Chromosomal rearrangements play a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions1. A recently discovered example is a fusion between the Echinoderm Microtubule-associated Protein-like 4 (EML4) and the Anaplastic Lymphoma Kinase (ALK) genes, generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC)2 and is clinically relevant because it confers sensitivity to ALK inhibitors3. Despite their importance, modeling such genetic events in mice has proven challenging and requires complex manipulation of the germline. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumors invariably harbor the Eml4-Alkinversion, express the Eml4-Alk fusion gene, display histo-pathologic and molecular features typical of ALK+ human NSCLCs, and respond to treatment with ALK-inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

The miR-17~92 cluster is frequently amplified or overexpressed in human cancers and has emerged as the prototypical oncogenic polycistron microRNA (miRNA). miR-17~92 is a direct transcriptional target of c-Myc, and experiments in a mouse model of B-cell lymphomas have shown cooperation between these two oncogenes. However, both the molecular mechanism underlying this cooperation and the individual miRNAs that are responsible for it are unknown. By using a conditional knockout allele of miR-17~92, we show here that sustained expression of endogenous miR-17~92 is required to suppress apoptosis in Myc-driven B-cell lymphomas. Furthermore, we show that among the six miRNAs that are encoded by miR-17~92, miR-19a and miR-19b are absolutely required and largely sufficient to recapitulate the oncogenic properties of the entire cluster. Finally, by combining computational target prediction, gene expression profiling, and an in vitro screening strategy, we identify a subset of miR-19 targets that mediate its prosurvival activity.Supplemental material is available at http://www.genesdev.org. The experiments presented in this study were designed to examine the role of the endogenous miR-17;92 allele in Myc-driven lymphomas, and to determine the relative contribution of each of the six constituent miRNAs to the overall oncogenic potential of the cluster.Our results show that, in the context of Myc-driven B-cell lymphomas, genetic ablation of the endogenous miR-17;92 locus leads to a dramatic reduction of tumor cell growth in vitro and suppresses tumorigenicity in vivo, two effects that are largely the consequence of increased cell death. We also demonstrate that, among the six miRNAs encoded by the miR-17;92 cluster, the members of the miR-19 family (miR-19a and miR-19b) are essential to mediate the oncogenic activity of the entire cluster, and that they do so at least in part by modulating the expression of the tumor suppressor gene Pten (phosphatase and tensin homologous). Results and DiscussionGeneration of miR-17;92 flox/flox ;Em-Myc miceTo investigate the role of miR-17;92 in Myc-induced cancers, we employed the Em-Myc mouse model of B-cell lymphomas (Adams et al. 1985). Em-Myc mice express a c-Myc transgene under the control of the B-cell-specific Em enhancer and develop B-cell lymphomas within 4-6 mo of age (Adams et al. 1985). Em-Myc mice were crossed to mice carrying a conditional miR-17;92 knockout allele (miR-17;92 fl ) ( Fig. 1B; Ventura et al. 2008). To temporally control the deletion of the floxed miR-17;92 allele, these mice were further crossed to mice carrying a 4-hydroxytamoxifen (4-OHT)-inducible Cre-recombinase estrogen receptor-T2 (Cre-ER T2 ) knock-in allele targeted to the ubiquitously expressed ROSA26 locus (R26-Cre-ER T2 mice, hereafter referred to as Cre-ER) (Ventura et al. 2007).As expected, Em-Myc; miR-17;92 fl/fl ; Cre-ER mice developed B-cell lymphomas with similar latency and phenotype as the parental Em-Myc strain (data not shown). From these mice, we derived two independent lymphoma lines (...

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34–deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34–deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc–initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.

The p53 family member TAp73 is a transcription factor that plays a key role in many biological processes, including neuronal development. In particular, we have shown that p73 drives the expression of miR-34a, but not miR-34b and c, in mouse cortical neurons. miR-34a in turn modulates the expression of synaptic targets including synaptotagmin-1 and syntaxin-1A. Here we show that this axis is retained in mouse ES cells committed to differentiate toward a neurological phenotype. Moreover, overexpression of miR-34a alters hippocampal spinal morphology, and results in electrophysiological changes consistent with a reduction in spinal function. Therefore, the TAp73/miR-34a axis has functional relevance in primary neurons. These data reinforce a role for miR-34a in neuronal development.cell death | synaptogenesis | neuronal differentiation | hippocampus M icro-RNAs (miRs) are one family of a number of small noncoding regulatory RNAs (1). They are initially transcribed as pri-miRs, which are processed by a nuclear RNase III enzyme to form stem-loop structured premiRs. The premiRs are transported to the cytosol, where another RNase III cleaves off double-stranded portions of the hairpin to generate a short-lived dsRNA of approximately 20 to 25 nt. This duplex becomes unwound, and one strand (forming the mature miR) becomes incorporated into miR-protein complexes. The mature miR within the miR-protein complex recognizes complementary sites in the 3′ UTR of target genes, resulting in translational inhibition or destabilization of the target mRNAs and down-regulation of the encoded protein. During development, a number of miRs show distinct expression patterns during maturation of the CNS (2). For example, microarray miR profiling of embryonic, early postnatal, and adult brain revealed differential changes in nine miRNAs, including miR-9 and -124, and the levels of both these miRs increase markedly during the transition from neuronal precursors to mature neurons. miR-124 has also been implicated in the differentiation of neuroblastoma cells induced by retinoic acid (3).p73 is a member of the p53 family. Two distinct promoters transcribe different isoforms containing-TAp73-or lackingΔNp73-the aminoterminal transactivation domain (4); furthermore, extensive alternative 3′-splicing produces additional isoforms (5, 6). Trp73-KO mice have significant developmental abnormalities of the central nervous system, including congenital hydrocephalus, hippocampal dysgenesis, and defects of pheromone detection (7). Isoform-selective KOs have shown both a distinct neuronal phenotype and altered tumor susceptibility (8, 9). p53 can regulate several miRs (10). Indeed, the miR-34 family (miR-34a-c) is a p53 target (11-13), which can mimic several p53 effects in a cell type-specific manner. miR-34a is ubiquitous with the highest expression in mouse brain, and overexpression of miR-34a in neuroblastoma cell lines modulates neuronal-specific genes (14), whereas miR-34b and c are mainly expressed in the lung (15). Less information is available ...

B cell receptor (BCR) signaling plays a critical role in B cell tolerance and activation. Here, we show that mice with B cell-specific ablation of both Cbl and Cbl-b (Cbl-/-Cblb-/-) manifested systemic lupus erythematosus (SLE)-like autoimmune disease. The Cbl double deficiency resulted in a substantial increase in marginal zone (MZ) and B1 B cells. The mutant B cells were not hyperresponsive in terms of proliferation and antibody production upon BCR stimulation; however, B cell anergy to protein antigen appeared to be impaired. Concomitantly, BCR-proximal signaling, including tyrosine phosphorylation of Syk tyrosine kinase, Phospholipase C-gamma2 (PLC-gamma2), and Rho-family GTP-GDP exchange factor Vav, and Ca2+ mobilization were enhanced, whereas tyrosine phosphorylation of adaptor protein BLNK was substantially attenuated in the mutant B cells. These results suggested that the loss of coordination between these pathways was responsible for the impaired B cell tolerance induction. Thus, Cbl proteins control B cell-intrinsic checkpoint of immune tolerance, possibly through coordinating multiple BCR-proximal signaling pathways during anergy induction.

Polycistronic microRNA clusters are a common feature of vertebrate genomes. The coordinated expression of miRNAs belonging to different seed families from a single transcription unit suggests functional cooperation, but this hypothesis has not been experimentally tested. Here we report the characterization of an allelic series of genetically engineered mice harboring selective targeted deletions of individual components of miR-17~92. Our results demonstrate the co-existence of functional cooperation and specialization among members of this cluster, identify a novel function for the miR-17 seed family in controlling axial patterning in vertebrates, and show that loss of miR-19 selectively impairs Myc-driven tumorigenesis in two models of human cancer. By integrating phenotypic analysis and gene expression profiling, we provide a genome-wide view of how components of a polycistronic miRNA-cluster affect gene expression in vivo. The reagents and datasets reported here will accelerate exploration of the complex biological functions of this important miRNA cluster.

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5' sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

Feingold syndrome (FS) is an autosomal dominant disorder characterized by microcephaly, short stature, digital anomalies, esophageal/duodenal atresia, facial dysmorphism, and various learning disabilities. Heterozygous deletion of the miR-17–92 cluster is responsible for a subset of FS (Feingold syndrome type 2, FS2), and the developmental abnormalities that characterize this disorder are partially recapitulated in mice that harbor a heterozygous deletion of this cluster (miR-17–92Δ/+ mice). Although Feingold patients develop a wide array of learning disabilities, no scientific description of learning/cognitive disabilities, intellectual deficiency, and brain alterations have been described in humans and animal models of FS2. The aim of this study was to draw a behavioral profile, during development and in adulthood, of miR-17–92Δ/+ mice, a genetic mouse model of FS2. Moreover, dopamine, norepinephrine and serotonin tissue levels in the medial prefrontal cortex (mpFC), and Hippocampus (Hip) of miR-17–92Δ/+ mice were analyzed. Our data showed decreased body growth and reduced vocalization during development. Moreover, selective deficits in spatial ability, social novelty recognition and memory span were evident in adult miR-17–92Δ/+ mice compared with healthy controls (WT). Finally, we found altered dopamine as well as serotonin tissue levels, in the mpFC and Hip, respectively, of miR-17–92Δ/+ in comparison with WT mice, thus suggesting a possible link between cognitive deficits and altered brain neurotransmission.