Functional characterization of two enhancers located downstream FOXP2

Background: Mutations in the coding region of FOXP2 are known to cause speech and language impairment. However, it is not clear how dysregulation of the gene contributes to language deficit. Interestingly, microdeletions of the region downstream the gene have been associated with cognitive deficits. Methods: Here, we investigate changes in FOXP2 expression in the SK-N-MC neuroblastoma human cell line after deletion by CRISPR-Cas9 of two enhancers located downstream of the gene. Results: Deletion of any of these two functional enhancers downregulates FOXP2, but also upregulates the closest 3′ gene MDFIC. Because this effect is not statistically significant in a HEK 293 cell line, derived from the human kidney, both enhancers might confer a tissue specific regulation to both genes. We have also found that the deletion of any of these enhancers downregulates six well-known FOXP2 target genes in the SK-N-MC cell line. Conclusions: We expect these findings contribute to a deeper understanding of how FOXP2 and MDFIC are regulated to pace neuronal development supporting cognition, speech and language.Spanish National Research and Development Plan PI14/01884Instituto de Salud Carlos III PI14/01884FEDER PI14/0188

Diseño y predicción de promotores específicos de neuroblastoma

El neuroblastoma es una enfermedad infantil caracterizada por el desarrollo de tumores a partir de células cancerígenas del tejido nervioso de las glándulas suprarrenales. Existen marcadores genéticos para caracterizar, clasificar y diagnosticar este tipo de tumores. Un ejemplo de estos marcadores es la amplificación de MYCN, frecuentemente utilizada como herramienta de pronóstico. A pesar de los tratamientos disponibles actualmente, el neuroblastoma es un tipo de cáncer que sigue estando caracterizado por una alta mortalidad. Por este motivo, es necesario el desarrollo de nuevas terapias que dirijan su acción específicamente a este tipo de células tumorales, maximizando su actividad en el tejido diana y reduciendo su toxicidad en otros tejidos. Un ejemplo de este tipo de terapia podría ser la edición génica. Para lograr una expresión específica en un tejido determinado, la expresión de genes terapéuticos debe estar controlada por promotores específicos de ese tejido. Para ello se deben analizar las secuencias promotoras que regulan la transcripción de genes cuya expresión sea restringida a neuroblastoma. Sin embargo, estas secuencias promotoras abarcan grandes regiones de ADN, y los vectores utilizados en terapia génica solamente pueden incorporar secuencias de un tamaño limitado. Por esta razón, se deben seleccionar los fragmentos esenciales que garantizan el funcionamiento del promotor. En este trabajo, se realiza un análisis y predicción de las secuencias fundamentales de cuatro promotores de proteínas expresadas diferencialmente en neuroblastoma para, posteriormente, evaluar su actividad mediante la medida de la expresión de una proteína reporter

Narrowing the Genetic Causes of Language Dysfunction in the 1q21.1 Microduplication Syndrome

The chromosome 1q21.1 duplication syndrome (OMIM# 612475) is characterized by head anomalies, mild facial dysmorphisms, and cognitive problems, including autistic features, mental retardation, developmental delay, and learning disabilities. Speech and language development are sometimes impaired, but no detailed characterization of language problems in this condition has been provided to date. We report in detail on the cognitive and language phenotype of a child who presents with a duplication in 1q21.1 (arr[hg19] 1q21.1q21.2(145,764,455-147,824,207) × 3), and who exhibits cognitive delay and behavioral disturbances. Language is significantly perturbed, being the expressive domain the most impaired area (with significant dysphemic features in absence of pure motor speech deficits), although language comprehension and use (pragmatics) are also affected. Among the genes found duplicated in the child, CDH1L is upregulated in the blood of the proband. ROBO1, a candidate for dyslexia, is also highly upregulated, whereas, TLE3, a target of FOXP2, is significantly downregulated. These changes might explain language, and particularly speech dysfunction in the proband

