Publications by authors named "Berkley E Gryder"

27 Publications

  • Page 1 of 1

BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma.

Nat Commun 2021 11 26;12(1):6924. Epub 2021 Nov 26.

Genetics Branch, NCI, NIH, Bethesda, MD, USA.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.
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http://dx.doi.org/10.1038/s41467-021-27176-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8626462PMC
November 2021

SNAI2-Mediated Repression of Protects Rhabdomyosarcoma from Ionizing Radiation.

Cancer Res 2021 11 30;81(21):5451-5463. Epub 2021 Aug 30.

Greehey Children's Cancer Research Institute (GCCRI), UT Health Science Center, San Antonio, Texas.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene . Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity and . SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of . Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-4191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8669772PMC
November 2021

Lysine Demethylase 5A is Required for MYC Driven Transcription in Multiple Myeloma.

Blood Cancer Discov 2021 07 10;2(4):370-387. Epub 2021 Apr 10.

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

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output and . Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265280PMC
July 2021

Neopetrothiazide: An Intriguing Pentacyclic Thiazide Alkaloid from the Sponge sp.

Org Lett 2021 05 13;23(9):3278-3281. Epub 2021 Apr 13.

Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States.

Neopetrothiazide (), a pentacyclic isoquinoline quinone, was isolated from a sp. sponge. The structure elucidation was facilitated by utilizing long-range heteronuclear single quantum multiple bond correlation (LR-HSQMBC) and heteronuclear multiple bond correlation (HMBC) nuclear magnetic resonance (NMR) pulse sequences optimized to detect four- and five-bond H-C heteronuclear correlations. These NMR experiments can help assign proton-deficient structural motifs like neopetrothiazide (), which has 14 contiguous nonprotonated centers (C, N, and S). Neopetrothiazide (), with an unprecedented thiazide-fused structural scaffold, is the first natural product containing a thiazide moiety.
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http://dx.doi.org/10.1021/acs.orglett.1c00743DOI Listing
May 2021

Translocations Constitute Ependymoma Chromatin Remodeling and Transcription Factors.

Cancer Discov 2021 Sep 19;11(9):2216-2229. Epub 2021 Mar 19.

Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, England.

()-a gene of unknown function-partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation-including ZFTA zinc fingers and partner gene transactivation domains-thereby unmasking vulnerabilities for therapeutic targeting. SIGNIFICANCE: Ependymomas are hard-to-treat brain tumors driven by translocations between and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1052DOI Listing
September 2021

FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma.

Oncogene 2021 03 24;40(12):2182-2199. Epub 2021 Feb 24.

Division of Pulmonary Biology, The Perinatal Institute of Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.
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http://dx.doi.org/10.1038/s41388-021-01694-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005492PMC
March 2021

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma.

Nat Commun 2021 01 8;12(1):192. Epub 2021 Jan 8.

Greehey Children's Cancer Research Institute, Department of Molecular Medicine, University of Texas Health Sciences Center, San Antonio, Texas, USA.

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.
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http://dx.doi.org/10.1038/s41467-020-20386-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794422PMC
January 2021

Denigrins and Dactylpyrroles, Arylpyrrole Alkaloids from a sp. Marine Sponge.

J Nat Prod 2020 11 5;83(11):3464-3470. Epub 2020 Nov 5.

Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States.

Seven new arylpyrrole alkaloids (-), along with four known compounds, were isolated from an extract of a sp. nov. marine sponge, and their structures were elucidated by interpretation of NMR and MS spectroscopic data. Denigrins D-G (-) have highly substituted pyrrole or pyrrolone rings in their core structures, while dactylpyrroles A-C (-) have tricyclic phenanthrene cores. Due to the proton-deficient nature of these scaffolds, key heteronuclear correlations from H-N HMBC and LR-HSQMBC NMR experiments were used in the structure assignment of denigrin D (). Dictyodendrin F (), a previously described co-metabolite, inhibited transcription driven by the oncogenic PAX3-FOXO1 fusion gene with an IC value of 13 μM.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01103DOI Listing
November 2020

NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency.

