngeneic mouse model of high-grade serous ovarian carcinoma, ΔVF virus lacking the viral thymidine kinase (TK), armed with granulocyte-macrophage colony-stimulating factor (GM-CSF), and expressing NeonGreen (ΔVFTK-NG-GM-CSF) is tumor-specific. A combination of the ΔVFTK-NG-GM-CSF virus with vinorelbine prolongs mouse survival compared to the treatment of mice with either agent alone. Our study suggests that vinorelbine is a promising agent to combine with oncolytic vaccinia virus for the management of ovarian cancer.

Vaccinia virus, known for its clinical safety has a tropism for primary and metastatic tumours as well as ovarian tissue. Consequently, oncolytic approaches with recombinant vaccinia viruses have emerged as attractive agents against ovarian cancer. Unfortunately, oncolytic vaccinia monotherapies are yet to live up to their potential promise. Given this, there is a need to identify combination agents that improve the effectiveness of vaccinia in ovarian cancer treatment. We screened 9,000 compounds to identify drugs that enhance the ability of a recombinant vaccinia virus lacking VGF and F1 (ΔVF) to induce death of ID8 Trp53-/- murine ovarian cancer cells. We identified a class of tubulin polymerisation inhibitors including vinorelbine. The combination of vinorelbine and vaccinia induces ID8 Trp53-/- cell death via apoptosis. In a syngeneic mouse model of high grade serous ovarian carcinoma, ΔVF virus lacking the viral thymidine kinase (TK), armed with GM-CSF and expressing NeonGreen (ΔVFTK-NG-GM-CSF) is tumour specific. A combination of the ΔVFTK-NG-GM-CSF virus with vinorelbine prolongs mouse survival compared to the treatment of mice with either agent alone. Our study suggests vinorelbine is a promising agent to combine with oncolytic vaccinia virus approaches for the management of ovarian cancer. ### Competing Interest Statement The authors have declared no competing interest.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.

Abstract Background Cancer immunotherapies show low activity in metastatic microsatellite-stable (MSS) colorectal cancer (CCR) patients. Arginase 1 inhibitors have shown activity in pre-clinical models associated with PD-1 inhibitors. Methods We established a platform with co-cultures of metastatic MSS CCR patient-derived organoids (PDOs) and expanded autologous tumor-infiltrating lymphocytes to evaluate the efficacy of retifanlimab (PD-1 inhibitor), CB-1158 (arginase inhibitor), and their combination. CB-1158, retifanlimab, and CB-1158 + retifanlimab were tested using two PDOs representative of the IMMETCOLS IMC3 signature. Results Increased T-cell PDOs recognition was observed in co-cultures versus T cells alone. CB-1158, retifanlimab and CB- 1158 plus retifanlimab failed to increase T-cell PDOs recognition and T-cell cytotoxic effects. CB-1158 inhibited arginase, but the impaired synthesis of endogenous ornithine, through arginase, decreased ornithine release into the co-culture medium and increased glutamine, histidine, and putrescine consumption as compensation to guarantee ornithine and polyamines synthesis. Conclusion These results demonstrate the lack of meaningful activity of both monotherapy and combination therapy in PDO and autologous tumor-infiltrating lymphocyte preclinical models and suggest modest clinical activity in metastatic CCR MSS, at least in tumors meeting IMC3 metabolic characteristics.

Reactive oxygen species (ROS) serve important homeostatic functions but must be constantly neutralized by an adaptive antioxidant response to prevent supraphysiological levels of ROS from causing oxidative damage to cellular components. Here, we report that the cellular plasticity transcription factors ZEB1 and ZEB2 modulate in opposing directions the adaptive antioxidant response to fasting in skeletal muscle. Using transgenic mice in which Zeb1 or Zeb2 were specifically deleted in skeletal myofibers, we show that in fasted mice, the deletion of Zeb1 , but not Zeb2 , increased ROS production and that the adaptive antioxidant response to fasting essentially requires ZEB1 and is inhibited by ZEB2. ZEB1 expression increased in fasted muscles and protected them from atrophy; conversely, ZEB2 expression in muscles decreased during fasting and exacerbated muscle atrophy. In fasted muscles, ZEB1 reduces mitochondrial damage and increases mitochondrial respiratory activity; meanwhile, ZEB2 did the opposite. Treatment of fasting mice with Zeb1 -deficient myofibers with the antioxidant triterpenoid 1[2-cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoro-ethylamide (CDDO-TFEA) completely reversed their altered phenotype to that observed in fasted control mice. These results set ZEB factors as potential therapeutic targets to modulate the adaptive antioxidant response in physiopathological conditions and diseases caused by redox imbalance.

