miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.

Tumorigenesis is a multi-step process that requires activation of oncogenes and inactivation of tumour suppressor genes. Mouse models of human cancers have recently demonstrated that continuous expression of a dominantly acting oncogene (for example, Hras, Kras and Myc) is often required for tumour maintenance; this phenotype is referred to as oncogene addiction. This concept has received clinical validation by the development of active anticancer drugs that specifically inhibit the function of oncoproteins such as BCR-ABL, c-KIT and EGFR. Identifying additional gene mutations that are required for tumour maintenance may therefore yield clinically useful targets for new cancer therapies. Although loss of p53 function is a common feature of human cancers, it is not known whether sustained inactivation of this or other tumour suppressor pathways is required for tumour maintenance. To explore this issue, we developed a Cre-loxP-based strategy to temporally control tumour suppressor gene expression in vivo. Here we show that restoring endogenous p53 expression leads to regression of autochthonous lymphomas and sarcomas in mice without affecting normal tissues. The mechanism responsible for tumour regression is dependent on the tumour type, with the main consequence of p53 restoration being apoptosis in lymphomas and suppression of cell growth with features of cellular senescence in sarcomas. These results support efforts to treat human cancers by way of pharmacological reactivation of p53.

MicroRNAs (miRNAs) may be important regulators of gene expression. By modulating oncogenic and tumor suppressor pathways they could, in principle, contribute to tumorigenesis. Consistent with this hypothesis, recurrent genetic and epigenetic alterations of individual miRNAs are found in some tumors. Functional studies are now elucidating the mechanism of action of putative oncogenic and tumor suppressor miRNAs.

We have generated two lentiviral vectors for conditional, Cre-loxregulated, RNA interference. One vector allows for conditional activation, whereas the other permits conditional inactivation of short hairpin RNA (shRNA) expression. The former is based on a strategy in which the mouse U6 promoter has been modified by including a hybrid between a LoxP site and a TATA box. The ability to efficiently control shRNA expression by using these vectors was shown in cell-based experiments by knocking down p53, nucleophosmin and DNA methyltransferase 1. We also demonstrate the usefulness of this approach to achieve conditional, tissue-specific RNA interference in Cre-expressing transgenic mice. Combined with the growing array of Cre expression strategies, these vectors allow spatial and temporal control of shRNA expression in vivo and should facilitate functional genetic analysis in mammals.

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 (...

Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not in¯uenced by p66Shc expression. In basal conditions, p66Shc7/7 and p537/7 cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

Despite their clear importance as a class of regulatory molecules, pinpointing the relevance of individual miRNAs has been challenging. Studies querying miRNA functions by overexpressing or silencing specific miRNAs have yielded data that are often at odds with those collected from loss-of-functions models. In addition, knockout studies suggest that many conserved miRNAs are dispensable for animal development or viability. In this review we discuss these observations in the context of our current knowledge of miRNA biology and review the evidence implicating miRNA-mediated gene regulation in the mechanisms that ensure biological robustness.