Multi-omics analysis identifies therapeutic vulnerabilities in triple-negative breast cancer subtypes.

Triple-negative breast cancer (TNBC) is a collection of biologically diverse cancers characterized by distinct transcriptional patterns, biology, and immune composition. TNBCs subtypes include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Through a comprehensive analysis of mutation, copy number, transcriptomic, epigenetic, proteomic, and phospho-proteomic patterns we describe the genomic landscape of TNBC subtypes. Mesenchymal subtype tumors display high mutation loads, genomic instability, absence of immune cells, low PD-L1 expression, decreased global DNA methylation, and transcriptional repression of antigen presentation genes. We demonstrate that major histocompatibility complex I (MHC-I) is transcriptionally suppressed by H3K27me3 modifications by the polycomb repressor complex 2 (PRC2). Pharmacological inhibition of PRC2 subunits EZH2 or EED restores MHC-I expression and enhances chemotherapy efficacy in murine tumor models, providing a rationale for using PRC2 inhibitors in PD-L1 negative mesenchymal tumors. Subtype-specific differences in immune cell composition and differential genetic/pharmacological vulnerabilities suggest additional treatment strategies for TNBC.

Polycomb-group proteins in the initiation and progression of cancer.

The Polycomb group (PcG) proteins are a family of chromatin regulators and critical for the maintenance of cellular identity. The PcG machinery can be categorized into at least three multi-protein complexes, namely Polycomb Repressive Complex 1 (PRC1), PRC2, and Polycomb Repressive DeUBiquitinase (PR-DUB). Their deregulation has been associated with human cancer initiation and progression. Here we review the updated understanding for PcG proteins in transcription regulation and DNA damage repair and highlight increasing links to the hallmarks in cancer. Accordingly, we discuss some of the recent advances in drug development or strategies against cancers caused by the gain or loss of PcG functions.

Polycomb Paralog Chromodomain Inhibitors Active against Both CBX6 and CBX8*.

Methyllysine reader proteins bind to methylated lysine residues and alter gene transcription by changing either the compaction state of chromatin or by the recruitment of other multiprotein complexes. The polycomb paralog family of methyllysine readers bind to trimethylated lysine on the tail of histone 3 (H3) via a highly conserved aromatic cage located in their chromodomains. Each of the polycomb paralogs are implicated in several disease states. CBX6 and CBX8 are members of the polycomb paralog family with two structurally similar chromodomains. By exploring the structure-activity relationships of a previously reported CBX6 inhibitor we have discovered more potent and cell permeable analogs. Our current report includes potent, dual-selective inhibitors of CBX6 and CBX8. We have shown that the -2 position in our scaffold is an important residue for selectivity amongst the polycomb paralogs. Preliminary cell-based studies show that the new inhibitors impact cell proliferation in a rhabdoid tumor cell line.

Discovery of an H3K36me3-Derived Peptidomimetic Ligand with Enhanced Affinity for Plant Homeodomain Finger Protein 1 (PHF1).

Plant homeodomain finger protein 1 (PHF1) is an accessory component of the gene silencing complex polycomb repressive complex 2 and recognizes the active chromatin mark, trimethylated lysine 36 of histone H3 (H3K36me3). In addition to its role in transcriptional regulation, PHF1 has been implicated as a driver of endometrial stromal sarcoma and fibromyxoid tumors. We report the discovery and characterization of UNC6641, a peptidomimetic antagonist of the PHF1 Tudor domain which was optimized through in silico modeling and incorporation of non-natural amino acids. UNC6641 binds the PHF1 Tudor domain with a Kd value of 0.96 ± 0.03 µM while also binding the related protein PHF19 with similar potency. A crystal structure of PHF1 in complex with UNC6641, along with NMR and site-directed mutagenesis data, provided insight into the binding mechanism and requirements for binding. Additionally, UNC6641 enabled the development of a high-throughput assay to identify small molecule binders of PHF1.

Inhibiting CBX4 efficiently protects hepatocellular carcinoma cells against sorafenib resistance.