Human embryonic stem cells (hESCs) can differentiate into any cell lineage. Here, we report that ZEB1 and ZEB2 promote and inhibit mesodermal-to-myogenic specification of hESCs, respectively. Knockdown and/or overexpression experiments of ZEB1, ZEB2, or PAX7 in hESCs indicate that ZEB1 is required for hESC Nodal/Activin-mediated mesodermal specification and PAX7+ human myogenic progenitor (hMuP) generation, while ZEB2 inhibits these processes. ZEB1 downregulation induces neural markers, while ZEB2 downregulation induces mesodermal/myogenic markers. Mechanistically, ZEB1 binds to and transcriptionally activates the PAX7 promoter, while ZEB2 binds to and activates the promoter of the neural OTX2 marker. Transplanting ZEB1 or ZEB2 knocked down hMuPs into the muscles of a muscular dystrophy mouse model, showing that hMuP engraftment and generation of dystrophin-positive myofibers depend on ZEB1 and are inhibited by ZEB2. The mouse model results suggest that ZEB1 expression and/or downregulating ZEB2 in hESCs may also enhance hESC regenerative capacity for human muscular dystrophy therapy.

Accumulation of lipid-laden macrophages within the arterial neointima is a critical step in atherosclerotic plaque formation. Here, we show that reduced levels of the cellular plasticity factor ZEB1 in macrophages increase atherosclerotic plaque formation and the chance of cardiovascular events. Compared to control counterparts ( Zeb1 WT / Apoe KO ), male mice with Zeb1 ablation in their myeloid cells ( Zeb1 ∆M / Apoe KO ) have larger atherosclerotic plaques and higher lipid accumulation in their macrophages due to delayed lipid traffic and deficient cholesterol efflux. Zeb1 ∆M / Apoe KO mice display more pronounced systemic metabolic alterations than Zeb1 WT / Apoe KO mice, with higher serum levels of low-density lipoproteins and inflammatory cytokines and larger ectopic fat deposits. Higher lipid accumulation in Zeb1 ∆M macrophages is reverted by the exogenous expression of Zeb1 through macrophage-targeted nanoparticles. In vivo administration of these nanoparticles reduces atherosclerotic plaque formation in Zeb1 ∆M / Apoe KO mice. Finally, low ZEB1 expression in human endarterectomies is associated with plaque rupture and cardiovascular events. These results set ZEB1 in macrophages as a potential target in the treatment of atherosclerosis.

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and is initiated by mesenchyme-driving transcription factors and actin cytoskeletal assembly. We show a cytoplasmic-to-nuclear transport gradient of the EMT transcription factor Zeb1 toward sites of invasion in lung adenocarcinoma (LUAD), driven by the EMT inducer Tgfb, which is expressed in M2 polarized macrophages. We show that Zeb1 binds free actin monomers and RhoA in the cytoplasm to inhibit actin polymerization, blocking cell migration and Yap1 nu-clear transport. Tgfb causes turnover of the scaffold protein Rassf1a, which targets RhoA. Release of this RhoA inhibition in response to Tgfb overcomes Zeb1's block of cytoskeleton assembly and frees it for nuclear transport. A ZEB1 nuclear transport signature highlights EMT progression, identifies dedifferentiated inva-sive/metastatic human LUADs, and predicts survival. Blocking Zeb1 nuclear transport with a small molecule identified in this study inhibits cytoskeleton assembly, cell migration, Yap1 nuclear transport, EMT, and pre -cancerous-to-malignant transition.

