Danielle Hulsman

Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice

We have used large-scale insertional mutagenesis to identify functional landmarks relevant to cancer in the recently completed mouse genome sequence. We infected Cdkn2a−/− mice with Moloney murine leukemia virus (MoMuLV) to screen for loci that can participate in tumorigenesis in collaboration with loss of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF. Insertional mutagenesis by the latent retrovirus was synergistic with loss of Cdkn2a expression, as indicated by a marked acceleration in the development of both myeloid and lymphoid tumors. We isolated 747 unique sequences flanking retroviral integration sites and mapped them against the mouse genome sequence databases from Celera and Ensembl. In addition to 17 insertions targeting gene loci known to be cancer-related, we identified a total of 37 new common insertion sites (CISs), of which 8 encode components of signaling pathways that are involved in cancer. The effectiveness of large-scale insertional mutagenesis in a sensitized genetic background is demonstrated by the preference for activation of MAP kinase signaling, collaborating with Cdkn2a loss in generating the lymphoid and myeloid tumors. Collectively, our results show that large-scale retroviral insertional mutagenesis in genetically predisposed mice is useful both as a system for identifying genes underlying cancer and as a genetic framework for the assignment of such genes to specific oncogenic pathways.

Ubiquitin E3 Ligase Ring1b/Rnf2 of Polycomb Repressive Complex 1 Contributes to Stable Maintenance of Mouse Embryonic Stem Cells

Background Polycomb repressive complex 1 (PRC1) core member Ring1b/Rnf2, with ubiquitin E3 ligase activity towards histone H2A at lysine 119, is essential for early embryogenesis. To obtain more insight into the role of Ring1b in early development, we studied its function in mouse embryonic stem (ES) cells. Methodology/Principal Findings We investigated the effects of Ring1b ablation on transcriptional regulation using Ring1b conditional knockout ES cells and large-scale gene expression analysis. The absence of Ring1b results in aberrant expression of key developmental genes and deregulation of specific differentiation-related pathways, including TGFbeta signaling, cell cycle regulation and cellular communication. Moreover, ES cell markers, including Zfp42/Rex-1 and Sox2, are downregulated. Importantly, retained expression of ES cell regulators Oct4, Nanog and alkaline phosphatase indicates that Ring1b-deficient ES cells retain important ES cell specific characteristics. Comparative analysis of our expression profiling data with previously published global binding studies shows that the genes that are bound by Ring1b in ES cells have bivalent histone marks, i.e. both active H3K4me3 and repressive H3K27me3, or the active H3K4me3 histone mark alone and are associated with CpG-‘rich’ promoters. However, deletion of Ring1b results in deregulation, mainly derepression, of only a subset of these genes, suggesting that additional silencing mechanisms are involved in repression of the other Ring1b bound genes in ES cells. Conclusions Ring1b is essential to stably maintain an undifferentiated state of mouse ES cells by repressing genes with important roles during differentiation and development. These genes are characterized by high CpG content promoters and bivalent histone marks or the active H3K4me3 histone mark alone.

The chromodomain helicase Chd4 is required for Polycomb‐mediated inhibition of astroglial differentiation

Polycomb group (PcG) proteins form transcriptional repressor complexes with well‐established functions during cell‐fate determination. Yet, the mechanisms underlying their regulation remain poorly understood. Here, we extend the role of Polycomb complexes in the temporal control of neural progenitor cell (NPC) commitment by demonstrating that the PcG protein Ezh2 is necessary to prevent the premature onset of gliogenesis. In addition, we identify the chromodomain helicase DNA‐binding protein 4 (Chd4) as a critical interaction partner of Ezh2 required specifically for PcG‐mediated suppression of the key astrogenic marker gene GFAP. Accordingly, in vivo depletion of Chd4 in the developing neocortex promotes astrogenesis. Collectively, these results demonstrate that PcG proteins operate in a highly dynamic, developmental stage‐dependent fashion during neural differentiation and suggest that target gene‐specific mechanisms regulate Polycomb function during sequential cell‐fate decisions.

Rapid and Robust Transgenic High-Grade Glioma Mouse Models for Therapy Intervention Studies

Purpose: To develop a transgenic mouse model of glioma that can be conveniently used for testing therapy intervention strategies. High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. Typical for high-grade glioma is that glioma cells infiltrate extensively into surrounding pivotal brain structures, thereby rendering current treatments largely ineffective. Evaluation of novel therapies requires the availability of appropriate glioma mouse models. Experimental Design: High-grade gliomas were induced by stereotactic intracranial injection of lentiviral GFAP-Cre or CMV-Cre vectors into compound LoxP-conditional mice, resulting in K-Rasv12 expression and loss of p16Ink4a/p19Arf with or without concomitant loss of p53 or Pten. Results: Tumors reproduced many of the features that are characteristic for human high-grade gliomas, including invasiveness and blood-brain barrier functionality. Especially, CMV-Cre injection into p53;Ink4a/Arf;K-Rasv12 mice resulted in high-grade glioma with a short tumor latency (2-3 weeks) and full penetrance. Early detection and follow-up was accomplished by noninvasive bioluminescence imaging, and the practical utility for therapy intervention was shown in a study with temozolomide. Conclusion: We have developed a realistic high-grade glioma model that can be used with almost the same convenience as traditional xenograft models, thus allowing its implementation at the forefront of preclinical evaluation of new treatments. Clin Cancer Res; 16(13); 3431–41. ©2010 AACR.

