A doxycycline- and light-inducible Cre recombinase mouse model for optogenetic genome editing.

The experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research.

A murine mesenchymal stem cell model for initiating events in osteosarcomagenesis points to CDK4/CDK6 inhibition as a therapeutic target.

Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15Ink4b, p16Ink4a, or p19Arf transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15Ink4b, p16Ink4a, or p19Arf were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20-23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.

Cellular senescence inhibits renal regeneration after injury in mice, with senolytic treatment promoting repair.

The ability of the kidney to regenerate successfully after injury is lost with advancing age, chronic kidney disease, and after irradiation. The factors responsible for this reduced regenerative capacity remain incompletely understood, with increasing interest in a potential role for cellular senescence in determining outcomes after injury. Here, we demonstrated correlations between senescent cell load and functional loss in human aging and chronic kidney diseases including radiation nephropathy. We dissected the causative role of senescence in the augmented fibrosis occurring after injury in aged and irradiated murine kidneys. In vitro studies on human proximal tubular epithelial cells and in vivo mouse studies demonstrated that senescent renal epithelial cells produced multiple components of the senescence-associated secretory phenotype including transforming growth factor ß1, induced fibrosis, and inhibited tubular proliferative capacity after injury. Treatment of aged and irradiated mice with the B cell lymphoma 2/w/xL inhibitor ABT-263 reduced senescent cell numbers and restored a regenerative phenotype in the kidneys with increased tubular proliferation, improved function, and reduced fibrosis after subsequent ischemia-reperfusion injury. Senescent cells are key determinants of renal regenerative capacity in mice and represent emerging treatment targets to protect aging and vulnerable kidneys in man.

Combined deletion of Bap1, Nf2, and Cdkn2ab causes rapid onset of malignant mesothelioma in mice.

We have generated mouse models of malignant mesothelioma (MM) based upon disruption of the Bap1, Nf2, and Cdkn2ab tumor suppressor loci in various combinations as also frequently observed in human MM. Inactivation of all three loci in the mesothelial lining of the thoracic cavity leads to a highly aggressive MM that recapitulates the histological features and gene expression profile observed in human patients. The tumors also show a similar inflammatory phenotype. Bap1 deletion alone does not cause MM but dramatically accelerates MM development when combined with Nf2 and Cdkn2ab (hereafter BNC) disruption. The accelerated tumor development is accompanied by increased Polycomb repression and EZH2-mediated redistribution of H3K27me3 toward promoter sites with concomitant activation of PI3K and MAPK pathways. Treatment of BNC tumor-bearing mice with cisplatin and pemetrexed, the current frontline treatment, prolongs survival. This makes the autochthonous mouse model described here very well suited to explore the pathogenesis of MM and validate new treatment regimens for MM, including immunotherapy.

A natural WNT signaling variant potently synergizes with Cdkn2ab loss in skin carcinogenesis.

Cdkn2ab knockout mice, generated from 129P2 ES cells develop skin carcinomas. Here we show that the incidence of these carcinomas drops gradually in the course of backcrossing to the FVB/N background. Microsatellite analyses indicate that this cancer phenotype is linked to a 20 Mb region of 129P2 chromosome 15 harboring the Wnt7b gene, which is preferentially expressed from the 129P2 allele in skin carcinomas and derived cell lines. ChIPseq analysis shows enrichment of H3K27-Ac, a mark for active enhancers, in the 5' region of the Wnt7b 129P2 gene. The Wnt7b 129P2 allele appears sufficient to cause in vitro transformation of Cdkn2ab-deficient cell lines primarily through CDK6 activation. These results point to a critical role of the Cdkn2ab locus in keeping the oncogenic potential of physiological levels of WNT signaling in check and illustrate that GWAS-based searches for cancer predisposing allelic variants can be enhanced by including defined somatically acquired lesions as an additional input.

SHP2 is required for growth of KRAS-mutant non-small-cell lung cancer in vivo.

