Topography of transcriptionally active chromatin in glioblastoma.

Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer-driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM.

MNK1 and MNK2 enforce expression of E2F1, FOXM1, and WEE1 to drive soft tissue sarcoma.

Soft tissue sarcoma (STS) is a heterogeneous disease that arises from connective tissues. Clinical outcome of patients with advanced tumors especially de-differentiated liposarcoma and uterine leiomyosarcoma remains unsatisfactory, despite intensive treatment regimens including maximal surgical resection, radiation, and chemotherapy. MAP kinase-interacting serine/threonine-protein kinase 1 and 2 (MNK1/2) have been shown to contribute to oncogenic translation via phosphorylation of eukaryotic translation initiation factor 4E (eIF4E). However, little is known about the role of MNK1/2 and their downstream targets in STS. In this study, we show that depletion of either MNK1 or MNK2 suppresses cell viability, anchorage-independent growth, and tumorigenicity of STS cells. We also identify a compelling antiproliferative efficacy of a novel, selective MNK inhibitor ETC-168. Cellular responsiveness of STS cells to ETC-168 correlates positively with that of phosphorylated ribosomal protein S6 (RPS6). Mirroring MNK1/2 silencing, ETC-168 treatment strongly blocks eIF4E phosphorylation and represses expression of sarcoma-driving onco-proteins including E2F1, FOXM1, and WEE1. Moreover, combination of ETC-168 and MCL1 inhibitor S63845 exerts a synergistic antiproliferative activity against STS cells. In summary, our study reveals crucial roles of MNK1/2 and their downstream targets in STS tumorigenesis. Our data encourage further clinical translation of MNK inhibitors for STS treatment.

Core transcriptional regulatory circuitries in cancer.

Transcription factors (TFs) coordinate the on-and-off states of gene expression typically in a combinatorial fashion. Studies from embryonic stem cells and other cell types have revealed that a clique of self-regulated core TFs control cell identity and cell state. These core TFs form interconnected feed-forward transcriptional loops to establish and reinforce the cell-type-specific gene-expression program; the ensemble of core TFs and their regulatory loops constitutes core transcriptional regulatory circuitry (CRC). Here, we summarize recent progress in computational reconstitution and biologic exploration of CRCs across various human malignancies, and consolidate the strategy and methodology for CRC discovery. We also discuss the genetic basis and therapeutic vulnerability of CRC, and highlight new frontiers and future efforts for the study of CRC in cancer. Knowledge of CRC in cancer is fundamental to understanding cancer-specific transcriptional addiction, and should provide important insight to both pathobiology and therapeutics.

TP63, SOX2, and KLF5 Establish a Core Regulatory Circuitry That Controls Epigenetic and Transcription Patterns in Esophageal Squamous Cell Carcinoma Cell Lines.

BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.

Bromodomain and extraterminal proteins foster the core transcriptional regulatory programs and confer vulnerability in liposarcoma.

Liposarcomas (LPSs) are a group of malignant mesenchymal tumors showing adipocytic differentiation. Here, to gain insight into the enhancer dysregulation and transcriptional addiction in this disease, we chart super-enhancer structures in both LPS tissues and cell lines. We identify a bromodomain and extraterminal (BET) protein-cooperated FUS-DDIT3 function in myxoid LPS and a BET protein-dependent core transcriptional regulatory circuitry consisting of FOSL2, MYC, and RUNX1 in de-differentiated LPS. Additionally, SNAI2 is identified as a crucial downstream target that enforces both proliferative and metastatic potentials to de-differentiated LPS cells. Genetic depletion of BET genes, core transcriptional factors, or SNAI2 mitigates consistently LPS malignancy. We also reveal a compelling susceptibility of LPS cells to BET protein degrader ARV-825. BET protein depletion confers additional advantages to circumvent acquired resistance to Trabectedin, a chemotherapy drug for LPS. Moreover, this study provides a framework for discovering and targeting of core oncogenic transcriptional programs in human cancers.

Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma.

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.