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.

The Inactivation of Enzymes Belonging to the Central Carbon Metabolism Is a Novel Mechanism of Developing Antibiotic Resistance.

Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate, that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from phosphoenolpyruvate to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly being used, mainly for treating infections caused by Gram-negative multidrug-resistant bacteria. The mechanisms of mutational resistance to fosfomycin in Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to commonly used antibiotics, were studied in the current work. None of the mechanisms reported so far for other organisms, which include the production of fosfomycin-inactivating enzymes, target modification, induction of an alternative peptidoglycan biosynthesis pathway, and the impaired entry of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Instead, the unique cause of resistance in the mutants studied is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants, showing that neither inactivation nor transport of the antibiotic is involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes or any related enzyme. Finally, the mutants did not present an increased phosphoenolpyruvate concentration that might compete with fosfomycin for its binding to MurA. On the basis of these results, we describe a completely novel mechanism of antibiotic resistance based on mutations of genes encoding metabolic enzymes.IMPORTANCE Antibiotic resistance has been largely considered a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated on mechanisms that affect the antibiotics (mutations in transporters, efflux pumps, and antibiotic-modifying enzymes, or their regulators) or their targets (i.e., target mutations, protection, or bypass). Usually, antibiotic resistance-associated metabolic changes were considered a consequence (fitness costs) and not a cause of antibiotic resistance. Herein, we show that alterations in the central carbon bacterial metabolism can also be the cause of antibiotic resistance. In the study presented here, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas pathway. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that antibiotic resistance can be under metabolic control, our results provide evidence that antibiotic resistance is intertwined with the bacterial metabolism.

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.

Differential development of large-cell neuroendocrine or small-cell lung carcinoma upon inactivation of 4 tumor suppressor genes.

High-grade neuroendocrine lung malignancies (large-cell neuroendocrine cell carcinoma, LCNEC, and small-cell lung carcinoma, SCLC) are among the most deadly lung cancer conditions with no optimal clinical management. The biological relationships between SCLC and LCNEC are still largely unknown and a current matter of debate as growing molecular data reveal high heterogeneity with potential therapeutic consequences. Here we describe murine models of high-grade neuroendocrine lung carcinomas generated by the loss of 4 tumor suppressors. In an Rbl1-null background, deletion of Rb1, Pten, and Trp53 floxed alleles after Ad-CMVcre infection in a wide variety of lung epithelial cells produces LCNEC. Meanwhile, inactivation of these genes using Ad-K5cre in basal cells leads to the development of SCLC, thus differentially influencing the lung cancer type developed. So far, a defined model of LCNEC has not been reported. Molecular and transcriptomic analyses of both models revealed strong similarities to their human counterparts. In addition, a 68Ga-DOTATOC-based molecular-imaging method provides a tool for detection and monitoring the progression of the cancer. These data offer insight into the biology of SCLC and LCNEC, providing a useful framework for development of compounds and preclinical investigations in accurate immunocompetent models.

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.

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.

The intrinsic resistome of Klebsiella pneumoniae.

Molecular epidemiology studies aiming at understanding the acquisition of antimicrobial resistance by clinical isolates of Klebsiella pneumoniae are regularly published; however, information on the genes that contribute to its characteristic phenotype of resistance to antibiotics (intrinsic resistome) is scarce. To fill this gap, a K. pneumoniae transposon mutant library was screened and 171 mutants presenting changes in their susceptibility to antibiotics were selected, in which the transposon insertion site was determined in 75. Twenty-seven mutants for which insertion points had been previously identified were included in the analysis. A total of 102 mutants were selected for further studies. In 70 mutants the transposon was inserted in a gene with a known function, whilst in 19 the insertion occurred in genes encoding proteins with unknown functions and 13 insertions occurred in intergenic regions. Moreover, 87 of the insertions were localised in the chromosome, with 15 insertions located in the two plasmids carried by this strain. Whereas some of the mutated genes are already known to be involved in antimicrobial resistance (ampG, acrB, tolC), several of them are involved in regular processes of bacterial physiology, including K. pneumoniae virulence. Together with results published for other organisms, these results support that determinants involved in basic processes of bacterial physiology may contribute to antimicrobial resistance. These findings also indicate that, besides acquired resistance genes, plasmids may harbour other genes belonging to their backbone that can also be involved in resistance.

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.

Genome-wide analysis shows that RNase G plays a global role in the stability of mRNAs in Stenotrophomonas maltophilia.

Gene expression is determined by critical processes such as RNA synthesis and degradation. Ribonucleases participate in the coordinated and differential decay of messenger RNAs. We describe a suitable method of normalization and calculation of mRNAs half-life values quantified by RNA-Seq. We determined the mRNA half-lives of more than 2000 genes in Stenotrophomonas maltophilia D457 and in an isogenic RNase G deficient mutant. Median half-lives were 2,74 and 3 min in the wild-type and the rng-deficient strain, respectively. The absence of RNase G resulted in an overall enhancement of mRNA half-life times, showing that many RNAs are targets of RNase G in S. maltophilia. Around 40 genes are likely to be regulated directly by RNase G since their half-lives were more than two-fold higher in the rng-deficient mutant. Gene length, GC content or expression levels did not correlate with mRNAs lifetimes, although groups of genes with different functions showed different RNA half-lives. Further, we predicted 1542 gene pairs to be part of the same operons in S. maltophilia. In contrast to what was described for other bacteria, our data indicate that RNase G has a global role in mRNA stability and consequently in the regulation of S. maltophilia gene expression.

Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

The analysis of the antibiotic resistome offers new opportunities for therapeutic intervention.

Most efforts in the development of antimicrobials have focused on the screening of lethal targets. Nevertheless, the constant expansion of antimicrobial resistance makes the antibiotic resistance determinants themselves suitable targets for finding inhibitors to be used in combination with antibiotics. Among them, inhibitors of antibiotic inactivating enzymes and of multidrug efflux pumps are suitable candidates for improving the efficacy of antibiotics. In addition, the application of systems biology tools is helping to understand the changes in bacterial physiology associated to the acquisition of resistance, including the increased susceptibility to other antibiotics displayed by some antibiotic-resistant mutants. This information is useful for implementing novel strategies based in metabolic interventions or combination of antibiotics for improving the efficacy of antibacterial therapy.

Use of phenotype microarrays to study the effect of acquisition of resistance to antimicrobials in bacterial physiology.

It is widely accepted that the acquisition of resistance to antimicrobials confers a fitness cost. Different works have shown that the effect of acquiring resistance in bacterial physiology may be more specific than previously thought. Study of these specific changes may help to predict the outcome of resistant organisms in different ecosystems. In addition to changing bacterial physiology, acquisition of resistance either increases or reduces susceptibility to other antimicrobials. In the current article, we review recent information on the effect of acquiring resistance upon bacterial physiology, with a specific focus on studies using phenotype microarray technology.

The inactivation of RNase G reduces the Stenotrophomonas maltophilia susceptibility to quinolones by triggering the heat shock response.

Quinolone resistance is usually due to mutations in the genes encoding bacterial topoisomerases. However, different reports have shown that neither clinical quinolone resistant isolates nor in vitro obtained Stenotrophomonas maltophilia mutants present mutations in such genes. The mechanisms so far described consist on efflux pumps' overexpression. Our objective is to get information on novel mechanisms of S. maltophilia quinolone resistance. For this purpose, a transposon-insertion mutant library was obtained in S. maltophilia D457. One mutant presenting reduced susceptibility to nalidixic acid was selected. Inverse PCR showed that the inactivated gene encodes RNase G. Complementation of the mutant with wild-type RNase G allele restored the susceptibility to quinolones. Transcriptomic and real-time RT-PCR analyses showed that several genes encoding heat-shock response proteins were expressed at higher levels in the RNase defective mutant than in the wild-type strain. In agreement with this situation, heat-shock reduces the S. maltophilia susceptibility to quinolone. We can then conclude that the inactivation of the RNase G reduces the susceptibility of S. maltophilia to quinolones, most likely by regulating the expression of heat-shock response genes. Heat-shock induces a transient phenotype of quinolone resistance in S. maltophilia.

