Histone demethylase KDM5D upregulation drives sex differences in colon cancer.

Sex exerts a profound impact on cancer incidence, spectrum and outcomes, yet the molecular and genetic bases of such sex differences are ill-defined and presumptively ascribed to X-chromosome genes and sex hormones1. Such sex differences are particularly prominent in colorectal cancer (CRC) in which men experience higher metastases and mortality. A murine CRC model, engineered with an inducible transgene encoding oncogenic mutant KRASG12D and conditional null alleles of Apc and Trp53 tumour suppressors (designated iKAP)2, revealed higher metastases and worse outcomes specifically in males with oncogenic mutant KRAS (KRAS*) CRC. Integrated cross-species molecular and transcriptomic analyses identified Y-chromosome gene histone demethylase KDM5D as a transcriptionally upregulated gene driven by KRAS*-mediated activation of the STAT4 transcription factor. KDM5D-dependent chromatin mark and transcriptome changes showed repression of regulators of the epithelial cell tight junction and major histocompatibility complex class I complex components. Deletion of Kdm5d in iKAP cancer cells increased tight junction integrity, decreased cell invasiveness and enhanced cancer cell killing by CD8+ T cells. Conversely, iAP mice engineered with a Kdm5d transgene to provide constitutive Kdm5d expression specifically in iAP cancer cells showed an increased propensity for more invasive tumours in vivo. Thus, KRAS*-STAT4-mediated upregulation of Y chromosome KDM5D contributes substantially to the sex differences in KRAS* CRC by means of its disruption of cancer cell adhesion properties and tumour immunity, providing an actionable therapeutic strategy for metastasis risk reduction for men afflicted with KRAS* CRC.

High enhancer activity is an epigenetic feature of HPV negative atypical head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with significant mortality and frequent recurrence. Prior efforts to transcriptionally classify HNSCC into groups of varying prognoses have identified four accepted molecular subtypes of the disease: Atypical (AT), Basal (BA), Classical (CL), and Mesenchymal (MS). Here, we investigate the active enhancer landscapes of these subtypes using representative HNSCC cell lines and identify samples belonging to the AT subtype as having increased enhancer activity compared to the other 3 HNSCC subtypes. Cell lines belonging to the AT subtype are more resistant to enhancer-blocking bromodomain inhibitors (BETi). Examination of nascent transcripts reveals that both AT TCGA tumors and cell lines express higher levels of enhancer RNA (eRNA) transcripts for enhancers controlling BETi resistance pathways, such as lipid metabolism and MAPK signaling. Additionally, investigation of higher-order chromatin structure suggests more enhancer-promoter (E-P) contacts in the AT subtype, including on genes identified in the eRNA analysis. Consistently, known BETi resistance pathways are upregulated upon exposure to these inhibitors. Together, our results identify that the AT subtype of HNSCC is associated with higher enhancer activity, resistance to enhancer blockade, and increased signaling through pathways that could serve as future targets for sensitizing HNSCC to BET inhibition.

Chromatin state dynamics confers specific therapeutic strategies in enhancer subtypes of colorectal cancer.

OBJECTIVE: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described. DESIGN: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin. RESULTS: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFßi, mTORi and SRCi) for EpiC groups. CONCLUSION: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.

Chromatin state profiling reveals PRC2 inhibition as a therapeutic target in NRAS-mutant melanoma.

Recently, we have generated 284 epigenomic maps in melanoma. Using chromatin state profiling we identify an association of NRAS-mutants with bivalent Histone H3 lysine 27 trimethylation (H3K27me3) and broad H3K4me3 domains. Reprogramming of bivalent H3K27me3 occurs on critical invasive-regulators and its resolution using Enhancer of Zeste Homolog 2 (EZH2) inhibition reduces invasive capacity and tumor burden in NRAS-mutant patient samples.