Elife 2020 08 3;9. Epub 2020 Aug 3.

Department of Oncology and Children's Research Center, University Children's Hospital, Zurich, Switzerland.

The NuRD complex subunit CHD4 is essential for fusion-positive rhabdomyosarcoma (FP-RMS) survival, but the mechanisms underlying this dependency are not understood. Here, a NuRD-specific CRISPR screen demonstrates that FP-RMS is particularly sensitive to CHD4 amongst the NuRD members. Mechanistically, NuRD complex containing CHD4 localizes to super-enhancers where CHD4 generates a chromatin architecture permissive for the binding of the tumor driver and fusion protein PAX3-FOXO1, allowing downstream transcription of its oncogenic program. Moreover, CHD4 depletion removes HDAC2 from the chromatin, leading to an increase and spread of histone acetylation, and prevents the positioning of RNA Polymerase 2 at promoters impeding transcription initiation. Strikingly, analysis of genome-wide cancer dependency databases identifies CHD4 as a general cancer vulnerability. Our findings describe CHD4, a classically defined repressor, as positive regulator of transcription and super-enhancer accessibility as well as establish this remodeler as an unexpected broad tumor susceptibility and promising drug target for cancer therapy.
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http://dx.doi.org/10.7554/eLife.54993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438112PMC
August 2020

MEIS1 down-regulation by MYC mediates prostate cancer development through elevated HOXB13 expression and AR activity.

Oncogene 2020 08 17;39(34):5663-5674. Epub 2020 Jul 17.

Laboratory of Genitourinary Cancer Pathogenesis, National Cancer Institute, NIH, Bethesda, MD, 20892, USA.

Localized prostate cancer develops very slowly in most men, with the androgen receptor (AR) and MYC transcription factors amongst the most well-characterized drivers of prostate tumorigenesis. Canonically, MYC up-regulation in luminal prostate cancer cells functions to oppose the terminally differentiating effects of AR. However, the effects of MYC up-regulation are pleiotropic and inconsistent with a poorly proliferative phenotype. Here we show that increased MYC expression and activity are associated with the down-regulation of MEIS1, a HOX-family transcription factor. Using RNA-seq to profile a series of human prostate cancer specimens laser capture microdissected on the basis of MYC immunohistochemistry, MYC activity, and MEIS1 expression were inversely correlated. Knockdown of MYC expression in prostate cancer cells increased the expression of MEIS1 and increased the occupancy of MYC at the MEIS1 locus. Finally, we show in laser capture microdissected human prostate cancer samples and the prostate TCGA cohort that MEIS1 expression is inversely proportional to AR activity as well as HOXB13, a known interacting protein of both AR and MEIS1. Collectively, our data demonstrate that elevated MYC in a subset of primary prostate cancers functions in a negative role in regulating MEIS1 expression, and that this down-regulation may contribute to MYC-driven development and progression.
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http://dx.doi.org/10.1038/s41388-020-01389-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441006PMC
August 2020

Miswired Enhancer Logic Drives a Cancer of the Muscle Lineage.

iScience 2020 May 29;23(5):101103. Epub 2020 Apr 29.

Genetics Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

Core regulatory transcription factors (CR TFs) establish enhancers with logical ordering during embryogenesis and development. Here we report that in fusion-positive rhabdomyosarcoma, a cancer of the muscle lineage, the chief oncogene PAX3-FOXO1 is driven by a translocated FOXO1 super enhancer (SE) restricted to a late stage of myogenesis. Using chromatin conformation capture techniques, we demonstrate that the extensive FOXO1 cis-regulatory domain interacts with PAX3. Furthermore, RNA sequencing and chromatin immunoprecipitation sequencing data in tumors bearing rare PAX translocations implicate enhancer miswiring across all fusion-positive tumors. HiChIP of H3K27ac showed connectivity between the FOXO1 SE, additional intra-domain enhancers, and the PAX3 promoter. We show that PAX3-FOXO1 transcription is diminished when this network of enhancers is ablated by CRISPR. Our data reveal a hijacked enhancer network that disrupts the stepwise CR TF logic of normal skeletal muscle development (PAX3 to MYOD to MYOG), replacing it with an "infinite loop" enhancer logic that locks rhabdomyosarcoma in an undifferentiated stage.
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http://dx.doi.org/10.1016/j.isci.2020.101103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226896PMC
May 2020

Targeting the PI3K/AKT Pathway Overcomes Enzalutamide Resistance by Inhibiting Induction of the Glucocorticoid Receptor.