Polycomb Repressive Complex 2 Is a Barrier to KRAS-Driven Inflammation and Epithelial-Mesenchymal Transition in Non-Small-Cell Lung Cancer

Polycomb repressive complexes (PRC) are frequently implicated in human cancer, acting either as oncogenes or tumor suppressors. Here, we show that PRC2 is a critical regulator of KRAS-driven non-small cell lung cancer progression. Modulation of PRC2 by either Ezh2 overexpression or Eed deletion enhances KRAS-driven adenomagenesis and inflammation, respectively. Eed-loss-driven inflammation leads to massive macrophage recruitment and marked decline in tissue function. Additional Trp53 inactivation activates a cell-autonomous epithelial-to-mesenchymal transition program leading to an invasive mucinous adenocarcinoma. A switch between methylated/acetylated chromatin underlies the tumor phenotypic evolution, prominently involving genes controlled by Hippo/Wnt signaling. Our observations in the mouse models were conserved in human cells. Importantly, PRC2 inactivation results in context-dependent phenotypic alterations, with implications for its therapeutic application.

Prolonged Ezh2 Depletion in Glioblastoma Causes a Robust Switch in Cell Fate Resulting in Tumor Progression

EZH2 is frequently overexpressed in glioblastoma (GBM), suggesting an oncogenic function that could be a target for therapeutic intervention. However, reduced EZH2 activity can also promote tumorigenesis, leading to concerns about the use of EZH2 inhibitors. Here, we provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and primary tumor-derived human GBM cell lines. Using doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor, we demonstrate that prolonged Ezh2 depletion causes a robust switch in cell fate, including significantly enhanced proliferation, DNA damage repair, and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression. Short-term Ezh2 depletion significantly improved survival without the tumor progression observed upon prolonged Ezh2 depletion, suggesting that precise dosing regiments are very important. These results could be of high clinical relevance with regard to how glioblastomas should be treated with epigenetic therapies.

Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells.

In vivo deletion of USP3, a deubiquitinating enzyme involved in DNA damage repair, increases the incidence of spontaneous cancer and impairs the proliferation and repopulation ability of HSCs.

Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

Histone ubiquitination at DNA breaks is required for activation of the DNA damage response (DDR) and DNA repair. How the dynamic removal of this modification by deubiquitinating enzymes (DUBs) impacts genome maintenance in vivo is largely unknown. To address this question, we generated mice deficient for Ub-specific protease 3 (USP3; Usp3{delta}/{delta}), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling. Notably, USP3 deletion increased the levels of histone ubiquitination in adult tissues, reduced the hematopoietic stem cell (HSC) reserves over time, and shortened animal life span. Mechanistically, our data show that USP3 is important in HSC homeostasis, preserving HSC self-renewal, and repopulation potential in vivo and proliferation in vitro. A defective DDR and unresolved spontaneous DNA damage contribute to cell cycle restriction of Usp3{delta}/{delta} HSCs. Beyond the hematopoietic system, Usp3{delta}/{delta} animals spontaneously developed tumors, and primary Usp3{delta}/{delta} cells failed to preserve chromosomal integrity. These findings broadly support the regulation of chromatin ubiquitination as a key pathway in preserving tissue function through modulation of the response to genotoxic stress.

In vivo shRNA screens in solid tumors

Loss-of-function (LOF) experiments targeting multiple genes during tumorigenesis can be implemented using pooled shRNA libraries. RNAi screens in animal models rely on the use of multiple shRNAs to simultaneously disrupt gene function, as well as to serve as barcodes for cell fate outcomes during tumorigenesis. Here we provide a protocol for performing RNAi screens in orthotopic mouse tumor models, referring to glioma and lung adenocarcinoma as specific examples. The protocol aims to provide guidelines for applying RNAi to a diverse spectrum of solid tumors and to highlight crucial considerations when designing and performing these studies. It covers shRNA library assembly and packaging into lentiviral particles, and transduction into tumor-initiating cells (TICs), followed by in vivo transplantation, tumor DNA recovery, sequencing and analysis. Depending on the target genes and tumor model, tumor suppressors and oncogenes can be identified or biological pathways can be dissected in 6–9 weeks.

Bmi1 deficient neural stem cells have increased integrin dependent adhesion to self-secreted matrix.

BACKGROUND Neural cells deficient for Polycomb group (PcG) protein Bmi1 are impaired in the formation and differentiation of high grade glioma, an incurable cancer of the brain. It was shown that mechanisms involved in cell adhesion and migration were specifically affected in these tumors. METHODS Using biochemical and cell biological approaches, we investigated the adhesive capacities of Bmi1;Ink4a/Arf deficient primary neural stem cells (NSCs). RESULTS Bmi1;Ink4a/Arf deficient NSCs have altered expression of Collagen-related genes, secrete increased amounts of extracellular matrix, and exhibit enhanced cell-matrix binding through the Beta-1 Integrin receptor. These traits are independent from the well described role of Bmi1 as repressor of the Ink4a/Arf tumor suppressor locus. CONCLUSION In addition to proliferative processes, Bmi1 controls the adhesive capacities of primary NSCs by modulating extracellular matrix secretion. GENERAL SIGNIFICANCE Since PcG protein Bmi1 is important for both normal development and tumorigenesis, it is vital to understand the complete network in which this protein acts. Whereas it is clear that control of Ink4a/Arf is a major Bmi1 function, there is evidence that other downstream mechanisms exist. Hence, our novel finding that Bmi1 also governs cell adhesion significantly contributes to our understanding of the PcG proteins.