Publications by authors named "Charles Y Lin"

60 Publications

ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma.

Cancer Discov 2021 Mar 19. Epub 2021 Mar 19.

Texas Children's Cancer Center.

Over 60% of supratentorial (ST) ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE driven ZRfus tumors by CUT&RUN, ChIP, ATAC, and RNA sequencing and compared to human ZRfus driven ependymoma. In addition to direct canonical NF-kB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with Plagl family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional co-activators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by Plagl transcription factor proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks.
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http://dx.doi.org/10.1158/2159-8290.CD-20-1066DOI Listing
March 2021

HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling.

Circulation 2021 May 8;143(19):1874-1890. Epub 2021 Mar 8.

Department of Medicine, Division of Cardiology (J.G.T., S.A.W., B.P., R.A.B., Y.-H.L., K.M.D.-D., M.A.C., L.M., K.C.W., M.P.Y.L., T.A.M.), University of Colorado Anschutz Medical Campus, Aurora.

Background: Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction.

Methods: Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts.

Results: HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements.

Conclusions: These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.046462DOI Listing
May 2021

Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity.

Cell Rep Med 2021 Jan 19;2(1):100188. Epub 2021 Jan 19.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop.
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http://dx.doi.org/10.1016/j.xcrm.2020.100188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817874PMC
January 2021

Impact of radiopacified bone cement on radiotherapy dose calculation.

Phys Imaging Radiat Oncol 2020 Apr 20;14:12-16. Epub 2020 May 20.

Cancer Care Services, Royal Brisbane & Women's Hospital, Herston, QLD 4029, Australia.

Background And Purpose: Radiopacifiers are introduced to bone cements to provide the appearance of bone in kilovoltage (kV) radiographic images. For higher energy megavoltage (MV) radiotherapy treatment beams, however, these radiopacifiers do not cause a bone-like perturbation of dose. This study therefore aimed to determine the impact of the barium-contrasted plastic-based cement materials on radiotherapy dose calculations.

Materials And Methods: The radiological properties of a physical sample of bone cement were characterised by computed tomography (CT) imaging and transmission measurements. Monte Carlo simulations of percentage depth-dose profiles were performed to determine the possible dose error for MV treatment beams. Dose differences were then investigated for clinical volumetric modulated radiotherapy treatment plans, with and without density overrides applied.

Results: Differences of up to 7% were observed at the downstream interface of a 0.6 cm thick bone cement layer, compared to bone. Differences in planning target volume dose-volume metrics varied between -0.5% and 2.0%.

Conclusion: Before planning radiotherapy treatments for patients who have undergone cranioplasty, every effort should be made to identify whether a radiopacified bone cement has been implanted. Density overrides should be applied to minimise dose calculation errors, whenever bone cement is used.
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http://dx.doi.org/10.1016/j.phro.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7807530PMC
April 2020

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer.

Cell 2021 Jan 14;184(2):384-403.e21. Epub 2021 Jan 14.

Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.
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http://dx.doi.org/10.1016/j.cell.2020.12.031DOI Listing
January 2021

In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice.

Nature 2021 01 6;589(7843):608-614. Epub 2021 Jan 6.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.
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http://dx.doi.org/10.1038/s41586-020-03086-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872200PMC
January 2021

Modulating Androgen Receptor-Driven Transcription in Prostate Cancer with Selective CDK9 Inhibitors.

Cell Chem Biol 2021 Feb 20;28(2):134-147.e14. Epub 2020 Oct 20.

Kronos Bio, Inc., Cambridge, MA 02139, USA.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.
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http://dx.doi.org/10.1016/j.chembiol.2020.10.001DOI Listing
February 2021

Computational Drug Repositioning Identifies Potentially Active Therapies for Chordoma.

Neurosurgery 2021 01;88(2):428-436

Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Background: Chordomas are aggressive bone tumors that often recur despite maximal resection and adjuvant radiation. To date there are no Food and Drug Administration (FDA)-approved chemotherapies. Computational drug repositioning is an expanding approach to identify pharmacotherapies for clinical trials.

Objective: To identify FDA-approved compounds for repurposing in chordoma.

