SARS-CoV-2 Proteins Induce Endotoxin Tolerance Hallmarks: A Demonstration in Patients with COVID-19.

According to a large number of reported cohorts, sepsis has been observed in nearly all deceased patients with COVID-19. We and others have described sepsis, among other pathologies, to be an endotoxin tolerance (ET)-related disease. In this study, we demonstrate that the culture of human blood cells from healthy volunteers in the presence of SARS-CoV-2 proteins induced ET hallmarks, including impairment of proinflammatory cytokine production, low MHC class II (HLA-DR) expression, poor T cell proliferation, and enhancing of both phagocytosis and tissue remodeling. Moreover, we report the presence of SARS-CoV-2 blood circulating proteins in patients with COVID-19 and how these levels correlate with an ET status, the viral RNA presence of SARS-CoV-2 in plasma, as well as with an increase in the proportion of patients with secondary infections.

The association between restricted intra-uterine growth and inadequate postnatal nutrition in very-low-birth-weight infants and their neurodevelopmental outcomes: a 50-month follow-up study.

Inadequate nutrition during a critical period of development - as is the case during gestation and the first days of life, especially in very-low-birth-weight (VLBW) infants, can impact on neurodevelopment and favour co-morbidities. In this study, we evaluate how neurodevelopment may be affected by intra-uterine growth (IUGR) restriction and by an inadequate intake of nutritional energy during the early neonatal period. A longitudinal cohort study was conducted to analyse the nutritional contributions received during the first week of life, among a population of 396 VLBW infants. Motor, cognitive, sensory and behavioural development was assessed at 14, 25, 33 and 50 months. The association between IUGR, postnatal energy restriction and neurodevelopment was examined using multivariate logistic regression techniques. Mild cognitive delay was observed in 35·6 % of neonates with IUGR and in 24 % of those with appropriate birth weight. IUGR is associated with behavioural disorder (OR 2·60; 95 % CI 1·25, 5·40) and delayed cognitive development (OR 2·64; 95 % CI 1·34, 5·20). Energy restriction during the first week of life is associated with visual deficiency (OR 2·96; 95 % CI 1·26, 6·84) and cerebral palsy (OR 3·05; CI 95 % 1·00, 9·54). In VLBW infants, IUGR is associated with behavioural disorder, while postnatal energy restriction is significantly associated with motor disorder, infantile cerebral palsy and sensory disorder.

Liquid Biopsy Biomarkers in Bladder Cancer: A Current Need for Patient Diagnosis and Monitoring.

Bladder Cancer (BC) represents a clinical and social challenge due to its high incidence and recurrence rates, as well as the limited advances in effective disease management. Currently, a combination of cytology and cystoscopy is the routinely used methodology for diagnosis, prognosis and disease surveillance. However, both the poor sensitivity of cytology tests as well as the high invasiveness and big variation in tumour stage and grade interpretation using cystoscopy, emphasizes the urgent need for improvements in BC clinical guidance. Liquid biopsy represents a new non-invasive approach that has been extensively studied over the last decade and holds great promise. Even though its clinical use is still compromised, multiple studies have recently focused on the potential application of biomarkers in liquid biopsies for BC, including circulating tumour cells and DNA, RNAs, proteins and peptides, metabolites and extracellular vesicles. In this review, we summarize the present knowledge on the different types of biomarkers, their potential use in liquid biopsy and clinical applications in BC.

EZH2 in Bladder Cancer, a Promising Therapeutic Target.

Bladder Cancer (BC) represents a current clinical and social challenge. The recent studies aimed to describe the genomic landscape of BC have underscored the relevance of epigenetic alterations in the pathogenesis of these tumors. Among the epigenetic alterations, histone modifications occupied a central role not only in cancer, but also in normal organism homeostasis and development. EZH2 (Enhancer of Zeste Homolog 2) belongs to the Polycomb repressive complex 2 as its catalytic subunit, which through the trimethylation of H3 (Histone 3) on K27 (Lysine 27), produces gene silencing. EZH2 is frequently overexpressed in multiple tumor types, including BC, and plays multiple roles besides the well-recognized histone mark generation. In this review, we summarize the present knowledge on the oncogenic roles of EZH2 and its potential use as a therapeutic target, with special emphasis on BC pathogenesis and management.

