CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity.

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation.

Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼ 5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.

Rewriting DNA Methylation Signatures at Will: The Curable Genome Within Reach?

Epigenetic regulation of gene expression is vital for the maintenance of genome integrity and cell phenotype. In addition, many different diseases have underlying epigenetic mutations, and understanding their role and function may unravel new insights for diagnosis, treatment, and even prevention of diseases. It was an important breakthrough when epigenetic alterations could be gene-specifically manipulated using epigenetic regulatory proteins in an approach termed epigenetic editing. Epigenetic editors can be designed for virtually any gene by targeting effector domains to a preferred sequence, where they write or erase the desired epigenetic modification. This chapter describes the tools for editing DNA methylation signatures and their applications. In addition, we explain how to achieve targeted DNA (de)methylation and discuss the advantages and disadvantages of this approach. Silencing genes directly at the DNA methylation level instead of targeting the protein and/or RNA is a major improvement, as repression is achieved at the source of expression, potentially eliminating the need for continuous administration. Re-expression of silenced genes by targeted demethylation might closely represent the natural situation, in which all transcript variants might be expressed in a sustainable manner. Altogether epigenetic editing, for example, by rewriting DNA methylation, will assist in realizing the curable genome concept.

Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer.

The transcription factor (TF) SOX2 is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition to its normal stem cell function, SOX2 over-expression is associated with cancer development. The ability to selectively target this and other oncogenic TFs in cells, however, remains a significant challenge due to the 'undruggable' characteristics of these molecules. Here, we employ a zinc finger (ZF)-based artificial TF (ATF) approach to selectively suppress SOX2 gene expression in cancer cells. We engineered four different proteins each composed of 6ZF arrays designed to bind 18 bp sites in the SOX2 promoter and enhancer region, which controls SOX2 methylation. The 6ZF domains were linked to the Kruppel Associated Box (SKD) repressor domain. Three engineered proteins were able to bind their endogenous target sites and effectively suppress SOX2 expression (up to 95% repression efficiencies) in breast cancer cells. Targeted down-regulation of SOX2 expression resulted in decreased tumor cell proliferation and colony formation in these cells. Furthermore, induced expression of an ATF in a mouse model inhibited breast cancer cell growth. Collectively, these findings demonstrate the effectiveness and therapeutic potential of engineered ATFs to mediate potent and long-lasting down-regulation of oncogenic TF expression in cancer cells.

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability.

BACKGROUND: Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. RESULTS: Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. CONCLUSIONS: These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated.

Click chemistry enables preclinical evaluation of targeted epigenetic therapies.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Breaking through an epigenetic wall: re-activation of Oct4 by KRAB-containing designer zinc finger transcription factors.

The gene Oct4 encodes a transcription factor critical for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition, improper re-activation of Oct4 contributes to oncogenic processes. Herein, we describe a novel designer zinc finger protein (ZFP) capable of upregulating the endogenous Oct4 promoter in a panel of breast and ovarian cell lines carrying a silenced gene. In some ovarian tumor lines, the ZFP triggered a strong reactivation of Oct4, with levels of expression comparable with exogenous Oct4 cDNA delivery. Surprisingly, the reactivation of Oct4 required a KRAB domain for effective upregulation of the endogenous gene. While KRAB-containing ZFPs are traditionally described as transcriptional repressors, our results suggest that these proteins could, in certain genomic contexts, function as potent activators and, thus, outline an emerging novel function of KRAB-ZFPs. In addition, we document a novel ZFP that could be used for the epigenetic reprograming of cancer cells.

Epigenetic reprogramming of cancer cells via targeted DNA methylation.

An obstacle in the treatment of human diseases such as cancer is the inability to selectively and effectively target historically undruggable targets such as transcription factors. Here, we employ a novel technology using artificial transcription factors (ATFs) to epigenetically target gene expression in cancer cells. We show that site-specific DNA methylation and long-term stable repression of the tumor suppressor Maspin and the oncogene SOX2 can be achieved in breast cancer cells via zinc-finger ATFs targeting DNA methyltransferase 3a (DNMT3a) to the promoters of these genes. Using this approach, we show Maspin and SOX2 downregulation is more significant as compared with transient knockdown, which is also accompanied by stable phenotypic reprogramming of the cancer cell. These findings indicate that multimodular Zinc Finger Proteins linked to epigenetic editing domains can be used as novel cell resources to selectively and heritably alter gene expression patterns to stably reprogram cell fate.

Modulation of gene expression using zinc finger-based artificial transcription factors.

Artificial transcription factors (ATFs) consist of a transcriptional effector domain fused to a DNA-binding domain such as an engineered zinc finger protein (ZFP). Depending on the effector domain, ATFs can up- or downregulate gene expression and thus represent powerful tools in biomedical research and allow novel approaches in clinical practice. Here, we describe the construction of ATFs directed against the promoter of the epithelial cell adhesion molecule and against the promoter of the RNA component of telomerase. Methods to assess DNA binding of the engineered ZFP as well as to determine and improve the cellular effect of ATFs on (endogenous) promoter activity are described.