Estudios fenotípicos y funcionales inducidos en células dendríticas humanas por vacunas de origen bacteriano

Las infecciones recurrentes de las vías respiratorias (RRTIs, recurrent respiratory tract infections) y de las vías urinarias (RUTIs, recurrent urinary tract infections) figuran como las enfermedades infecciosas más comunes a nivel mundial con una alta tasa de mortalidad. Por ello, las RRTIs y RUTIs representan un problema de salud global muy serio con un gran impacto socioeconómico. Hasta la fecha, el tratamiento de referencia para combatir estas infecciones consiste en la administración repetida y prolongada de numerosos ciclos de antibióticos. Estos tratamientos han demostrado eficacia clínica en muchos casos pero su uso indiscriminado conlleva una de serie efectos adversos. Entre ellos, cabe destacar el incremento de resistencias a antibióticos asociado a un consumo abusivo de los mismos o su efecto perjudicial en el microbioma, lo que favorece las infecciones por hongos y bacterias. Por ello, según los últimos informes de la Organización Mundial de la Salud (OMS) y otras instituciones competentes existe y urge la necesidad de desarrollar nuevos tratamientos alternativos para prevenir y tratar las RRTIs y RUTIs. En este sentido, las vacunas bacterianas administradas a través de la mucosa sublingual han demostrado una alta eficacia, ya que son capaces de potenciar el sistema inmune asociado a las mucosas de manera local y en órganos distantes, mejorando así los mecanismos de defensa del organismo frente a patógenos que afectan distintos tipos de tejidos. Estudios recientes indican que la inmunización sublingual con MV130 (Bactek®) o MV140 (Uromune®), preparaciones bacterianas polivalentes (PBPs) compuestas por bacterias enteras inactivadas por calor comercializadas por Inmunotek S.L., reducen drásticamente la tasa de infecciones en los pacientes que sufren RRTIs y RUTIs, respectivamente. MV130 está formada por bacterias enteras inactivadas por calor causantes del mayor número de infecciones respiratorias en Europa (90% Gram positivas: 60% Streptococcus pneumoniae, 15% Staphylococcus aureus y 15% Staphylococcus epidermidis; y 10% Gram negativas: 4% Klebsiella pneumoniae, 3% Moraxella catarrhalis y 3% Haemophilus influenzae). Por otro lado, MV140 está formada por bacterias enteras inactivadas por calor causantes de un gran número de infecciones urinarias (75% Gram negativas: 25% Escherichia coli, 25% Proteus vulgaris, 25% Klebsiella pneumoniae; y 25% Gram positivas: Enterococcus faecalis)..

FGFR1 Cooperates with EGFR in Lung Cancer Oncogenesis, and Their Combined Inhibition Shows Improved Efficacy

Introduction: There is substantial evidence for the onco- genic effects of fi broblast growth factor receptor 1 (FGFR1) in many types of cancer, including lung cancer, but the role of this receptor has not been addressed speci fi cally in lung adenocarcinoma. Methods: We performed FGFR1 and EGFR overexpression and co-overexpression assays in adenocarcinoma and in inmortalized lung cell lines, and we also carried out surrogateandinteractionassays.Weperformedmono- therapy and combination EGFR /FGFR inhibitor sensitivity assays in vitro and in vivo in cell line – and patient- derived xenografts. We determined FGFR1 mRNA expression in a cohort of patients with anti – EGFR ther- apy – treated adenocarcinoma. Results: We have reported a cooperative interaction between FGFR1 and EGFR in this context, resulting in increased EGFR activation and oncogenic signaling. We have provided in vitro and in vivo evidence indicating that FGFR1 expression in- creases tumorigenicity in cells with high EGFR activation in EGFR-mutated and EGFR wild-type models. At the clinical level, we have shown that high FGFR1 expression levels pre- dict higher resistance to erlotinib or ge fi tinib in a cohort of patients with tyrosine kinase inhibitor – treated EGFR-mutated and EGFR wild-type lung adenocarcinoma. Dual EGFR and FGFR inhibition in FGFR1-over expressing, EGFR-activated models shows synergistic effects on tumor growth in vitro and in cell line – and patient-derived xenografts, suggesting that patients with tumors bearing these characteristics may bene fi t from combined EGFR/FGFR inhibition. Conclusion: These results support the extended the use of EGFR inhibitors beyond monotherapy in the EGFR-mutated adenocarcinoma setting in combination with FGFR in- hibitors for selected patients with increased FGFR1 over- expression and EGFR activation.ISCIII PI14/01964 PIE15/00076 PI17/00778 DTS17/00089 PI15/00045 PI17/00033 PI16/01311 FI12/00429CIBERONC CD16/12/00442FEDER CD16/12/00442 PI16/01311Spanish Ministry of Economy and Competitiveness PI15/00045Ministry of Health and Social Welfare of Junta de Andalucía PI-0046-2012 C-0040-2016Ministry of Equality, Health and Social Policies of the Junta de Andalucía PI- 0029-2013Comunidad de Madrid B2017/BMD3884Ministry of Education, Culture and Sports FPU13/0259

Identification of novel synthetic lethal vulnerability in non small cell lung cancer by co targeting TMPRSS4 and DDR1

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (~40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4