Late Neonatal Sepsis in Very-low-birth-weight Premature Newborns Is Associated With Alterations in Neurodevelopment at Twenty-five Months of Age.

AIM: To evaluate the impact of late-onset sepsis (LOS) on the neurodevelopment of very-low-birth-weight (VLBW) premature infants. METHODS: This is a retrospective cohort study of VLBW premature infants. The Mental Development Index (MDI) was determined for a population of 546 VLBW infants, at 14 and 25 months of age, and evaluated using the Bayley test. A history of meningitis or early neonatal sepsis was considered an exclusion criterion. The study parameters analyzed included perinatal variables, the development of neonatal comorbidities and a history of LOS. Multivariate linear regression and multinomial logistic regression analyses were performed. RESULTS: LOS was observed in 115 newborns, among whom microbiological testing showed that 65.0% presented Gram-positive bacteria, with Staphylococcus epidermidis being responsible for 55.4%. There was a significant association between the 25-month MDI and a history of LOS. This represents a decrease of 7.9 points in the MDI evaluation of newborns with a history of LOS. The latter history is also associated with the following neurodevelopmental alternations: mild motor disorders [odds ratio (OR): 2.75; 95% confidence intervals (CI): 1.07-7.05], moderate cognitive delay (OR: 3.07; 95% CI: 1.17-8.00) and cerebral palsy (OR: 2.41; 95% CI: 1.09-5.35). CONCLUSIONS: In our study cohort, LOS was associated with alterations in neurodevelopment, including reduced MDI, together with motor and cognitive disorders and cerebral palsy. To improve neurodevelopmental outcomes in this group of newborns, neonatal intensive care unit personnel should focus attention on preventing hospital-acquired infections.

Modulation of tumor microenvironment by targeting histone acetylation in bladder cancer.

Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

CD44v6, STn & O-GD2: promising tumor associated antigens paving the way for new targeted cancer therapies.

Targeted therapies are the state of the art in oncology today, and every year new Tumor-associated antigens (TAAs) are developed for preclinical research and clinical trials, but few of them really change the therapeutic scenario. Difficulties, either to find antigens that are solely expressed in tumors or the generation of good binders to these antigens, represent a major bottleneck. Specialized cellular mechanisms, such as differential splicing and glycosylation processes, are a good source of neo-antigen expression. Changes in these processes generate surface proteins that, instead of showing decreased or increased antigen expression driven by enhanced mRNA processing, are aberrant in nature and therefore more specific targets to elicit a precise anti-tumor therapy. Here, we present promising TAAs demonstrated to be potential targets for cancer monitoring, targeted therapy and the generation of new immunotherapy tools, such as recombinant antibodies and chimeric antigen receptor (CAR) T cell (CAR-T) or Chimeric Antigen Receptor-Engineered Natural Killer (CAR-NK) for specific tumor killing, in a wide variety of tumor types. Specifically, this review is a detailed update on TAAs CD44v6, STn and O-GD2, describing their origin as well as their current and potential use as disease biomarker and therapeutic target in a diversity of tumor types.

Immune Predictors of Response after Bacillus Calmette-Guérin Treatment in Non-Muscle-Invasive Bladder Cancer.

Bacillus Calmette-Guérin (BCG) has been the standard of care for the treatment of high-risk, non-muscle-invasive bladder cancer (NMIBC) for decades, but 49.6% of high-risk and very-high-risk patients will experience progression to muscle-invasive disease in five years. Furthermore, cytology and cystoscopy entail a high burden for both patients and health care systems due to the need for very long periods of follow-up. Subsequent adjuvant treatment using intravesical immunotherapy with BCG has been shown to be effective in reducing tumor recurrence and progression, but it is not free of severe adverse effects that ultimately diminish patients' quality of life. Because not all patients benefit from BCG treatment, it is of paramount importance to be able to identify responders and non-responders to BCG as soon as possible in order to offer the best available treatment and prevent unnecessary adverse events. The tumor microenvironment (TME), local immune response, and systemic immune response (both adaptive and innate) seem to play an important role in defining responders, although the way they interact remains unclear. A shift towards a proinflammatory immune response in TME is thought to be related to BCG effectiveness. The aim of this review is to collect the most relevant data available regarding BCG's mechanism of action, its role in modulating innate and adaptive immune responses and the secretion of certain cytokines, and their potential use as immunological markers of response; the aim is also to identify promising lines of investigation.

Neuropsychological evaluation of high-risk children from birth to seven years of age.

UNLABELLED: High Risk Children (HRC) are those with an increased risk of abnormal development due to any factor affecting neurological growth. Those factors have been the focus of most studies in this area. However, little is known about their long-term consequences over the course of child development. OBJECTIVES: The goal was to study the cognitive, emotional and academic outcomes of 7-year-old children diagnosed as HRC at birth. METHOD: We compared 14 HRC and 20 healthy children using the WISC-IV, BASC and Brunet-Lezine tests. RESULTS: HRC showed cognitive, emotional and academic deficits compared with healthy children. However, Brunet-Lezine scores obtained over the course of development (6, 12, 18 and 24 months) were not predictive of the children's' current psychological status. CONCLUSIONS: Long-term follow-up with HRC should be maintained until 7 years of age, at which point an appropriate treatment should be implemented.

Anti-inflammatory effects of α-humulene and ß-caryophyllene on pterygium fibroblasts.

AIM: To investigate the anti-inflammatory effects of the sesquiterpenes α-humulene and ß-caryophyllene on pterygium fibroblasts. METHODS: Primary cultures of pterygium fibroblasts were established. Third passage pterygium fibroblasts were exposed to α-humulene and ß-caryophyllene separately and together. The cell viability was assessed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay at 12, 24, 48, and 72h after exposure. The levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α and IL-10 in the conditioned culture medium were assessed by enzyme-linked immunosorbent assay (ELISA) at 12, 24 and 48h after exposure. Data were statistically analyzed using Friedman repeated measures analysis of variances on ranks. RESULTS: The 25 µmol/L ß-caryophyllene induced significant decrease in the IL-6 production by pterygium fibroblasts 48h after the exposure (P=0.041). The levels of IL-1ß, IL-8, IL-10, and TNF-α were very low and had no statistically significant variations after exposure to α-humulene, ß-caryophyllene, or both compounds together. CONCLUSION: The exposure to 25 µmol/L of ß-caryophyllene significantly reduce the production of IL-6 by pterygium fibroblasts after 48h. This sesquiterpene may be a potential alternative adjuvant agent for the treatment of pterygium.

Genomic Landscape of Vinflunine Response in Metastatic Urothelial Cancer.

BACKGROUND AND AIMS: Metastatic urothelial carcinoma (mUC) remains an incurable disease with limited treatment options after platinum-based chemotherapy and immune checkpoint blockade (ICB). Vinflunine has shown a modest increase in overall survival and remains a therapeutic option for chemo- and immunotherapy refractory tumours. However, biomarkers that could identify responding patients to vinflunine and possible alternative therapies after failure to treatment are still missing. In this study, we aimed to identify potential genomic biomarkers of vinflunine response in mUC patient samples and potential management alternatives. METHODS: Formalin-fixed paraffin-embedded samples of mUC patients (n = 23) from three university hospitals in Spain were used for genomic targeted-sequencing and transcriptome (using the Immune Profile panel by NanoString) analyses. Patients who received vinflunine after platinum-based chemotherapy failure were classified in non-responders (NR: progressive disease ≤ 3 months; n= 11) or responders (R: response ≥ 6 months; n = 12). RESULTS: Genomic characterization revealed that the most common alteration, TP53 mutations, had comparable frequency in R (6/12; 50%) and NR (4/11; 36%). Non-synonymous mutations in KTM2C (4/12; 33.3%), PIK3CA (3/12; 25%) and ARID2 (3/12; 25%) were predominantly associated with response. No significant difference was observed in tumour mutational burden (TMB) between R and NR patients. The NR tumours showed increased expression of diverse immune-related genes and pathways, including various interferon gamma-related genes. We also identified increased MAGEA4 expression as a potential biomarker of non-responding tumours to vinflunine treatment. CONCLUSIONS: Our data may help to identify potential genomic biomarkers of response to vinflunine. Moreover, tumours refractory to vinflunine showed immune signatures potentially associated with response to ICB. Extensive validation studies, including longitudinal series, are needed to corroborate these findings.