Mol Cancer Ther 2020 07 5;19(7):1436-1447. Epub 2020 May 5.

Laboratory for Genitourinary Cancer Pathogenesis, Center for Cancer Research, NCI, NIH, Bethesda, Maryland.

The PI3K-AKT pathway has pleiotropic effects and its inhibition has long been of interest in the management of prostate cancer, where a compensatory increase in PI3K signaling has been reported following androgen receptor (AR) blockade. Prostate cancer cells can also bypass AR blockade through induction of other hormone receptors, in particular the glucocorticoid receptor (GR). Here we demonstrate that AKT inhibition significantly decreases cell proliferation through both cytostatic and cytotoxic effects. The cytotoxic effect is enhanced by AR inhibition and is most pronounced in models that induce compensatory GR expression. AKT inhibition increases canonical AR activity and remodels the chromatin landscape, decreasing enhancer interaction at the GR gene () locus. Importantly, it blocks induction of GR expression and activity following AR blockade. This is confirmed in multiple models, where AKT inhibition of established xenografts leads to increased canonical AR activity, decreased GR expression, and marked antitumor activity. Overall, our results demonstrate that inhibition of the PI3K/AKT pathway can block GR activity and overcome GR-mediated resistance to AR-targeted therapy. Ipatasertib is currently in clinical development, and GR induction may be a biomarker to identify responsive patients or a responsive disease state.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8522555PMC
July 2020

Outcome-Related Signatures Identified by Whole Transcriptome Sequencing of Resectable Stage III/IV Melanoma Evaluated after Starting Hu14.18-IL2.

Clin Cancer Res 2020 07 9;26(13):3296-3306. Epub 2020 Mar 9.

Department of Human Oncology, University of Wisconsin-Madison, Madison, Wisconsin.

Purpose: We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, to identify predictive immune and/or tumor biomarkers in patients with melanoma at high risk for recurrence.

Experimental Design: Patients were randomly assigned to receive the first of three monthly courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known diseases. Tumors were evaluated by histology and whole transcriptome sequencing.

Results: Tumor-infiltrating lymphocyte (TIL) levels directly associated with relapse-free survival (RFS) and overall survival (OS) in resected tumors from Group A, where early responses to the immunotherapy agent could be assessed. TIL levels directly associated with a previously reported immune signature, which associated with RFS and OS, particularly in Group A tumors. In Group A tumors, there were decreased cell-cycling gene RNA transcripts, but increased RNA transcripts for repair and growth genes. We found that outcome (RFS and OS) was directly associated with several immune signatures and immune-related RNA transcripts and inversely associated with several tumor growth-associated transcripts, particularly in Group A tumors. Most of these associations were not seen in Group B tumors.

Conclusions: We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNA-sequencing analyses detected shortly after initiation of hu14.18-IL2 therapy, are associated with long-term survival and could potentially be used as prognostic biomarkers in tumor resection specimens obtained after initiating neoadjuvant immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334053PMC
July 2020

Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP).

Nat Protoc 2020 03 12;15(3):1209-1236. Epub 2020 Feb 12.

Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.

