Alcohol-use disorder and alcohol-associated liver disease (ALD) are major causes of death and liver transplantation1. The gut-liver axis has a crucial yet poorly understood role in ALD pathogenesis, which depends on microbial translocation. Intestinal goblet cells (GCs) educate the immune system by forming GC-associated antigen passages (GAPs) on activation of muscarinic acetylcholine receptor M4 (mAChR4, also known as M4), enabling sampling of luminal antigens by lamina propria antigen-presenting cells. Here we show that chronic alcohol use in humans and mice downregulates small intestinal mAChR4 and reduces GAP formation, disrupting antimicrobial immunity. This is reversed on activation of intestinal IL-6 signal transducer (IL6ST, also known as glycoprotein 130; gp130), which restores mAChR4 expression and GAP formation, enabling induction of downstream type-3 innate lymphoid cell-derived IL-22 and antimicrobial REG3 proteins. This blunts translocation of enteric bacteria to the liver, thereby conferring ALD resistance. GAP induction by GC-specific mAChR4 activation was essential and sufficient for prevention of ethanol-induced steatohepatitis. These results lay the foundation for a therapeutic approach using mAChR4 or IL6ST agonists to promote GAP formation and prevent ALD by inhibiting microbial translocation.

The growing recognition of the role of the gut microbiome's impact on alcohol-associated diseases, especially in alcohol-associated liver disease, emphasizes the need to understand molecular mechanisms involved in governing organ-organ communication to identify novel avenues to combat alcohol-associated diseases. The gut-liver axis refers to the bidirectional communication and interaction between the gut and the liver. Intestinal microbiota plays a pivotal role in maintaining homeostasis within the gut-liver axis, and this axis plays a significant role in alcohol-associated liver disease. The intricate communication between intestine and liver involves communication between multiple cellular components in each organ that enable them to carry out their physiological functions. In this review, we focus on novel approaches to understanding how chronic alcohol exposure impacts the microbiome and individual cells within the liver and intestine, as well as the impact of ethanol on the molecular machinery required for intraorgan and interorgan communication.

Goblet cells (GCs) are specialised guardians lining the intestine. They play a critical role in gut defence and immune regulation. GCs continuously secrete mucus creating a physical barrier to protect from pathogens while harbouring symbiotic gut bacteria adapted to live within the mucus. GCs also form specialised GC-associated passages in a dynamic and regulated manner to deliver luminal antigens to immune cells, promoting gut tolerance and preventing inflammation. The composition of gut bacteria directly influences GC function, highlighting the intricate interplay between these components of a healthy gut. Indeed, imbalances in the gut microbiome can disrupt GC function, contributing to various gastrointestinal diseases like colorectal cancer, inflammatory bowel disease, cystic fibrosis, pathogen infections and liver diseases. This review explores the interplay between GCs and the immune system. We delve into the underlying mechanisms by which GC dysfunction contributes to the development and progression of gastrointestinal diseases. Finally, we examine current and potential treatments that target GCs and represent promising avenues for further investigation.

Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research.

Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts.

