Publications by authors named "Behnam Nabet"

28 Publications

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Balancing cohesin eviction and retention prevents aberrant chromosomal interactions, Polycomb-mediated repression, and X-inactivation.

Mol Cell 2021 Mar 8. Epub 2021 Mar 8.

Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Depletion of architectural factors globally alters chromatin structure but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Cohesin loss disrupts the Xi superstructure, unveiling superloops between escapee genes with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure, compromises spreading of Xist RNA-Polycomb complexes, and attenuates Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss creates an Xi superloop, and cohesin retention creates Xi megadomains on the active X chromosome. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X inactivation and inter-chromosomal interactions across the genome.
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http://dx.doi.org/10.1016/j.molcel.2021.02.031DOI Listing
March 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

Discovery of a Pyrimidothiazolodiazepinone as a Potent and Selective Focal Adhesion Kinase (FAK) Inhibitor.

ACS Med Chem Lett 2021 Jan 14;12(1):30-38. Epub 2020 Dec 14.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, United States.

Focal adhesion kinase (FAK) is a tyrosine kinase with prominent roles in protein scaffolding, migration, angiogenesis, and anchorage-independent cell survival and is an attractive target for the development of cancer therapeutics. However, current FAK inhibitors display dual kinase inhibition and/or significant activity on several kinases. Although multitargeted activity is at times therapeutically advantageous, such behavior can also lead to toxicity and confound chemical-biology studies. We report a novel series of small molecules based on a tricyclic pyrimidothiazolodiazepinone core that displays both high potency and selectivity for FAK. Structure-activity relationship (SAR) studies explored modifications to the thiazole, diazepinone, and aniline "tail," which identified lead compound BJG-03-025. BJG-03-025 displays potent biochemical FAK inhibition (IC = 20 nM), excellent kinome selectivity, activity in 3D-culture breast and gastric cancer models, and favorable pharmacokinetic properties in mice. BJG-03-025 is a valuable chemical probe for evaluation of FAK-dependent biology.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812608PMC
January 2021

Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase.

Proc Natl Acad Sci U S A 2021 Jan;118(4)

Department of Microbiology, Blavatnik Institute of Harvard Medical School, Boston, MA 02115;

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT's myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT's enzymatic activities for cell viability. Using genetic complementation, we found that OGT's glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT's catalytic and noncatalytic activities affect protein abundance, we found that OGT's noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.
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http://dx.doi.org/10.1073/pnas.2016778118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848692PMC
January 2021

Charting a New Path Towards Degrading Every Protein.

Authors:
Behnam Nabet

Chembiochem 2021 Feb 26;22(3):483-484. Epub 2020 Oct 26.

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

Strategies to directly alter protein abundance such as small-molecule-induced targeted protein degradation (TPD) are innovative pharmacological modalities with promising clinical potential. Herein, I describe my experience with the development of the degradation tag (dTAG) system, which is a chemical biology strategy to induce rapid and precise degradation of any target protein. Open-source collaborative discovery has been critical for advancing the versatility and accessibility of the dTAG system and will be necessary to understand the benefits and limits of TPD-based strategies in the clinic.
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http://dx.doi.org/10.1002/cbic.202000531DOI Listing
February 2021

Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules.

Nat Commun 2020 09 18;11(1):4687. Epub 2020 Sep 18.

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

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAG-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12-tagged proteins. dTAG-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.
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http://dx.doi.org/10.1038/s41467-020-18377-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501296PMC
September 2020

Identification of a potent and selective covalent Pin1 inhibitor.

Nat Chem Biol 2020 09 1;16(9):979-987. Epub 2020 Jun 1.

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

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.
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http://dx.doi.org/10.1038/s41589-020-0550-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442691PMC
September 2020

Selective Mediator dependence of cell-type-specifying transcription.

Nat Genet 2020 07 1;52(7):719-727. Epub 2020 Jun 1.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.
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http://dx.doi.org/10.1038/s41588-020-0635-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610447PMC
July 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 Cancer Biology, 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

Exploring Targeted Degradation Strategy for Oncogenic KRAS.

