M. van Lohuizen

Retroviral insertional mutagenesis: past, present and future

Retroviral insertion mutagenesis screens in mice are powerful tools for efficient identification of oncogenic mutations in an in vivo setting. Many oncogenes identified in these screens have also been shown to play a causal role in the development of human cancers. Sequencing and annotation of the mouse genome, along with recent improvements in insertion site cloning has greatly facilitated identification of oncogenic events in retrovirus-induced tumours. In this review, we discuss the features of retroviral insertion mutagenesis screens, covering the mechanisms by which retroviral insertions mutate cellular genes, the practical aspects of insertion site cloning, the identification and analysis of common insertion sites, and finally we address the potential for use of somatic insertional mutagens in the study of nonhaematopoietic and nonmammary tumour types.

Mice bearing the E mu-myc and E mu-pim-1 transgenes develop pre-B-cell leukemia prenatally.

Previously, it has been shown that E mu-pim-1 transgenic mice are predisposed to T-cell lymphomas, whereas E mu-myc transgenic mice are predisposed to pre-B-cell lymphomas. Here we show that double-transgenic E mu-myc E mu-pim-1 mice exhibit pre-B-cell leukemia in utero. Upon transplantation into recipient mice, embryo-derived double-transgenic leukemic cells frequently progressed to highly malignant monoclonal tumors, indicating that additional (epi)genetic events had occurred during the progression of the disease.

Functional analysis of mouse Polycomb group genes

Two groups of genes, the Polycomb group (Pc-G) and trithorax group (trx-G), have been identified in Drosophila to provide a transcriptional memory mechanism. They ensure the maintenance of transcription patterns of key regulators such as the Hox genes and thereby the correct execution of developmental programmes. Recent data suggest that this memory mechanism is conserved in vertebrates and plants. Here we discuss current insights into the role of mouse Pc-G genes, with a particular focus on the best-studied Bmi1, Mel18 and M33 genes, as representative examples. Common phenotypes observed in knockout mice mutant for each of these genes indicate an important role for Pc-G genes not only in regulation of Hox gene expression and axial skeleton development but also in control of proliferation and survival of haematopoietic cell lineages. Proliferation defects are also observed in other cell lineages derived from these null-mutant mice, and provide new tools to study the impact of Pc-G deregulation on cell cycle control.Abstract. Two groups of genes, the Polycomb group (Pc-G) and trithorax group (trx-G), have been identified in Drosophila to provide a transcriptional memory mechanism. They ensure the maintenance of transcription patterns of key regulators such as the Hox genes and thereby the correct execution of developmental programmes. Recent data suggest that this memory mechanism is conserved in vertebrates and plants. Here we discuss current insights into the role of mouse Pc-G genes, with a particular focus on the best-studied Bmi1, Mel18 and M33 genes, as representative examples. Common phenotypes observed in knockout mice mutant for each of these genes indicate an important role for Pc-G genes not only in regulation of Hox gene expression and axial skeleton development but also in control of proliferation and survival of haematopoietic cell lineages. Proliferation defects are also observed in other cell lineages derived from these null-mutant mice, and provide new tools to study the impact of Pc-G deregulation on cell cycle control.

In vitro genetic screen identifies a cooperative role for LPA signaling and c-Myc in cell transformation

c-Myc drives uncontrolled cell proliferation in various human cancers. However, in mouse embryo fibroblasts (MEFs), c-Myc also induces apoptosis by activating the p19Arf tumor suppressor pathway. Tbx2, a transcriptional repressor of p19Arf, can collaborate with c-Myc by suppressing apoptosis. MEFs overexpressing c-Myc and Tbx2 are immortal but not transformed. We have performed an unbiased genetic screen, which identified 12 oncogenes that collaborate with c-Myc and Tbx2 to transform MEFs in vitro. One of them encodes the LPA2 receptor for the lipid growth factor lysophosphatidic acid (LPA). We find that LPA1 and LPA4, but not LPA3, can reproduce the transforming effect of LPA2. Using pharmacological inhibitors, we show that the in vitro cell transformation induced by LPA receptors is dependent on the Gi-linked ERK and PI3K signaling pathways. The transforming ability of LPA1, LPA2 and LPA4 was confirmed by tumor formation assays in vivo and correlated with prolonged ERK1/2 activation in response to LPA. Our results reveal a direct role for LPA receptor signaling in cell transformation and tumorigenesis in conjunction with c-Myc and reduced p19Arf expression.

Context-dependent actions of Polycomb repressors in cancer.

Polycomb Group (PcG) proteins form Polycomb Repressive Complexes (PRCs) that function as epigenetic repressors of gene expression. The large variety of PcG proteins, in addition to the high number of paralogs, allows for the formation of diverse PRCs with different properties, providing fine-tuned control over cell specification. Initially identified as being oncogenes, a small number of PcG genes are involved in tumor development in part through the repression of the CDKN2A locus. Therefore, enhanced PcG-mediated repression has long been assumed to be cancer promoting. However, recent data have revealed that for some cancers, PcG proteins act as tumor suppressors, indicating that this traditional view is oversimplified. In this review, we present an overview of the roles of PcG genes in oncogenesis and how the nature of their role is context dependent.