Enhancer reprogramming in PRC2-deficient malignant peripheral nerve sheath tumors induces a targetable de-differentiated state.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that frequently harbor genetic alterations in polycomb repressor complex 2 (PRC2) components-SUZ12 and EED. Here, we show that PRC2 loss confers a dedifferentiated early neural-crest phenotype which is exclusive to PRC2-mutant MPNSTs and not a feature of neurofibromas. Neural crest phenotype in PRC2 mutant MPNSTs was validated via cross-species comparative analysis using spontaneous and transgenic MPNST models. Systematic chromatin state profiling of the MPNST cells showed extensive epigenomic reprogramming or chromatin states associated with PRC2 loss and identified gains of active enhancer states/super-enhancers on early neural crest regulators in PRC2-mutant conditions around genomic loci that harbored repressed/poised states in PRC2-WT MPNST cells. Consistently, inverse correlation between H3K27me3 loss and H3K27Ac gain was noted in MPNSTs. Epigenetic editing experiments established functional roles for enhancer gains on DLX5-a key regulator of neural crest phenotype. Consistently, blockade of enhancer activity by bromodomain inhibitors specifically suppressed this neural crest phenotype and tumor burden in PRC2-mutant PDXs. Together, these findings reveal accumulation of dedifferentiated neural crest like state in PRC2-mutant MPNSTs that can be targeted by enhancer blockade.

Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients.

Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma.

Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.

Atypical plant homeodomain of UBR7 functions as an H2BK120Ub ligase and breast tumor suppressor.

The roles of Plant Homeodomain (PHD) fingers in catalysis of histone modifications are unknown. We demonstrated that the PHD finger of Ubiquitin Protein Ligase E3 Component N-Recognin7 (UBR7) harbors E3 ubiquitin ligase activity toward monoubiquitination of histone H2B at lysine120 (H2BK120Ub). Purified PHD finger or full-length UBR7 monoubiquitinated H2BK120 in vitro, and loss of UBR7 drastically reduced H2BK120Ub genome-wide binding sites in MCF10A cells. Low UBR7 expression was correlated with occurrence of triple-negative breast cancer and metastatic tumors. Consistently, UBR7 knockdown enhanced the invasiveness, induced epithelial-to-mesenchymal transition and promoted metastasis. Conversely, ectopic expression of UBR7 restored these cellular phenotypes and reduced tumor growth. Mechanistically, UBR7 loss reduced H2BK120Ub levels on cell adhesion genes, including CDH4, and upregulated the Wnt/ß-Catenin signaling pathway. CDH4 overexpression could partially revert UBR7-dependent cellular phenotypes. Collectively, our results established UBR7 as a histone H2B monoubiquitin ligase that suppresses tumorigenesis and metastasis of triple-negative breast cancer.

Author Correction: Mutations in the SWI/SNF complex induce a targetable dependence on oxidative phosphorylation in lung cancer.

In the version of this article originally published, information regarding several funding sources was omitted from the Acknowledgements section. The following sentences should have been included: "This work was supported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shots Program, the UT Lung SPORE 5 P50 CA07090, and the MD Anderson Cancer Center Support Grant P30CA01667. V.P is supported by R01CA155196-01A1 from the National Cancer Institute." Also, reference 18 was incorrect. The original reference was: Kim, E. S. et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov. 1, 44-53 (2011). It should have been: Papadimitrakopoulou, V. et al. The BATTLE-2 study: a biomarker-integrated targeted therapy study in previously treated patients with advanced non-small-cell lung cancer. J Clin. Oncol. 34, 3638-3647 (2016). The errors have been corrected in the HTML and PDF versions of this article.

KMT2D/MLL2 inactivation is associated with recurrence in adult-type granulosa cell tumors of the ovary.

Adult-type granulosa cell tumors of the ovary (aGCTs) are rare gynecologic malignancies that exhibit a high frequency of somatic FOXL2 c.C402G (p.Cys134Trp) mutation. Treatment of relapsed aGCT remains a significant clinical challenge. Here we show, using whole-exome and cancer gene panel sequencing of 79 aGCTs from two independent cohorts, that truncating mutation of the histone lysine methyltransferase gene KMT2D (also known as MLL2) is a recurrent somatic event in aGCT. Mono-allelic KMT2D-truncating mutations are more frequent in recurrent (10/44, 23%) compared with primary (1/35, 3%) aGCTs (p = 0.02, two-sided Fisher's exact test). IHC detects additional non-KMT2D-mutated aGCTs with loss of nuclear KMT2D expression, suggesting that non-genetic KMT2D inactivation may occur in this tumor type. These findings identify KMT2D inactivation as a novel driver event in aGCTs and suggest that mutation of this gene may increase the risk of disease recurrence.

