Ewa Malgorzata Michalak

Chromatin is a macromolecular complex predominantly comprising DNA, histone proteins and RNA. The methylation of chromatin components is highly conserved as it helps coordinate the regulation of gene expression, DNA repair and DNA replication. Dynamic changes in chromatin methylation are essential for cell-fate determination and development. Consequently, inherited or acquired mutations in the major factors that regulate the methylation of DNA, RNA and/or histones are commonly observed in developmental disorders, ageing and cancer. This has provided the impetus for the clinical development of epigenetic therapies aimed at resetting the methylation imbalance observed in these disorders. In this Review, we discuss the cellular functions of chromatin methylation and focus on how this fundamental biological process is corrupted in cancer. We discuss methylation-based cancer therapies and provide a perspective on the emerging data from early-phase clinical trial therapies that target regulators of DNA and histone methylation. We also highlight promising therapeutic strategies, including monitoring chromatin methylation for diagnostic purposes and combination epigenetic therapy strategies that may improve immune surveillance in cancer and increase the efficacy of conventional and targeted anticancer drugs.

Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type–specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states. The formation of mammary glands requires the tight regulation of many genes that govern cell fate decisions in the cells that form them. However, most of these genes remain undefined. The Polycomb repressive complex 2 (PRC2) has a role in gene silencing, and it is comprised of several subunits, which include either Enhancer of Zeste homolog 2 (Ezh2) or Ezh1 in combination with Suppressor of Zeste 12 protein homolog (Suz12) and embryonic ectoderm development (EED). Dysregulation of these subunits can lead to breast cancer. Although previous studies have analyzed the contribution of these complexes in mammary epithelium, failure to inactivate all canonical PRC2 complexes has made this task difficult. Deletion of Suz12 resulted in nonfunctional PRC2 and led to growth defects in mammary epithelial cells in vivo and in vitro. Here, we have used a 3D mammary organoid system to circumvent the lethality associated with Suz12 loss and studied chromatin dynamics and gene expression of PRC2 complexes. Our results suggest that loss of all canonical PRC2 complexes results in failure to repress transcriptional programs associated with early commitment and differentiation of mammary epithelial cells.

The mammary stem cell field wakes up to hibernating cells

Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues on histone tails and non‐histone targets. These important post‐translational modifications are exquisitely regulated and affect chromatin compaction and transcriptional programs leading to diverse biological outcomes. There is accumulating evidence that genetic alterations of several HMTs impinge on oncogenic or tumor‐suppressor functions and influence both cancer initiation and progression. HMTs therefore represent an opportunity for therapeutic targeting in those patients with tumors in which HMTs are dysregulated, to reverse the histone marks and transcriptional programs associated with aggressive tumor behavior. In this review, we describe the known histone methyltransferases and their emerging roles in breast cancer tumorigenesis.

Preclinical in vivo validation of target genes for therapeutic intervention requires careful selection and characterization of the most suitable animal model in order to assess the role of these genes in a particular process or disease. To this end, genetically engineered mouse models (GEMMs) are typically used. However, the appropriate engineering of these models is often cumbersome and time consuming. Recently, we and others described a modular approach for fast-track modification of existing GEMMs by re-derivation of embryonic stem cells (ESCs) that can be modified by recombinase-mediated transgene insertion and subsequently used for the production of chimeric mice. This 'GEMM-ESC strategy' allows for rapid in vivo analysis of gene function in the chimeras and their offspring. Moreover, this strategy is compatible with CRISPR/Cas9-mediated genome editing. This protocol describes when and how to use the GEMM-ESC strategy effectively, and it provides a detailed procedure for re-deriving and manipulating GEMM-ESCs under feeder- and serum-free conditions. This strategy produces transgenic mice with the desired complex genotype faster than traditional methods: generation of validated GEMM-ESC clones for controlled transgene integration takes 9–12 months, and recombinase-mediated transgene integration and chimeric cohort production takes 2–3 months. The protocol requires skills in embryology, stem cell biology and molecular biology, and it is ideally performed within, or in close collaboration with, a transgenic facility.

Significance: Poly(ADP-ribose) polymerase (PARP) inhibitors hold promise for patients with breast cancer 1 ( BRCA1 )-associatated cancers but are anticipated to give rise to resistance. We show that mouse mammary tumors resembling BRCA1-associated metaplastic breast carcinoma display intrinsic resistance to the PARP inhibitor olaparib as a result of increased P-glycoprotein drug efflux transporter expression. These findings may have implications for ongoing clinical trials.

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.

Caloric restriction (CR) is proposed to decrease tumorigenesis through a variety of mechanisms including effects on glycolysis. However, the understanding of how CR affects the response to cancer therapy is still rudimentary. Here, using the Eµ-Myc transgenic mouse model of B-cell lymphoma, we report that by reducing protein translation, CR can reduce expression of the prosurvival Bcl-2 family member Mcl-1 and sensitize lymphomas to ABT-737–induced death in vivo. By using Eµ-Myc lymphoma cells lacking p53, we showed that CR mimetics such as 2-deoxyglucose led to a decrease in Mcl-1 expression and sensitized lymphoma cells to ABT-737–induced death independently of p53. In keeping with this, Eµ-Myc lymphoma cells lacking the BH3-only proapoptotic members Noxa, Puma, or Bim were also sensitized by CR mimetics to ABT-737–induced death. Remarkably, neither the loss of both Puma and Noxa, the loss of both Puma and Bim, nor the loss of all three BH3-only proteins prevented sensitization to ABT-737 induced by CR mimetics. Thus, CR can influence Mcl-1 expression and sensitize cells to BH3 mimetic–induced apoptosis, independently of the main BH3-only proteins and of p53. Exploiting this may improve the efficiency of, or prevent resistance to, cancer therapy.

Specification of the cellular hierarchy in the mammary gland involves complex signaling that remains poorly defined. Polycomb group proteins are known to contribute to the maintenance of stem cell identity through epigenetic modifications, leading to stable alterations in gene expression. The polycomb protein family member EZH2 is known to be important for stem cell maintenance in multiple tissues, but its role in mammary gland development and differentiation remains unknown. Our analyses show that EZH2 is predominantly expressed in luminal cells of the mouse mammary epithelium. As mammary gland development occurs mostly after birth, the analysis of EZH2 gene function in postnatal development is precluded by embryonic lethality of conventional EZH2 knockout mice. To investigate the role of EZH2 in normal mammary gland epithelium, we have generated novel transgenic mice that express doxycycline‐regulatable short hairpin (sh) RNAs directed against Ezh2. Knockdown of EZH2 results in delayed outgrowth of the mammary epithelium during puberty, due to impaired terminal end bud formation and ductal elongation. Furthermore, our results demonstrate that EZH2 is required to maintain the luminal cell pool and may limit differentiation of luminal progenitors into CD61⁺ differentiated luminal cells, suggesting a role for EZH2 in mammary luminal cell fate determination. Consistent with this, EZH2 knockdown reduced lobuloalveolar expansion during pregnancy, suggesting EZH2 is required for the differentiation of luminal progenitors to alveolar cells.Stem Cells 2013;31:1910‐1920