Methods: Previously identified highly differentially expressed genes from chordoma tissue samples at our institution were compared with pharmacogenomic interactions in the Comparative Toxicogenomics Database (CTD) using ksRepo, a drug-repositioning platform. Compounds selected by ksRepo were then validated in CH22 and UM-Chor1 human chordoma cells in Vitro.

Results: A total of 13 chemical compounds were identified in silico from the CTD, and 6 were selected for preclinical validation in human chordoma cell lines based on their clinical relevance. Of these, 3 identified drugs are FDA-approved chemotherapies for other malignancies (cisplatin, cytarabine, and lucanthone). Cytarabine, a deoxyribonucleic acid polymerase inhibitor approved for the treatment of various leukemias, exhibited a significant concentration-dependent effect against CH22 and UM-Chor1 cells when compared to positive (THZ1) and negative (venetoclax) controls. Tretinoin exhibited a significant concentration-dependent cytotoxic effect in CH22, sacral chordoma-derived cell lines but to a much lesser extent in UM-Chor1, a cell line derived from skull base chordoma.

Conclusion: Cytarabine administration reduces the viability of human chordoma cells. The equally effective reduction in viability seen with tretinoin seems to be cell line dependent. Based on our findings, we recommend the evaluation of cytarabine and tretinoin in an expanded set of human chordoma cell lines and animal models.
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http://dx.doi.org/10.1093/neuros/nyaa398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803434PMC
January 2021

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

J Clin Invest 2020 11;130(11):5875-5892

Division of Cancer Therapeutics, Institute of Cancer Research (ICR), London and Royal Marsden NHS Trust, Sutton, United Kingdom.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.
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http://dx.doi.org/10.1172/JCI134132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598076PMC
November 2020

Discovery of a selective inhibitor of doublecortin like kinase 1.

Nat Chem Biol 2020 06 6;16(6):635-643. Epub 2020 Apr 6.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.
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http://dx.doi.org/10.1038/s41589-020-0506-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246176PMC
June 2020

Springing an evolutionary trap on cancer.

Authors:
Charles Y Lin

Nat Genet 2020 04;52(4):361-362

Kronos Bio, Cambridge, MA, USA.

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http://dx.doi.org/10.1038/s41588-020-0608-3DOI Listing
April 2020

Mechanistic basis and efficacy of targeting the β-catenin-TCF7L2-JMJD6-c-Myc axis to overcome resistance to BET inhibitors.

Blood 2020 04;135(15):1255-1269

The University of Texas MD Anderson Cancer Center, Houston TX.

The promising activity of BET protein inhibitors (BETi's) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin-TCF7L2-JMJD6-c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin-TCF7L2-JMJD6-c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.
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http://dx.doi.org/10.1182/blood.2019002922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146021PMC
April 2020

AMP-activated protein kinase links acetyl-CoA homeostasis to BRD4 recruitment in acute myeloid leukemia.

Blood 2019 12;134(24):2183-2194

Program in Developmental Biology.

Altered metabolism fuels 2 hallmark properties of cancer cells: unlimited proliferation and differentiation blockade. Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of bioenergetics crucial for glucose metabolism in acute myeloid leukemia (AML), and its inhibition delays leukemogenesis, but whether the metabolic function of AMPK alters the AML epigenome remains unknown. Here, we demonstrate that AMPK maintains the epigenome of MLL-rearranged AML by linking acetyl-coenzyme A (CoA) homeostasis to Bromodomain and Extra-Terminal domain (BET) protein recruitment to chromatin. AMPK deletion reduced acetyl-CoA and histone acetylation, displacing BET proteins from chromatin in leukemia-initiating cells. In both mouse and patient-derived xenograft AML models, treating with AMPK and BET inhibitors synergistically suppressed AML. Our results provide a therapeutic rationale to target AMPK and BET for AML therapy.
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http://dx.doi.org/10.1182/blood.2019001076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908829PMC
December 2019

IRF2 is a master regulator of human keratinocyte stem cell fate.

Nat Commun 2019 10 14;10(1):4676. Epub 2019 Oct 14.

Chemical Biology & Therapeutics, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Forum 1 Novartis Campus, CH-4056, Basel, Switzerland.

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.
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http://dx.doi.org/10.1038/s41467-019-12559-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791852PMC
October 2019

High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program.

Nat Commun 2019 09 25;10(1):4358. Epub 2019 Sep 25.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Systemic metabolic alterations associated with increased consumption of saturated fat and obesity are linked with increased risk of prostate cancer progression and mortality, but the molecular underpinnings of this association are poorly understood. Here, we demonstrate in a murine prostate cancer model, that high-fat diet (HFD) enhances the MYC transcriptional program through metabolic alterations that favour histone H4K20 hypomethylation at the promoter regions of MYC regulated genes, leading to increased cellular proliferation and tumour burden. Saturated fat intake (SFI) is also associated with an enhanced MYC transcriptional signature in prostate cancer patients. The SFI-induced MYC signature independently predicts prostate cancer progression and death. Finally, switching from a high-fat to a low-fat diet, attenuates the MYC transcriptional program in mice. Our findings suggest that in primary prostate cancer, dietary SFI contributes to tumour progression by mimicking MYC over expression, setting the stage for therapeutic approaches involving changes to the diet.
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http://dx.doi.org/10.1038/s41467-019-12298-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761092PMC
September 2019

PAX8 activates metabolic genes via enhancer elements in Renal Cell Carcinoma.

Nat Commun 2019 08 20;10(1):3739. Epub 2019 Aug 20.

Disease Area Oncology, Novartis Institute for Biomedical Research, Basel, Switzerland.

Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity.
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http://dx.doi.org/10.1038/s41467-019-11672-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702156PMC
August 2019

Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation.

Circ Res 2019 09 14;125(7):662-677. Epub 2019 Aug 14.

From the Department of Medicine, Division of Cardiology (M.S.S., R.A.B., M.B.F., A.S.R., B.Y.E., K.A.K., M.A.C., K.S., M.P.Y.L., T.A.M.), University of Colorado Anschutz Medical Campus, Aurora.

Rationale: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown.

Objective: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart.

Methods And Results: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-β (transforming growth factor-β) signaling to stimulate conversion of quiescent cardiac fibroblasts into ()-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for in TGF-β-mediated cardiac fibroblast activation.

Conclusions: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for . The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310347PMC
September 2019

A C19MC-LIN28A-MYCN Oncogenic Circuit Driven by Hijacked Super-enhancers Is a Distinct Therapeutic Vulnerability in ETMRs: A Lethal Brain Tumor.

Cancer Cell 2019 07;36(1):51-67.e7

Children's Brain Tumor Research Centre, Queen's Medical Centre University of Nottingham, Nottingham NG72UH, UK.

Embryonal tumors with multilayered rosettes (ETMRs) are highly lethal infant brain cancers with characteristic amplification of Chr19q13.41 miRNA cluster (C19MC) and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a C19MC-LIN28A-MYCN circuit fueled by multiple complex regulatory loops including an MYCN core transcriptional network and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. Our data show that this powerful oncogenic circuit, which entraps an early neural lineage network, is potently abrogated by bromodomain inhibitor JQ1, leading to ETMR cell death.
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http://dx.doi.org/10.1016/j.ccell.2019.06.002DOI Listing
July 2019

Chromatin landscapes reveal developmentally encoded transcriptional states that define human glioblastoma.

J Exp Med 2019 05 4;216(5):1071-1090. Epub 2019 Apr 4.

Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA

Glioblastoma is an incurable brain cancer characterized by high genetic and pathological heterogeneity. Here, we mapped active chromatin landscapes with gene expression, whole exomes, copy number profiles, and DNA methylomes across 44 patient-derived glioblastoma stem cells (GSCs), 50 primary tumors, and 10 neural stem cells (NSCs) to identify essential super-enhancer (SE)-associated genes and the core transcription factors that establish SEs and maintain GSC identity. GSCs segregate into two groups dominated by distinct enhancer profiles and unique developmental core transcription factor regulatory programs. Group-specific transcription factors enforce GSC identity; they exhibit higher activity in glioblastomas versus NSCs, are associated with poor clinical outcomes, and are required for glioblastoma growth in vivo. Although transcription factors are commonly considered undruggable, group-specific enhancer regulation of the MAPK/ERK pathway predicts sensitivity to MEK inhibition. These data demonstrate that transcriptional identity can be leveraged to identify novel dependencies and therapeutic approaches.
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http://dx.doi.org/10.1084/jem.20190196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504206PMC
May 2019

Development of a Selective CDK7 Covalent Inhibitor Reveals Predominant Cell-Cycle Phenotype.

Cell Chem Biol 2019 06 21;26(6):792-803.e10. Epub 2019 Mar 21.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biology Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.
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http://dx.doi.org/10.1016/j.chembiol.2019.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588464PMC
June 2019

Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription.

Cell Chem Biol 2019 05 14;26(5):711-723.e14. Epub 2019 Mar 14.

Division of Oncology, Departments of Medicine and Pathology Stanford School of Medicine, Stanford, CA 94305, USA.

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in vivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.
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http://dx.doi.org/10.1016/j.chembiol.2019.02.009DOI Listing
May 2019

Small-molecule targeting of brachyury transcription factor addiction in chordoma.

Nat Med 2019 02 21;25(2):292-300. Epub 2019 Jan 21.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Chordoma is a primary bone cancer with no approved therapy. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T (brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors. In chordoma, we find that T is associated with a 1.5-Mb region containing 'super-enhancers' and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of T gene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.
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http://dx.doi.org/10.1038/s41591-018-0312-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633917PMC
February 2019

Non-overlapping Control of Transcriptome by Promoter- and Super-Enhancer-Associated Dependencies in Multiple Myeloma.

Cell Rep 2018 12;25(13):3693-3705.e6

Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA; VA Boston Healthcare System, Boston, MA, USA. Electronic address:

The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy.
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http://dx.doi.org/10.1016/j.celrep.2018.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407615PMC
December 2018

Trisomy of a Down Syndrome Critical Region Globally Amplifies Transcription via HMGN1 Overexpression.

Cell Rep 2018 11;25(7):1898-1911.e5

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of Harvard and MIT, Cambridge, MA, USA. Electronic address:

Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted.
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http://dx.doi.org/10.1016/j.celrep.2018.10.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321629PMC
November 2018

Targeting MYC dependency in ovarian cancer through inhibition of CDK7 and CDK12/13.

Elife 2018 11 13;7. Epub 2018 Nov 13.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, United States.

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.
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http://dx.doi.org/10.7554/eLife.39030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251623PMC
November 2018

Mutant NPM1 Maintains the Leukemic State through HOX Expression.

Cancer Cell 2018 09;34(3):499-512.e9

Stem Cell and Regenerative Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Texas Children's Hospital and Houston Methodist Hospital, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

NPM1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (AML). In AML cells, NPM1 mutations result in abnormal cytoplasmic localization of the mutant protein (NPM1c); however, it is unknown whether NPM1c is required to maintain the leukemic state. Here, we show that loss of NPM1c from the cytoplasm, either through nuclear relocalization or targeted degradation, results in immediate downregulation of homeobox (HOX) genes followed by differentiation. Finally, we show that XPO1 inhibition relocalizes NPM1c to the nucleus, promotes differentiation of AML cells, and prolongs survival of Npm1-mutated leukemic mice. We describe an exquisite dependency of NPM1-mutant AML cells on NPM1c, providing the rationale for the use of nuclear export inhibitors in AML with mutated NPM1.
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http://dx.doi.org/10.1016/j.ccell.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159911PMC
September 2018

Combinatorial inhibition of PTPN12-regulated receptors leads to a broadly effective therapeutic strategy in triple-negative breast cancer.

Nat Med 2018 05 26;24(4):505-511. Epub 2018 Mar 26.

Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer diagnosed in more than 200,000 women each year and is recalcitrant to targeted therapies. Although TNBCs harbor multiple hyperactive receptor tyrosine kinases (RTKs), RTK inhibitors have been largely ineffective in TNBC patients thus far. We developed a broadly effective therapeutic strategy for TNBC that is based on combined inhibition of receptors that share the negative regulator PTPN12. Previously, we and others identified the tyrosine phosphatase PTPN12 as a tumor suppressor that is frequently inactivated in TNBC. PTPN12 restrains several RTKs, suggesting that PTPN12 deficiency leads to aberrant activation of multiple RTKs and a co-dependency on these receptors. This in turn leads to the therapeutic hypothesis that PTPN12-deficient TNBCs may be responsive to combined RTK inhibition. However, the repertoire of RTKs that are restrained by PTPN12 in human cells has not been systematically explored. By methodically identifying the suite of RTK substrates (MET, PDGFRβ, EGFR, and others) inhibited by PTPN12, we rationalized a combination RTK-inhibitor therapy that induced potent tumor regression across heterogeneous models of TNBC. Orthogonal approaches revealed that PTPN12 was recruited to and inhibited these receptors after ligand stimulation, thereby serving as a feedback mechanism to limit receptor signaling. Cancer-associated mutation of PTPN12 or reduced PTPN12 protein levels diminished this feedback mechanism, leading to aberrant activity of these receptors. Restoring PTPN12 protein levels restrained signaling from RTKs, including PDGFRβ and MET, and impaired TNBC survival. In contrast with single agents, combined inhibitors targeting the PDGFRβ and MET receptors induced the apoptosis in TNBC cells in vitro and in vivo. This therapeutic strategy resulted in tumor regressions in chemo-refractory patient-derived TNBC models. Notably, response correlated with PTPN12 deficiency, suggesting that impaired receptor feedback may establish a combined addiction to these proto-oncogenic receptors. Taken together, our data provide a rationale for combining RTK inhibitors in TNBC and other malignancies that lack receptor-activating mutations.
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http://dx.doi.org/10.1038/nm.4507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675908PMC
May 2018

NRL and CRX Define Photoreceptor Identity and Reveal Subgroup-Specific Dependencies in Medulloblastoma.

Cancer Cell 2018 03;33(3):435-449.e6

Institut Curie - Recherche, Laboratoire 110, Centre Universitaire, Orsay Cedex 91405, France; INSERM U1021, Centre Universitaire, Orsay 91405, France; CNRS UMR 3347, Centre Universitaire, Orsay 91405, France; Université Paris Sud-11, 91405 Orsay, France; PSL Research University, Paris, France. Electronic address:

Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.
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http://dx.doi.org/10.1016/j.ccell.2018.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368680PMC
March 2018

BET bromodomain proteins regulate enhancer function during adipogenesis.

Proc Natl Acad Sci U S A 2018 02 14;115(9):2144-2149. Epub 2018 Feb 14.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115;

Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARγ and C/EBPα, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARγ and C/EBPα expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARγ and C/EBPα. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation. Inhibition of the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins impedes BRD4 occupancy at these de novo enhancers and disrupts transcription of and , thereby blocking adipogenesis. Furthermore, silencing of these BRD4-occupied distal regulatory elements at the locus by CRISPRi demonstrates a critical role for these enhancers in the control of gene expression and adipogenesis in 3T3L1s. Together, these data establish BET bromodomain proteins as time- and context-dependent coactivators of the adipocyte cell state transition.
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http://dx.doi.org/10.1073/pnas.1711155115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834672PMC
February 2018

Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma.

Nat Genet 2018 04 29;50(4):515-523. Epub 2018 Jan 29.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Amplification of the locus encoding the oncogenic transcription factor MYCN is a defining feature of high-risk neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of MYCN perturbation in neuroblastoma. At oncogenic levels, MYCN associates with E-box binding motifs in an affinity-dependent manner, binding to strong canonical E-boxes at promoters and invading abundant weaker non-canonical E-boxes clustered at enhancers. Loss of MYCN leads to a global reduction in transcription, which is most pronounced at MYCN target genes with the greatest enhancer occupancy. These highly occupied MYCN target genes show tissue-specific expression and are linked to poor patient survival. The activity of genes with MYCN-occupied enhancers is dependent on the tissue-specific transcription factor TWIST1, which co-occupies enhancers with MYCN and is required for MYCN-dependent proliferation. These data implicate tissue-specific enhancers in defining often highly tumor-specific 'MYC target gene signatures' and identify disruption of the MYCN enhancer regulatory axis as a promising therapeutic strategy in neuroblastoma.
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http://dx.doi.org/10.1038/s41588-018-0044-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310397PMC
April 2018