Cell Chem Biol 2020 01 26;27(1):19-31.e6. Epub 2019 Dec 26.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

KRAS is the most frequently mutated oncogene found in pancreatic, colorectal, and lung cancers. Although it has been challenging to identify targeted therapies for cancers harboring KRAS mutations, KRAS can be targeted by small-molecule inhibitors that form covalent bonds with cysteine 12 (C12). Here, we designed a library of C12-directed covalent degrader molecules (PROTACs) and subjected them to a rigorous evaluation process to rapidly identify a lead compound. Our lead degrader successfully engaged CRBN in cells, bound KRASin vitro, induced CRBN/KRAS dimerization, and degraded GFP-KRAS in reporter cells in a CRBN-dependent manner. However, it failed to degrade endogenous KRAS in pancreatic and lung cancer cells. Our data suggest that inability of the lead degrader to effectively poly-ubiquitinate endogenous KRAS underlies the lack of activity. We discuss challenges for achieving targeted KRAS degradation and proposed several possible solutions which may lead to efficient degradation of endogenous KRAS.
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http://dx.doi.org/10.1016/j.chembiol.2019.12.006DOI Listing
January 2020

Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.

Cell Rep 2019 10;29(1):118-134.e8

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA; Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, MA. Electronic address:

The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.
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http://dx.doi.org/10.1016/j.celrep.2019.08.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918830PMC
October 2019

A Mutation in Histone H2B Represents a New Class of Oncogenic Driver.

Cancer Discov 2019 10 23;9(10):1438-1451. Epub 2019 Jul 23.

Division of Hematology/Oncology, University of Florida Health Cancer Center, Gainesville, Florida.

By examination of the cancer genomics database, we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K), found particularly in bladder and head and neck cancers, that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with mutant to promote colony formation, and caused a significant drift in gene expression and fundamental changes in chromatin accessibility, particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression. SIGNIFICANCE: Mutations in the core histones frequently occur in cancer and represent a new mechanism of epigenetic dysfunction that involves destabilization of the nucleosome, deregulation of chromatin accessibility, and alteration of gene expression to drive cellular transformation...
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http://dx.doi.org/10.1158/2159-8290.CD-19-0393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774836PMC
October 2019

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

It Takes Two To Target: A Study in KRAS Dimerization.

Biochemistry 2018 04 12;57(16):2289-2290. Epub 2018 Apr 12.

Department of Cancer Biology , Dana-Farber Cancer Institute , Boston , Massachusetts 02215 , United States.

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http://dx.doi.org/10.1021/acs.biochem.8b00376DOI Listing
April 2018

The dTAG system for immediate and target-specific protein degradation.

Nat Chem Biol 2018 05 26;14(5):431-441. Epub 2018 Mar 26.

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

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12 with expression of FKBP12 in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRAS loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.
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http://dx.doi.org/10.1038/s41589-018-0021-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295913PMC
May 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

YY1 Is a Structural Regulator of Enhancer-Promoter Loops.

Cell 2017 Dec 7;171(7):1573-1588.e28. Epub 2017 Dec 7.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.
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http://dx.doi.org/10.1016/j.cell.2017.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785279PMC
December 2017

UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition.

Cell Rep 2017 Oct;21(3):628-640

Division of Hematology/Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Division of Hematology/Oncology, University of Florida Health Cancer Center, Gainesville, FL 2033, USA. Electronic address:

Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.
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http://dx.doi.org/10.1016/j.celrep.2017.09.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706555PMC
October 2017

MELK is not necessary for the proliferation of basal-like breast cancer cells.

Elife 2017 09 19;6. Epub 2017 Sep 19.

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

Thorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth. This discrepancy to previous findings illuminated selectivity issues of the widely used MELK inhibitor OTSSP167, and potential off-target effects of MELK-targeting short hairpins. The different genetic and chemical tools developed here allow for the identification and validation of any causal roles MELK may play in cancer biology, which will be required to guide future MELK drug discovery efforts. Furthermore, our study provides a general framework for preclinical target validation.
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http://dx.doi.org/10.7554/eLife.26693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605198PMC
September 2017

Transcription control by the ENL YEATS domain in acute leukaemia.

Nature 2017 03 1;543(7644):270-274. Epub 2017 Mar 1.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.
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http://dx.doi.org/10.1038/nature21688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497220PMC
March 2017

Deregulation of the Ras-Erk Signaling Axis Modulates the Enhancer Landscape.

Cell Rep 2015 Aug 13;12(8):1300-13. Epub 2015 Aug 13.

Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Unrestrained receptor tyrosine kinase (RTK) signaling and epigenetic deregulation are root causes of tumorigenesis. We establish linkage between these processes by demonstrating that aberrant RTK signaling unleashed by oncogenic HRas(G12V) or loss of negative feedback through Sprouty gene deletion remodels histone modifications associated with active typical and super-enhancers. However, although both lesions disrupt the Ras-Erk axis, the expression programs, enhancer signatures, and transcription factor networks modulated upon HRas(G12V) transformation or Sprouty deletion are largely distinct. Oncogenic HRas(G12V) elevates histone 3 lysine 27 acetylation (H3K27ac) levels at enhancers near the transcription factor Gata4 and the kinase Prkcb, as well as their expression levels. We show that Gata4 is necessary for the aberrant gene expression and H3K27ac marking at enhancers, and Prkcb is required for the oncogenic effects of HRas(G12V)-driven cells. Taken together, our findings demonstrate that dynamic reprogramming of the cellular enhancer landscape is a major effect of oncogenic RTK signaling.
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http://dx.doi.org/10.1016/j.celrep.2015.06.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551578PMC
August 2015

The RNA binding protein RBM38 (RNPC1) regulates splicing during late erythroid differentiation.

PLoS One 2013 18;8(10):e78031. Epub 2013 Oct 18.

Department of Medicine (Hematology-Oncology Division), Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71(+) erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078031PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820963PMC
June 2014

A compendium of RNA-binding motifs for decoding gene regulation.

Nature 2013 Jul;499(7457):172-7

Donnelly Centre, University of Toronto, Toronto M5S 3E1, Canada.

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.
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http://dx.doi.org/10.1038/nature12311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929597PMC
July 2013

Sprouty proteins are negative regulators of interferon (IFN) signaling and IFN-inducible biological responses.

J Biol Chem 2012 Dec 16;287(50):42352-60. Epub 2012 Oct 16.

Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

Interferons (IFNs) have important antiviral and antineoplastic properties, but the precise mechanisms required for generation of these responses remain to be defined. We provide evidence that during engagement of the Type I IFN receptor (IFNR), there is up-regulation of expression of Sprouty (Spry) proteins 1, 2, and 4. Our studies demonstrate that IFN-inducible up-regulation of Spry proteins is Mnk kinase-dependent and results in suppressive effects on the IFN-activated p38 MAP kinase (MAPK), the function of which is required for transcription of interferon-stimulated genes (ISGs). Our data establish that ISG15 mRNA expression and IFN-dependent antiviral responses are enhanced in Spry1,2,4 triple knock-out mouse embryonic fibroblasts, consistent with negative feedback regulatory roles for Spry proteins in IFN-mediated signaling. In other studies, we found that siRNA-mediated knockdown of Spry1, Spry2, or Spry4 promotes IFN-inducible antileukemic effects in vitro and results in enhanced suppressive effects on malignant hematopoietic progenitors from patients with polycythemia vera. Altogether, our findings demonstrate that Spry proteins are potent regulators of Type I IFN signaling and negatively control induction of Type I IFN-mediated biological responses.
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http://dx.doi.org/10.1074/jbc.M112.400721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516778PMC
December 2012

Identification of a putative network of actin-associated cytoskeletal proteins in glomerular podocytes defined by co-purified mRNAs.

PLoS One 2009 Aug 4;4(8):e6491. Epub 2009 Aug 4.

Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

The glomerular podocyte is a highly specialized and polarized kidney cell type that contains major processes and foot processes that extend from the cell body. Foot processes from adjacent podocytes form interdigitations with those of adjacent cells, thereby creating an essential intercellular junctional domain of the renal filtration barrier known as the slit diaphragm. Interesting parallels have been drawn between the slit diaphragm and other sites of cell-cell contact by polarized cells. Notably mutations in several genes encoding proteins localized to the foot processes can lead to proteinuria and kidney failure. Mutations in the Wilm's tumor gene (WT1) can also lead to kidney disease and one isoform of WT1, WT1(+KTS), has been proposed to regulate gene expression post-transcriptionally. We originally sought to identify mRNAs associated with WT1(+KTS) through an RNA immunoprecipitation and microarray approach, hypothesizing that the proteins encoded by these mRNAs might be important for podocyte morphology and function. We identified a subset of mRNAs that were remarkably enriched for transcripts encoding actin-binding proteins and other cytoskeletal proteins including several that are localized at or near the slit diaphragm. Interestingly, these mRNAs included those of alpha-actinin-4 and non-muscle myosin IIA that are mutated in genetic forms of kidney disease. However, isolation of the mRNAs occurred independently of the expression of WT1, suggesting that the identified mRNAs were serendipitously co-purified on the basis of co-association in a common subcellular fraction. Mass spectroscopy revealed that other components of the actin cytoskeleton co-purified with these mRNAs, namely actin, tubulin, and elongation factor 1alpha. We propose that these mRNAs encode a number of proteins that comprise a highly specialized protein interactome underlying the slit diaphragm. Collectively, these gene products and their interactions may prove to be important for the structural integrity of the actin cytoskeleton in podocytes as well as other polarized cell types.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006491PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2714980PMC
August 2009

ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing.

Mol Cell 2009 Mar;33(5):591-601

Department of Medicine, Renal Division, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Cell-type-specific expression of epithelial and mesenchymal isoforms of Fibroblast Growth Factor Receptor 2 (FGFR2) is achieved through tight regulation of mutually exclusive exons IIIb and IIIc, respectively. Using an application of cell-based cDNA expression screening, we identified two paralogous epithelial cell-type-specific RNA-binding proteins that are essential regulators of FGFR2 splicing. Ectopic expression of either protein in cells that express FGFR2-IIIc caused a switch in endogenous FGFR2 splicing to the epithelial isoform. Conversely, knockdown of both factors in cells that express FGFR2-IIIb by RNA interference caused a switch from the epithelial to mesenchymal isoform. These factors also regulate splicing of CD44, p120-Catenin (CTNND1), and hMena (ENAH), three transcripts that undergo changes in splicing during the epithelial-to-mesenchymal transition (EMT). These studies suggest that Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are coordinators of an epithelial cell-type-specific splicing program.
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http://dx.doi.org/10.1016/j.molcel.2009.01.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2702247PMC
March 2009

Predictors of prognosis in patients with olfactory disturbance.

Ann Neurol 2008 Feb;63(2):159-66

Department of Otorhinolaryngology: Head and Neck Surgery, Smell and Taste Center, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Objective: Although olfaction is often compromised by such factors as head trauma, viruses, and toxic agents, the olfactory epithelium and sectors of the olfactory bulb have the potential for regeneration. This study assessed the degree to which olfactory function changes over time in patients presenting to a university-based smell and taste center with complaints of olfactory dysfunction and the influences of etiology (eg, head trauma, upper respiratory infection), sex, age, smoking behavior, degree of initial dysfunction, and other factors on such change.

Methods: Well-validated odor identification tests were administered to 542 patients on 2 occasions separated from one another by 3 months to 24 years. Multivariable regression and chi2 analyses assessed the influences of the variables on the longitudinal changes in olfactory test scores.

Results: On average, smell test scores improved modestly over time. Patient age, severity of initial olfactory loss, and the duration of dysfunction at first testing were significant predictors of the amount of the change. Etiology, sex, time between the two test administrations, and initial smoking behavior were not significant predictors. The percentage of anosmic and microsmic patients exhibiting statistically significant change in function was 56.72 and 42.86%, respectively. However, only 11.31% of anosmic and 23.31% of microsmic patients regained normal age-related function over time.

Interpretation: Some recovery can be expected in a significant number of patients who experience smell loss. The amount of recovery depends on the degree of initial loss, age, and the duration of loss. Etiology, per se, is not a significant determinant of prognosis, in contrast with what is commonly believed.
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http://dx.doi.org/10.1002/ana.21293DOI Listing
February 2008