Retroviral insertional mutagenesis in murine mammary cancer

We are attempting to identify cellular oncogenes activated in mammary tumours by using the mouse mammary tumour virus (MMTV) as an insertional mutagen. MMTV, a retrovirus lacking a host cell-derived viral oncogene, induces adenocarcinomas of the mammary gland after a long latency period. The tumours are clonal outgrowths of cells carrying one or more integrated MMTV proviral copies. We have cloned an integrated MMTV provirus with its adjacent chromosomal DNA and we have established that the insertion site was part of a domain of the mouse genome in which MMTV proviruses are inserted in many different tumours. A gene within this domain, called int-1 is transcriptionally activated as a consequence of proviral integration. We have proposed that int-1 is a cellular oncogene for mammary tumours. Proviral activation of int-1 occurs in cis, over distances of up to 10 kilobases and is presumably caused by the transcriptional enhancer present on the MMTV long terminal repeat. The putative int-1 mammary oncogene has been subjected to a detailed structural analysis by S1 mapping and DNA sequencing. It encodes a protein that is highly conserved between mouse and man. The protein encoding domain of the gene is distributed over four exons which are demarcated by the insertion sites of MMTV proviruses found in mammary tumours. Some insertions, however, are found in the transcriptional unit of int-1, but these insertions do not disrupt the protein encoding domain of the gene.

Combination therapy of the EZH2 inhibitor GSK126 and the PARP inhibitor Olaparib shows in vivo synergy in a patient-derived xenograft model of BRCA1-deficient breast cancer

Background: Current treatment options for BRCA1-deficient breast cancer are limited highlighting the need for novel targeted therapies. Our group previously demonstrated that EZH2 is overexpressed in BRCA1-deficient breast tumors and a promising target for this subtype. EZH2 is the catalytic subunit of polycomb repressive complex 2 and involved in gene silencing through methylation of histone H3K27. Recently, GSK126, a highly selective inhibitor of EZH2 methyltransferase activity, was discovered. Additionally, PARP inhibitors are effective in breast tumors with defects in homologous recombination due to BRCA1 mutations. Methods: To study a possible synergy between the EZH2 inhibitor GSK126 and PARP inhibitor Olaparib we made use of a patient-derived xenograft (PDX) model from triple negative breast cancer with BRCA1 mutation. Cell lines were derived from a genetically modified mouse model for hereditary breast cancer (K14cre;Brca1F/F;p53F/F). Results: Combined inhibition of EZH2 and PARP in BRCA1-deficient cell lines resulted in delayed cell growth compared to single treatments. In the BRCA1-deficient PDX model the EZH2 inhibitor GSK126 alone attenuated the tumor growth modestly. Treatment with Olaparib shows tumor stasis. However, dual EZH2 and PARP inhibition with GSK126 and Olaparib reduced the tumor volume substantially. Western blot analysis of tumor lysates demonstrate target inhibition of GSK126 by significantly reduced H3K27me3-levels. Conclusion: Here, we report that the combination of EZH2 inhibition with a PARP inhibitor provides in vivo synergy in a PDX-mouse model for BRCA1-deficient breast tumors. Our findings suggest that this candidate combination might be an effective treatment of BRCA1-related tumors to be tested in further trials.

Polycomb group proteins control neural stem cells and brain development, and contribute to gliomagenesis in an Ink4a/Arf independent manner

ATF5 is a bZIP transcription factor that is a member of the ATF/ CREB family. Recent studies have indicated a role for ATF5 in neural, astrocyte and oligodendrocyte progenitor proliferation. Studies done in vivo and in vitro indicate that ATF5 promotes neuroprogenitor cell proliferation and that down-regulation of ATF5 is necessary for such cells to exit the cell cycle and differentiate. Furthermore, expression of ATF5 is high in glioblastomas where its expression appears to be required for survival. Though existing data shed light on biological functions of ATF5, there is little information regarding mechanisms that govern the induction and maintenance of ATF5 expression in proliferating neuroprogenitors and in tumor cells. Because of its well characterized properties, we chose the developing mouse cerebellum as a model system to address these issues. ATF5 protein is highly expressed by cerebellar granule neuron progenitors (GNPs) in the EGL and expression diminishes as GNPs differentiate into granule neurons. Such expression coincides with regions of Sonic Hedgehog (Shh)-driven proliferative activity in the EGL. We therefore assessed in GNP cultures whether ATF5 might be under the control of Shh, a major GNP mitogen. Without added Shh, both ATF5 expression and GNP proliferation rapidly fell. In Shh-treated cultures, in contrast there was a robust increase in the proportion of ATF5 positive cells. Childhood medulloblastomas are thought to arise largely from GNPs and examination of a number of such tumors revealed high expression of ATF5. In summary, our in vivo and in vitro observations are consistent with the idea that ATF5 plays a required role in cerebellar neuroprogenitor cell proliferation and suggest that Shh signaling is involved in ATF5 regulation in this population. Our findings also suggest a potential role for ATF5 in medulloblastomas.

Structural and Biological Properties of the INT-1 Mammary Oncogene

We are interested in defining genes implicated in the development of mammary tumors. In mice, mammary tumors can be induced by a variety of methods, including hormones, chemical carcinogens, gene transfer into germ line and a retrovirus (1.2).A direct view on specific genetic alterations in mammary tumorigenesis is nevertheless not always obtained. The identification of oncogenes specific for mammary tumors could eventually be helpful in, for example, examining what the mechanism of action of hormonal carcinogenesis is. The large majority of the cellular oncogenes has been discovered with the aid of retroviruses (3, 4); thus, the Mouse Mammary Tumor Virus (MMTV) could be the system of choice to define mammary oncogenes.