Introduction: Mantle cell lymphoma (MCL) is one of the most aggressive mature B-cell neoplasms. MCL frequently responds to initial treatments although later the development of resistance is common, relapsing with a more aggressive disease. Several groups have isolated MCL-cancer stem cells (CSC), presenting self-renewal, clonogenic growth, tumorigenic capabilities, and resistance to standard therapies. SRY-related HMG-box gene 11 (SOX11) is expressed in progenitors and embryonic stem cells (ESC). Its overexpression has been observed in undifferentiated tumor cell populations with CSC features. In MCL, its overexpression has been associated with more aggresive behavior and worse patient’s outcome. SOX11 is regulating several oncogenic mechanisms in MCL. However, nothing is known about its possible stemness role in MCL. Methods: To search for stem-cell related genes regulated by SOX11 that may contribute to MCL biological and clinical evolution, we compared SOX11+ and SOX11- MCL primary cases differential gene expression profiling (GEP). We analyzed the prognostic value of stem-cell related genes directly regulated by SOX11 and their involvement in stemness features in MCL, using several cellular and molecular approaches. Results: We observed a significant enrichment of leukemic- and hematopoietic stem cells (HSC)-related genes in the SOX11+ compared to SOX11- MCCL subtype. The RNA-binding protein Musashi-2 (MSI2), that maintains self-renewal and prevents differentiation in ESC and HSC, emerged as one of the most significant stem cell-related gene upregulated in SOX11+ MCL primary cases. Moreover, we have demonstrated that SOX11 binds to MSI2 promoter and activates its expression. However, MSI2 intronic superenhancers might be also responsible for MSI2 upregulation in MCL. Moreover, we found that higher expression of MSI2 is significantly associated with poor overall survival, independently of other knows high-risk features in MCL. MSI2 knockdown (KD) or MSI2 function inhibition with Ro 08-2750 (Ro) changed the GEP, downregulating genes involved in CSC-related pathways whereas upregulating pro-apoptotic genes in MCL cells. MSI2KD or MSI2 Ro-inhibition led to suppress stemness phenotypic features, such as clonogenic growth, chemoresistance and cell survival. Moreover, MSI2KD MCL cells have reduced tumorigenic engraftment into mice bone marrow and spleen compared to control cell lines in vivo, suggesting that MSI2 is an important tumorigenic factor in MCL. Conclusions: Our findings suggest that MSI2 expression in MCL is upregulated by SOX11 binding to its promoter and to several active MSI2 intronic superenhancers. MSI2 upregulation might contribute to sustain stemness and chemoresistance to MCL cells, through the post-transcriptional regulation of stem cell-related genes, representing a novel target for therapeutic interventions in aggressive MCL. Keywords: Genomics, Epigenomics, and Other -Omics, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

SOX11 overexpression has been associated with aggressive behavior of mantle cell lymphomas (MCL). SOX11 is overexpressed in embryonic and cancer stem cells (CSC) of some tumors. Although CSC have been isolated from primary MCL, their relationship to SOX11 expression and contribution to MCL pathogenesis and clinical evolution remain unknown. Here, we observed enrichment in leukemic and hematopoietic stem cells gene signatures in SOX11+ compared to SOX11- MCL primary cases. Musashi-2 (MSI2) emerged as one of the most significant upregulated stem cell-related genes in SOX11+ MCLs. SOX11 is directly bound to the MSI2 promoter upregulating its expression in vitro. MSI2 intronic enhancers were strongly activated in SOX11+ MCL cell lines and primary cases. MSI2 upregulation was significantly associated with poor overall survival independently of other high-risk features of MCL. MSI2 knockdown decreased the expression of genes related to apoptosis and stem cell features and significantly reduced clonogenic growth, tumor cell survival and chemoresistance in MCL cells. MSI2-knockdown cells had reduced tumorigenic engraftment into mice bone marrow and spleen compared to control cells in xenotransplanted mouse models. Our results suggest that MSI2 might play a key role in sustaining stemness and tumor cell survival, representing a possible novel target for therapeutic interventions in MCL.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a multi-organ damage that includes hepatic dysfunction, which has been observed in over 50% of COVID-19 patients. Liver injury in COVID-19 could be attributed to the cytopathic effects, exacerbated immune responses or treatment-associated drug toxicity. Herein we demonstrate that hepatocytes are susceptible to infection in different models: primary hepatocytes derived from humanized angiotensin-converting enzyme-2 mice (hACE2) and primary human hepatocytes. Pseudotyped viral particles expressing the full-length spike of SARS-CoV-2 and recombinant receptor binding domain (RBD) bind to ACE2 expressed by hepatocytes, promoting metabolic reprogramming towards glycolysis but also impaired mitochondrial activity. Human and hACE2 primary hepatocytes, where steatosis and inflammation were induced by methionine and choline deprivation, are more vulnerable to infection. Inhibition of the renin-angiotensin system increases the susceptibility of primary hepatocytes to infection with pseudotyped viral particles. Metformin, a common therapeutic option for hyperglycemia in type 2 diabetes patients known to partially attenuate fatty liver, reduces the infection of human and hACE2 hepatocytes. In summary, we provide evidence that hepatocytes are amenable to infection with SARS-CoV-2 pseudovirus, and we propose that metformin could be a therapeutic option to attenuate infection by SARS-CoV-2 in patients with fatty liver.

