Inactivation of Capicua in adult mice causes T-cell lymphoblastic lymphoma

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4 Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy.We are grateful to Carol MacKintosh (University of Dundee, UK) for the pcDNA5/FRT/TO-GFP-CIC plasmid, and Huda Zoghbi (Baylor College of Medicine, Houston, TX) and Yoontae Lee (University of Pohang, Korea) for Cic antisera. We thank Scott Brown and Robert Holt (University of Vancouver, Canada) for their help with TCR abundance calculations. We also thank Carmen G. Lechuga, Marta San Roman, Raquel Villar, Beatriz Jimenez, and Nuria Cabrera for excellent technical assistance. We value the support of Sagrario Ortega (Transgenic Mice Core Unit, CNIO) for help in generating the Cic mutant mice, Orlando Dominguez (Genomics Core Unit, CNIO) for the RNA-seq analysis, and the Histopathology Core Unit. This work was supported by grants from the Fundacio La Marato de TV3 (20131730/1) to G.J. and M.B., and the European Research Council (ERC-AG/250297-RAS AHEAD), the EU-Framework Programme (HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010-260791/EUROCANPLATFORM), the Spanish Ministry of Economy and Competitiveness (SAF2014-59864-R), the Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE), and the Asociacion Espanola contra el Cancer (AECC) (GC16173694BARB) to M.B. M.B. is the recipient of an Endowed Chair from the AXA Research Fund. L.S.-C. was supported by a fellowship from the Programa de Formacion de Personal Investigator (FPI) of the Spanish Ministry of Economy and Competitiveness. M.D. and M.B. conceived and designed the study. L.S.-C., O.G., G.J., M.D., and M.B. developed the methodology. L.S.-C., O.G., M.S., and M.D. acquired the data. L.S.-C., O.G., M.S., H.K.C.J., G.J., M.D., and M.B. analyzed and interpreted the data. L.S-C., O.G., G.J., M.D., and M.B. wrote, reviewed, and/or revised the manuscript. G.J. provided material support. A. G. analyzed the T-ALL sequencing. M.D. and M.B. supervised the study.S

KRAS4A induces metastatic lung adenocarcinomas in vivo in the absence of the KRAS4B isoform.

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a Kras FSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras +/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras +/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences.We thank Marta San Roman, Raquel Villar, and Nuria Cabrera for excellent technical assistance; Mayte Lamparero and Isabel Blanco (Animal Facility) for mouse work; the Histopathology Unit for processing of mouse tissues; Lola Martinez (Flow Cytometry Unit) for her help with flow cytometry analyses; Diego Megias and Manuel Perez (Confocal Microscopy Unit) for assistance with confocal microscopy; and the Mouse Genome Editing Unit for support with the generation of the mouse strains described here. We also thank Ignacio Perez de Castro (Instituto de Salud Carlos III, Madrid, Spain) for sharing the EGFP-KRAS4B plasmid and Orlando Dominguez (Genomics Unit) and Pedro P. Lopez-Casas (Clinical Research Program) for their advice on exome sequencing. This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6576-1), and the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM); a grant from the CRIS Cancer Foundation (to M.B.); and a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00, to M.B. and M.M.). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. M.S. was supported by predoctoral contract "Severo Ochoa" (BES-2016-079096) from the SpanishMinistry of Science, Innovation and Universities. G.P. was a recipient of a "Young Ph.D." grant from the Government of the Community of Madrid. F.F.-G. was supported by a formacion de profesorado universitario (FPU) fellowship from the Spanish Ministry of Science, Innovation and Universities.S

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

Analysis of genetic ablation of mutant Kras versus its inhibition in lung adenocarcinoma

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Bioquímica. Fecha de Lectura: 14-07-2022Esta Tesis tiene embargado el acceso al texto completo hasta el 14-01-2024Los recientes avances en la estructura de KRAS han llevado a la identificación de inhibidores específicos frente a KRASG12C, que hasta ahora se consideraba inabordable. A pesar de unos prometedores resultados iniciales, existe cierta evidencia de resistencia intrínseca o adquirida en los primeros ensayos clínicos, por lo que se necesitarían tratamientos combinados para maximizar el potencial terapéutico de las terapias dirigidas frente a KRAS. Con el fin de profundizar en este aspecto, hemos desarrollado dos modelos de ratón genéticamente modificados de adenocarcinoma de pulmón inducidos por KRAS que nos permitieron evaluar tanto la eliminación genética como la inhibición farmacológica de KRAS en tumores establecidos. En esta tesis hemos descrito una regresión tumoral completa y prolongada tras la eliminación genética de KrasG12V, sin observar recurrencia tumoral en ausencia del oncogén, a pesar de carecer del gen supresor tumoral Trp53. La regresión tumoral estuvo mediada por apoptosis y un rápido arresto de la proliferación, acompañada de la infiltración de linfocitos T CD8+ y células NK. De forma similar, la eliminación genética de Kras en tumores de pulmón inducidos por uretano, un modelo de tumores con una mayor carga mutacional, se asoció con una regresión tumoral completa en todos los casos, sin conseguir escapar de la adicción oncogénica. Por el contrario, la inhibición farmacológica de KRASG12C con sotorasib derivó en la adquisición de resistencia en todos los ratones del estudio tras una primera respuesta inicial. Los tumores resistentes a sotorasib mostraron una elevada amplificación de la región genómica donde se encuentra el alelo de Kras y una sobreexpresión de vías relacionadas con el metabolismo de fármacos, que presuntamente podrían estar mediando la detoxificación directa en células tumorales. Estos resultados sugieren la existencia de una fuerte presión selectiva que impide al sotorasib actuar frente a KRASG12C, bien mediante la amplificación del alelo de KrasG12C, mediante el metabolismo del fármaco o por una acción conjunta de ambas. Aún así, fuimos capaces de aislar clones individuales en cultivo que sobrevivieron tras la eliminación genética de KrasG12V y crecieron en completa ausencia del oncogén. La mayoría de los clones resistentes mostraron niveles bajos de actividad de RAS, sugiriendo la presencia de mecanismos alternativos que sustenten su crecimiento de forma independiente a KRAS. Detectamos una elevada activación de las vías de NF-kB y STAT3 en los clones resistentes, y por consiguiente, el bloqueo simultáneo de ambas vías inhibió notablemente su crecimiento. Asimismo, observamos resultados similares al inhibir el co-activador transcripcional YAP1 y la proteína anti-apoptótica BIRC5, lo que sugiere la activación de un complejo programa transcripcional para eludir la apoptosis tras la eliminación de Kras. Además, los clones resistentes también mostraron un aumento de EGFR, que podría estar impulsando la reactivación de la vía MAPK, representando una potencial diana para evitar sortear al oncogén. Estos resultados revelan las ventajas de la eliminación genética de KRAS respecto a su inhibición y ponen de manifiesto la importancia de las intervenciones terapéuticas simultáneas al bloquear KRAS con el fin de evitar posibles recurrencias tumorale

