Combination of EZH2 and ATM inhibition in BAP1-deficient mesothelioma.

Background: More than half of mesothelioma tumours show alterations in the tumour suppressor gene BAP1. BAP1-deficient mesothelioma is shown to be sensitive to EZH2 inhibition in preclinical settings but only showed modest efficacy in clinical trial. Adding a second inhibitor could potentially elevate EZH2i treatment efficacy while preventing acquired resistance at the same time.

Combined Inhibition of EZH2 and FGFR is Synergistic in BAP1-deficient Malignant Mesothelioma.

Unlabelled: Malignant mesothelioma is a highly aggressive tumor with a survival of only 4-18 months after diagnosis. Treatment options for this disease are limited. Immune checkpoint blockade using ipilimumab and nivolumab has recently been approved as a frontline therapy, but this led to only a small improvement in overall patient survival.

Genetic screens reveal new targetable vulnerabilities in BAP1-deficient mesothelioma.

More than half of patients with malignant mesothelioma show alterations in the BAP1 tumor-suppressor gene. Being a member of the Polycomb repressive deubiquitinating (PR-DUB) complex, BAP1 loss results in an altered epigenome, which may create new vulnerabilities that remain largely unknown. Here, we performed a CRISPR-Cas9 kinome screen in mesothelioma cells that identified two kinases in the mevalonate/cholesterol biosynthesis pathway.

Functional antagonism of chromatin modulators regulates epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to modulate proliferation, migration, and stress response. Whereas kinase signaling is believed to be an EMT driver, the molecular mechanisms underlying epithelial-mesenchymal interconversion are incompletely understood. Here, we show that the impact of chromatin regulators on EMT interconversion is broader than that of kinases.

USP15 Deubiquitinase Safeguards Hematopoiesis and Genome Integrity in Hematopoietic Stem Cells and Leukemia Cells.

Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens.

Phenotypic Mapping of Pathologic Cross-Talk between Glioblastoma and Innate Immune Cells by Synthetic Genetic Tracing.

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, and .

Ezh2 inhibition in Kras-driven lung cancer amplifies inflammation and associated vulnerabilities.

Kras-driven non-small-cell lung cancers (NSCLCs) are a leading cause of death with limited therapeutic options. Many NSCLCs exhibit high levels of Ezh2, the enzymatic subunit of polycomb repressive complex 2 (PRC2). We tested Ezh2 inhibitors as single agents or before chemotherapy in mice with orthotopic Kras-driven NSCLC grafts, which homogeneously express Ezh2.

TRIM28 is an Epigenetic Barrier to Induced Pluripotent Stem Cell Reprogramming.

Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin.

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.

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.

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.

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; Usp3Δ/Δ), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling.

In vivo RNAi screen for BMI1 targets identifies TGF-β/BMP-ER stress pathways as key regulators of neural- and malignant glioma-stem cell homeostasis.

In mouse and human neural progenitor and glioblastoma "stem-like" cells, we identified key targets of the Polycomb-group protein BMI1 by combining ChIP-seq with in vivo RNAi screening. We discovered that Bmi1 is important in the cellular response to the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) and endoplasmic reticulum (ER) stress pathways, in part converging on the Atf3 transcriptional repressor. We show that Atf3 is a tumor-suppressor gene inactivated in human glioblastoma multiforme together with Cbx7 and a few other candidates.

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.

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.

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).

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.

Bmi1 controls tumor development in an Ink4a/Arf-independent manner in a mouse model for glioma.

The Polycomb group and oncogene Bmi1 is required for the proliferation of various differentiated cells and for the self-renewal of stem cells and leukemic cancer stem cells. Repression of the Ink4a/Arf locus is a well described mechanism through which Bmi1 can exert its proliferative effects. However, we now demonstrate in an orthotopic transplantation model for glioma, a type of cancer harboring cancer stem cells, that Bmi1 is also required for tumor development in an Ink4a/Arf-independent manner.

Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice.

The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We show that Arf is a general target of Bmi1, however particularly in neural stem cells, derepression of Ink4a contributes to Bmi1(-/-) phenotypes.

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.