Mantle cell lymphoma (MCL) is a mature B cell neoplasm with two distinct biological subtypes regarding clinical, molecular and pathological features. Conventional MCL (cMCL) subtype is characterized by high aggressiveness and poor outcome with frequent relapses after initial response to treatment, suggesting the presence of a population of cancer stem cells (CSC) able to self-renew and responsible for drug resistance. SOX11 embryonic transcription factor is aberrantly overexpressed mostly in cMCL and has an oncogenic role in MCL. MCL-CSC have been isolated from MCL cases by different groups. However, its relationship to SOX11 expression and contribution to MCL clinical features and aggressive behavior remains unknown. We hypothesized that SOX11 may promote stem cell-like properties through the activation of stem cell-related genes, leading to an aggressive and incurable tumor in MCL patients. Here, we integrated the differential gene expression profile (GEP) between SOX11+ and SOX11- MCL primary cases with stem cell-related genes, and specific SOX11 ChIP-chip and epigenetic data in MCL cell lines and primary cases, to identify potential mediators of CSC directly regulated by SOX11 in MCL. We observed an enrichment of hematopoietic (HSC) and leukemic stem cells (LSC) gene signatures in SOX11+ compared to SOX11- MCL primary cases. MSI2, an RNA binding protein that maintains self-renewal and prevents differentiation in HSC, emerged as one of the most significant upregulated CSC-related gene in SOX11+ compared to SOX11- MCLs, positively correlating with SOX11 expression in MCL primary cases. Our in vitro experiments showed that SOX11 binds to MSI2 regulatory regions increasing its expression in MCL cells. MSI2 enhancers located in intronic regions were activated in SOX11+ but not in SOX11- MCL cell lines and primary cases. MSI2 upregulation significantly associated with poor overall survival, independently of common risk factors (SOX11 expression, high copy number alterations, 17p/TP53, 11q/ATM and 9p/CDKN2A alterations), in MCL patients. MSI2 knockdown MCL cell lines had a gene expression profile enriched in proapoptotic-related genes; whereas HSC and LSC gene signatures were downregulated compared to its control cells. MSI2 knockdown or MSI2 inhibition with Ro 08-2750 treatment decreased clonogenic growth, tumor survival and chemoresistance in MCL cells. SOX11+/MSI2 high MCL primary leukemic samples showed higher percentage of ALDH+ cells compared to SOX11-/MSI2 low samples, which decreased after Ro 08-2750 treatment. Besides, MSI2 silencing delayed tumor growth in vivo in MCL xenograft mice models. Overall, our results suggest that SOX11+ MCL cases acquire stemness features through the upregulation of MSI2 expression, which promotes chemoresistance, self-renewal and tumor survival. MSI2 represents a novel biomarker for MCL-CSC and therapeutic target for relapsed MCL. Citation Format: Marta Sureda-Gómez, Patricia Balsas, Marta Leonor Rodríguez, Ferran Nadeu, Anna De Bolòs, Álvaro Eguileor, Marta Kulis, Giancarlo Castellano, José Ignacio Martin-Subero, Santiago Demajo, Pedro Jares, Eva Giné, Elias Campo, Virginia Amador. Deciphering the role of MSI2 as a regulator of mantle cell lymphoma stem-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 777.