Methods developed to capture protein-anchored chromatin interactions (chromatin interaction analysis by paired-end tag sequencing and HiChIP) have yielded tremendous insights into the 3D folding principles of the genome, but are normalized by sequencing depth and therefore unable to accurately measure global changes in chromatin interactions and contact domain organization. We herein describe the protocol for absolute quantification of chromatin architecture (AQuA)-HiChIP, an advance that allows the absolute differences in protein-anchored chromatin interactions between samples to be determined. With our method, defined ratios of mouse and human fixed nuclei are mixed and subjected to endonuclease digestion. Chromatin contacts are captured by biotin-dATP incorporation and proximity ligation, followed by gentle shearing, ChIP, biotin capture and paired-end sequencing. 3D contacts are counted from paired-end tags (PETs) from the human genome and are normalized to the total PETs from the mouse genome. As orthogonal normalization allows observation of global changes, the approach will enable more quantitative insights into the topological determinants of transcriptional control and tissue-specific epigenetic memory. With our approach, we have discovered that rapid histone deacetylase inhibition disrupts super enhancer function by creating many new aberrant contacts. The code for data analysis is available at https://github.com/GryderArt/AQuA-HiChIP. This protocol reports both experimental and bioinformatic details to perform AQuA-HiChIP, going from cell culture to ranking chromatin interactions within 6 d.
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http://dx.doi.org/10.1038/s41596-019-0285-9DOI Listing
March 2020

Histone hyperacetylation disrupts core gene regulatory architecture in rhabdomyosarcoma.

Nat Genet 2019 12 29;51(12):1714-1722. Epub 2019 Nov 29.

Genetics Branch, NCI, NIH, Bethesda, MD, USA.

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.
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http://dx.doi.org/10.1038/s41588-019-0534-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886578PMC
December 2019

Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery.

Nat Biotechnol 2020 01 11;38(1):50-55. Epub 2019 Nov 11.

Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, Chapel Hill, NC, USA.

Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.
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http://dx.doi.org/10.1038/s41587-019-0296-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954327PMC
January 2020

Chemical genomics reveals histone deacetylases are required for core regulatory transcription.

Nat Commun 2019 07 8;10(1):3004. Epub 2019 Jul 8.

Genetics Branch, NCI, NIH, Bethesda, MD, 20892, USA.

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.
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http://dx.doi.org/10.1038/s41467-019-11046-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614369PMC
July 2019

Insights into pediatric rhabdomyosarcoma research: Challenges and goals.

Pediatr Blood Cancer 2019 10 21;66(10):e27869. Epub 2019 Jun 21.

Fred Hutchinson Cancer Research Center, Seattle, Washington.

Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.
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http://dx.doi.org/10.1002/pbc.27869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707829PMC
October 2019

MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma.

Sci Transl Med 2018 07;10(448)

Oncogenomics Section, Genetics Branch, National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD 20892, USA.

The RAS isoforms are frequently mutated in many types of human cancers, including PAX3/PAX7 fusion-negative rhabdomyosarcoma. Pediatric RMS arises from skeletal muscle progenitor cells that have failed to differentiate normally. The role of mutant RAS in this differentiation blockade is incompletely understood. We demonstrate that oncogenic RAS, acting through the RAF-MEK [mitogen-activated protein kinase (MAPK) kinase]-ERK (extracellular signal-regulated kinase) MAPK effector pathway, inhibits myogenic differentiation in rhabdomyosarcoma by repressing the expression of the prodifferentiation myogenic transcription factor, MYOG. This repression is mediated by ERK2-dependent promoter-proximal stalling of RNA polymerase II at the locus. Small-molecule screening with a library of mechanistically defined inhibitors showed that RAS-driven RMS is vulnerable to MEK inhibition. MEK inhibition with trametinib leads to the loss of ERK2 at the promoter and releases the transcriptional stalling of expression. MYOG subsequently opens chromatin and establishes super-enhancers at genes required for late myogenic differentiation. Furthermore, trametinib, in combination with an inhibitor of IGF1R, potently decreases rhabdomyosarcoma cell viability and slows tumor growth in xenograft models. Therefore, this combination represents a potential therapeutic for RAS-mutated rhabdomyosarcoma.
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http://dx.doi.org/10.1126/scitranslmed.aan4470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054766PMC
July 2018

EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis.