BACKGROUND: This study aimed to investigate the possible role of inhibiting chromobox protein homologue 4 (CBX4) to deregulate of cancer stem cells (CSCs) and to evaluate the contribution of these molecules to sorafenib resistance in advanced hepatocellular carcinoma (HCC). METHODS: HCC cell lines and a xenograft mouse model with resistance to sorafenib were employed to analyse the effects of miR424 on CSC characteristics. RNA expression was analysed by RT-PCR and next-generation sequencing in a cohort of HCC cancer patients and sorafenib-resistant (SR) cell lines, respectively, to validate the key microRNAs and targets in the network. RESULTS: MicroRNA and mRNA profiles of SR cell lines identified miR424 and its direct target CBX4 as significantly associated with stem-cell-like properties, poor survival, and clinical characteristics. Functional experiments demonstrated that miR424 suppressed CBX4 and CBX4 induced nuclear translocation of YAP1 protein but was not associated with protein production. When YAP1 and CBX4 were modulated with CA3 and UNC3866, tumorigenicity and stem-like properties were extremely inhibited, thus indicating that these compounds exerted a strong anti-tumour effect in vivo against SR HCC cells. CONCLUSIONS: Our results revealed that blocking CBX4 expression is critical in response to sorafenib resistance with advanced HCC.

Pathogenic Impacts of Dysregulated Polycomb Repressive Complex Function in Hematological Malignancies.

Polycomb repressive complexes (PRCs) are epigenetic regulators that mediate repressive histone modifications. PRCs play a pivotal role in the maintenance of hematopoietic stem cells through repression of target genes involved in cell proliferation and differentiation. Next-generation sequencing technologies have revealed that various hematologic malignancies harbor mutations in PRC2 genes, such as EZH2, EED, and SUZ12, and PRC1.1 genes, such as BCOR and BCORL1. Except for the activating EZH2 mutations detected in lymphoma, most of these mutations compromise PRC function and are frequently associated with resistance to chemotherapeutic agents and poor prognosis. Recent studies have shown that mutations in PRC genes are druggable targets. Several PRC2 inhibitors, including EZH2-specific inhibitors and EZH1 and EZH2 dual inhibitors have shown therapeutic efficacy for tumors with and without activating EZH2 mutations. Moreover, EZH2 loss-of-function mutations appear to be attractive therapeutic targets for implementing the concept of synthetic lethality. Further understanding of the epigenetic dysregulation associated with PRCs in hematological malignancies should improve treatment outcomes.

Targeting the CK1α/CBX4 axis for metastasis in osteosarcoma.

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.

Pan-specific and partially selective dye-labeled peptidic inhibitors of the polycomb paralog proteins.

Epigenetic regulation of gene expression is in part controlled by post-translational modifications on histone proteins. Histone methylation is a key epigenetic mark that controls gene transcription and repression. There are five human polycomb paralog proteins (Cbx2/4/6/7/8) that use their chromodomains to recognize trimethylated lysine 27 on histone 3 (H3K27me3). Recognition of the methyllysine side chain is achieved through multiple cation-pi interactions within an 'aromatic cage' motif. Despite high structural similarity within the chromodomains of this protein family, they each have unique functional roles and are linked to different cancers. Selective inhibition of different CBX proteins is desirable for both fundamental studies and potential therapeutic applications. We report here on a series of peptidic inhibitors that target certain polycomb paralogs. We have identified peptidic scaffolds with sub-micromolar potency, and will report examples that are pan-specific and that are partially selective for individual members within the family. These results highlight important structure-activity relationships that allow for differential binding to be achieved through interactions outside of the methyllysine-binding aromatic cage motif.

Polycomb/Trithorax Antagonism: Cellular Memory in Stem Cell Fate and Function.

Stem cells are continuously challenged with the decision to either self-renew or adopt a new fate. Self-renewal is regulated by a system of cellular memory, which must be bypassed for differentiation. Previous studies have identified Polycomb group (PcG) and Trithorax group (TrxG) proteins as key modulators of cellular memory. In this Perspective, we draw from embryonic and adult stem cell studies to discuss the complex roles played by PcG and TrxG in maintaining cell identity while allowing for microenvironment-mediated alterations in cell fate. Finally, we discuss the potential for targeting these proteins as a therapeutic approach in cancer.

Brassinosteroid Signaling Recruits Histone 3 Lysine-27 Demethylation Activity to FLOWERING LOCUS C Chromatin to Inhibit the Floral Transition in Arabidopsis.

The steroid hormone brassinosteroid (BR) plays a broad role in plant growth and development. As the retarded growth in BR-insensitive and BR-deficient mutants causes a strong delay in days to flowering, BR signaling has been thought to promote the floral transition in Arabidopsis. In this study, using a developmental measure of flowering time, we show that BR signaling inhibits the floral transition and promotes vegetative growth in the Arabidopsis accessions Columbia and Enkheim-2. We found that BR signaling promotes the expression of the potent floral repressor FLOWERING LOCUS C (FLC) and three FLC homologs to inhibit flowering. In the presence of BR, the transcription factor BRASSINAZOLE-RESISTANT1 (BZR1), together with BES1-INTERACTING MYC-like proteins (BIMs), specifically binds a BR- responsive element in the first intron of FLC and further recruits a histone 3 lysine 27 (H3K27) demethylase to downregulate levels of the repressive H3K27 trimethylation mark and thus antagonize Polycomb silencing at FLC, leading to its activation. Taken together, our findings demonstrate that BR signaling inhibits the floral transition in Arabidopsis by a novel molecular mechanism in which BR signals are transduced into FLC activation and consequent floral repression.