RAS mutations are frequent in human cancer, especially in pancreatic, colorectal and non-small-cell lung cancers (NSCLCs)1-3. Inhibition of the RAS oncoproteins has proven difficult4, and attempts to target downstream effectors5-7 have been hampered by the activation of compensatory resistance mechanisms8. It is also well established that KRAS-mutant tumors are insensitive to inhibition of upstream growth factor receptor signaling. Thus, epidermal growth factor receptor antibody therapy is only effective in KRAS wild-type colon cancers9,10. Consistently, inhibition of SHP2 (also known as PTPN11), which links receptor tyrosine kinase signaling to the RAS-RAF-MEK-ERK pathway11,12, was shown to be ineffective in KRAS-mutant or BRAF-mutant cancer cell lines13. Our data also indicate that SHP2 inhibition in KRAS-mutant NSCLC cells under normal cell culture conditions has little effect. By contrast, SHP2 inhibition under growth factor-limiting conditions in vitro results in a senescence response. In vivo, inhibition of SHP2 in KRAS-mutant NSCLC also provokes a senescence response, which is exacerbated by MEK inhibition. Our data identify SHP2 inhibition as an unexpected vulnerability of KRAS-mutant NSCLC cells that remains undetected in cell culture and can be exploited therapeutically.

DCC Receptors Drive Prefrontal Cortex Maturation by Determining Dopamine Axon Targeting in Adolescence.

BACKGROUND: Dopaminergic input to the prefrontal cortex (PFC) increases throughout adolescence and, by establishing precisely localized synapses, calibrates cognitive function. However, why and how mesocortical dopamine axon density increases across adolescence remains unknown. METHODS: We used a developmental application of axon-initiated recombination to label and track the growth of dopamine axons across adolescence in mice. We then paired this recombination with cell-specific knockdown of the netrin-1 receptor DCC to determine its role in adolescent dopamine axon growth. We then assessed how altering adolescent PFC dopamine axon growth changes the structural and functional development of the PFC by quantifying pyramidal neuron morphology and cognitive performance. RESULTS: We show, for the first time, that dopamine axons continue to grow from the striatum to the PFC during adolescence. Importantly, we discover that DCC, a guidance cue receptor, controls the extent of this protracted growth by determining where and when dopamine axons recognize their final target. When DCC-dependent adolescent targeting events are disrupted, dopamine axons continue to grow ectopically from the nucleus accumbens to the PFC and profoundly change PFC structural and functional development. This leads to alterations in cognitive processes known to be impaired across psychiatric conditions. CONCLUSIONS: The prolonged growth of dopamine axons represents an extraordinary period for experience to influence their adolescent trajectory and predispose to or protect against psychopathology. DCC receptor signaling in dopamine neurons is a molecular link where genetic and environmental factors may interact in adolescence to influence the development and function of the prefrontal cortex.

Rejuvenation by Therapeutic Elimination of Senescent Cells.

In this issue of Cell, Baar et al. show how FOXO4 protects senescent cell viability by keeping p53 sequestered in nuclear bodies, preventing it from inducing apoptosis. Disrupting this interaction with an all-D amino acid peptide (FOXO4-DRI) restores p53's apoptotic role and ameliorates the consequences of senescence-associated loss of tissue homeostasis.

Wnt Down, Tumors Wind Up?

In mouse intestinal tumors induced by the inhibition of APC, the restoration of APC function causes complete tumor regression with normal differentiation and return of stem cell function irrespective of whether tumors also carried mutations in Kras and p53. These findings by Dow et al. validate the Wnt pathway as an exquisite target for intervention.

Progressive disorganization of paranodal junctions and compact myelin due to loss of DCC expression by oligodendrocytes.

Paranodal axoglial junctions are critical for maintaining the segregation of axonal domains along myelinated axons; however, the proteins required to organize and maintain this structure are not fully understood. Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are proteins enriched at paranodes that are expressed by neurons and oligodendrocytes. To identify the specific function of DCC expressed by oligodendrocytes in vivo, we selectively eliminated DCC from mature myelinating oligodendrocytes using an inducible cre regulated by the proteolipid protein promoter. We demonstrate that DCC deletion results in progressive disruption of the organization of axonal domains, myelin ultrastructure, and myelin protein composition. Conditional DCC knock-out mice develop balance and coordination deficits and exhibit decreased conduction velocity. We conclude that DCC expression by oligodendrocytes is required for the maintenance and stability of myelin in vivo, which is essential for proper signal conduction in the CNS.

Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer.

Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.

Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells.

Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

DCC expression by neurons regulates synaptic plasticity in the adult brain.

The transmembrane protein deleted in colorectal cancer (DCC) and its ligand, netrin-1, regulate synaptogenesis during development, but their function in the mature central nervous system is unknown. Given that DCC promotes cell-cell adhesion, is expressed by neurons, and activates proteins that signal at synapses, we hypothesized that DCC expression by neurons regulates synaptic function and plasticity in the adult brain. We report that DCC is enriched in dendritic spines of pyramidal neurons in wild-type mice, and we demonstrate that selective deletion of DCC from neurons in the adult forebrain results in the loss of long-term potentiation (LTP), intact long-term depression, shorter dendritic spines, and impaired spatial and recognition memory. LTP induction requires Src activation of NMDA receptor (NMDAR) function. DCC deletion severely reduced Src activation. We demonstrate that enhancing NMDAR function or activating Src rescues LTP in the absence of DCC. We conclude that DCC activation of Src is required for NMDAR-dependent LTP and certain forms of learning and memory.

Deleted in colorectal carcinoma suppresses metastasis in p53-deficient mammary tumours.

Since its discovery in the early 1990s the deleted in colorectal cancer (DCC) gene, located on chromosome 18q21, has been proposed as a tumour suppressor gene as its loss is implicated in the majority of advanced colorectal and many other cancers. DCC belongs to the family of netrin 1 receptors, which function as dependence receptors as they control survival or apoptosis depending on ligand binding. However, the role of DCC as a tumour suppressor remains controversial because of the rarity of DCC-specific mutations and the presence of other tumour suppressor genes in the same chromosomal region. Here we show that in a mouse model of mammary carcinoma based on somatic inactivation of p53, additional loss of DCC promotes metastasis formation without affecting the primary tumour phenotype. Furthermore, we demonstrate that in cell cultures derived from p53-deficient mouse mammary tumours DCC expression controls netrin-1-dependent cell survival, providing a mechanistic basis for the enhanced metastatic capacity of tumour cells lacking DCC. Consistent with this idea, in vivo tumour-cell survival is enhanced by DCC loss. Together, our data support the function of DCC as a context-dependent tumour suppressor that limits survival of disseminated tumour cells.

Rapid validation of cancer genes in chimeras derived from established genetically engineered mouse models.

Recent technological advances have opened the door for the fast and cost-effective generation of genetically engineered mouse models (GEMMs) to study cancer. We describe here a conceptually novel approach for the generation of chimeric GEMMs based on the controlled introduction of various genetic elements in embryonic stem cells (ESCs) that are derived from existing mouse strains with a predisposition for cancer. The isolation of GEMM-derived ESC lines is greatly facilitated by the availability of the newly defined culture media containing inhibitors that effectively preserve ESC pluripotency. The feasibility of the GEMM-ESC approach is discussed in light of current literature and placed into the context of existing models. This approach will allow for fast and flexible validation of candidate cancer genes and drug targets and will result in a repository of GEMM-ESC lines and corresponding vector collections that enable easy distribution and use of preclinical models to the wider scientific community.

Mammary-specific inactivation of E-cadherin and p53 impairs functional gland development and leads to pleomorphic invasive lobular carcinoma in mice.

