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The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates., Author summary Non-melanoma skin cancers, the most common cancers in the US, are caused by UV skin exposure. Nearly 1 million cases of cutaneous squamous cell carcinoma (cuSCC) are diagnosed in the US each year. While most cuSCCs are highly treatable, more than twice as many individuals die from this disease as from melanoma. The high burden of UV-induced DNA damage in human skin poses a challenge for identifying initiating and cooperating mutations that promote cuSCC development and for defining potential therapeutic targets. Here, we describe a genetic screen in mice using a DNA transposon system to mutagenize the genome of keratinocytes and drive squamous cell carcinoma in the absence of UV. By sequencing where the transposons selectively integrated in the genomes of normal skin, skin with pre-cancerous lesions and skin with fully developed cuSCCs from our mouse model, we were able to identify frequently mutated genes likely important for this disease. Our analysis also defined cooperation between sets of genes not previously appreciated in cuSCC. Our mouse model and ensuing data provide a framework for understanding the genetics of cuSCC and for defining the molecular changes that may lead to the future therapies for patients.

Yu et al. demonstrate that the transcription factor Bcl11b is specifically expressed in mouse innate lymphoid progenitors committed to the ILC2 lineage and is required for their development. Bcl11b-deficient mice exhibit a complete lack of ILC2 development, which is confirmed by immune challenges with either papain treatment or influenza virus infection., Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.

Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated β-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers. ASTAR (Agency for Sci., Tech. and Research, S’pore)

SignificanceTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Despite extensive cancer genome-sequencing efforts, there is still an incomplete understanding of the genetic networks driving TNBC. Here, we usedSleeping Beautytransposon mutagenesis to identify genes that cooperate with mutantPtenin the induction of TNBC. We identified 12 candidate TNBC trunk drivers and a larger number of progression genes. Subsequent functional validation studies identified eight human TNBC tumor suppressor genes, including the GATA-like transcriptional repressorTRPS1, which was shown to inhibit lung metastasis by deregulating the expression of multiple serpin and epithelial-to-mesenchymal transition (EMT) pathway genes. Our study provides a better understanding of the genetic forces driving TNBC and discovered genes with clinical importance in TNBC.

Contains fulltext : 167380.pdf (Publisher’s version ) (Open Access) Intellectual disability (ID) is a common condition with considerable genetic heterogeneity. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes. 22 p.

Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here, we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies., Triple-negative breast cancers (TNBCs) tend to have poor prognosis; however, the mechanisms underlying TNBC pathology are not well understood. Here the authors utilize epidemiologic data and animal models to demonstrate an important role for BCL11A in the genesis and propagation of TNBCs.

One Two TT cells develop in the thymus, where they proceed through several developmental stages, losing alternative lineage potential as they progress. The molecular regulation of this developmental process, however, is not fully understood (see the Perspective byDi Santo).P. Liet al.(p.85, published online 10 June),L. Liet al.(p.89), andIkawaet al.(p.93) now identify expression of the zinc finger transcription factorBcl11bas the earliest checkpoint in T cell development in mice. Genetic deletion ofBcl11bin developing T cells inhibited commitment to the T cell lineage. Under conditions that should have stimulated T lineage differentiation,Bcl11b-deficient T cell progenitors failed to up-regulate genes associated with lineage-committed T cells and maintained stem cell– and progenitor cell–associated gene expression. In both developing and committed T cells, loss ofBcl11bresulted in the generation of cells that resembled natural killer (NK) cells in both phenotype and function. These NK-like cells could be expanded easily in vitro and possessed antitumor cytotoxicity, but they did not exhibit cytotoxicity against normal cells and were not tumorigenic. Because T cells are much easier to obtain from human patients than NK cells, deletion ofBcl11bin T cells may thus provide a source of easy-to-grow NK cells for cell-based antitumor therapies.

INTRODUCTIONThe critical step in generating a knockout mouse is gene-targeting vector construction. Recombineering technology has greatly streamlined the vector construction process. This protocol describes a method for making conditional knockout (cko) targeting vectors using the pSim18 plasmid. This plasmid carries the three λ phage Red genes (Gam, Bet, and Exo) under the control of the pL promoter, which is in turn regulated by the temperature-sensitive CI857 repressor. Hence, the heat-inducible recombineering functions can be easily delivered to bacterial artificial chromosomes (BACs) using a simple plasmid transformation, allowing one to manipulate any cloned mouse genomic region in Escherichia coli for cko vector construction. The conditional targeting vectors described in this protocol generate a flexible reporter/null/conditional allele in the mouse.

Cre-loxP recombination is widely used for genetic manipulation of the mouse genome. Here, we report generation and characterization of a new Cre line, Stella-Cre, where Cre expression cassette was targeted to the 3′ UTR of the Stella locus. Stella is specifically expressed in preimplantation embryos and in the germline. Cre-loxP recombination efficiency in Stella-Cre mice was investigated at several genomic loci including Rosa26, Jak2, and Npm1. At all the loci examined, we observed 100% Cre-loxP recombination efficiency in the embryos and in the germline. Thus, Stella-Cre mice serve as a very efficient deleter line. genesis 49:689–695, 2011. © 2011 Wiley-Liss, Inc.

Recombineering is a technology that utilizes the efficient homologous recombination functions encoded by gamma phage to manipulate DNA in Escherichia coli. Construction of knockout vectors has been greatly facilitated by recombineering as it allows one to choose any genomic region to manipulate. We describe here an efficient recombineering-based protocol for making mouse conditional knockout targeting vectors.