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

TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells.

The epigenetic regulator TET2 is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type-specific enhancers. We find that deletion of Tet2 in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix-loop-helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.K.D.R. was supported by a postdoctoral fellowship from the Danish Medical Research Council (FSS 1333-00120B), K.N. was supported by Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers, JSPS (S2704), and L.S.-C. was supported by a Marie Sklodowska-Curie individual fellowship (Horizon 2020 Framework Programme, grant agreement no. H2020-MSCA-IF-2017-796341). The work in the Helin laboratory was supported by grants to K.H. from The European Research Council (294666_DNAMET), the Danish Cancer Society, the Danish National Research Foundation (DNRF82), and through a center grant from the Novo Nordisk Foundation (NNF17CC0027852). This work was also supported by the National Institutes of Health (NIH, P30 CA008748)

Genetic characterization of CIC: a tumor suppressor regulated by RAS signaling

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-02-2017Esta tesis tiene embargado el acceso al texto completo hasta el 13-08-2018CIC es un represor transcripcional regulado negativamente por la vía de MAPK (mitogen-activated protein kinases, proteínas quinasas activadas por mitógenos). En Drosophila, Cic tiene un importante papel en el establecimiento de los ejes del embrión. Además, su inactivación anula el requerimiento de la señalización mitogénica mediada por Ras. De forma notoria, recientemente se han identificado mutaciones en CIC en cáncer humano, principalmente en oligodendroglioma, lo que sugiere un posible papel como supresor tumoral. Con el fin de determinar el papel de CIC en la señalización homeostática por RAS así como en el desarrollo de tumores hemos generado una estirpe de ratón condicional de pérdida de función en la cual hemos rodeado con sitios LoxP el dominio de unión a ADN, una región requerida para la represión transcripcional, de las dos isoformas de CIC (Cic-L y Cic-S). La expresión en línea germinal de la proteína defectuosa CICΔ2-6 provoca letalidad perinatal, lo que indica que CIC posee una función clave en el desarrollo del ratón. Se extrajeron embriones a día embrionario 18.5 y en ese punto los ratones homocigotos estaban presentes en proporciones mendelianas. Sin embargo, la mayoría de ellos presentaba onfalocele, un defecto en la pared abdominal por el cual los intestinos y/o el hígado permanecen fuera del abdomen dentro del espacio umbilical. Con el objetivo de estudiar si la pérdida de función de CIC podía abolir el requerimiento de las proteínas RAS para la proliferación también en células de mamíferos, generamos una línea de ratón compuesta con el alelo condicional de Cic y la línea knockout condicional para las tres isoformas de RAS (Hras-/-, Nras-/-, Kraslox/lox). Abordamos esta pregunta tanto in vitro, utilizando MEFs (fibroblastos embrionarios murinos), como in vivo, con ratones adultos, pero la pérdida de función de CIC no pudo compensar la falta de RAS en ninguno de los sistemas mencionados. Asimismo, para estudiar el papel de CIC en ratones adultos, hemos cruzado la línea condicional de Cic con ratones que expresan una CreERT2 recombinasa inducible por tamoxifeno bajo un promotor ubicuo (el promotor de la Ubiquitina C humana) para determinar el papel de CIC en ratones adultos. La exposición de los ratones Ciclox/lox; Tg.UBC-CreERT2+/T a 4-Hidroxitamoxifeno en el destete resultó en el desarrollo de linfoma linfoblástico de las células T (T-ALL) durante el primer año de vida. Algunos de estos ratones presentaban también esplenomegalia causada por la infiltración de células tumorales. Estos tumores sobreexpresan los bien caracterizados genes diana de CIC, Etv4 y Etv5. Estos resultados generaron pruebas experimentales apoyando que CIC tiene un papel en la supresión tumoral. Además, la secuenciación de ARN de tumores T-ALL CicΔ2-6/Δ2-6 nos ha permitido generar una firma de expresión génica de pérdida de función de CIC, que proponemos se puede usar como indicativa de la inactivación de CIC. Hemos demostrado que tumores T-ALL producidos por un RAS oncogénico presentan un enriquecimiento en dicha firma, lo que sugiere que RAS podría inducir T-ALL mediante la inactivación de CIC.CIC is a transcriptional repressor negatively regulated by the MAPK (mitogen-activated protein kinases) pathway. In Drosophila, Cic plays an important role in embryo patterning. Moreover, its inactivation overcomes the need for Ras-mediated mitogenic signaling. Interestingly, mutations in CIC have recently been identified in human cancer, most notably in oligodendroglioma, suggesting a potential role as a tumor suppressor. To determine the role of CIC in homeostatic mammalian RAS signaling as well as in tumor development we generated a conditional loss-of-function allele in which we have targeted the DNA-binding domain, a region required for transcriptional repression, of the two CIC isoforms (Cic-L and Cic-S). Germline expression of the defective CICΔ2-6 proteins leads to perinatal lethality, thus indicating a key role for CIC in mouse development. Embryos were extracted at embryonic day 18.5 and, at this time point, homozygous mice were present at Mendelian rate. However, most of them presented omphalocele, an abdominal wall defect in which the intestines and/or liver remain outside of the abdomen inside the umbilical space. To study if CIC loss-of-function could bypass the requirement for RAS in proliferation also in mammalian cells, a compound strain carrying the Cic conditional allele with a conditional knockout strain for the three RAS isoforms (Hras-/-, Nras-/-, Kraslox/lox) was generated. We addressed the question both in vitro, using MEFs, and in vivo, with adult mice. Nevertheless, the absence of CIC function could not compensate for the lack of RAS in any of the mentioned systems. Furthermore, to assess the role of CIC in adult mice, we crossed the conditional Cic strain with mice expressing an inducible CreERT2 recombinase under the control of a ubiquitous promoter (human ubiquitin C promoter). Importantly, activation of Cre by exposure of Ciclox/lox; Tg.UBC-CreERT2+/T mice to 4- Hydroxytamoxifen at weaning resulted in the development of T-cell acute lymphoblastic lymphoma (T-ALL) within the first year of life. In addition, some of these mice showed associated splenomegaly caused by the infiltration of tumoral cells. These tumors overexpress the well know CIC targets Etv4 and Etv5. These results provide experimental evidence supporting a role for CIC in tumor suppression. Moreover, RNA sequencing of CicΔ2-6/Δ2-6 T-ALL tumors has allowed us to generate a CIC loss-of-function gene expression signature, that functions as readout of CIC inactivation. We have demonstrated that T-ALL driven by an oncogenic RAS show enrichment in this signature, suggesting that RAS may drive T-ALL by inactivating CIC

The Capicua tumor suppressor: a gatekeeper of Ras signaling in development and cancer

The transcriptional repressor Capicua (CIC) has emerged as an important rheostat of cell growth regulated by RAS/MAPK signaling. Cic was originally discovered in Drosophila, where it was shown to be inactivated by MAPK signaling downstream of the RTKs Torso and EGFR, which results in signal-dependent responses that are required for normal cell fate specification, proliferation and survival of developing and adult tissues. CIC is highly conserved in mammals, where it is also negatively regulated by MAPK signaling. Here, we review the roles of CIC during mammalian development, tissue homeostasis, tumor formation and therapy resistance. Available data indicate that CIC is involved in multiple biological processes, including lung development, liver homeostasis, autoimmunity and neurobehavioral processes. Moreover, CIC has been shown to be involved in tumor development as a tumor suppressor, both in human as well as in mouse models. Finally, several lines of evidence implicate CIC as a determinant of sensitivity to EGFR and MAPK pathway inhibitors, suggesting that CIC may play a broader role in human cancer than originally anticipated.This work has been supported by grants from the Fundacio La Marato de TV3 (20131730/1) to G.J. and M.B., from the European Research Council (ERC-AG/250297-RAS AHEAD and ERC-AG/695566-THERACAN), the Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE) and the Foundation of the Asociacion Espanola contra el Cancer (AECC) (GC16173694BARB) to M.B, and the Spanish Ministry of Economy and Competitiveness (SAF2014-59864-R to M.B. and BFU2014-52863-P to G.J.). M.B. is the recipient of an Endowed Chair from the AXA Research Fund. L.S.C. has been supported by a fellowship from the Programa de Formacion de Personal Investigator (FPI) of the Spanish Ministry of Economy and CompetitivenessS

Integrated analysis of <i>FHIT</i> gene alterations in cancer

The Fragile Histidine Triad Diadenosine Triphosphatase (FHIT) gene is located in the Common Fragile Site FRA3B and encodes an enzyme that hydrolyzes the dinucleotide Ap3A. Although FHIT loss is one of the most frequent copy number alterations in cancer, its relevance for cancer initiation and progression remains unclear. FHIT is frequently lost in cancers from the digestive tract, which is compatible with being a cancer driver event in these tissues. However, FHIT loss could also be a passenger event due to the inherent fragility of the FRA3B locus. Moreover, the physiological relevance of FHIT enzymatic activity and the levels of Ap3A is largely unclear. We have conducted here a systematic pan-cancer analysis of FHIT status in connection with other mutations and phenotypic alterations, and we have critically discussed our findings in connection with the literature to provide an overall view of FHIT implications in cancer.</p

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

A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer

<div><p>HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout <i>Drosophila</i> development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.</p></div

Patterns of <i>CIC</i> mutations in human OD and <i>CIC-DUX4</i> sarcomas.

<p>(A) Diagram of the CIC protein showing a set of curated mutations from the COSMIC database (<a href="http://cancer.sanger.ac.uk/cosmic" target="_blank">http://cancer.sanger.ac.uk/cosmic</a>). Only mutations corresponding to gliomas are shown. The tumor suppressor role of CIC in OD is thought to involve the repression of CIC targets such as the <i>ETV/PEA3</i> family genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref029" target="_blank">29</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref030" target="_blank">30</a>]. Note that missense mutations tend to cluster in the HMG-box and C1 domains. In contrast, nonsense and frameshift mutations (indicated as ‘Other mutations’) are distributed along the entire length of the protein, which is also consistent with a requirement for an intact C-terminal region. (B) Structure and function of oncogenic CIC-DUX4 fusions, which usually include most of the CIC protein (including the C1 domain) coupled to the C-terminal trans-activation domain of DUX4 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref031" target="_blank">31</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref066" target="_blank">66</a>]. The double homeodomain region of DUX4 is indicated by boxes.</p