Publications by authors named "Silvia Pomella"

18 Publications

  • Page 1 of 1

DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma.

Cells 2021 10 30;10(11). Epub 2021 Oct 30.

Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Recently, we demonstrated the overexpression of both DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B) in RMS tumour biopsies and cell lines compared to normal skeletal muscle. Radiotherapy may often fail due to the abnormal expression of some molecules able to drive resistance mechanisms. The aim of this study was to analyse the involvement of DNMT3A and DNMT3B in radioresistance in RMS. RNA interference experiments against DNMT3A/3B were performed in embryonal RMS cells, upon ionizing radiation (IR) exposure and the effects of the combined treatment on RMS cells were analysed. DNMT3A and DNMT3B knocking down increased the sensitivity of RMS cells to IR, as indicated by the drastic decrease of colony formation ability. Interestingly, DNMT3A/3B act in two different ways: DNMT3A silencing triggers the cellular senescence program by up-regulating p16 and p21, whilst DNMT3B depletion induces significant DNA damage and impairs the DNA repair machinery (ATM, DNA-PKcs and Rad51 reduction). Our findings demonstrate for the first time that DNMT3A and DNMT3B overexpression may contribute to radiotherapy failure, and their inhibition might be a promising radiosensitizing strategy, mainly in the treatment of patients with metastatic or recurrent RMS tumours.
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http://dx.doi.org/10.3390/cells10112956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616246PMC
October 2021

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetic effects.

J Exp Clin Cancer Res 2021 Nov 16;40(1):364. Epub 2021 Nov 16.

Unit of Molecular Genetics of Complex Phenotypes, Bambino Gesù Children's Hospital, IRCCS, Via S. Paolo, 15, 00146, Rome, Italy.

Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC.

Methods: The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods.

Results: TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation.

Conclusions: Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.
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http://dx.doi.org/10.1186/s13046-021-02154-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8597092PMC
November 2021

MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells.

Int J Mol Sci 2021 Oct 1;22(19). Epub 2021 Oct 1.

Department of Radiotherapy, Policlinico Umberto I, Sapienza University of Rome, 00161 Rome, Italy.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated , , and anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.
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http://dx.doi.org/10.3390/ijms221910671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8508838PMC
October 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

Romidepsin (FK228) fails in counteracting the transformed phenotype of rhabdomyosarcoma cells but efficiently radiosensitizes, in vitro and in vivo, the alveolar phenotype subtype.

Int J Radiat Biol 2021 4;97(7):943-957. Epub 2021 Jun 4.

Department of Radiotherapy, Policlinico Umberto I, "Sapienza" University of Rome, Rome, Italy.

Purpose: Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS).

Methods: RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts.

Results: Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts.

Conclusion: FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.
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http://dx.doi.org/10.1080/09553002.2021.1928786DOI Listing
September 2021

Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor.

Cell Death Dis 2021 05 6;12(5):452. Epub 2021 May 6.

Centre for Neuroscience and Nanotechnology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, 41125, Modena, Italy.

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.
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http://dx.doi.org/10.1038/s41419-021-03737-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102500PMC
May 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

FAK Signaling in Rhabdomyosarcoma.

Int J Mol Sci 2020 Nov 10;21(22). Epub 2020 Nov 10.

Department of Oncohematology, Bambino Gesù Children's Hospital, IRCCS, 00165 Rome, Italy.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children and adolescents. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 is at high risk. The fusion negative subgroup, FN-RMS, has a good prognosis when non-metastatic. Despite a multimodal therapeutic approach, FP-RMS and metastatic FN-RMS often show a dismal prognosis with 5-year survival of less than 30%. Therefore, novel targets need to be discovered to develop therapies that halt tumor progression, reducing long-term side effects in young patients. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates focal contacts at the cellular edges. It plays a role in cell motility, survival, and proliferation in response to integrin and growth factor receptors' activation. FAK is often dysregulated in cancer, being upregulated and/or overactivated in several adult and pediatric tumor types. In RMS, both in vitro and preclinical studies point to a role of FAK in tumor cell motility/invasion and proliferation, which is inhibited by FAK inhibitors. In this review, we summarize the data on FAK expression and modulation in RMS. Moreover, we give an overview of the approaches to inhibit FAK in both preclinical and clinical cancer settings.
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http://dx.doi.org/10.3390/ijms21228422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697003PMC
November 2020

CDK9 as a Valuable Target in Cancer: From Natural Compounds Inhibitors to Current Treatment in Pediatric Soft Tissue Sarcomas.

Front Pharmacol 2020 13;11:1230. Epub 2020 Aug 13.

Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy.

Cyclin-Dependent Kinases (CDKs) are well-known reliable targets for cancer treatment being often deregulated. Among them, since the transcription-associated CDK9 represents the sentry of cell transcriptional homeostasis, it can be a valuable target for managing cancers in which the transcriptional machinery is dysregulated by tumor-driver oncogenes. Here we give an overview of some natural compounds identified as CDK inhibitors with reported activity also against CDK9, that were taken as a model for the development of highly active synthetic anti-CDK9 agents. After, we summarize the data on CDK9 inhibition in a group of rare pediatric solid tumors such as rhabdomyosarcoma, Ewing's sarcoma, synovial sarcoma and malignant rhabdoid tumors (soft tissue sarcomas), highlighting the more recent results in this field. Finally, we discuss the perspective and challenge of CDK9 modulation in cancer.
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http://dx.doi.org/10.3389/fphar.2020.01230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438590PMC
August 2020

Design of First-in-Class Dual EZH2/HDAC Inhibitor: Biochemical Activity and Biological Evaluation in Cancer Cells.

ACS Med Chem Lett 2020 May 19;11(5):977-983. Epub 2020 Mar 19.

Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.

Since the histone modifying enzymes EZH2 and HDACs control a number of epigenetic-dependent carcinogenic pathways, we designed the first-in-class dual EZH2/HDAC inhibitor displaying (sub)micromolar inhibition against both targets. When tested in several cancer cell lines, the hybrid impaired cell viability at low micromolar level and in leukemia U937 and rhabdomyosarcoma RH4 cells provided G1 arrest, apoptotic induction, and increased differentiation, associated with an increase of acetyl-H3 and acetyl-α-tubulin and a decrease of H3K27me3 levels. In glioblastoma U87 cells, hampered epithelial to mesenchymal transition by increasing the E-cadherin expression, thus proposing itself as a useful candidate for anticancer therapy.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236245PMC
May 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

The CRISP(Y) Future of Pediatric Soft Tissue Sarcomas.

Front Chem 2020 13;8:178. Epub 2020 Mar 13.

Department of Oncohematology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

The RNA-guided clustered regularly interspaced palindromic repeats (CRISPR)/associated nuclease 9 (Cas9)-based genome editing technology has increasingly become a recognized method for translational research. In oncology, the ease and versatility of CRISPR/Cas9 has made it possible to obtain many results in the identification of new target genes and in unravel mechanisms of resistance to therapy. The majority of the studies have been made on adult tumors so far. In this mini review we present an overview on the major aspects of CRISPR/Cas9 technology with a focus on a group of rare pediatric malignancies, soft tissue sarcomas, on which this approach is having promising results.
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http://dx.doi.org/10.3389/fchem.2020.00178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083251PMC
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

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

In vitro and in vivo single-agent efficacy of checkpoint kinase inhibition in acute lymphoblastic leukemia.

J Hematol Oncol 2015 Nov 5;8:125. Epub 2015 Nov 5.

Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology "L. e A. Seragnoli", University of Bologna, Bologna, Italy.

Background: Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2).

Methods: The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent N-ethyl-N-nitrosourea.

Results: Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose- and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia.

Conclusions: In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation.
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http://dx.doi.org/10.1186/s13045-015-0206-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635624PMC
November 2015

MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma.

Clin Epigenetics 2015 6;7:82. Epub 2015 Aug 6.

Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.

Background: Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells.

Results: Herein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells.

Conclusions: Altogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma.
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http://dx.doi.org/10.1186/s13148-015-0107-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4527101PMC
August 2015
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