Live-cell imaging reveals the dynamics of PRC2 and recruitment to chromatin by SUZ12-associated subunits.

Polycomb-repressive complex 2 (PRC2) is a histone methyltransferase that promotes epigenetic gene silencing, but the dynamics of its interactions with chromatin are largely unknown. Here we quantitatively measured the binding of PRC2 to chromatin in human cancer cells. Genome editing of a HaloTag into the endogenous EZH2 and SUZ12 loci and single-particle tracking revealed that ∼80% of PRC2 rapidly diffuses through the nucleus, while ∼20% is chromatin-bound. Short-term treatment with a small molecule inhibitor of the EED-H3K27me3 interaction had no immediate effect on the chromatin residence time of PRC2. In contrast, separation-of-function mutants of SUZ12, which still form the core PRC2 complex but cannot bind accessory proteins, revealed a major contribution of AEBP2 and PCL homolog proteins to chromatin binding. We therefore quantified the dynamics of this chromatin-modifying complex in living cells and separated the contributions of H3K27me3 histone marks and various PRC2 subunits to recruitment of PRC2 to chromatin.

Polycomb protein family member CBX7 regulates intrinsic axon growth and regeneration.

Neurons in the central nervous system (CNS) lose their intrinsic ability and fail to regenerate, but the underlying mechanisms are largely unknown. Polycomb group (PcG) proteins, which include PRC1 and PRC2 complexes function as gene repressors and are involved in many biological processes. Here we report that PRC1 components (polycomb chromobox (CBX) 2, 7, and 8) are novel regulators of axon growth and regeneration. Especially, knockdown of CBX7 in either embryonic cortical neurons or adult dorsal root ganglion (DRG) neurons enhances their axon growth ability. Two important transcription factors GATA4 and SOX11 are functional downstream targets of CBX7 in controlling axon regeneration. Moreover, knockdown of GATA4 or SOX11 in cultured DRG neurons inhibits axon regeneration response from CBX7 downregulation in DRG neurons. These findings suggest that targeting CBX signaling pathway may be a novel approach for promoting the intrinsic regenerative capacity of damaged CNS neurons.

Enhancer of polycomb maintains germline activity and genome integrity in Drosophila testis.

Tissue homeostasis depends on the ability of tissue-specific adult stem cells to maintain a balance between proliferation and differentiation, as well as ensure DNA damage repair. Here, we use the Drosophila male germline stem cell system to study how a chromatin factor, enhancer of polycomb [E(Pc)], regulates the proliferation-to-differentiation (mitosis-to-meiosis) transition and DNA damage repair. We identified two critical targets of E(Pc). First, E(Pc) represses CycB transcription, likely through modulating H4 acetylation. Second, E(Pc) is required for accumulation of an important germline differentiation factor, Bag-of-marbles (Bam), through post-transcriptional regulation. When E(Pc) is downregulated, increased CycB and decreased Bam are both responsible for defective mitosis-to-meiosis transition in the germline. Moreover, DNA double-strand breaks (DSBs) accumulate upon germline inactivation of E(Pc) under both physiological condition and recovery from heat shock-induced endonuclease expression. Failure of robust DSB repair likely leads to germ cell loss. Finally, compromising the activity of Tip60, a histone acetyltransferase, leads to germline defects similar to E(Pc) loss-of-function, suggesting that E(Pc) acts cooperatively with Tip60. Together, our data demonstrate that E(Pc) has pleiotropic roles in maintaining male germline activity and genome integrity. Our findings will help elucidate the in vivo molecular mechanisms of E(Pc).

CBX4 exhibits oncogenic activities in breast cancer via Notch1 signaling.

Polycomb chromobox (CBX) proteins are involved in gene silencing to function as oncogenes or tumor suppressors through the polycomb repressive complex (PRC1). CBX4 has been implicated in the progression of human cancers, but its role and clinical significance in breast cancer remain unclear. Here, we show that CBX4 is up-regulated in breast cancer and exerts oncogenic activities via miR-137-mediated activation of Notch1 signaling pathway. CBX4 expression was increased in breast cancer, compared with the nontumorous tissues. High CBX4 expression was closely correlated with tumor metastasis, advanced clinical stage and poor overall survival in a cohort of 179 patients with breast cancer. In vitro studies demonstrated that CBX4 overexpression enhanced, whereas CBX4 knockdown inhibited cell growth and migration. Mechanistically, in a PRC1-dependent manner, CBX4 inhibited the promoter activity of miR-137 and suppressed its expression. miR-137 decreased the expression of Notch1, Jag1 and Hey2 via targeting their 3'-UTRs. The suppression of Notch1 by siRNA or overexpression of miR-137 markedly attenuated CBX4-promoted phenotypes. Collectively, these findings indicate that CBX4 promotes breast cancer via miR-137-mediated Notch1 signaling. Our data, therefore, suggest that CBX4 serve as a prognostic biomarker and that targeting CBX4/miR-137 axis may provide therapeutic potent in the treatment of breast cancer.

Elevated expression of a pharmacologic Polycomb signature predicts poor prognosis in gastric and breast cancer.

AIM: Polycomb Group complexes are epigenetic repressors that silence tumor suppressive genes. Studies demonstrated that pharmacologic inhibition of Polycomb Group complexes with 3-deazaneplanocin A (DZNeP) induces cancer cell death by re-expressing silenced genes. Here we evaluate the prognostic significance of DZNeP target genes in gastric and breast cancer. Patients & methods/materials: The prognostic impact of a DZNeP-regulated gene signature was investigated using the KM Plotter and cBio Portal resources containing microarray data from tumor tissue. RESULTS: We report that elevated expression of DZNeP targets is associated with poor clinical outcome in gastric and breast cancer. In gastric cancer, elevated expression of DZNeP signature is inversely correlated with decreased overall survival. In breast cancer, DZNeP signature predicted poor prognosis in HER2+ tumors but not in HER2- neoplasms. CONCLUSION: These findings demonstrate that DZNeP target genes are not predictive of better but rather of poor clinical outcome in gastric and breast cancer.

Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B-cell lymphoma cells harboring gain-of-function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL-AF9, MLL-AF4, and AML1-ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.

Hematopoiesis during development, aging, and disease.

Hematopoietic stem cells were once considered identical. However, in the mid-1990s, it became apparent that stem cells from a person's early developmental phases are superior to those from adults, and aged stem cells are defective compared with young stem cells. It has since become clear that polycomb group proteins are important regulators of stem cell functioning. Polycomb group proteins are chromatin-associated proteins involved in writing or reading epigenetic histone modifications. Polycomb group proteins are involved in normal blood cell formation, in cancer, and possibly in aging. In this review, we describe how the different phases of hematopoietic stem cells-birth, maintenance, functional decline, derailment, and death-are continuous processes that may be controlled by polycomb group proteins.

Targeting EZH2 in cancer.

Recent genomic studies have resulted in an emerging understanding of the role of chromatin regulators in the development of cancer. EZH2, a histone methyl transferase subunit of a Polycomb repressor complex, is recurrently mutated in several forms of cancer and is highly expressed in numerous others. Notably, both gain-of-function and loss-of-function mutations occur in cancers but are associated with distinct cancer types. Here we review the spectrum of EZH2-associated mutations, discuss the mechanisms underlying EZH2 function, and synthesize a unifying perspective that the promotion of cancer arises from disruption of the role of EZH2 as a master regulator of transcription. We further discuss EZH2 inhibitors that are now showing early signs of promise in clinical trials and also additional strategies to combat roles of EZH2 in cancer.

SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression.

Polycomb group (PcG) complexes regulate cellular identity through epigenetic programming of chromatin. Here, we show that SSX2, a germline-specific protein ectopically expressed in melanoma and other types of human cancers, is a chromatin-associated protein that antagonizes BMI1 and EZH2 PcG body formation and derepresses PcG target genes. SSX2 further negatively regulates the level of the PcG-associated histone mark H3K27me3 in melanoma cells, and there is a clear inverse correlation between SSX2/3 expression and H3K27me3 in spermatogenesis. However, SSX2 does not affect the overall composition and stability of PcG complexes, and there is no direct concordance between SSX2 and BMI1/H3K27me3 presence at regulated genes. This suggests that SSX2 antagonizes PcG function through an indirect mechanism, such as modulation of chromatin structure. SSX2 binds double-stranded DNA in a sequence non-specific manner in agreement with the observed widespread association with chromatin. Our results implicate SSX2 in regulation of chromatin structure and function.