Health conditions of Hitnü indigenous people potentially exposed to crude oil in Arauca, Colombia / Condiciones de salud de los indígenas hitnü potencialmente expuestos a petróleo crudo (Arauca, Colombia)

Introduction: The Hitnü indigenous people live in precarious sanitary conditions, with food insecurity and being victims of sociopolitical violence in Arauca, Colombia. In addition, it is possible that they may be affected by exposure to hydrocarbons found in oil. Objective: To identify the health outcomes of morbidity and mortality profiles of the Hitnü people that could be associated with the exposure to crude oil. Materials and methods: A cross-sectional study was carried out with Hitnü indigenous people, during February and March, 2021, time of drought. A household questionnaire was applied, and one individual to collect data from the environment around the house, occupations and other activities, as well as data from sociodemographic, signs, symptoms, and findings of a medical examination. The potential association with hydrocarbons was explored considering three groups: inhabitants in Arauca city, Aspejaná reserve (not exposed), and San José del Lipa and La Vorágine reserves (exposed by the Ele river and tributaries). With free listings, causes of death were explored. The study incorporated a rigorous intercultural management in all its components. Results: A total of 576 indigenous people from 16 settlements participated. The water consumed could serve as means of exposure to hydrocarbons. Health problems were very varied, including infectious and chronic diseases, malnutrition, and trauma. The masses on the neck were associated with residing in the ancestral reserves (PR = 3.86; CI95% 1.77-8.39), territories with potential exposure to crude-oil. The most relevant causes of death were homicide, tumors, and tuberculosis. Conclusion: For its possible association with exposure to hydrocarbons, it is a priority to start the intercultural study of lymphadenopathies in indigenous communities potentially exposed to crude oil.

Introducción. El pueblo hitnü vive en condiciones sanitarias precarias, con inseguridad alimentaria y víctima de la violencia sociopolítica en Arauca (Colombia). Además, se sospecha que pueden estar afectados por la exposición a los hidrocarburos del petróleo. Objetivo. Identificar los eventos de salud del perfil de morbilidad y mortalidad de los indígenas hitnü que podrían asociarse con la exposición a petróleo crudo. Materiales y métodos. Se realizó un estudio transversal con indígenas hitnü, durante febrero y marzo de 2021, época de sequía. Se aplicó un cuestionario de hogares y uno individual para recolectar datos del ambiente peridomiciliario, ocupaciones y otras actividades, así como datos sociodemográficos, signos, síntomas y hallazgos de un examen médico. La potencial asociación con los hidrocarburos se exploró considerando tres grupos, según su localización: cabecera de Arauca, resguardo Aspejená (no expuestos) y resguardos de San José del Lipa y La Vorágine (expuestos por su cercanía al río Ele y afluentes). Con listados libres, se exploraron las causas de muerte. El estudio incorporó un riguroso manejo intercultural en todos sus componentes. Resultados. Participaron 576 indígenas de 16 asentamientos. El agua consumida pudo servir como medio de exposición a los hidrocarburos. Los problemas de salud fueron muy variados e incluían enfermedades infecciosas y crónicas, malnutrición y trauma. Las masas en el cuello se asociaron con residir en los resguardos ancestrales (RP=3,86; IC95% 1,77-8,39), territorios potencialmente expuestos al petróleo. Las causas de muerte más relevantes fueron el homicidio, los tumores y la tuberculosis. Conclusión: Por su posible asociación con los hidrocarburos, es prioritario el estudio intercultural de linfoadenopatías entre indígenas potencialmente expuestos al petróleo.

Toward Tumor Fight and Tumor Microenvironment Remodeling: PBA Induces Cell Cycle Arrest and Reduces Tumor Hybrid Cells' Pluripotency in Bladder Cancer.

Bladder cancer (BC) is the second most frequent cancer of the genitourinary system. The most successful therapy since the 1970s has consisted of intravesical instillations of Bacillus Calmette-Guérin (BCG) in which the tumor microenvironment (TME), including macrophages, plays an important role. However, some patients cannot be treated with this therapy due to comorbidities and severe inflammatory side effects. The overexpression of histone deacetylases (HDACs) in BC has been correlated with macrophage polarization together with higher tumor grades and poor prognosis. Herein we demonstrated that phenylbutyrate acid (PBA), a HDAC inhibitor, acts as an antitumoral compound and immunomodulator. In BC cell lines, PBA induced significant cell cycle arrest in G1, reduced stemness markers and increased PD-L1 expression with a corresponding reduction in histone 3 and 4 acetylation patterns. Concerning its role as an immunomodulator, we found that PBA reduced macrophage IL-6 and IL-10 production as well as CD14 downregulation and the upregulation of both PD-L1 and IL-1ß. Along this line, PBA showed a reduction in IL-4-induced M2 polarization in human macrophages. In co-cultures of BC cell lines with human macrophages, a double-positive myeloid-tumoral hybrid population (CD11b+EPCAM+) was detected after 48 h, which indicates BC cell-macrophage fusions known as tumor hybrid cells (THC). These THC were characterized by high PD-L1 and stemness markers (SOX2, NANOG, miR-302) as compared with non-fused (CD11b-EPCAM+) cancer cells. Eventually, PBA reduced stemness markers along with BMP4 and IL-10. Our data indicate that PBA could have beneficial properties for BC management, affecting not only tumor cells but also the TME.

Dried Blood Specimens as an Alternative Specimen for Immune Response Monitoring During HIV Infection: A Proof of Concept and Simple Method in a Pediatric Cohort.

Programs to prevent mother-to-child HIV transmission do not reduce the number of infants exposed during pregnancy and breastfeeding. HIV-exposed but uninfected children (HEU) present higher risk of morbidity and mortality than HIV-unexposed and uninfected children (UU). In this line, the study of immune biomarkers in HIV could improve prediction of disease progression, allowing to diminish comorbidity risk. Dried blood specimens (DBS) are an alternative to serum for collecting and transporting samples in countries with limited infrastructure and especially interesting for groups such as pediatrics, where obtaining a high sample volume is challenging. This study explores the usefulness of DBS for immune profile monitoring in samples from 30 children under clinical follow-up in Kinshasa: 10 HIV-infected (HIV+), 10 HEU, and 10 UU. We have measured the gene expression levels of 12 immune and inflammatory markers (CD14, IL-6, TNFα, HVEM, B7.1, HIF-1α, Siglec-10, IRAK-M, CD163, B7H5, PD-L1, and Galectin-9) in DBS samples by reverse transcription of total RNA and RT-qPCR. Principal component analysis, Kruskal-Wallis test, and Mann-Whitney test were performed in order to study group differences. HIV+ children presented significantly higher levels of seven biomarkers (CD14, IL-6 HVEM, B7.1, Siglec-10, HIF-1α, and CD163) than the UU group. In HEU, we found seven biomarkers significantly elevated (CD14, IL-6, HVEM, B7.1, Siglec-10, HIF-1α, and IRAK-M) vs. UU. Six biomarkers (CD14, IL-6, HVEM, B7.1, Siglec-10, and HIF-1α) showed a significantly higher expression in both HIV+ and HEU vs. UU, with HVEM and CD14 being significantly overexpressed among HIV+ vs. HEU. Our data reveal the utility of DBS for immune response monitoring. Moreover, significant differences in specific biomarker expression across groups strongly suggest the effect of HIV infection and/or HIV exposure on these immune biomarkers' expressions.

Potential Role of the Galectin-9/TIM-3 Axis in the Disparate Progression of SARS-CoV-2 in a Married Couple: A Case Report.

We report the disparate clinical progression of a couple infected by SARS-CoV-2 based on their immune checkpoint (IC) levels and immune cell distribution in blood from admission to exitus in patient 1 and from admission to discharge and recovery in patient 2. A detailed clinical follow-up accompanied by a longitudinal analysis of immune phenotypes and IC levels is shown. The continuous increase in the soluble IC ligand galectin-9 (Gal-9) and the increment in T-cell immunoglobulin and mucin domain-containing 3 (TIM-3) protein in T cells in patient 1 suggests an activation of the Gal-9/TIM-3 axis and, subsequently, a potential cell exhaustion in this patient that did not occur in patient 2. Our data indicate that the Gal-9/TIM-3 axis could be a potential target in this clinical setting, along with a patent effector memory T-cell reduction.

Tumor stem cells fuse with monocytes to form highly invasive tumor-hybrid cells.

The 'cancer cell fusion' theory is controversial due to the lack of methods available to identify hybrid cells and to follow the phenomenon in patients. However, it seems to be one of the best explanations for both the origin and metastasis of primary tumors. Herein, we co-cultured lung cancer stem cells with human monocytes and analyzed the dynamics and properties of tumor-hybrid cells (THC), as well as the molecular mechanisms beneath this fusion process by several techniques: electron-microscopy, karyotyping, CRISPR-Cas9, RNA-seq, immunostaining, signaling blockage, among others. Moreover, mice models were assessed for in vivo characterization of hybrids colonization and invasiveness. Then, the presence of THCs in bloodstream and samples from primary and metastatic lesions were detected by FACS and immunofluorescence protocols, and their correlations with TNM stages established. Our data indicate that the generation of THCs depends on the expression of CD36 on tumor stem cells and the oxidative state and polarization of monocytes, the latter being strongly influenced by microenvironmental fluctuations. Highly oxidized M2-like monocytes show the strongest affinity to fuse with tumor stem cells. THCs are able to proliferate, colonize and invade organs. THC-specific cell surface signature CD36+CD14+PANK+ allows identifying them in matched primary tumor tissues and metastases as well as in bloodstream from patients with lung cancer, thus functioning as a biomarker. THCs levels in circulation correlate with TNM classification. Our results suggest that THCs are involved in both origin and spread of metastatic cells. Furthermore, they might set the bases for future therapies to avoid or eradicate lung cancer metastasis.

Molecular characterization of Complement Factor I deficiency in two Spanish families.

Complement Factor I (CFI) is a regulator of the classical and alternative pathways. CFI has enzymatic activity and is able to cleave C3b and C4b. Homozygous Factor I deficiency is associated with infectious and/or autoimmune diseases. Here we describe the biochemical and genetic characterization in two Spanish families with complete Factor I deficiency. In Family 1, the propositus suffered from several episodes of meningitis for more than a year. Biochemical complement studies showed undetectable Factor I levels in the propositus and in her sister, while their parents and a brother had partial Factor I deficiency and were healthy. In Family 2, three out of five children were homozygous for Factor I deficiency, two of whom suffered from meningitis and the third one from several infections. The parents and the other two siblings were healthy and heterozygous for Factor I deficiency. Molecular studies showed that the two families had different mutations at exon 5 of the Factor I gene, which codifies for module LDLr1. One mutation corresponds to a 772G>A change at the donor splice site that was originally found in a family from Northern England. The second is a new missense mutation 739T>G, that generates a Cys to Gly change.

Epigenetics of Bladder Cancer: Where Biomarkers and Therapeutic Targets Meet.

Bladder cancer (BC) is the most common neoplasia of the urothelial tract. Due to its high incidence, prevalence, recurrence and mortality, it remains an unsolved clinical and social problem. The treatment of BC is challenging and, although immunotherapies have revealed potential benefit in a percentage of patients, it remains mostly an incurable disease at its advanced state. Epigenetic alterations, including aberrant DNA methylation, altered chromatin remodeling and deregulated expression of non-coding RNAs are common events in BC and can be driver events in BC pathogenesis. Accordingly, these epigenetic alterations are now being used as potential biomarkers for these disorders and are being envisioned as potential therapeutic targets for the future management of BC. In this review, we summarize the recent findings in these emerging and exciting new aspects paving the way for future clinical treatment of this disease.

CDK4/6 Inhibitor as a Novel Therapeutic Approach for Advanced Bladder Cancer Independently of RB1 Status.

PURPOSE: Bladder cancer is a clinical and social problem due to its high incidence and recurrence rates. It frequently appears in elderly patients showing other medical comorbidities that hamper the use of standard chemotherapy. We evaluated the activity of CDK4/6 inhibitor as a new therapy for patients unfit for cisplatin (CDDP). EXPERIMENTAL DESIGN: Bladder cancer cell lines were tested for in vitro sensitivity to CDK4/6 inhibition. A novel metastatic bladder cancer mouse model was developed and used to test its in vivo activity. RESULTS: Cell lines tested were sensitive to CDK4/6 inhibition, independent on RB1 gene status. Transcriptome analyses and knockdown experiments revealed a major role for FOXM1 in this response. CDK4/6 inhibition resulted in reduced FOXM1 phosphorylation in vitro and in vivo and showed synergy with CDDP, allowing a significant tumor regression. FOXM1 exerted important oncogenic roles in bladder cancer. CONCLUSIONS: CDK4/6 inhibitors, alone or in combination, are a novel therapeutic strategy for patients with advanced bladder cancer previously classified as unfit for current treatment options.

Inhibition of a G9a/DNMT network triggers immune-mediated bladder cancer regression.

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5-8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/-) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.