Inactivation of capicua in adult mice causes T-cell lymphoblastic lymphoma

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4. Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancyThis work was supported by grants from the Fundació La Marató de TV3 (20131730/1) to G.J. and M.B., and the European Research Council (ERC-AG/ 250297-RAS AHEAD), the EU-Framework Programme (HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010- 260791/EUROCANPLATFORM), the Spanish Ministry of Economy and Competitiveness (SAF2014-59864-R), the Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE), and the Asociación Española contra el Cáncer (AECC) (GC16173694BARB) to M.B. M.B. is the recipient of an Endowed Chair from the AXA Research Fund. L.S.-C. was supported by a fellowship from the Programa de Formación de Personal Investigator (FPI) of the Spanish Ministry of Economy and Competitiveness.Peer reviewe

Effects of Bentonite on p-Methoxybenzyl Acetate: A Theoretical Model for Oligomerization via an Electrophilic-Substitution Mechanism

Tonsil Actisil FF, a commercial bentonitic clay, promotes the formation of a series of electrophilic-aromatic-substitution products from para-methoxybenzyl acetate in carbon disulfide. The molecules obtained correspond to linear isomeric dimers, trimers, tetramers and a pentamer, according to their spectroscopic data. A clear indication of the title mechanistic pathway for the oligomerization growth was obtained from the analysis of a set of computational-chemistry calculations using the density-functional-theory level B3LYP/6-311++G(d,p). The corresponding conclusions were based on the computed dipole moments, the HOMO/LUMO distributions, and a natural-populations analysis of the studied molecules

Caracterización espectroscópica, química y morfológica y propiedades superficiales de una montmorillonita mexicana

Se describe la caracterización mediante técnicas espectroscópicas de una montmorillonita natural colectada en Cuencamé, Durango. La composición de las capas tetraédricas y octaédricas fue analizada por Resonancia Magnética Nuclear (RMN) de ángulo mágico para los núcleos de 29Si y 27Al. Las propiedades superfi ciales de la arcilla se cuantifi caron utilizando técnicas de adsorción-desorción de nitrógeno y mediante la aplicación de los métodos de Brunahuer-Emmett-Teller (BET) y Barrett-Joyner- Halenda (BJH). Los resultados indican que la arcilla analizada es una montmorillonita sódica con una capa molecular de agua adsorbida entre sus láminas, cuyos átomos de Si en la estructura de fi losilicato Q3 presentan diferente composición Si-nAl (n = 0, 1 y 2). El 3.3% de los átomos de Al correspondientes a la estructura se encuentran sustituyendo a los de Si en la capa. La presencia de los iones paramagnéticos Fe+3 y Mn+2 en la montmorillonita fue determinada por resonancia paramagnética electrónica (RPE), después de ser tratada con HCl y CF3SO3H. Adicionalmente, se discuten los resultados adquiridos por las técnicas de absorción atómica, microscopía de barrido electrónico, infrarrojo y análisis térmico gravimétrico

Desertomycin G, a New Antibiotic with Activity against <i>Mycobacterium tuberculosis</i> and Human Breast Tumor Cell Lines Produced by <i>Streptomyces althioticus</i> MSM3, Isolated from the Cantabrian Sea Intertidal Macroalgae <i>Ulva</i> sp.

The isolation and structural elucidation of a structurally new desertomycin, designated as desertomycin G (1), with strong antibiotic activity against several clinically relevant antibiotic resistant pathogens are described herein. This new natural product was obtained from cultures of the marine actinomycete Streptomyces althioticus MSM3, isolated from samples of the intertidal seaweed Ulva sp. collected in the Cantabrian Sea (Northeast Atlantic Ocean). Particularly interesting is its strong antibiotic activity against Mycobacterium tuberculosis clinical isolates, resistant to antibiotics in clinical use. To the best of our knowledge, this is the first report on a member of the desertomycin family displaying such activity. Additionally, desertomycin G shows strong antibiotic activities against other relevant Gram-positive clinical pathogens such as Corynebacterium urealyticum, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Clostridium perfringens. Desertomycin G also displays moderate antibiotic activity against relevant Gram-negative clinical pathogens such as Bacteroides fragilis, Haemophilus influenzae and Neisseria meningitidis. In addition, the compound affects viability of tumor cell lines, such as human breast adenocarcinoma (MCF-7) and colon carcinoma (DLD-1), but not normal mammary fibroblasts