Breast cancer is the most common malignancy in women of the Western world. Even though a large percentage of breast cancer patients show pathological complete remission after standard treatment regimes, approximately 30-40% are non-responsive and ultimately develop metastatic disease. To generate a good preclinical model of invasive breast cancer, we have taken a tissue-specific approach to somatically inactivate p53 and E-cadherin, the cardinal cell-cell adhesion receptor that is strongly associated with tumor invasiveness. In breast cancer, E-cadherin is found mutated or otherwise functionally silenced in invasive lobular carcinoma (ILC), which accounts for 10-15% of all breast cancers. We show that mammary-specific stochastic inactivation of conditional E-cadherin and p53 results in impaired mammary gland function during pregnancy through the induction of anoikis resistance of mammary epithelium, resulting in loss of epithelial organization and a dysfunctional mammary gland. Moreover, combined inactivation of E-cadherin and p53 induced lactation-independent development of invasive and metastatic mammary carcinomas, which showed strong resemblance to human pleomorphic ILC. Dissemination patterns of mouse ILC mimic the human malignancy, showing metastasis to the gastrointestinal tract, peritoneum, lung, lymph nodes and bone. Our results confirm that loss of E-cadherin contributes to both mammary tumor initiation and metastasis, and establish a preclinical mouse model of human ILC that can be used for the development of novel intervention strategies to treat invasive breast cancer.

Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes.

The cyclin dependent kinase (CDK) inhibitors p15, p16, p21, and p27 are frequently deleted, silenced, or downregulated in many malignancies. Inactivation of CDK inhibitors predisposes mice to tumor development, showing that these genes function as tumor suppressors. Here, we describe high-throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific genetic context of predisposing germline and somatic mutations. We also found associations between these loci with gender, age of tumor onset, and lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites with single nucleotide polymorphisms associated with chronic lymphocytic leukemia revealed a significant overlap between the datasets. Together, our findings highlight the importance of genetic context within large-scale mutation detection studies, and they show a novel use for insertional mutagenesis data in prioritizing disease-associated genes that emerge from genome-wide association studies.

TCR down-regulation controls virus-specific CD8+ T cell responses.

The CD3gamma di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3gamma di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8(+) T cells is impaired in mice with a mutated CD3gamma di-leucine-based motif. The CD3gamma mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8(+) T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3gamma-mediated TCR down-regulation in virus-specific CD8(+) T cell responses.

A conditional mouse model for malignant mesothelioma.

Malignant mesothelioma is a devastating disease that has been associated with loss of Neurofibromatosis type 2 (NF2) and genetic lesions affecting RB and P53 pathways. We introduced similar lesions in the mesothelial lining of the thoracic cavity of mice. Mesothelioma developed at high incidence in Nf2;Ink4a/Arf and Nf2;p53 conditional knockout mice with median survival times of approximately 30 and 20 weeks, respectively. Murine mesothelioma closely mimicked human malignant mesothelioma. Conditional Nf2;Ink4a/Arf mice showed increased pleural invasion compared to conditional Nf2;p53 mice. Interestingly, upon Ink4a loss in the latter mice median survival was significantly reduced and all tumors were highly invasive, suggesting that Ink4a loss substantially contributes to the poor clinical outcome of malignant mesothelioma.

p15Ink4b is a critical tumour suppressor in the absence of p16Ink4a.

The CDKN2b-CDKN2a locus on chromosome 9p21 in human (chromosome 4 in mouse) is frequently lost in cancer. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame of CDKN2a (ref. 1). Whereas the tumour suppressor functions for p16INK4a and p14ARF have been firmly established, the role of p15INK4b remains ambiguous. However, many 9p21 deletions also remove CDKN2b, so we hypothesized a synergistic effect of the combined deficiency for p15INK4b, p14ARF and p16INK4a. Here we report that mice deficient for all three open reading frames (Cdkn2ab-/-) are more tumour-prone and develop a wider spectrum of tumours than Cdkn2a mutant mice, with a preponderance of skin tumours and soft tissue sarcomas (for example, mesothelioma) frequently composed of mixed cell types and often showing biphasic differentiation. Cdkn2ab-/- mouse embryonic fibroblasts (MEFs) are substantially more sensitive to oncogenic transformation than Cdkn2a mutant MEFs. Under conditions of stress, p15Ink4b protein levels are significantly elevated in MEFs deficient for p16Ink4a. Our data indicate that p15Ink4b can fulfil a critical backup function for p16Ink4a and provide an explanation for the frequent loss of the complete CDKN2b-CDKN2a locus in human tumours.