WIP regulates persistence of cell migration and ruffle formation in both mesenchymal and amoeboid modes of motility.

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.

The intrinsic resistome of bacterial pathogens.

Intrinsically resistant bacteria have emerged as a relevant health problem in the last years. Those bacterial species, several of them with an environmental origin, present naturally low-level susceptibility to several drugs. It has been proposed that intrinsic resistance is mainly the consequence of the impermeability of cellular envelopes, the activity of multidrug efflux pumps or the lack of appropriate targets for a given family of drugs. However, recently published articles indicate that the characteristic phenotype of susceptibility to antibiotics of a given bacterial species depends on the concerted activity of several elements, what has been named as intrinsic resistome. These determinants comprise not just classical resistance genes. Other elements, several of them involved in basic bacterial metabolic processes, are of relevance for the intrinsic resistance of bacterial pathogens. In the present review we analyze recent publications on the intrinsic resistomes of Escherichia coli and Pseudomonas aeruginosa. We present as well information on the role that global regulators of bacterial metabolism, as Crc from P. aeruginosa, may have on modulating bacterial susceptibility to antibiotics. Finally, we discuss the possibility of searching inhibitors of the intrinsic resistome in the aim of improving the activity of drugs currently in use for clinical practice.

[Neonatal cholestasis and hypoglycemia like form of congenital hypopituitarism presentation]. / Colestasis neonatal e hipoglucemia como forma de presentación de hipopituitarismo congénito.

Congenital hypopituitarism is a rare disease, of variable clinic. The neonatal hypoglycemia is one of the habitual forms of presentation; the cholestasis is a rare symptom of this disease. This is the case of a 2-months-old infant hospitalized for cholestatic jaundice. He added repeated episodes of severe hypoglycemia. We investigated metabolic and endocrine causes. The etiology was clarified by obtaining a critical sample that demonstrated the counterregulatory hormone deficiency. The diagnosis of congenital hypopituitarism was completed with confirmation of thyroid hormone and growth hormone deficiencies. It was confirmed the neuro-anatomical defect of "syndrome of pituitary stalk section" determined by pituitary stalk agenesis, pituitary hipoplasia, and ectopic neurohypophysis. Hormone replacement therapy was started with good response and outcome.

Colestasis neonatal e hipoglucemia como forma de presentación de hipopituitarismo congénito / Neonatal cholestasis and hypoglycemia like form of congenital hypopituitarism presentation

El hipopituitarismo congénito es una enfermedad poco frecuente, de clínica variable. La hipoglucemia neonatal es una de las formas habituales de presentación; la colestasis es un síntoma raro de esta enfermedad. Se presenta el caso de un lactante de 2 meses hospitalizado por ictericia colestática. Agregó episodios reiterados de hipoglucemia grave. Se investigaron causas metabólicas y endocrinológicas. Se arribó a la etiología a través de la obtención de una muestra crítica que demostró el déficit de hormonas contrarreguladoras. Se completó el diagnóstico de hipopituitarismo congénito con la demostración del déficit de hormona tiroidea y hormona de crecimiento. Se confirmó el defecto neuroanatómico de "síndrome de sección del tallo hipofisario", determinado por agenesia del tallo pituitario, hipoplasia hipofisaria y neurohipófisis ectópica. Se inició tratamiento hormonal restitutivo, con una buena respuesta y una buena evolución posterior.

Congenital hypopituitarism is a rare disease, of variable clinic. The neonatal hypoglycemia is one of the habitual forms of presentation; the cholestasis is a rare symptom of this disease. This is the case of a 2-months-old infant hospitalized for cholestatic jaundice. He added repeated episodes of severe hypoglycemia. We investigated metabolic and endocrine causes. The etiology was clarified by obtaining a critical sample that demonstrated the counterregulatory hormone deficiency. The diagnosis of congenital hypopituitarism was completed withconfirmation of thyroid hormone and growth hormone deficiencies. It was confirmed the neuro-anatomical defect of "syndrome of pituitary stalk section" determined by pituitary stalkagenesis, pituitary hipoplasia, and ectopic neurohypophysis. Hormone replacement therapy was started with good response and outcome.