Next-generation sequencing (NGS) provides a molecular rationale to inform prognostic stratification and guide personalized treatment in cancer patients. Here, we aimed at determining the prognostic and predictive value of actionable mutated genes in metastatic colorectal cancer (mCRC). A total of 294 mCRC patients that underwent targeted NGS-based testing were selected, of whom 200 receiving first-line treatments were included for prognostic analyses. Cox proportional hazards models were fitted to assess risks of individual and coexistent mutated genes on progression-free and overall survival (PFS and OS), adjusting for baseline clinical variables. Discriminative performance of biomarkers was assessed by time-dependent estimates of the area under the curve (AUC). The most recurrently mutated genes were TP53 (64%), KRAS or NRAS (49%), PIK3CA (15%), SMAD4 (14%), BRAF (13%) and FBXW7 (9.5%). Mutations in FBXW7 correlated with worse OS rates (P = 0.036; HR, 2.24) and SMAD4 mutations predicted negative PFS (P = 0.0015; HR, 2.63) independently of clinical factors. Concurrent mutations in TP53 and FBXW7 were associated with increased risk of death (P = 0.02; HR, 3.31) as well as double mutated TP53 and SMAD4 (P = 0.03; HR, 2.91). These associations showed a trend towards statistical significance in a second cohort of 1095 patients sequenced with the MSK-IMPACT gene-panel. Addition of the previous three mutated genes upon clinical factors discriminated those patients at high risk with an AUC of 0.87. Gene set enrichment analysis revealed specific functions associated with SMAD4 and FBXW7 mutated tumors in samples with altered TP53. Mutated SMAD4 and FBXW7 in TP53-driven tumors predict a negative prognostic outcome in mCRC, which could be useful for patient clinical management.

Next–generation sequencing (NGS) provides a molecular rationale to inform prognostic stratification and to guide personalized treatment in cancer patients. Here, we determined the prognostic and predictive value of actionable mutated genes in metastatic colorectal cancer (mCRC). Among a total of 294 mCRC tumors examined by targeted NGS, 200 of them derived from patients treated with first–line chemotherapy plus/minus monoclonal antibodies were included in prognostic analyses. Discriminative performance was assessed by time–dependent estimates of the area under the curve (AUC). The most recurrently mutated genes were TP53 (64%), KRAS or NRAS (49%), PIK3CA (15%), SMAD4 (14%), BRAF (13%), and FBXW7 (9.5%). Mutations in FBXW7 correlated with worse OS rates (p = 0.036; HR, 2.24) independently of clinical factors. Concurrent mutations in TP53 and FBXW7 were associated with increased risk of death (p = 0.02; HR, 3.31) as well as double–mutated TP53 and SMAD4 (p = 0.03; HR, 2.91). Analysis of the MSK–IMPACT mCRC cohort (N = 1095 patients) confirmed the same prognostic trend for the previously identified mutated genes. Addition of the mutational status of these genes upon clinical factors resulted in a time–dependent AUC of 87%. Gene set enrichment analysis revealed specific molecular pathways associated with SMAD4 and FBXW7 mutations in TP53–defficient tumors. Conclusively, SMAD4 and FBXW7 mutations in TP53–altered tumors were predictive of a negative prognostic outcome in mCRC patients treated with first–line regimens.

Existing immune signatures and tumor mutational burden have only modest predictive capacity for the efficacy of immune check point inhibitors. In this study, we developed an immune-metabolic signature suitable for personalized ICI therapies. A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 77 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here, we reveal that the IMMETCOLS signature classifies tumors into three distinct immune-metabolic clusters. Cluster 1 displays markers of enhanced glycolisis, hexosamine byosinthesis and epithelial-to-mesenchymal transition. On multivariate analysis, cluster 1 tumors were enriched in pro-immune signature but not in immunophenoscore and were associated with the poorest median survival. Its predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival and preferential use of alternative carbon sources, including glutamine and lipid uptake/β-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic but also has a solid mitochondrial function, with concomitant upregulation of glutamine and essential amino acid transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together, these findings suggest that the IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches.

The PDL1-PD1 immune checkpoint inhibits T cell activation, and its blockade is effective in a subset of patients. Studies are investigating how checkpoints are hijacked by cancer cells and why most patients remain resistant to immunotherapy. Epithelial mesenchymal transition (EMT), which drives tumor cell invasion via the Zeb1 transcription factor, is linked to immunotherapy resistance. In addition, M2-polarized tumor-associated macrophages (TAMs), which inhibit T cell migration and activation, may also cause immunotherapy resistance. How EMT in invading cancer cells is linked to therapy resistance and events driving TAM M2 polarization are therefore important questions. We show that Zeb1 links these two resistance pathways because it is required for PDL1 expression on invading lung cancer cells, and it also induces CD47 on these invading cells, which drives M2 polarization of adjacent TAMs. Resulting reprogramming of the microenvironment around invading cells shields them from the hostile inflammatory environment surrounding tumors.

Abstract A significant proportion of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) patients harbor a gain-of-function, heterozygous somatic mutations of the methyltransferase gene EZH2. Despite acceptable safety profile and early signs of activity in clinical trials, single agent treatment with EZH2 inhibitors is unlikely to be curative in aggressive lymphomas. In an effort to established novel rational combinations, we have evaluated the activity and mechanism of action of the EZH2 small molecule inhibitor CPI169 as single agent and in combination with BET bromodomain inhibition, using preclinical models of DLBCL and FL with distinct EZH2 mutational status. CPI169 anti-tumor activity and specificity was assessed in vitro in 10 DLBCL and FL cell lines, including cells expressing basal or ectopic EZH2mut. Molecular bases of its activity were determined by gene expression profiling (GEP), qPCR and western blot, followed by automated exploratory data analysis. Biomarkers validation was made in vitro and in vivo, using a mouse xenotransplant model of EZH2mut DLBCL, considering both exposure to CPI169 single agent and/or combination treatment with a BRD4 inhibitor, CPI203. CPI169 induced dose-dependent proliferation blockade in EZH2mut, but not EZH2wt DLBCL and FL cell lines, independently of EZH2 expression level or basal methyltransferase activity. Loss of H3K27me3 mark upon CPI169 treatment was associated with upregulation of gene sets related to G1 cell cycle blockade, mTOR and P53 pathways, and MYC signaling. Accordingly, combination with the MYC-interfering drug, BET inhibitor CPI203, achieved a synergistic anti-proliferating activity in EZH2 mutated cases and in mice bearing EZH2mut DLBCL tumors. Activity of EZHi/BRD4i combo was characterized by lower mitotic index, increased loss of H3K27me3 mark, in association with MYC downregulation. GEP analysis, followed by automated exploratory data analysis and validation by a siRNA screening, further identified the PI3K/AKT-regulated gene and mitosis regulator, YPEL2, and the regulator of innate-like B lymphocyte maturation, KLHL14, as crucial factors involved in the efficacy of MYC/EZH2 dual targeting. In conclusion, CPI169 shows significant activity and safety as single agent in EZH2 mutated-DLBCL and FL cases and displays synergistic interaction in vitro and in vivo with BRD4 inhibition, mediated by the modulation of a limited set of EZH2-regulated genes. Citation Format: Aranzazu Chamorro-Jorganes, Marcelo L. Ribeiro, Nuria Profitos-Peleja, Diana Reyes-Garau, Clara Recasens-Zorzo, Juan G. Valero, Marc Armengol, Patricia Perez-Galan, Ray Butler, Antonio Postigo, Francesc Bosch, Gael Roue. Safety and efficacy of EZH2 and BRD4 dual targeting in EZH2Y641mut germinal centre-derived lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2925.

ObjectiveChronic inflammation is a risk factor in colorectal cancer (CRC) and reactive oxygen species (ROS) released by the inflamed stroma elicit DNA damage in epithelial cells. We sought to identify new drivers of ulcerative colitis (UC) and inflammatory CRC.DesignThe study uses samples from patients with UC, mouse models of colitis and CRC and mice deficient for the epithelial-to-mesenchymal transition factor ZEB1 and the DNA repair glycosylase N-methyl-purine glycosylase (MPG). Samples were analysed by immunostaining, qRT-PCR, chromatin immunoprecipitation assays, microbiota next-generation sequencing and ROS determination.ResultsZEB1 was induced in the colonic epithelium of UC and of mouse models of colitis. Compared with wild-type counterparts,Zeb1-deficient mice were partially protected from experimental colitis and, in a model of inflammatory CRC, they developed fewer tumours and exhibited lower levels of DNA damage (8-oxo-dG) and higher expression of MPG. Knockdown of ZEB1 in CRC cells inhibited 8-oxo-dG induction by oxidative stress (H2O2) and inflammatory cytokines (interleukin (IL)1β). ZEB1 bound directly to the MPG promoter whose expression inhibited. This molecular mechanism was validated at the genetic level and the crossing ofZeb1-deficient andMpg-deficient mice reverted the reduced inflammation and tumourigenesis in the former. ZEB1 expression in CRC cells induced ROS and IL1β production by macrophages that, in turn, lowered MPG in CRC cells thus amplifying a positive loop between both cells to promote DNA damage and inhibit DNA repair.ConclusionsZEB1 promotes colitis and inflammatory CRC through the inhibition of MPG in epithelial cells, thus offering new therapeutic strategies to modulate inflammation and inflammatory cancer.

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration.