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Structural and functional characterization of 4-1BBN/CEGFR after conjugation with p-SCN-Bn-deferoxamine (Df).

Oligonucleotides used in this study

Purpose:The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity.Experimental Design:Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo.Results:In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.Conclusions:These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.

Background: Microtubule targeting compounds are a successful class of anticancer agents in the clinic. Although highly potent, the currently approved antitumor agents targeting tubulin present some drawbacks such as the development of acquired resistances, which remain an obstacle for an effective prolonged anticancer treatment. In this work, we present a novel microtubule destabilizing agent with an optimized interaction for the colchicine site and a novel mechanism of action which confers it a high affinity binding for tubulin. Methods and Results: We have determined the X-ray crystallographic structure of the complex T2R-TTL with PM534 at a resolution of 2.45 Å. We unequivocally found PM534 ligand density at the intra-dimer interface between α and β tubulin of both tubulin dimers (the colchicine domain) within the complex. The colchicine domain of tubulin can be divided into three zones: a central pocket and two accessories. Whereas other colchicine site-ligands only bind to two of the zones, we found that PM534 binds extensively along the three zones, making multiple contacts with β-tubulin N terminal domain, central hinge helix, and the intermediate domain. PM534 interaction did not affect the overall curved conformation of tubulin and its mechanism of action consists on precluding the curve-to-straight conformational transition required for tubulin to assemble intro microtubules and thus, to perform its cellular functions. In competition assays we found that the compound displaces a bona fide high-affinity colchicine-probe with a binding affinity of 5.1±0.3 × 107 M−1 at 25̊C. Moreover, PM534 shows potent antineoplastic activity in vitro, with GI50 values in the low nanomolar range, observed in different human tumor cancer cell lines. An in vivo Proof of the Concept (PoC) was performed in athymic nu/nu mice bearing H460 (NSCLC) tumors that were treated (intravenous, on days 0, 7, 14) with different doses (ranging from 0.75 mg/kg to 2.5 mg/kg) of PM534. Results demonstrated strong, dose-related PM534-induced antitumor activity in this in vivo model. Conclusions: The mechanism of action of PM534 involves an optimized interaction with the colchicine site, which is reflected in its high affinity for the substrate, being this the ultimate reason for the observed cellular activity. Additionally, PM534 presents a potent antitumor activity in vitro that was translated into a clear positive in vivo PoC that resulted in strong antitumor effect. Citation Format: Maria Ángela Oliva, Beatriz Álvarez-Bernad, Daniel Lucena-Agell, Marta Martínez Diez, María José Guillén, Gema Santamaría Nuñez, María José Muñoz-Alonso, Eva M. Garrido-Martin, Pablo Avilés, Carmen Cuevas, J. Fernando Díaz. PM534 is a novel microtubule-destabilizing agent with high affinity and potent antineoplastic properties. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6239.

Background Intestinal macrophages are key immune cells in the maintenance of intestinal immune homeostasis and have a role in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms by which macrophages exert a pathological influence in both ulcerative colitis (UC) and Crohn disease (CD) are not yet well understood. Methods We purified intestinal macrophages from gastrointestinal mucosal biopsies (patients with UC, patients with CD, and healthy donors) and analyzed their transcriptome by RNA sequencing and bioinformatics, confirming results with quantitative polymerase chain reaction and immunohistochemistry. Results Compared with those of healthy donors, intestinal macrophages in patients with UC and with CD showed cellular reprograming of 1287 and 840 dysregulated genes, respectively (false discovery rate ≤ 0.1). The UC and CD intestinal macrophages showed an activated M1 inflammatory phenotype and the downregulation of genes engaged in drug/xenobiotic metabolism. Only macrophages from CD showed, concomitant to an M1 phenotype, a significant enrichment in the expression of M2 and fibrotic and granuloma-related genes. For the first time, we showed (and validated by quantitative polymerase chain reaction and immunohistochemistry) that intestinal macrophages in patients with IBD present both M1 and M2 features, as recently described for tumor-associated macrophages, that affect key pathways for IBD pathology, represented by key markers such as MMP12 (fibrosis), CXCL9 (T-cell attraction), and CD40 (T-cell activation). Conclusions Our data support the therapeutic targeting of macrophages to maintain remission in IBD but also indicate that a shift toward an M2 program—as proposed by some reports—may not limit the recruitment and activation of T cells because M2 features do not preclude M1 activation in patients with UC or CD and could exacerbate M2-related CD-specific features such as fibrosis and the formation of granulomas.

SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis.Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell- and patient-derived xenografts and metastasis. YES1 levels were evaluated in mouse and patient plasma-derived exosomes.Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell- and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/monitoring.Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.

Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent histology. While immunotherapy with checkpoint inhibitors has shown outstanding results in NSCLC, the precise identification of responders remains a major challenge. Most studies attempting to overcome this handicap have focused on adenocarcinomas or squamous cell carcinomas. Among NSCLC subtypes, the molecular and immune characteristics of lung large cell carcinoma (LCC), which represents 10% of NSCLC cases, are not well defined. We hypothesized that specific molecular aberrations may impact the immune microenvironment in LCC and, consequently, the response to immunotherapy. To that end, it is particularly relevant to thoroughly describe the molecular genotype–immunophenotype association in LCC–to identify robust predictive biomarkers and improve potential benefits from immunotherapy. We established a cohort of 18 early-stage, clinically annotated, LCC cases. Their molecular and immune features were comprehensively characterized by genomic and immune-targeted sequencing panels along with immunohistochemistry of immune cell populations. Unbiased clustering defined two novel subgroups of LCC. Pro-immunogenic tumors accumulated certain molecular alterations, showed higher immune infiltration and upregulated genes involved in potentiating immune responses when compared to pro-tumorigenic samples, which favored tumoral progression. This classification identified a set of biomarkers that could potentially predict response to immunotherapy. These results could improve patient selection and expand potential benefits from immunotherapy.

Small cell lung cancer (SCLC) is a high-grade neuroendocrine subtype of lung cancer. This tumor is characterized by its aggressiveness, refractoriness to therapies, poor prognosis, and early dissemination. The first line of treatment consists of platinum-based chemotherapy, and although patients initially respond very well to this therapy, they promptly develop resistance. Currently, there are no targeted therapies approved for the treatment of SCLC patients. Here, we identified YES1, a non-receptor tyrosine kinase, as a novel targetable candidate with a relevant role in SCLC. In silico analysis revealed that YES1 is overexpressed in SCLC patients compared to non-malignant lung tissues. In a multi-institutional cohort of 80 SCLC patients, 31% of cases showed high YES1 protein expression and 26% of all specimens had YES1 gain/amplification (CNV≥2.5), analyzed by immunohistochemistry and FISH, respectively. In addition, high expression of YES1 predicted both lower overall survival (OS) and progression free survival (PFS) (n=80). In accordance, YES1 mRNA expression significantly correlated with YES1 CNV in SCLC cell lines. We knocked-down YES1 in two human SCLC cell lines (DMS53 and H209) with high expression and amplification of YES1. Depletion of YES1 almost totally abrogated cell proliferation and the formation of cell line-derived organoids in 3D. Accordingly, inhibition of YES1 arrested the cell cycle and promoted cell apoptosis in vitro. Consistent with these results, knockdown of YES1 promoted a dramatic reduction of subcutaneous tumor growth in both models with high levels of YES1, leading to a rate of partial regression of 54% and 23% complete regression. A metastasis assay, performed by intracardiac injection of H209 cells, showed that inhibition of YES1 led to complete disappearance of metastatic lung, leg and liver tumors in 4 out of 5 mice. Gene set enrichment analysis (GSEA) of RNA sequencing data performed with YES1-depleted DMS53 cells revealed that cells lacking YES1 had a downregulation in DNA replication and DNA repair signatures. We next explored the effect of pharmacological inhibition of YES1 using the multityrosine kinase inhibitor dasatinib and the selective YES1 inhibitor CH6953755. Dasatinib and CH6953755 effectively reduced cell proliferation in vitro at doses 10µM). More interestingly, dasatinib and CH6953755 decreased DMS53, H209 and a high-YES1 PDX (YU-16)-derived organoid growth. Pharmacological inhibition of YES1 significantly reduced the tumor growth of high-YES1 models: DMS53 and H209 xenografts, and TP135 and YU-16 PDXs, in contrast to low-YES1 models (H69 xenograft and YU-34 PDX). Our results provide evidence that YES1 is a new druggable oncogenic target in SCLC and that its inhibition could be a promising therapeutic strategy for a subset of patients with high YES1 expression or copy number gain/amplification. Citation Format: Esther Redin, Eva M. Garrido-Martin, Karmele Valencia, Miriam Redrado, Jose Luis Solorzano, Rafael Carias, Franscisco Exposito, Diego Serrano, Nerea Otegui, Luis Paz Ares, Katerina Politi, Luis M. Montuenga, Alfonso Calvo. YES1 is a novel druggable oncogene in small cell lung cancer [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 839.

32 p.-4 fig., Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity., Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo., Results: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer., Conclusions: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions., This work was supported by grants from the European Union [IACT Project (602262), H2020-iNEXT (1676)]; the Spanish Ministry of Science, Innovation and Universities and the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, CTQ2017-83810-R, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018); and the Spanish Association Against Cancer (AECC, 19084). C. Domínguez-Alonso was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (PRE2018-083445). M. Zonca was supported by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness, co-founded by the European Social Fund (PTQ-16-08340).

BackgroundThe role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor’s anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer.MethodsMacrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+tissue-resident memory T cells (TRM) in tumors and survival data from an independent cohort of 393 patients with lung cancer.ResultsTAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hotTAMs and TRMcells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1hotTAMs may recruit TRMcells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRMdepend.ConclusionsWe showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hotTAMs was associated with a strong TRMtumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hotphenotype are likely to augment the adaptive antitumor responses.

Background The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor’s anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer. Methods Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+ tissue-resident memory T cells (TRM) in tumors and survival data from an independent cohort of 393 patients with lung cancer. Results TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hot TAMs and TRM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1hot TAMs may recruit TRM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRM depend. Conclusions We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hot TAMs was associated with a strong TRM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hot phenotype are likely to augment the adaptive antitumor responses.

To determine the nature of CD4+ T cells that provide ‘help’ for generating robust anti-tumor CD8+ cytotoxic T cell (CTL) responses, we profiled the transcriptomes of patient-matched CD4+ and CD8+ T cells present in the tumor micro-environment (TME) and analyzed them jointly using integrated weighted gene correlation network analysis. We found the follicular helper T cell (TFH) program in CD4+ T cells was strongly associated with proliferation and tissue-residency in CD8+ CTLs. Single-cell analysis demonstrated the presence of TFH-like cells and features linked to cytotoxic function and their provision of CD8+ T cell ‘help’. Tumor-infiltrating TFH-like cells expressed PD-1 and were enriched in tumors following checkpoint blockade, suggesting that they may respond to anti-PD-1 therapy. Adoptive transfer or induction of TFH cells in mouse models resulted in augmented CD8+ CTL responses and impairment of tumor growth, indicating an important role of TFH-like CD4+ T cells in anti-tumor immunity.

Stratification of patients for targeted and immune-based therapies requires extensive genomic profiling that enables sensitive detection of clinically relevant variants and interrogation of biomarkers, such as tumor mutational burden (TMB) and microsatellite instability (MSI). Detection of single and multiple nucleotide variants, copy number variants, MSI, and TMB was evaluated using a commercially available next-generation sequencing panel containing 523 cancer-related genes (1.94 megabases). Analysis of formalin-fixed, paraffin-embedded tissue sections and cytologic material from 45 tumor samples showed that all previously known MSI-positive samples (n = 7), amplifications (n = 9), and pathogenic variants (n = 59) could be detected. TMB and MSI scores showed high intralaboratory and interlaboratory reproducibility (eight samples tested in 11 laboratories). For reliable TMB analysis, 20 ng DNA was shown to be sufficient, even for relatively poor-quality samples. A minimum of 20% neoplastic cells was required to minimize variations in TMB values induced by chromosomal instability or tumor heterogeneity. Subsequent analysis of 58 consecutive lung cancer samples in a diagnostic setting was successful and revealed sufficient somatic mutations to generate mutational signatures in 14 cases. In conclusion, the 523-gene assay can be applied for evaluation of multiple DNA-based biomarkers relevant for treatment selection.

MicroRNAs are known to regulate important pathways in asthma pathology including the IL-6 and IFN pathways. MicroRNAs have been found not only within cells but also within extracellular vesicles such as exosomes. In this study, we particularly focused on microRNA cargo of nanovesicles in bronchoalveolar lavage of severe asthmatic patients. We extracted nanovesicle RNA using a serial filtration method. RNA content was analyzed with small RNA sequencing and mapped to pathways affected using WebGestalt 2017 Software. We report that severe asthma patients have deficient loading of microRNAs into their airway luminal nanovesicles and an altered profile of small RNA nanovesicle content (i.e., ribosomal RNA and broken transcripts, etc.). This decrease in microRNA cargo is predicted to increase the expression of genes by promoting inflammation and remodeling. Consistently, a network of microRNAs was associated with decreased FEV1 and increased eosinophilic and neutrophilic inflammation in severe asthma. MicroRNAs in airway nanovesicles may, thus, be valid biomarkers to define abnormal biological disease processes in severe asthma and monitor the impact of interventional therapies.

The European Respiratory Society (ERS) International Congress is the largest respiratory congress and brings together leading experts in all fields of respiratory medicine and research. ERS Assembly 3 shapes the basic and translational science aspects of this congress, aiming to combine cutting-edge novel developments in basic research with novel clinical findings. In this article, we summarise a selection of the scientific highlights from the perspective of the three groups within Assembly 3. In particular, we discuss new insights into the pathophysiology of the human alveolus, novel tools in organoid development and (epi)genome editing, as well as insights from the presented abstracts on novel therapeutic targets being identified for idiopathic pulmonary fibrosis., The amount of basic and translational science presented at #ERSCongress is steadily increasing, showing novel cutting-edge technologies and models. http://bit.ly/2GgXIJi

BackgroundTumor mutational burden (TMB) is a recently proposed predictive biomarker for immunotherapy in solid tumors, including non-small cell lung cancer (NSCLC). Available assays for TMB determination differ in horizontal coverage, gene content and algorithms, leading to discrepancies in results, impacting patient selection. A harmonization study of TMB assessment with available assays in a cohort of patients with NSCLC is urgently needed.MethodsWe evaluated the TMB assessment obtained with two marketed next generation sequencing panels: TruSight Oncology 500 (TSO500) and Oncomine Tumor Mutation Load (OTML) versus a reference assay (Foundation One, FO) in 96 NSCLC samples. Additionally, we studied the level of agreement among the three methods with respect to PD-L1 expression in tumors, checked the level of different immune infiltrates versus TMB, and performed an inter-laboratory reproducibility study. Finally, adjusted cut-off values were determined.ResultsBoth panels showed strong agreement with FO, with concordance correlation coefficients (CCC) of 0.933 (95% CI 0.908 to 0.959) for TSO500 and 0.881 (95% CI 0.840 to 0.922) for OTML. The corresponding CCCs were 0.951 (TSO500-FO) and 0.919 (OTML-FO) in tumors with =55), and 0.861 (TSO500-FO) and 0.722 (OTML-FO) in tumors with PD-L1≥1% (N=41). Inter-laboratory reproducibility analyses showed higher reproducibility with TSO500. No significant differences were found in terms of immune infiltration versus TMB. Adjusted cut-off values corresponding to 10 muts/Mb with FO needed to be lowered to 7.847 muts/Mb (TSO500) and 8.380 muts/Mb (OTML) to ensure a sensitivity >88%. With these cut-offs, the positive predictive value was 78.57% (95% CI 67.82 to 89.32) and the negative predictive value was 87.50% (95% CI 77.25 to 97.75) for TSO500, while for OTML they were 73.33% (95% CI 62.14 to 84.52) and 86.11% (95% CI 74.81 to 97.41), respectively.ConclusionsBoth panels exhibited robust analytical performances for TMB assessment, with stronger concordances in patients with negative PD-L1 expression. TSO500 showed a higher inter-laboratory reproducibility. The cut-offs for each assay were lowered to optimal overlap with FO.

Interleukin-11 (IL11) has been associated with tumorigenesis in a wide variety of tumors, including lung cancer, and it has been proposed as a diagnostic biomarker for lung adenocarcinoma. However, it is still not clear how IL11 affects the tumorigenesis. It is possible that, as other cytokines, it has a dual role in the tumor cell and tumoral microenvironment. Thus, the inhibition of IL11 could be an interesting therapeutic option to test in these patients. First, we confirmed the pro-tumorigenic effect of IL11 in patients and mouse models of lung adenocarcinoma (cancer cell lines xenografts, patient derived xenografts; PDXs and genetically engineered mouse models; GEMMs). Later, we knocked-down the expression of IL11 or its specific receptor IL11RA in adenocarcinoma cell lines in order to analyze their tumorigenic properties in vitro and in vivo. We confirmed that fibroblasts are a relevant source of IL11, so we knocked-down IL11 expression in order to analyze how it affects the fibroblasts´ properties, including the secretion of other pro-tumorigenic cytokines and growth factors. We reported that an increased expression of IL11 correlates with a poorer survival in lung adenocarcinoma patients and that IL11 stimulation increases the proliferation rates in xenografts, PDXs and GEMMs. On the contrary, IL11 or IL11RA knockdown in adenocarcinoma cell lines reduces their pro-tumorigenic properties in vitro and in vivo. Finally, the silencing of IL11 in fibroblasts reduces their proliferation, migration and secretion of pro-tumorigenic cytokines and growth factors. In conclusion, we propose that IL11 plays a direct pro-oncogenic role in lung adenocarcinoma tumoral cell and an indirect role in tumoral microenvironment. The genetic ablation of IL11 has an anti-tumoral effect, so it could be interesting to develop a pharmacological tool which neutralizes the IL11-IL11RA signaling to test as a therapeutic strategy in preclinical models. Thus, IL11 could represent a potential therapeutic target that deserves to be more exhaustively studied in the clinical settings. Citation Format: Laura Ojeda, Cristina Cirauqui, Sonia Molina-Pinelo, Eva M Garrido-Martín, Javier Ramos-Paradas, Patricia Yagüe, Alba Santos, Nuria Carrizo, Ana B. Enguita, Maria Teresa Muñoz, Jose Luis Solorzano, Luis Paz-Ares, Irene Ferrer. Interleukin-11 could be a novel therapeutic target for lung adenocarcinoma patients [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 5247.

Lung cancer leads cancer mortality, being Non-Small-Cell Lung Cancer (NSCLC) the most prevalent subtype. Immunotherapy with checkpoint inhibitors has shown promising results in NSCLC, yet this benefit is restricted to a subset of patients. Robust predictive biomarkers of response are lacking. We seek a multiparametric biomarker signature. We hypothesize that specific molecular alterations of NSCLC tumor cells have a deep impact on the immune microenvironment. Describing this relationship is critical to predict response to immunotherapy. We evaluated 178 samples from a retrospective cohort of clinically annotated early stage NSCLC patients. We analyzed their immune profile by immunohistochemistry and a RNAseq immune panel. In parallel, we defined their molecular aberrations and Tumor Mutational Burden by Next Generation Sequencing. We integrated our multiparametric data with Multi-Omics Factor Analysis. Adenocarcinomas (N=80) cluster in 4 groups with two main immunophenotypes. Key factors of this clustering are TOP2A, MKI67, CCNB2, CDK1, JCHAIN, VEGFA, PCLAF, KRT7, FOXM1, BUB1. The proimmune tumors upregulate genes related to immune activation (CD40LG, CD28, MS4A1), signaling (CD79B), chemoattraction (CXCR2), antigen presentation (CD1C, HLA-DQA2), inflammatory cytokines (IL2, IL1B), and immune cells (CD19). Oppositely, the protumoral samples overexpress genes involved in cell cycle (CCNB2, FOXM1, CDKN2A, CDKN3), immune inhibition (CD276), angiogenesis (VEGFA) and proliferation (AKT1, EGFR, PIK3CA, MTOR, MKI67). Regarding molecular characterization, KRAS mutation is enriched in the proimmune subset, while the protumoral one gathers mutations in ARID1A and ALK. Squamous cell carcinomas (N=98) cluster in 4 groups with two main immunophenotypes. Critical factors of this clustering are TRIM29, FOXM1, KRT5, TOP2A, MKI67, TFRC, CCNB2, CD44, EGFR, KREMEN1. The proimmune tumors upregulate genes involved in immune signaling (CD3D, CD3E, CD3G, CD79A, CD79B), activation (CD40LG, CD27, MS4A1), chemoattraction (CXCR5, CXCL13), antigen presentation (CD1C, HLA-DMA/DPB1/DRA/DMB/DOB), inflammatory cytokines (IFNG, IL17F, IL21), immune cells (CD8A, CD19) and immunomodulation (ICOS). Contrarily, the protumoral samples upregulate genes related to cell cycle (FOXM1, BUB1, CCNB2, CDK1), immune cells (FOXP3), angiogenesis (VEGFA), apoptosis (BCL2), immunomodulation (PD-L1) and proliferation (AKT1, MTOR, MYC, PIK3CA, MKI67). Of note, the proimmune subset has higher infiltration of CD8+, CD4+ and B cells. The protumoral one accrues mutations in TFRC, PIK3CA, PIK3CB, FGF3, FGF10. In conclusion, we performed a comprehensive description of novel NSCLC subgroups based on multiparametric data that will improve prediction of response to immunotherapy. Citation Format: Javier Ramos-Paradas, David Gomez-Sanchez, Aranzazu Rosado, Irene Ferrer, Nuria Carrizo, Ana B. Enguita, Maria T. Muñoz, Urbicio Perez-Gonzalez, Ivan Martinez, Luis Paz-Ares, Eva M. Garrido-Martin. Comprehensive multiparametric analysis of non-small cell lung cancer describes novel genotype-immunophenotype relationships and provides putative biomarker signatures of response to checkpoint blockade [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 2006.

Tumour associated macrophages (TAMs) are thought to be biased towards an M2 phenotype, suppressing anti-tumour immune responses and thus therapies to eradicate macrophages have been proposed. However, current knowledge on TAMs in human cancer is based on a limited number of markers; their comprehensive M1 (anti-tumour) or M2 (pro-tumour) signatures, as well as their role in the anti-tumour immune response, have not yet been determined. Here, for the first time, we have generated transcriptomic data from TAMs isolated from human lung cancer, in comparison with non-tumour lung macrophages from the same patients. We found out that TAMs in lung cancer show a broad pro-tumour M2 signature, but that this signature is not associated with a weaker anti-immune response. However, it is the activation of an M1 signature in TAMs that determines whether TAMs can promote or not a strong anti-tumour immune response. We describe a novel set of M1high TAMs that simultaneously activate M1 and M2 signatures. M1high TAMs strongly activate antigen-presenting genes and express high levels of T-cell chemoattractants (CXCL9, 10 and 11), associating with a higher infiltration of anti-tumour CD8+ T-cells and a better clinical outcome. AIM2 and IL1beta levels are increased in M1high TAMs, indicating a direct involvement in pyroptosis and clearance of cancer cells. We provide a unique transcriptomic resource of TAMs in lung cancer and propose that TAMs can be used to improve immunotherapy due to a novel and pivotal role for M1high TAMs in orchestrating the immune adaptive anti-tumour responses.

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103(hi) tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

High tumor infiltrating lymphocyte (TIL) density predicts for good prognosis in several cancers and therapies that boost the anti-tumor responses of cytotoxic lymphocytes (CTLs) have shown promise in the clinic. However, clinical responses to currently available immunotherapeutic agents vary considerably, the molecular basis of which is unclear. We performed global transcriptional profiling of CTLs in tumors and adjacent non-tumor tissue from 36 patients with early stage lung cancer to define the molecular features associated with robustness and heterogeneity of anti-tumor immune responses. We observed major differences in the transcriptional program of tumor-infiltrating CTLs (CD8+ TILs) that is shared across tumor subtypes. Pathway analysis revealed enrichment of genes in cell cycle, TCR activation and co-stimulation pathways, indicating tumor-driven expansion of presumed tumor antigen-specific CTLs. We also observed marked heterogeneity in the expression of molecules associated with TCR activation and immune checkpoints, and their expression was positively correlated with the density of tumor-infiltrating CTLs. Interestingly, TILhigh tumors were also enriched for a distinct CD8+ tumor infiltrating lymphocyte subset that appeared to have enhanced cytotoxicity and independently predicted improved survival in patients with lung cancer. In summary, we define the molecular fingerprint of tumor-infiltrating CTLs and identify a number of novel targets that may be important in modulating the magnitude and specificity of anti-tumor immune responses in lung cancer. Citation Format: Anusha Preethi Ganesan, Oliver Wood, Eva Garrido-Martin, Serena Chee, Toby Mellows, James Clarke, Daniela Samaniego-Castruita, Divya Singh, Gregory Seumois, Aiman Altezani, Edwin Woo, Peter Friedman, Gareth Thomas, Emma King, Tilman Sanchez-Elsner, Pandurangan Vijayanand, Christian H. Ottensmeier. A distinct CD8+ tumor infiltrating lymphocyte subset is associated with high TIL density, enhanced cytotoxicity and improved survival in patients with lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2948. doi:10.1158/1538-7445.AM2017-2948

Objective— Hereditary hemorrhagic telangiectasia is a genetic disorder characterized by visceral and mucocutaneous arteriovenous malformations (AVMs). Clinically indistinguishable hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 are caused by mutations in ENG and ALK1 , respectively. In this study, we have compared the development of visceral and mucocutaneous AVMs in adult stages between Eng - and Alk1 -inducible knockout (iKO) models. Approach and Results— Eng or Alk1 were deleted from either vascular endothelial cells (ECs) or smooth muscle cells in adult stages using Scl-CreER and Myh11-CreER lines, respectively. Latex perfusion and intravital spectral imaging in a dorsal skinfold window chamber system were used to visualize remodeling vasculature during AVM formation. Global Eng deletion resulted in lethality with visceral AVMs and wound-induced skin AVMs. Deletion of Alk1 or Eng in ECs, but not in smooth muscle cells, resulted in wound-induced skin AVMs. Visceral AVMs were observed in EC-specific Alk1 -iKO but not in Eng -iKO. Intravital spectral imaging revealed that Eng -iKO model exhibited more dynamic processes for AVM development when compared with Alk1 -iKO model. Conclusions— Both Alk1 - and Eng -deficient models require a secondary insult, such as wounding, and ECs are the primary cell type responsible for the pathogenesis. However, Alk1 but not Eng deletion in ECs results in visceral AVMs.

29 p.-8 fig, RATIONALE: Activin receptor-like kinase-1 (ALK1) is an endothelial transforming growth factor β receptor involved in angiogenesis. ALK1 expression is high in the embryo vasculature, becoming less detectable in the quiescent endothelium of adult stages. However, ALK1 expression becomes rapidly increased after angiogenic stimuli such as vascular injury. OBJECTIVE: To characterize the molecular mechanisms underlying the regulation of ALK1 on vascular injury. METHODS AND RESULTS: Alk1 becomes strongly upregulated in endothelial (EC) and vascular smooth muscle cells of mouse femoral arteries after wire-induced endothelial denudation. In vitro denudation of monolayers of human umbilical vein ECs also leads to an increase in ALK1. Interestingly, a key factor in tissue remodeling, Krüppel-like factor 6 (KLF6) translocates to the cell nucleus during wound healing, concomitantly with an increase in the ALK1 gene transcriptional rate. KLF6 knock down in human umbilical vein ECs promotes ALK1 mRNA downregulation. Moreover, Klf6(+/-) mice have lower levels of Alk1 in their vasculature compared with their wild-type siblings. Chromatin immunoprecipitation assays show that KLF6 interacts with ALK1 promoter in ECs, and this interaction is enhanced during wound healing. We demonstrate that KLF6 is transactivating ALK1 gene, and this transactivation occurs by a synergistic cooperative mechanism with specificity protein 1. Finally, Alk1 levels in vascular smooth muscle cells are not directly upregulated in response to damage, but in response to soluble factors, such as interleukin 6, released from ECs after injury. CONCLUSIONS: ALK1 is upregulated in ECs during vascular injury by a synergistic cooperative mechanism between KLF6 and specificity protein 1, and in vascular smooth muscle cells by an EC-vascular smooth muscle cell paracrine communication during vascular remodeling., This work was supported by the Ministerio de Ciencia e Innovación of Spain (MICINN grants SAF2007-61827 and SAF2010-19222 to C.B. and SAF2008-01218 to L.M.B., and predoctoral fellowship BES-2005–7974 to E.M.G-M), Genoma España (MEICA), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Red HERACLES (RD6/009/0008 to M.R.), and National Institutes of Health & National Center for Research Resources (DK56621 and DK37340 to S.L.F.).

Testicular germ cell tumors (TGCTs) are highly responsive to and curable by cisplatin-based chemotherapy even in advanced stages. We have studied the molecular mechanisms involved in the induction of apoptosis in response to cisplatin, and found that proapoptotic Noxa is transcriptionally up-regulated following cisplatin exposure, even in the absence of p53, in NTERA2 cisplatin-sensitive cells but not in 1411HP-resistant cells. Blockade of Noxa reduced the apoptotic response of embryonal carcinoma (EC) NTERA2 cells to cisplatin.Adetailed analysis of the Noxa promoter revealed that p73 and Sp1-like factors, Sp1 and KLF6, played key roles in the transcriptional control of this gene. Overexpression of TAp73 induced Noxa whereas the dominant negative isoform ΔNp73, reduced the levels of Noxa after cisplatin exposure in NTERA2 and 2102EP. Interestingly, down-regulation of Sp1 increased Noxa expression in response to cisplatin. However, blockade of KLF6 decreased cisplatin-induced up-regulation of Noxa in EC cell lines. In addition, tissue microarray analyses of TGCTs revealed that expression of Noxa correlates with good clinical prognosis in patients with embryonal carcinoma. Thus, our data show the transcriptional network that regulates Noxa in EC cells, which is key for their apoptotic response to cisplatin-based chemotherapy, and propose Noxa as a predictive factor of therapeutic response. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by Instituto de Salud Carlos III Grants RD06/0020/0074 (to J.L. F.-L.), RD06/0020/0059 (to E.A.), RD06/0020/0017 (to M.D.D.) (Red Tematica de Investigacion Cooperativa en Cancer), PI11/00397 (to M.D.D.), and PI081491 (to X.G.); and a grant from Instituto de Formacion e Investigación Marqués de Valdecilla (IFIMAV), API2011-04 (to J.L. F.-L.).

9 páginas, 2 figuras, 1 tabla -- PAGS nros. 169-177 Hereditary haemorrhagic telangiectasia (HHT) is a vascular hereditary autosomic dominant disease associatedwith epistaxis, telangiectases, gastrointestinal haemorrhages and arteriovenous malformations in lung, liverand brain. It affects 1–2 in 10,000 people. There are at least three different genes mutated in HHT, ENG,ACVRL1 and MADH4 that encode endoglin, activin receptor-like kinase (ALK1) and Smad4 proteins,respectively. These proteins are involved in the transforming growth factor (TGF)-β superfamily signallingpathway of vascular endothelial cells. Mutations in ENG (HHT1) and ACVRL1 (HHT2) account for more than90% of all HHT mutations. In this article, we review the underlying molecular and cellular bases and thetherapeutic approaches that have been addressed in our laboratory in recent years

10 páginas, 6 figuras.-- El pdf del artículo es la versión pre-print., Endoglin is a coreceptor for transforming growth factor-β (TGF-β) that acts as a suppressor of malignancy during mouse skin carcinogenesis. Because in this model system H-Ras activation drives tumor initiation and progression, we have assessed the effects of endoglin on the expression of H-Ras in transformed keratinocytes. We found that TGF-β1 increases the expression of H-Ras at both messenger RNA and protein levels. The TGF-β1-induced H-Ras promoter transactivation was Smad4 independent but mediated by the activation of the TGF-β type I receptor ALK5 and the Ras-mitogen-activated protein kinase (MAPK) pathway. Endoglin attenuated stimulation by TGF-β1 of both MAPK signaling activity and H-Ras gene expression. Moreover, endoglin inhibited the Ras/MAPK pathway in transformed epidermal cells containing an H-Ras oncogene, as evidenced by the levels of Ras-guanosine triphosphate, phospho-MAPK kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK) as well as the expression of c-fos, a MAPK downstream target gene. Interestingly, in spindle carcinoma cells, that have a hyperactivated Ras/MAPK pathway, endoglin inhibited ERK phosphorylation without affecting MEK or Ras activity. The mechanism for this effect is unknown but strongly depends on the endoglin extracellular domain. Because the MAPK pathway is a downstream mediator of the transforming potential of Ras, the effect of endoglin on the oncogenic function of H-Ras was assessed. Endoglin inhibited the transforming capacity of H-Ras(Q61K) and H-Ras(G12V) oncogenes in a NIH3T3 focus formation assay. The ability to interfere with the expression and oncogenic potential of H-Ras provides a new face of the suppressor role exhibited by endoglin in H-Ras-driven carcinogenesis., Spanish Ministry of Science and Innovation (SAF2007-61827 to C.B., SAF2007-63821 to M.Q.); Science and Technology of Chile (FONDECYT #1050476 to J.F.S.).

13 p.-6 fig.-3 fig. supl., KLF6 (Krüppel-like factor 6) is a ranscription factor and tumour suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of TGF-β1 (transforming growth factor-β1). KLF6 can be alternatively spliced, generating lower-molecular-mass isoforms that antagonize the full-length WT (wild-type) protein and promote growth. A key target gene of full-length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-β auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-β on KLF6 expression and splicing, and to define the contribution of TGF-β on promoters regulated by co-operation between KLF6 and Sp1 (specificity protein 1). Using co-transfection, co-immunoprecipitation and fluorescence resonance energy transfer, our data demonstrate that KLF6 co-operates with Sp1 in transcriptionally regulating KLF6-responsive genes and that this co-operation is further enhanced by TGF-β1 through at least two mechanisms. First, in specific cell types, TGF-β1 may decrease KLF6 alternative splicing, resulting in a net increase in full-length, growth-suppressive KLF6 activity. Secondly, KLF6–Sp1 co-operation is further enhanced by the TGF-β–Smad (similar to mothers against decapentaplegic) pathway via the likely formation of a tripartite KLF6–Sp1–Smad3 complex in which KLF6 interacts indirectly with Smad3 through Sp1, which may serve as a bridging molecule to co-ordinate this interaction. These findings unveil a finely tuned network of interactions between KLF6, Sp1 and TGF-β to regulate target genes

10 p.-6 fig., Aims Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-β type I membrane receptor restricted almost entirely to endothelial cells (ECs) and involved in vascular remodelling and angiogenesis. Previous reports have shown that the ubiquitous TGF-β type I receptor ALK5 and the type II receptor are located in cholesterol-rich membrane microdomains named caveolae. The aim of this work was to assess the location of ALK1 in endothelial caveolae as well as to study the role of caveolin-1 on the TGF-β/ALK1 signalling pathway. Methods and results The subcellular distribution of ALK1 was analysed by confocal microscopy and co-fractionation experiments in human ECs. The association between human ALK1 and caveolin-1 was studied in caveolin-1-deficient human epithelial cells by co-immunoprecipitation. The functional role of caveolin-1 on the ALK1-mediated TGF-β signalling was elucidated using ALK1-specific luciferase reporters in human ECs, caveolin-1−/−mouse embryonic fibroblasts, and rat myoblasts. Confocal microscopy analyses, as well as cholesterol depletion experiments in the presence of cholesterol-depleting agents such as nystatin or methyl-β-cyclodextrin, demonstrated that ALK1 is located in endothelial caveolae. Also, co-immunoprecipitation assays showed that ALK1 associates with the main caveolae component caveolin-1. Mapping of the ALK1/caveolin-1 interaction revealed that the caveolin-1 scaffolding domain and the caveolin-1 binding motif in ALK1 are responsible for this association. Moreover, this hitherto not reported interaction had a functional consequence for the ALK1-dependent signalling. In contrast with the previously published ALK5/caveolin-1 interaction, caveolin-1 enhances the TGF-β/ALK1 signalling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signalling pathway. Conclusion ALK1 is located in endothelial caveolae where it functionally interacts with caveolin-1 through its scaffolding domain, suggesting a joint contribution of ALK1 and caveolin-1 as key mediators of the TGF-β pathway in angiogenesis., Ministerio de Educación y Ciencia (grants SAF2004-01390 and SAF2007-61827 to CB, and predoctoral fellowship BES-2005-7974 to EMG-M

19 páginas, 8 figuras, 4 tablas -- PAGS nros. 1515-1533, Hereditary hemorrhagic telangiectasia (HHT) or Osler–Weber–Rendu syndrome is an autosomal dominant vascular disorder characterized by telangiectases and internal arteriovenous malformations. It is caused by mutations in elements of the transforming growth factor-β (TGF-β) receptor complex: endoglin, a co-receptor, responsible for HHT1, or ALK1 (activin receptor-like kinase 1), a type I receptor leading to HHT2. Recently, we have established cultures of HHT endothelial cells, primary targets of the disease. These cells showed deficient TGF-β signaling and angiogenesis, representing a useful human model to study the molecular mechanism of this disease. To understand the pathogenic mechanism underlying HHT, we have used total RNA probes to compare HHT versus non-HHT cells by expression microarrays. This work represents a systematic study to identify target genes affected in HHT cells. Given the similarity of symptoms in HHT1 and HHT2, special interest has been put on the identification of common targets for both HHT types. As a result, 277 downregulated and 63 upregulated genes were identified in HHT versus control cells. These genes are involved in biological processes relevant to the HHT pathology, such as angiogenesis, cytoskeleton, cell migration, proliferation and NO synthesis. The type of misregulated genes found in HHT endothelial cells lead us to propose a model of HHT pathogenesis, opening new perspectives to understand this disorder. Moreover, as the disease is originated by mutations in proteins of the TGF-β receptor complex, these results may be useful to find out targets of the TGF-β pathway in endothelium, This work was supported by grants Fondo de Investigación Sanitaria (PI020200) to CB and Ministerio de Educación y Ciencia (SAF05–01090 to LMB and SAF2004–01390 to CB, GEN2003–20649-C06–06/NAC to MAV, BFU2004–00285/BFI to JML-N), and HHT Foundation International Inc to CB. LMB, MAV and CB were supported by Genoma España to the development of the microarray experiments. Fernandez-L is a predoctoral fellow of I3P CSIC, MP is a predoctoral fellow of Junta de Castilla y Leon and EMG-M is a predoctoral fellow of Ministerio de Educacion y Ciencia. ARB is the recipient of a fellowship from ‘Ramon y Cajal’ program

9 páginas, 5 figuras, 2 tablas -- PAGS nros. 254-262, Recurrent epistaxis is the most frequent clinical manifestation of hereditary haemorrhagic telangiectasia (HHT). Its treatment is difficult. Our objective was to assess the use of tranexamic acid (TA), an antifibrinolytic drug, for the treatment of epistaxis in HHT patients and to investigate in vitro the effects of TA over endoglin and ALK-1 expression and activity in endothelial cells.A prospective study was carried out on patients with epistaxis treated with oral TA in the HHT Unit of Sierrallana Hospital (Cantabria, Spain). Primary cultures of endothelial cells were treated with TA to measure the levels of endoglin and ALK-1 at the cell surface by flow cytometry. RNA levels were also measured by real-time PCR, and the transcriptional effects ofTA on reporters for endoglin, ALK-1 and the endoglin/ALK-1 TGF beta pathway were assessed.The results showed that the fourteen HHT patients treated orally withTA improved, and the frequency and severity of their epistaxis were decreased. No complications derived from the treatment were observed. Cultured endothelial cells incubated with TA exhibited increased levels of endoglin and ALK-1 at the protein and mRNA levels, enhanced TGF-β signaling, and improved endothelial cell functions like tubulogenesis and migration. In summary, oral administration of TA proved beneficial for epistaxis treatment in selected patients with HHT. In addition to its already reported antifibrinolytic effects, TA stimulates the expression ofALK-1 and endoglin,as well as the activity of the ALK-1/endoglin pathway, Grants from Fondo de Investigacion Sanitaria (PI020200) to CB and MEC (SAF05–01090 to LB and SAF2004–01390 to CB).

Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic vascular disease in which arteriovenous malformations (AVMs) manifest in skin and multiple visceral organs. HHT is caused by heterozygous mutations in endoglin (ENG), activin receptor-like kinase 1 (ALK1), or SMAD4. ALK1 regulates angiogenesis, but the precise function of ALK1 in endothelial cells (ECs) remains elusive. Since most blood vessels of HHT patients do not produce pathological vascular lesions, ALK1 heterozygous ECs may be normal unless additional genetic or environmental stresses are imposed. To investigate the cellular and biochemical phenotypes of Alk1-null versus Alk1-heterozygous ECs, we have generated pulmonary EC lines in which a genotype switch from the Alk1-conditional allele (Alk1 (2f)) to the Alk1-null allele (Alk1 (1f)) can be induced by tamoxifen treatment. Alk1-null (1 f/1 f) ECs displayed increased migratory properties in vitro in response to bFGF compared with Alk1-het (2 f/1 f) ECs. The 1 f/1 f-ECs formed a denser and more persistent tubular network as compared with their parental 2 f/1 f-ECs. Interestingly, the response to BMP-9 on SMAD1/5 phosphorylation was impaired in both 2 f/1 f- and 1 f/1 f-ECs at a comparable manner, suggesting that other factors in addition to SMADs may play a crucial role for enhanced angiogenic activity in 1 f/1 f-ECs. We also demonstrated in vivo that Alk1-deficient ECs exhibited high migratory and invasive properties. Taken together, these data suggest that enhanced responses to angiogenic cues in ALK1-deficient ECs underlie the pathogenesis of HHT2.

Fontalba, Ana et al. 8 p.-1 fig., Background Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder characterised mainly by mutations in the Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes. Methods Here, we have identified 22 ALK1 mutations and 15 ENG mutations, many of which had not previously been reported, in independent Spanish families afflicted with HHT. Results We identified mutations in thirty-seven unrelated families. A detailed analysis of clinical symptoms was recorded for each patient analyzed, with a higher significant presence of pulmonary arteriovenous malformations (PAVM) in HHT1 patients over HHT2. Twenty-two mutations in ALK1 and fifteen in ENG genes were identified. Many of them, almost half, represented new mutations in ALK1 and in ENG. Missense mutations in ENG and ALK1 were localized in a tridimensional protein structure model. Conclusion Overall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1) in our Spanish population, in agreement with previous data from our country and other Mediterranean countries (France, Italy), but different to Northern Europe or North America. There was a significant increase of PAVM associated with HHT1 over HHT2 in these families., Ministerio de Educación y Ciencia (SAF2007-61827 to CB and; SAF05-01090 to LMB); Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII-CIBER CB/06/07/0038 and ISCIII-RETICC RD06/0020) Fundacion Ramón Areces of Spain.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

22 páginas, 9 figuras, 2 tablas.-- et al., [Background]: Activin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-β (TGF-β) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1. [Results]: We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210) was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1. [Conclusions]: Our results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into the molecular mechanisms that contribute to the expression of ACVRL1 gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of ACVRL1 gene transcriptional activity., This work was supported by grants from Ministerio de Ciencia e Innovación of Spain (SAF2007-61827 to CB; SAF08-01218 to LMB, and predoctoral fellowship BES-2005-7974 to EMG-M), National Institutes of Health & National Center for Research Resources (P20-RR-15555 and HL083151 to CPV; DK56621 and DK37340 to SLF) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).