A human genome editing-based MLL::AF4 B-cell ALL model recapitulates key cellular and molecular leukemogenic features

The cellular ontogeny and location of the MLL-breakpoint influence the capacity of MLL-edited CD34+ HSPCs to initiate pro-B-ALL, and recapitulate the molecular features of MLL-AF4+ infant B-ALL patients. We provide key insights into the cellular-molecular leukemogenic determinants of MLL-AF4+ infant B-ALL

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.Acknowledgments: This work was supported by MINECO (SAF2017-89643-R, SAF2014-57243-R, SAF2015-62547-ERC) (M.V.), Fundacion FERO (IX FERO Grant for Research in Oncology) (M.V.), Fundacio La Marato de TV3 (141) (M.V.), Melanoma Research Alliance (Bristol-Myers Squibb-Melanoma Research Alliance Young Investigator Award 2017 (https://doi.org/10.48050/pc.gr.75716)) (M.V.), Beug Foundation (Prize for Metastasis Research 2017) (M.V.), Fundacion Ramon Areces (CIVP19S8163) (M.V.) and CIVP20S10662 (E.O.P.), Worldwide Cancer Research (19-0177) (M.V.), H2020-FETOPEN (828972) (M.V.), Cancer Research Institute (Clinic and Laboratory Integration Program CRI Award 2018 (54545)) (M.V.), AECC (Coordinated Translational Groups 2017 (GCTRA16015SEOA) (M.V.), LAB AECC 2019 (LABAE19002-VALI) (M.V.), ERC CoG (864759) (M.V.), Sophien-Stiftung zur Förderung der klinischen Krebsforschung (T.W.), Promedica Stiftung (T.W.), Stiftung f€ur angewandte Krebsforschung (T.W.), Forschungskredit of the University of Zurich (FK-18-054) (T.W.), Betty and David Koetser Foundation for Brain Research (T.W.), Foundation for Applied Cancer Research in Zurich (T.W., M.W.), Comunidad de Madrid (S2017/BMD-3867 RENIM-CM and Y2018/NMT-4949 NanoLiver-CM) and European structural and investment funds (M.D.), ISCIII (PT20/00044) co-funded by FEDER “A way of making Europe” (M.D.), Ministero dell’Istruzione, dell’Universita e della Ricerca-MIUR, “Dipartimenti di Eccellenza 2018-2022”, (D15D18000410001) (L.B. and P.C.), Science Foundation Ireland Frontiers for the Future Award (19/FFP/6443) (L.Y.), Science Foundation Ireland Strategic Partnership Programme, Precision Oncology Ireland (18/SPP/3522) (L.Y.), Breast Cancer Now Fellowship Award/ with the generous support of Walk the Walk (2019AugSF1310) (D.V.), La Caixa-Severo Ochoa International PhD Program Fellowship (LCF/BQ/SO16/52270014) (L.Z.), La Caixa International PhD Program Fellowship-Marie Sklodowska-Curie (LCF/BQ/DI17/11620028) (P.G-G), MINECO-Severo Ochoa PhD Fellowship (BES-2017-081995) (L.A-E.), AECC Postdoctoral Fellowship (POSTD19016PRIE) (N.P.), Boehringer Ingelheim Fonds MD fellowship (L.M.). The contribution of the Experimental Therapeutics Programme was supported by core funding from the Spanish National Cancer Research Center (CNIO). CNIO is supported by the ISCIII, the Ministerio de Ciencia e Innovacion, and is a Severo Ochoa Center of Excellence (SEV-2015-0510). The CNIC is supported by the ISCIII, the Ministerio de Ciencia e Innovacion and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). M.V. was named Ramon y Cajal Investigator (RYC-2013-13365) and is member of EMBO YIP (4053)