Mol Carcinog 2018 10 19;57(10):1342-1357. Epub 2018 Jun 19.

Functional Genetics Section, Genetics Branch, Center for Cancer Research (CCR), National Cancer Institute (NCI), Bethesda, Maryland.

Ewing sarcoma (EWS) is a soft tissue and bone tumor that occurs primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The epigenome of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Here, we demonstrate that EWS-FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS-FLI1 activates expression of PHGDH, PSAT1, PSPH, and SHMT2. Using cell-based studies, we also establish that EWS cells are dependent on glutamine for cell survival and that EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. Furthermore, retrospective analysis of an independent data set generated a significant stratification of the overall survival of EWS patients into low- and high-risk groups based on the expression of PHGDH, PSAT1, PSPH, SHMT2, SLC1A5, MTHFD2, and MTHFD1L. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism or glutamine uptake are potential targetable vulnerabilities in this tumor type.
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http://dx.doi.org/10.1002/mc.22849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175245PMC
October 2018

Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.

Oncogene 2018 05 28;37(20):2630-2644. Epub 2018 Feb 28.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4 overexpression as murine myoblasts expressing FGFR4 undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4 were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4 tumor-derived cell and human RMS cell viability. FGFR4-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.
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http://dx.doi.org/10.1038/s41388-017-0122-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054765PMC
May 2018

PAX3-FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability.

Cancer Discov 2017 08 26;7(8):884-899. Epub 2017 Apr 26.

Genetics Branch, NCI, NIH, Bethesda, Maryland.

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the -regulatory landscape by inducing super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy. PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. .
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http://dx.doi.org/10.1158/2159-8290.CD-16-1297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802885PMC
August 2017

Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma.

Oncotarget 2015 Nov;6(34):35247-62

Oncogenomics Section, Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.
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http://dx.doi.org/10.18632/oncotarget.6208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742102PMC
November 2015

Selectively targeting prostate cancer with antiandrogen equipped histone deacetylase inhibitors.

ACS Chem Biol 2013 Nov 20;8(11):2550-60. Epub 2013 Sep 20.

Parker H. Petit Institute for Bioengineering & Biosciences, Department of Chemistry and Biochemistry, Georgia Institute of Technology , 315 Ferst Dr. NW, Atlanta, Georgia 30332-0230, United States.

Diverse cellular processes relevant to cancer progression are regulated by the acetylation status of proteins. Among such processes is chromatin remodeling via histone proteins, controlled by opposing histone deacetylase (HDAC) and histone acetyltransferase (HAT) enzymes. Histone deacetylase inhibitors (HDACi) show great promise in preclinical cancer models, but clinical trials treating solid tumors have failed to improve patient survival. This is due in part to an inability of HDACi to effectively accumulate in cancerous cells. To address this problem we designed HDACi with secondary pharmacophores to facilitate selective accumulation in malignant cells. We present the first example of HDACi compounds targeted to prostate tumors by equipping them with the additional ability to bind the androgen receptor (AR) with nonsteroidal antiandrogen moieties. Leads among these new dual-acting molecules bind to the AR and halt AR transcriptional activity at lower concentrations than clinical antiandrogens. They inhibit key isoforms of HDAC with low nanomolar potency. Fluorescent microscopy reveals varying degrees of AR nuclear localization in response to these compounds that correlates with their HDAC activity. These biological properties translate into potent anticancer activity against hormone-dependent (AR+) LNCaP and to a lesser extent against hormone-independent (AR-) DU145 prostate cancer, while having greatly reduced toxicity in noncancerous cells. This illustrates that engaging multiple biological targets with a single chemical probe can achieve both potent and cell-type-selective responses.
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http://dx.doi.org/10.1021/cb400542wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836611PMC
November 2013

Histone deacetylase inhibitors equipped with estrogen receptor modulation activity.

J Med Chem 2013 Jul 3;56(14):5782-96. Epub 2013 Jul 3.

School of Chemistry and Biochemistry, Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology , Atlanta, Georgia, 30332-0400, United States.

We describe a set of novel histone deacetylase inhibitors (HDACi) equipped with either an antagonist or an agonist of the estrogen receptor (ER) to confer selective activity against breast cancers. These bifunctional compounds potently inhibit HDAC at nanomolar concentrations and either agonize or antagonize ERα and ERβ. The ER antagonist activities of tamoxifen-HDACi conjugates (Tam-HDACi) are nearly identical to those of tamoxifen. Conversely, ethynyl-estradiol-HDACi conjugates (EED-HDACi) have attenuated ER agonist activities relative to the parent ethynyl-estradiol. In silico docking analysis provides structural basis for the trends of ER agonism/antagonism and ER subtype selectivity. Excitingly, lead Tam-HDACi conjugates show anticancer activity that is selectively more potent against MCF-7 (ERα positive breast cancer) compared to MDA-MB-231 (triple negative breast cancer), DU145 (prostate cancer), or Vero (noncancerous cell line). This dual-targeting approach illustrates the utility of designing small molecules with an emphasis on cell-type selectivity, not merely improved potency, working toward a higher therapeutic index at the earliest stages of drug development.
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http://dx.doi.org/10.1021/jm400467wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812312PMC
July 2013

Antiandrogen gold nanoparticles dual-target and overcome treatment resistance in hormone-insensitive prostate cancer cells.

Bioconjug Chem 2012 Aug 12;23(8):1507-12. Epub 2012 Jul 12.

Department of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332-0400, USA.

Prostate cancer is the most commonly diagnosed cancer among men in the developed countries.(1) One in six males in the U.S.(2) and one in nine males in the U.K.(3) will develop the disease at some point during their lifetime. Despite advances in prostate cancer screening, more than a quarter of a million men die from the disease every year(1) due primarily to treatment-resistance and metastasis. Colloidal nanotechnologies can provide tremendous enhancements to existing targeting/treatment strategies for prostate cancer to which malignant cells are less sensitive. Here, we show that antiandrogen gold nanoparticles--multivalent analogues of antiandrogens currently used in clinical therapy for prostate cancer--selectively engage two distinct receptors, androgen receptor (AR), a target for the treatment of prostate cancer, as well as a novel G-protein coupled receptor, GPRC6A, that is also upregulated in prostate cancer. These nanoparticles selectively accumulated in hormone-insensitive and chemotherapy-resistant prostate cancer cells, bound androgen receptor with multivalent affinity, and exhibited greatly enhanced drug potency versus monovalent antiandrogens currently in clinical use. Further, antiandrogen gold nanoparticles selectively stimulated GPRC6A with multivalent affinity, demonstrating that the delivery of nanoscale antiandrogens can also be facilitated by the transmembrane receptor in order to realize increasingly selective, increasingly potent therapy for treatment-resistant prostate cancers.
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http://dx.doi.org/10.1021/bc300158kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434689PMC
August 2012

Targeted cancer therapy: giving histone deacetylase inhibitors all they need to succeed.

Future Med Chem 2012 Mar;4(4):505-24

School of Chemistry & Biochemistry, Parker H. Petit Institute for Bioengineering & Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.

Histone deacetylase inhibitors (HDACis) have now emerged as a powerful new class of small-molecule therapeutics acting through the regulation of the acetylation states of histone proteins (a form of epigenetic modulation) and other non-histone protein targets. Over 490 clinical trials have been initiated in the last 10 years, culminating in the approval of two structurally distinct HDACis - SAHA (vorinostat, Zolinza™) and FK228 (romidepsin, Istodax™). However, the current HDACis have serious limitations, including ineffectively low concentrations in solid tumors and cardiac toxicity, which is hindering their progress in the clinic. Herein, we review the primary paradigms being pursued to overcome these hindrances, including HDAC isoform selectivity, localized administration, and targeting cap groups to achieve selective tissue and cell type distribution.
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http://dx.doi.org/10.4155/fmc.12.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3341130PMC
March 2012
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