SOX11 overexpression in mantle cell lymphoma (MCL) has been associated with more aggressive behavior and worse outcome. However, SOX11 oncogenic pathways driving MCL tumor progression are poorly understood. Here, we demonstrate that SOX11 binds to regulatory regions of 2 important genes for microenvironment signals in cancer: (C-X-C motif) chemokine receptor 4 (CXCR4) and PTK2 (encoding for focal adhesion kinase [FAK]). Moreover, SOX11(+) xenograft and human primary MCL tumors overexpress cell migration and stromal stimulation gene signatures compared with their SOX11(-) counterparts. We show that SOX11 directly upregulates CXCR4 and FAK expression, activating PI3K/AKT and ERK1/2 FAK-downstream pathways in MCL. Concordantly, SOX11(+) MCL cells have higher cell migration, transmigration through endothelial cells, adhesion to stromal cells, and cell proliferation and display an increased resistance to conventional drug therapies compared with SOX11(-) MCL cells. Specific FAK inhibition blocks downstream PI3K/AKT- and ERK1/2-mediated phosphorylation. Additionally, specific FAK and PI3K inhibitors reduce SOX11-enhanced MCL cell migration and stromal interactions and revert cell adhesion-mediated drug resistance (CAM-DR) to the same levels as SOX11(-) MCL cells. In intravenous MCL xenograftmodels, SOX11(+) MCL cells display higher cell migration, invasion, and growth compared with SOX11-knockdown cells, and specific FAK and CXCR4 inhibitors impair SOX11-enhanced MCL engraftment in bone marrow. Overall, our results suggest that SOX11 promotes MCL homing and invasion and increases CAM-DR through the direct regulation of CXCR4 and FAK expression and FAK/PI3K/AKT pathway activation, contributing to a more aggressive phenotype. Inhibition of this pathway may represent an efficient strategy to overcome stromal-mediated chemotherapy refractoriness in aggressive MCL.

Abstract The chromosomal translocation t(7;11)(p15,p15), that results in the oncogenic fusion protein Nup98-Hoxa9 (NH), appears in 1% of patients with AML and is associated with very poor prognosis and short overall survival. Despite the large severity of the leukemia induced by this fusion protein, the oncogenic events triggered by NH are poorly understood, although a potential role as an aberrant transcription factor has been proposed. We have generated a human Hematopoietic Progenitors (hHP) cellular model expressing NH constitutively to identify the molecular mechanisms supporting the malignancy of this fusion protein, facilitating the search for therapeutic targets. We identified the DNA binding sites of NH by performing ChIP-seq experiments, which were validated by qRT-PCR analysis on ChIP selected DNA and Luciferase assays. Expression profiling was performed in hHP-NH and co-Immunoprecipitations (Co-IPs) were done to demonstrate the interaction of NH with different transcriptional regulators. Specific drug sensitivity of the hHP-NH model was assessed in cell proliferation assays. Our work provides the first description of the DNA binding sites of NH, most of which are regulatory regions of genes involved in the development of AML. In particular, we demonstrate that NH induces the overexpression of MEIS1, HOXA9 and PBX3, transcription factors forming an activator complex that is a key element in the leukemic onset driven by other chromosome rearrangements. Interestingly, we show that NH directly interacts with this complex through Pbx3. To evaluate the biological relevance of the interaction of the MEIS1-HOXA9-PBX3 complex with NH, we have analyzed the sensitivity of hHP-NH to the HXR9 peptide (an inhibitor of the HOXA9-PBX3 interaction). Supporting our hypothesis, we observed an inhibitory effect on hHP-NH viability after HXR9 treatment. Finally, by combining the expression profile data from hHP-NH and the ChIP-seq results using GSEA analysis, we show that NH is able to induce both overexpression and down-regulation of its target genes. To provide evidences of the activator-repressor role of NH, we performed different Co-IPs that demonstrated its direct interaction with both p300 (transcriptional activator) and HDAC1 (transcriptional inhibitor). Taken together, we show that the direct overexpression of the complex MEIS1-HOXA9-PBX3 is one of the pathogenic mechanisms induced by NH. As expected, the disruption of this complex with the HXR9 peptide in the hHP-NH model has a direct effect on cell viability. Furthermore, we show that NH interacts with this complex via PBX3 and also with p300 and HDAC1. The features and architecture of these interactions need to be further explored, but these findings allow us to consider the use of the HXR9 peptide or some HDAC inhibitors as possible treatments for these patients. Citation Format: Ana Rio-Machin, Alba Maiques-Diaz, Sandra Rodriguez-Perales, Sara Alvarez, Rocio N. Salgado, Álvaro Eguileor, Raul Torres, Juan C. Ramirez, Juan C. Cigudosa. Interactions of the fusion protein Nup98-Hoxa9 with Pbx3, p300 and HDAC1: widening the targeted therapy window in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2014-472

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasia derived from mature B cells genetically characterized by the presence of the t(11;14)(q13;q32) translocation causing cyclin D1 overexpression. Furthermore, other secondary genetic alterations also contribute to the development and aggressiveness of MCL. However, recent studies have identifieda subset ofMCLwith indolent clinical behavior that tends to present with leukemic disease instead of extensive nodal infiltration and patients may not need chemotherapy for long periods. Recently, molecular studies have identified SOX11 (SRY [sex determining region-Y]-box11), as one of the best characterized discriminatory genes between these 2 clinical subtypes of MCL tumors. SOX11, togetherwith SOX4 and SOX12, belongs to the subgroupC of the SOX gene family encoding for transcription factors which play a critical role in embryonic development and cell differentiation. SOX11 plays an important role in the regulation of neuronal cell survival and neurite growth, and is highly expressed in different central nervous systemmalignancies, solid tumors, aggressiveMCL, and at lower levels in a subgroup of Burkitt and lymphoblastic lymphomas. However, the oncogenic mechanisms of SOX11 contributing to the development and progression of these tumors are largely unknown. We have recently demonstrated the in vivo tumorigenic potential of SOX11 in a MCL xenograft model. SOX11 blocks the terminal B-cell differentiation through direct positive regulation of PAX5 but the specific mechanisms regulated by SOX11 promoting the oncogenic and rapid tumor growth of aggressiveMCL still remain to be elucidated. To further characterize the potential oncogenic mechanisms regulated by SOX11 in MCL, we have investigated the gene and protein expression profiling of SOX11-positive and -knockdown MCL xenograft tumors, cell lines, and primary SOX11-positive and SOX11-negativeMCL.We have identified that SOX11modulates angiogenesis in MCL, and this mechanism is mediated by the upregulation of several proangiogencic factors, principally platelet-derived growth factor A (PDGFA). The inhibition of the PDGFA pathway not only impairs angiogenic development both in vitro and in vivo but also MCL tumor growth in vivo, offering a promising novel therapeutic strategy for the treatment of aggressive MCL.

SOX11 is overexpressed in several solid tumors and in the vast majority of aggressive mantle cell lymphomas (MCLs). We have recently proven that SOX11 silencing reduces tumor growth in a MCL xenograft model, consistent with the indolent clinical course of the human SOX11-negative mantle cell lymphoma (MCL). However, the direct oncogenic mechanisms and downstream effector pathways implicated in SOX11-driven transformation remain poorly understood. Here, we observed that SOX11-positive xenograft and human primary MCL tumors overexpressed angiogenic gene signatures and had a higher microvascular density compared with their SOX11-negative counterparts. Conditioned media of SOX11-positive MCL cell lines induced in vitro endothelial cell proliferation, migration, tube formation, and activation of downstream angiogenic pathways. We identified PDGFA as a SOX11 direct target gene upregulated in MCL cells whose inhibition impaired SOX11-enhanced in vitro angiogenic effects on endothelial cells. In addition, platelet-derived growth factor A (PDGFA) was overexpressed in SOX11-positive but not in SOX11-negative MCL. In vivo, imatinib impaired tumor angiogenesis and lymphoma growth in SOX11-positive MCL xenograft tumors. Overall, our results demonstrate a prominent role for SOX11 as a driver of proangiogenic signals in MCL, and highlight the SOX11-PDGFA axis as a potential therapeutic target for the treatment of this aggressive disease.