MV140, a sublingual polyvalent bacterial preparation to treat recurrent urinary tract infections, licenses human dendritic cells for generating Th1, Th17, and IL-10 responses via Syk and MyD88

Recurrent urinary tract infections (RUTIs) are one of the most common bacterial infectious diseases, especially in women. Antibiotics remain the mainstay of treatment, but their overuse is associated with antibiotic-resistant infections and deleterious effects in the microbiota. Therefore, alternative approaches are fully demanded. Sublingual immunization with MV140 (Uromune), a polyvalent bacterial preparation (PBP) of whole heat-inactivated bacteria, demonstrated clinical efficacy for the treatment of RUTIs, but the involved immunological mechanisms remain unknown. Herein, we demonstrated that MV140 endorses human dendritic cells (DCs) with the capacity to generate Th1/Th17 and IL-10-producing T cells by mechanisms depending on spleen tyrosine kinase (Syk)- and myeloid differentiation primary response gene 88 (MyD88)-mediated pathways. MV140-induced activation of nuclear factor κB (NF-κB) and p38 in human DCs is essential for the generated Th1/Th17 and IL-10 immune responses whereas c-Jun N-terminal Kinase (JNK) and extracellular-signal regulated kinase (ERK) contribute to Th1 and IL-10 responses, respectively. Sublingual immunization of BALB/c mice with MV140 also induces potent systemic Th1/Th17 and IL-10 responses in vivo. We uncover immunological mechanisms underlying the way of action of MV140, which might well also contribute to understand the rational use of specific PBPs in other clinical conditions with potential high risk of recurrent infections.MINECODepto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia

BACKGROUND: Protein phosphatase 2A is a novel potential therapeutic target in several types of chronic and acute leukemia, and its inhibition is a common event in acute myeloid leukemia. Upregulation of SET is essential to inhibit protein phosphatase 2A in chronic myeloid leukemia, but its importance in acute myeloid leukemia has not yet been explored. DESIGN AND METHODS: We quantified SET expression by real time reverse transcriptase polymerase chain reaction in 214 acute myeloid leukemia patients at diagnosis. Western blot was performed in acute myeloid leukemia cell lines and in 16 patients' samples. We studied the effect of SET using cell viability assays. Bioinformatics analysis of the SET promoter, chromatin immunoprecipitation, and luciferase assays were performed to evaluate the transcriptional regulation of SET. RESULTS: SET overexpression was found in 60/214 patients, for a prevalence of 28%. Patients with SET overexpression had worse overall survival (P<0.01) and event-free survival (P<0.01). Deregulation of SET was confirmed by western blot in both cell lines and patients' samples. Functional analysis showed that SET promotes proliferation, and restores cell viability after protein phosphatase 2A overexpression. We identified EVI1 overexpression as a mechanism involved in SET deregulation in acute myeloid leukemia cells. CONCLUSIONS: These findings suggest that SET overexpression is a key mechanism in the inhibition of PP2A in acute myeloid leukemia, and that EVI1 overexpression contributes to the deregulation of SET. Furthermore, SET overexpression is associated with a poor outcome in acute myeloid leukemia, and it can be used to identify a subgroup of patients who could benefit from future treatments based on PP2A activators

Integration of SNP and mRNA arrays with microRNA profiling reveals that MiR-370 is upregulated and targets NF1 in acute myeloid leukemia.

BACKGROUND: Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied. RESULTS: Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1. CONCLUSIONS: NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors

Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia

BACKGROUND: Protein phosphatase 2A is a novel potential therapeutic target in several types of chronic and acute leukemia, and its inhibition is a common event in acute myeloid leukemia. Upregulation of SET is essential to inhibit protein phosphatase 2A in chronic myeloid leukemia, but its importance in acute myeloid leukemia has not yet been explored. DESIGN AND METHODS: We quantified SET expression by real time reverse transcriptase polymerase chain reaction in 214 acute myeloid leukemia patients at diagnosis. Western blot was performed in acute myeloid leukemia cell lines and in 16 patients' samples. We studied the effect of SET using cell viability assays. Bioinformatics analysis of the SET promoter, chromatin immunoprecipitation, and luciferase assays were performed to evaluate the transcriptional regulation of SET. RESULTS: SET overexpression was found in 60/214 patients, for a prevalence of 28%. Patients with SET overexpression had worse overall survival (P<0.01) and event-free survival (P<0.01). Deregulation of SET was confirmed by western blot in both cell lines and patients' samples. Functional analysis showed that SET promotes proliferation, and restores cell viability after protein phosphatase 2A overexpression. We identified EVI1 overexpression as a mechanism involved in SET deregulation in acute myeloid leukemia cells. CONCLUSIONS: These findings suggest that SET overexpression is a key mechanism in the inhibition of PP2A in acute myeloid leukemia, and that EVI1 overexpression contributes to the deregulation of SET. Furthermore, SET overexpression is associated with a poor outcome in acute myeloid leukemia, and it can be used to identify a subgroup of patients who could benefit from future treatments based on PP2A activators

Integration of SNP and mRNA arrays with microRNA profiling reveals that MiR-370 is upregulated and targets NF1 in acute myeloid leukemia

Abstract Background: Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied. Results: Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1. Conclusions: NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR- 370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors