Harald Mikkers

High-throughput retroviral tagging to identify components of specific signaling pathways in cancer

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (EμMyc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from EμMyc Pim1−/− Pim2−/− mice and 27 tumors from EμMyc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of EμMyc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.

Mice Deficient for All PIM Kinases Display Reduced Body Size and Impaired Responses to Hematopoietic Growth Factors

ABSTRACT The Pim family of proto-oncogenes encodes a distinct class of serine/threonine kinases consisting of PIM1, PIM2, and PIM3. Although the Pim genes are evolutionarily highly conserved, the contribution of PIM proteins to mammalian development is unclear. PIM1-deficient mice were previously described but showed only minor phenotypic aberrations. To assess the role of PIM proteins in mammalian physiology, compound Pim knockout mice were generated. Mice lacking expression of Pim1, Pim2, and Pim3 are viable and fertile. However, PIM-deficient mice show a profound reduction in body size at birth and throughout postnatal life. In addition, the in vitro response of distinct hematopoietic cell populations to growth factors is severely impaired. In particular, PIM proteins are required for the efficient proliferation of peripheral T lymphocytes mediated by synergistic T-cell receptor and interleukin-2 signaling. These results indicate that members of the PIM family of proteins are important but dispensable factors for growth factor signaling.

Retroviral insertional mutagenesis:Tagging cancer pathways

Slow transforming retroviruses, such as the Moloney murine leukemia virus (M-MuLV), induce tumors upon infection of a host after a relatively long latency period. The underlying mechanism leading to cell transformation is the activation of proto-oncogenes or inactivation of tumor suppressor genes as a consequence of proviral insertions into the host genome. Cells carrying proviral insertions that confer a selective advantage will preferentially grow out. This means that proviral insertions mark genes contributing to tumorigenesis, as was demonstrated by the identification of numerous proto-oncogenes in retrovirally induced tumors in the past. Since cancer is a complex multistep process, the proviral insertions in one clone of tumor cells also represent oncogenic events that cooperate in tumorigenesis. Novel advances, such as the launch of the complete mouse genome, high-throughput isolation of proviral flanking sequences, and genetically modified animals have revolutionized proviral tagging into an elegant and efficient approach to identify signaling pathways that collaborate in cancer.

Differentiation of Induced Pluripotent Stem Cells Into Functional Oligodendrocytes

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell‐based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in‐vitro (co‐culture with DRG neurons) as well as in‐vivo (after implantation in the demyelinated corpus callosum of cuprizone‐treated mice). We report the importance of complete purification of the iPS‐derived OPC suspension to prevent the contamination with teratoma‐forming iPS cells.

Pim kinase expression is induced by LTP stimulation and required for the consolidation of enduring LTP.

In animals and several cellular models of synaptic plasticity, long‐lasting changes in synaptic strength are dependent on gene transcription and translation. Here we demonstrate that Pim‐1, a serine/threonine kinase closely related to Pim‐2 and Pim‐3, is induced in hippocampus in response to stimuli that evoke long‐term potentiation (LTP). Mice deficient for Pim‐1 show normal synaptic transmission and short‐term plasticity. However, they fail to consolidate enduring LTP even though Pim‐2 and Pim‐3 are constitutively expressed in the hippocampus and Pim‐3 expression is similarly induced by synaptic activity. Thus, expression of Pim‐1 is required for LTP. Its level of expression and, consequently, its capacity to phosphorylate target proteins in dendritic and nuclear compartments of stimulated neurons might be a determining factor for the establishment of long‐lasting changes in synaptic strength.

Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation.

Targeted ectopic expression of genes in the adult brain is an invaluable approach for studying many biological processes. This can be accomplished by generating transgenic mice or by virally mediated gene transfer, but these methods are costly and labor intensive. We devised a rapid strategy that allows localized in vivo transfection of plasmid DNA within the adult neurogenic niches without detectable brain damage. Injection of plasmid DNA into the ventricular system or directly into the hippocampus of adult mice, followed by application of electrical current via external electrodes, resulted in transfection of neural stem or progenitor cells and mature neurons. We showed that this strategy can be used for both fate mapping and gain- or loss-of-function experiments. Using this approach, we identified an essential role for cadherins in maintaining the integrity of the lateral ventricle wall. Thus, in vivo electroporation provides a new approach to study the adult brain.

Tumors Originating from Induced Pluripotent Stem Cells and Methods for Their Prevention

Pluripotent stem cells represent an almost unlimited source of most somatic cell types, providing them with great potential for cell‐based therapies. The earliest methods used for generating human pluripotent stem cells as embryonic stem cells from human embryos suffered from ethical and technical drawbacks. These problems have been solved in part through the efficient induction of pluripotency in somatic cells using forced expression of a tetrad of factors. Here, we describe the formation of rhabdomyosarcomas originating from factor‐induced pluripotent stem (iPS) cells derived from mouse neural stem cells. This underscores the commonly accepted notion that the use of retroviral delivery methods for inducing pluripotency will not be suited for clinical applications. However, the iPS cell field is developing rapidly. Safer protocols are now available for producing pluripotent stem cells. Here the current state‐of‐the‐art in this field will be discussed.

Induced pluripotency with endogenous and inducible genes

The recent discovery that two partly overlapping sets of four genes induce nuclear reprogramming of mouse and even human cells has opened up new possibilities for cell replacement therapies. Although the combination of genes that induce pluripotency differs to some extent, Oct4 and Sox2 appear to be a prerequisite. The introduction of four genes, several of which been linked with cancer, using retroviral approaches is however unlikely to be suitable for future clinical applications. Towards developing a safer reprogramming protocol, we investigated whether cell types that express one of the most critical reprogramming genes endogenously are predisposed to reprogramming. We show here that three of the original four pluripotency transcription factors (Oct4, Klf4 and c-Myc or MYCER(TAM)) induced reprogramming of mouse neural stem (NS) cells exploiting endogenous SoxB1 protein levels in these cells. The reprogrammed neural stem cells differentiated into cells of each germ layer in vitro and in vivo, and contributed to mouse development in vivo. Thus a combinatorial approach taking advantage of endogenously expressed genes and inducible transgenes may contribute to the development of improved reprogramming protocols.

Long non-coding RNAs: Guardians of development

Two decades ago, the existence of long non-coding RNAs (lncRNAs) was discovered. In the following genomics era more transcribed non-coding genomic regions were identified. These were initially regarded as transcriptional noise and did not receive a lot of attention. Emerging data on several of these long non-coding transcripts have refuted this hypothesis by demonstrating that non-coding RNAs (ncRNAs) are important for regulating transcription and cell signaling. A special subset of the lncRNAs affecting gene transcription appears to orchestrate major developmental programs. Here, we discuss the mechanisms by which lncRNAs regulate transcription, and review the evidence that links this class of lncRNAs to a role in development.

Proviral activation of the tumor suppressor E2a contributes to T cell lymphomagenesis in EμMyc transgenic mice

The basic helix–loop–helix factor E2A plays an important role in the development of B and T lymphocytes. In addition, E2a has been implicated as a gene with tumor suppressor activity, since mice deficient for E2a succumb to T cell lymphomas. We have performed retroviral tagging in EμMyc transgenic mice to identify genes that contribute to lymphomagenesis. The EμMyc transgenic mouse is a well-established model of a common translocation in human B cell lymphomas. Analyses of the proviral insertion sites in the MuLV-induced lymphomas revealed that a number of T cell lymphomas carried proviral insertions in the promoter region of E2a. These proviral insertions yield hybrid viral-E2a mRNAs resulting in a marked rise in E2A protein levels. The proviral insertions in E2a were predominantly of clonal origin indicating that E2a insertions are early events in these T cell lymphomas. The primary oncogenic effect of E2A is likely to be associated with enhancement of transcription of the c-Myc transgene via binding to the regulatory immunoglobulin enhancers. The results herein thus provide the first evidence that in a specific setting E2A overexpression can contribute to T-lymphomagenesis. This implies that E2a contains oncogenic features in addition to the previously described tumor suppressive properties.