Mutations in the SWI/SNF complex induce a targetable dependence on oxidative phosphorylation in lung cancer.

Lung cancer is a devastating disease that remains a top cause of cancer mortality. Despite improvements with targeted and immunotherapies, the majority of patients with lung cancer lack effective therapies, underscoring the need for additional treatment approaches. Genomic studies have identified frequent alterations in components of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A. To understand the mechanisms of tumorigenesis driven by mutations in this complex, we developed a genetically engineered mouse model of lung adenocarcinoma by ablating Smarca4 in the lung epithelium. We demonstrate that Smarca4 acts as a bona fide tumor suppressor and cooperates with p53 loss and Kras activation. Gene expression analyses revealed the signature of enhanced oxidative phosphorylation (OXPHOS) in SMARCA4 mutant tumors. We further show that SMARCA4 mutant cells have enhanced oxygen consumption and increased respiratory capacity. Importantly, SMARCA4 mutant lung cancer cell lines and xenograft tumors have marked sensitivity to inhibition of OXPHOS by a novel small molecule, IACS-010759, that is under clinical development. Mechanistically, we show that SMARCA4-deficient cells have a blunted transcriptional response to energy stress creating a therapeutically exploitable synthetic lethal interaction. These findings provide the mechanistic basis for further development of OXPHOS inhibitors as therapeutics against SWI/SNF mutant tumors.

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues.

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Systematic Epigenomic Analysis Reveals Chromatin States Associated with Melanoma Progression.

The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression.

Global Developmental Gene Programing Involves a Nuclear Form of Fibroblast Growth Factor Receptor-1 (FGFR1).

Genetic studies have placed the Fgfr1 gene at the top of major ontogenic pathways that enable gastrulation, tissue development and organogenesis. Using genome-wide sequencing and loss and gain of function experiments the present investigation reveals a mechanism that underlies global and direct gene regulation by the nuclear form of FGFR1, ensuring that pluripotent Embryonic Stem Cells differentiate into Neuronal Cells in response to Retinoic Acid. Nuclear FGFR1, both alone and with its partner nuclear receptors RXR and Nur77, targets thousands of active genes and controls the expression of pluripotency, homeobox, neuronal and mesodermal genes. Nuclear FGFR1 targets genes in developmental pathways represented by Wnt/ß-catenin, CREB, BMP, the cell cycle and cancer-related TP53 pathway, neuroectodermal and mesodermal programing networks, axonal growth and synaptic plasticity pathways. Nuclear FGFR1 targets the consensus sequences of transcription factors known to engage CREB-binding protein, a common coregulator of transcription and established binding partner of nuclear FGFR1. This investigation reveals the role of nuclear FGFR1 as a global genomic programmer of cell, neural and muscle development.

NGF-induced cell differentiation and gene activation is mediated by integrative nuclear FGFR1 signaling (INFS).

Nerve growth factor (NGF) is the founding member of the polypeptide neurotrophin family responsible for neuronal differentiation. To determine whether the effects of NGF rely upon novel Integrative Nuclear FGF Receptor-1 (FGFR1) Signaling (INFS) we utilized the PC12 clonal cell line, a long-standing benchmark model of sympathetic neuronal differentiation. We demonstrate that NGF increases expression of the fgfr1 gene and promotes trafficking of FGFR1 protein from cytoplasm to nucleus by inhibiting FGFR1 nuclear export. Nuclear-targeted dominant negative FGFR1 antagonizes NGF-induced neurite outgrowth, doublecortin (dcx) expression and activation of the tyrosine hydroxylase (th) gene promoter, while active constitutive nuclear FGFR1 mimics the effects of NGF. NGF increases the expression of dcx, th, ßIII tubulin, nurr1 and nur77, fgfr1and fibroblast growth factor-2 (fgf-2) genes, while enhancing binding of FGFR1and Nur77/Nurr1 to those genes. NGF activates transcription from isolated NurRE and NBRE motifs. Nuclear FGFR1 transduces NGF activation of the Nur dimer and raises basal activity of the Nur monomer. Cooperation of nuclear FGFR1 with Nur77/Nurr1 in NGF signaling expands the integrative functions of INFS to include NGF, the first discovered pluripotent neurotrophic factor.