Kras oncogene ablation prevents resistance in advanced lung adenocarcinomas

KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited antitumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS-signaling pathways. Instead, sotorasib-resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.This work was supported by grants from the European Research Council (ERC-GA 695566, THERACAN); the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033) (grant RTC2017-6576-1), cofunded by ERDF “A way of making Europe”; the Autonomous Community of Madrid (B2017/BMD-3884 iLung-CM), cofunded by FSE and ERDF “A way of making Europe”; the CRIS Cancer Foundation, the Scientific Foundation of the Spanish Association Against Cancer (GC166173694BARB); an ERA PerMed grant, funded by the Instituto de Salud Carlos III (AC20/00114), the Scientific Foundation of the Spanish Association Against Cancer (PERME20707BARB) and the European Union’s Horizon 2020 program (779282) to MB; and the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (grant RTI2018-094664-B-I00), cofunded by ERDF “A way of making Europe” to MM and MB. Additional funding included grants from the Spanish National Research and Development Plan, Instituto de Salud Carlos III, ERDF “A way of making Europe” (PI20/01837 and DTS19/00111); the Scientific Foundation of the Spanish Association Against Cancer (LABAE20049RODR) to SRP; the Instituto de Salud Carlos III (PI19/00514), cofunded by ERDF “A way of making Europe” to CG; the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (grant PID2020-116705RB-I00); and the Scientific Foundation of the Spanish Association Against Cancer (LABAE211678DROS) to MD. MB is a recipient of an endowed chair from the AXA Research Fund. M Salmón was supported by a predoctoral contract “Severo Ochoa” (BES-2016-079096) from the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación. OB is a recipient of a fellowship from the Formación de Personal Investigador (FPI) program of the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación. FFG was supported by a Formación de Profesorado Universitario (FPU) fellowship from the Ministerio de Universidades

Activation of the Unfolded Protein Response (UPR) Is Associated with Cholangiocellular Injury, Fibrosis and Carcinogenesis in an Experimental Model of Fibropolycystic Liver Disease

Fibropolycystic liver disease is characterized by hyperproliferation of the biliary epithelium and the formation of multiple dilated cysts, a process associated with unfolded protein response (UPR). In the present study, we aimed to understand the mechanisms of cyst formation and UPR activation in hepatocytic c-Jun N-terminal kinase 1/2 (Jnk1/2) knockout mice. Floxed JNK1/2 (Jnkf/f) and Jnk∆hepa animals were sacrificed at different time points during progression of liver disease. Histological examination of specimens evidenced the presence of collagen fiber deposition, increased α-smooth muscle actin (αSMA), infiltration of CD45, CD11b and F4/80 cells and proinflammatory cytokines (Tnf, Tgfβ1) and liver injury (e.g., ALT, apoptosis and Ki67-positive cells) in Jnk∆hepa compared with Jnkf/f livers from 32 weeks of age. This was associated with activation of effectors of the UPR, including BiP/GRP78, CHOP and spliced XBP1. Tunicamycin (TM) challenge strongly induced ER stress and fibrosis in Jnk∆hepa animals compared with Jnkf/f littermates. Finally, thioacetamide (TAA) administration to Jnk∆hepa mice induced UPR activation, peribiliary fibrosis, liver injury and markers of biliary proliferation and cholangiocarcinoma (CCA). Orthoallografts of DEN/CCl4-treated Jnk∆hepa liver tissue triggered malignant CCA. Altogether, these results suggest that activation of the UPR in conjunction with fibrogenesis might trigger hepatic cystogenesis and early stages of CCA

Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis.

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease

Impaired Condensin Complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL

B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, and high-hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD is an initiating oncogenic event affiliated to childhood B-ALL, the mitotic and chromosomal defects associated to HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated to chromosome alignment defects at the metaphase plate leading to robust chromosome segregation defects and non-modal karyotypes. Mechanistically, biochemical, functional and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness and mis-localization of the chromosome passenger complex proteins Aurora B Kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and impaired spindle assembly checkpoint (SAC) thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated to defective condensin complex, AURKB and SAC

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

We report a medium‐throughput drug‐screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood–brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug‐screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere