Publications by authors named "Olena Barbash"

26 Publications

  • Page 1 of 1

Phase 1 Study of Molibresib (GSK525762), a Bromodomain and Extra-Terminal Domain Protein Inhibitor, in NUT Carcinoma and Other Solid Tumors.

JNCI Cancer Spectr 2020 Apr 6;4(2):pkz093. Epub 2019 Nov 6.

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

Background: Bromodomain and extra-terminal domain proteins are promising epigenetic anticancer drug targets. This first-in-human study evaluated the safety, recommended phase II dose, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the bromodomain and extra-terminal domain inhibitor molibresib (GSK525762) in patients with nuclear protein in testis (NUT) carcinoma (NC) and other solid tumors.

Methods: This was a phase I and II, open-label, dose-escalation study. Molibresib was administered orally once daily. Single-patient dose escalation (from 2 mg/d) was conducted until the first instance of grade 2 or higher drug-related toxicity, followed by a 3 + 3 design. Pharmacokinetic parameters were obtained during weeks 1 and 3. Circulating monocyte chemoattractant protein-1 levels were measured as a pharmacodynamic biomarker.

Results: Sixty-five patients received molibresib. During dose escalation, 11% experienced dose-limiting toxicities, including six instances of grade 4 thrombocytopenia, all with molibresib 60-100 mg. The most frequent treatment-related adverse events of any grade were thrombocytopenia (51%) and gastrointestinal events, including nausea, vomiting, diarrhea, decreased appetite, and dysgeusia (22%-42%), anemia (22%), and fatigue (20%). Molibresib demonstrated an acceptable safety profile up to 100 mg; 80 mg once daily was selected as the recommended phase II dose. Following single and repeat dosing, molibresib showed rapid absorption and elimination (maximum plasma concentration: 2 hours; t: 3-7 hours). Dose-dependent reductions in circulating monocyte chemoattractant protein-1 levels were observed. Among 19 patients with NC, four achieved either confirmed or unconfirmed partial response, eight had stable disease as best response, and four were progression-free for more than 6 months.

Conclusions: Once-daily molibresib was tolerated at doses demonstrating target engagement. Preliminary data indicate proof-of-concept in NC.
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http://dx.doi.org/10.1093/jncics/pkz093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165800PMC
April 2020

Limited antitumor activity of combined BET and MEK inhibition in neuroblastoma.

Pediatr Blood Cancer 2020 06 19;67(6):e28267. Epub 2020 Apr 19.

Aflac Cancer and Blood Disorders Center of Children's Healthcare of Atlanta and Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia.

Background: The treatment of high-risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors can inhibit MYCN expression and suppress MYCN-amplified neuroblastoma in vivo. Furthermore, alterations within RAS-MAPK (mitogen-activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS-MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I-BET726 or I-BET762 and trametinib in high-risk neuroblastoma.

Procedure: Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models.

Results: Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non-MYCN-amplified, NRAS-mutated neuroblastoma xenograft model, but had no efficacy in an MYCN-amplified model harboring a loss-of-function mutation in NF1.

Conclusions: Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high-risk neuroblastoma.
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http://dx.doi.org/10.1002/pbc.28267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188563PMC
June 2020

Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation.

Cancer Cell 2019 08;36(2):194-209.e9

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A(∗)STAR), Singapore 138673, Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119077, Singapore; Department of Oncological Sciences and Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Pharmacological Sciences and Mount Sinai Center for Therapeutics Discovery, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.
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http://dx.doi.org/10.1016/j.ccell.2019.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194031PMC
August 2019

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Cancer Cell 2019 07 27;36(1):100-114.e25. Epub 2019 Jun 27.

Epigenetics Research Unit, GlaxoSmithKline, Collegeville, PA 19426, USA. Electronic address:

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.
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http://dx.doi.org/10.1016/j.ccell.2019.05.014DOI Listing
July 2019

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Nat Commun 2019 06 20;10(1):2723. Epub 2019 Jun 20.

Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
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http://dx.doi.org/10.1038/s41467-019-10652-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586637PMC
June 2019

Targeting epigenetic modifications in cancer therapy: erasing the roadmap to cancer.

Nat Med 2019 03 6;25(3):403-418. Epub 2019 Mar 6.

Epigenetics Department, Oncology, GlaxoSmithKline, Collegeville, PA, USA.

Epigenetic dysregulation is a common feature of most cancers, often occurring directly through alteration of epigenetic machinery. Over the last several years, a new generation of drugs directed at epigenetic modulators have entered clinical development, and results from these trials are now being disclosed. Unlike first-generation epigenetic therapies, these new agents are selective, and many are targeted to proteins which are mutated or translocated in cancer. This review will provide a summary of the epigenetic modulatory agents currently in clinical development and discuss the opportunities and challenges in their development. As these drugs advance in the clinic, drug discovery has continued with a focus on both novel and existing epigenetic targets. We will provide an overview of these efforts and the strategies being employed.
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http://dx.doi.org/10.1038/s41591-019-0376-8DOI Listing
March 2019

"Z4" Complex Member Fusions in NUT Carcinoma: Implications for a Novel Oncogenic Mechanism.

Mol Cancer Res 2018 12 23;16(12):1826-1833. Epub 2018 Aug 23.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Nuclear protein in testis (NUT) carcinoma (NC) is a rare, distinctly aggressive subtype of squamous carcinoma defined by the presence of -fusion oncogenes resulting from chromosomal translocation. In most cases, the gene () is fused to bromodomain containing 4 () forming the oncogene. Here, a novel fusion partner to was discovered using next-generation sequencing and FISH from a young patient with an undifferentiated malignant round cell tumor. Interestingly, the NUT fusion identified involved ZNF592, a zinc finger containing protein, which was previously identified as a component of the BRD4-NUT complex. In BRD4-NUT-expressing NC cells, wild-type ZNF592 and other associated "Z4" complex proteins, including ZNF532 and ZMYND8, colocalize with BRD4-NUT in characteristic nuclear foci. Furthermore, ectopic expression of BRD4-NUT in a non-NC cell line induces sequestration of Z4 factors to BRD4-NUT foci. Finally, the data demonstrate the specific dependency of NC cells on Z4 modules, ZNF532 and ZNF592. IMPLICATIONS: This study establishes the oncogenic role of Z4 factors in NC, offering potential new targeted therapeutic strategies in this incurable cancer. http://mcr.aacrjournals.org/content/molcanres/16/12/1826/F1.large.jpg.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279489PMC
December 2018

Activation of the p53-MDM4 regulatory axis defines the anti-tumour response to PRMT5 inhibition through its role in regulating cellular splicing.

Sci Rep 2018 06 26;8(1):9711. Epub 2018 Jun 26.

Epigenetics Discovery Performance Unit, Oncology R&D, GlaxoSmithKline, Collegeville, PA, USA.

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.
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http://dx.doi.org/10.1038/s41598-018-28002-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018746PMC
June 2018

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers.

Oncogenesis 2018 Apr 20;7(4):35. Epub 2018 Apr 20.

Cancer Epigenetics DPU, Oncology R&D, GlaxoSmithKline, Collegeville, PA, USA.

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.
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http://dx.doi.org/10.1038/s41389-018-0043-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908790PMC
April 2018

PRMT5 Is a Critical Regulator of Breast Cancer Stem Cell Function via Histone Methylation and FOXP1 Expression.

Cell Rep 2017 Dec;21(12):3498-3513

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

Breast cancer progression, treatment resistance, and relapse are thought to originate from a small population of tumor cells, breast cancer stem cells (BCSCs). Identification of factors critical for BCSC function is therefore vital for the development of therapies. Here, we identify the arginine methyltransferase PRMT5 as a key in vitro and in vivo regulator of BCSC proliferation and self-renewal and establish FOXP1, a winged helix/forkhead transcription factor, as a critical effector of PRMT5-induced BCSC function. Mechanistically, PRMT5 recruitment to the FOXP1 promoter facilitates H3R2me2s, SET1 recruitment, H3K4me3, and gene expression. Our findings are clinically significant, as PRMT5 depletion within established tumor xenografts or treatment of patient-derived BCSCs with a pre-clinical PRMT5 inhibitor substantially reduces BCSC numbers. Together, our findings highlight the importance of PRMT5 in BCSC maintenance and suggest that small-molecule inhibitors of PRMT5 or downstream targets could be an effective strategy eliminating this cancer-causing population.
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http://dx.doi.org/10.1016/j.celrep.2017.11.096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746596PMC
December 2017

Targeting Histone Methylation in Cancer.

Cancer J 2017 Sep/Oct;23(5):292-301

From the Cancer Epigenetics Discovery Performance Unit, Oncology R&D, GlaxoSmithKline, Collegeville, PA.

Most, if not all, human cancers exhibit altered epigenetic signatures that promote aberrant gene expression that contributes to cellular transformation. Historically, attempts to pharmacologically intervene in this process have focused on DNA methylation and histone acetylation. More recently, genome-wide studies have identified histone and chromatin regulators as one of the most frequently dysregulated functional classes in a wide range of cancer types. These findings have provided numerous potential therapeutic targets including many that affect histone methylation. These include histone lysine methyltransferases such as enhancer of zeste homolog 2 and DOT1L, protein arginine methyltransferases such as protein arginine methyltransferase 5, and histone lysine demethylases such as lysine-specific demethylase 1. This review presents the rationale for targeting histone methylation in oncology and provides an update on a few key targets that are being investigated in the clinic.
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http://dx.doi.org/10.1097/PPO.0000000000000283DOI Listing
May 2018

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Nat Chem Biol 2015 Jun 27;11(6):432-7. Epub 2015 Apr 27.

Departments of Biology and Molecular Discovery, Epizyme, Inc., Cambridge, Massachusetts, USA.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.
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http://dx.doi.org/10.1038/nchembio.1810DOI Listing
June 2015

SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.

Nature 2014 Jun 21;510(7504):283-7. Epub 2014 May 21.

1] Department of Biology, Stanford University, California 94305, USA [2].

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.
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http://dx.doi.org/10.1038/nature13320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122675PMC
June 2014

Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762.

Blood 2014 Jan 13;123(5):697-705. Epub 2013 Dec 13.

Centre for Haematology, Department of Medicine, Hammersmith Hospital, Imperial College London, London, United Kingdom;

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.
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http://dx.doi.org/10.1182/blood-2013-01-478420DOI Listing
January 2014

Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer.

Oncotarget 2013 Dec;4(12):2419-29

Cancer Epigenetics DPU, Oncology R and D GlaxoSmithKline, Collegeville, PA, USA.

BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926837PMC
http://dx.doi.org/10.18632/oncotarget.1572DOI Listing
December 2013

The FBXO4 tumor suppressor functions as a barrier to BRAFV600E-dependent metastatic melanoma.

Mol Cell Biol 2013 Nov 9;33(22):4422-33. Epub 2013 Sep 9.

The Leonard and Madlyn Abramson Family Cancer Research Institute.

Cyclin D1-cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16(INK4A), an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16(INK4A) deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy.
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http://dx.doi.org/10.1128/MCB.00706-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838190PMC
November 2013

BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in neuroblastoma tumor models.

PLoS One 2013 23;8(8):e72967. Epub 2013 Aug 23.

Cancer Epigenetics Discovery Performance Unit, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072967PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751846PMC
April 2014

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Epigenetics 2012 Apr 1;7(4):340-3. Epub 2012 Apr 1.

GlaxoSmithKline, Collegeville, PA, USA.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.
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http://dx.doi.org/10.4161/epi.19506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368817PMC
April 2012

The Fbx4 tumor suppressor regulates cyclin D1 accumulation and prevents neoplastic transformation.

Mol Cell Biol 2011 Nov 12;31(22):4513-23. Epub 2011 Sep 12.

The Leonard and Madlyn Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G(1)/S transition, SCF(Fbx4) targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4(+/-) and Fbx4(-/-) mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4(+/-) and Fbx4(-/-) mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4(-/-) MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCF(Fbx4) E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.
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http://dx.doi.org/10.1128/MCB.05733-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209253PMC
November 2011

SCF(Fbx4/alphaB-crystallin) E3 ligase: when one is not enough.

Cell Cycle 2008 Oct 12;7(19):2983-6. Epub 2008 Oct 12.

The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, Philadelphia, Pennsylvania 19104, USA.

Cell cycle progression is determined by the balance of positive regulators, cyclin-dependent-protein kinases (cdks) relative to negative regulators, cyclin-dependent kinase inhibitors (ckis). D-type cyclins, (D1, D2, D3) are expressed in a tissue-specific manner and are the first cyclins to be expressed during the cell cycle. Of the three D-type cyclins, cyclin D1 is most frequently overexpressed in human cancer. The mechanisms of cyclin D1 overexpression can be attributed to gene amplification, transcriptional activation and altered protein degradation; of these, inhibition of ubiquitin-dependent proteolysis of cyclin D1 is thought to be a primary mechanism of cyclin D1 overexpression in human tumors. Because the identity of the regulators of cyclin D1 proteolysis were largely undefined until recently, it had not been possible to determine whether this regulatory network was directly targeted in primary cancer. Cyclin D1 proteolysis requires phosphorylation by GSK3beta at Thr-286; additional work recently established that p286-D1 is a substrate for the SCF(Fbx4/alphaB-crystallin) E3 ligase. This discovery has facilitated an analysis of SCF(Fbx4/alphaB-crystallin) ligase in human cancers. This recent work revealed that Fbx4 is subject to mutational inactivation in human cancer, resulting in the accumulation of cyclin D1. Molecular analysis of this ligase has revealed striking regulatory features that contribute to regulated cyclin D1 accumulation and support the idea that Fbx4 is a bona fide tumor suppressor.
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http://dx.doi.org/10.4161/cc.7.19.6775DOI Listing
October 2008

Genotoxic stress-induced cyclin D1 phosphorylation and proteolysis are required for genomic stability.

Mol Cell Biol 2008 Dec 22;28(23):7245-58. Epub 2008 Sep 22.

Department of Cancer Biology, The Abramson Family Cancer Research Institute, 454 BRB II/III, Philadelphia, PA 19104-6140, USA.

While mitogenic induction of cyclin D1 contributes to cell cycle progression, ubiquitin-mediated proteolysis buffers this accumulation and prevents aberrant proliferation. Because the failure to degrade cyclin D1 during S-phase triggers DNA rereplication, we have investigated cellular regulation of cyclin D1 following genotoxic stress. These data reveal that expression of cyclin D1 alleles refractory to phosphorylation- and ubiquitin-mediated degradation increase the frequency of chromatid breaks following DNA damage. Double-strand break-dependent cyclin D1 degradation requires ATM and GSK3beta, which in turn mediate cyclin D1 phosphorylation. Phosphorylated cyclin D1 is targeted for proteasomal degradation after ubiquitylation by SCF(Fbx4-alphaBcrystallin). Loss of Fbx4-dependent degradation triggers radio-resistant DNA synthesis, thereby sensitizing cells to S-phase-specific chemotherapeutic intervention. These data suggest that failure to degrade cyclin D1 compromises the intra-S-phase checkpoint and suggest that cyclin D1 degradation is a vital cellular response necessary to prevent genomic instability following genotoxic insult.
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http://dx.doi.org/10.1128/MCB.01085-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2593367PMC
December 2008

Mutations in Fbx4 inhibit dimerization of the SCF(Fbx4) ligase and contribute to cyclin D1 overexpression in human cancer.

Cancer Cell 2008 Jul;14(1):68-78

Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

SCF(Fbx4) was recently identified as the E3 ligase for cyclin D1. We now describe cell-cycle-dependent phosphorylation and dimerization of Fbx4 that is regulated by GSK3beta and is defective in human cancer. We present data demonstrating that a pathway involving Ras-Akt-GSK3beta controls the temporal phosphorylation and dimerization of the SCF(Fbx4) E3 ligase. Inhibition of Fbx4 activity results in accumulation of nuclear cyclin D1 and oncogenic transformation. The importance of this regulatory pathway for normal cell growth is emphasized by the prevalence of mutations in Fbx4 in human cancer that impair dimerization. Collectively, these data reveal that inactivation of the cyclin D1 E3 ligase likely contributes to cyclin D1 overexpression in a significant fraction of human cancer.
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http://dx.doi.org/10.1016/j.ccr.2008.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597358PMC
July 2008

SCF Fbx4/alphaB-crystallin cyclin D1 ubiquitin ligase: a license to destroy.

Cell Div 2007 Jan 15;2. Epub 2007 Jan 15.

Cyclin D1 is an allosteric regulator for cyclin-dependent kinases 4 and 6 (CDK4/6). The cyclin D/CDK4 kinase promotes G1/S transition through the posttranslational modification and the subsequent inactivation of the retinoblastoma (Rb) protein and related family members (p107 and p130). Accumulation of cyclin D1 is tightly regulated through various mechanisms including transcription, protein localization and ubiquitin-dependent proteolysis. While regulators of cyclin D1 gene expression have been under considerable scrutiny, the identity of the protein complex that targets cyclin D1 protein for degradation, the putative E3 ubiquitin ligase, has remained obscure. In a recent report, Lin et al describe the identification and characterization of a novel SCF, wherein FBX4 and alphaB-crystallin serve as specificity factors that direct ubiquitination of phosphorylated cyclin D1. As cyclin D1 overexpression in human cancer has been postulated to occur through the loss of degradation machinery, the identification of the SCFFbx4/alphaB-crystallin ligase will allow new experimental approaches that address mechanisms of cyclin D1 overexpression in human cancer.
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http://dx.doi.org/10.1186/1747-1028-2-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1790704PMC
January 2007

Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex.

Mol Cell 2006 Nov;24(3):355-66

The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.
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http://dx.doi.org/10.1016/j.molcel.2006.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1702390PMC
November 2006

The oxidative stressor arsenite activates vascular endothelial growth factor mRNA transcription by an ATF4-dependent mechanism.

J Biol Chem 2005 May 23;280(21):20331-9. Epub 2005 Mar 23.

Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131-0001, USA.

Aberrant retinal expression of vascular endothelial growth factor (VEGF) leading to neovascularization is a central feature of age-related macular degeneration and diabetic retinopathy, two leading causes of vision loss. Oxidative stress is suggested to occur in retinal tissue during age-related macular degeneration and diabetic retinopathy and is suspected in the mechanism of VEGF expression in these diseases. Arsenite, a thiol-reactive oxidative stressor, induces VEGF expression by a HIF-1alpha-independent mechanism. Previously, we demonstrated that homocysteine, an endoplasmic reticulum stressor, increases VEGF transcription by a mechanism dependent upon activating transcription factor ATF4. Because ATF4 is expressed in response to oxidative stress, we hypothesized that ATF4 was also responsible for increased VEGF transcription in response to arsenite. We now show that arsenite increased steady state levels of VEGF mRNA and activated transcription from a VEGF promoter construct. Arsenite induced eIF2alpha phosphorylation, resulting in increased ATF4 protein levels. Inactivation or loss of ATF4 greatly diminished the VEGF response to arsenite treatment. Overexpression of ATF4 was sufficient to activate the VEGF promoter, and arsenite cooperated with exogenous ATF4 to further activate the promoter. A complex containing ATF4 binds a DNA element at +1767 bp relative to the VEGF transcription start site, and DNA binding activity is increased by arsenite treatment. In addition, the ability of a thiol antioxidant, N-acetylcysteine, to inhibit the effect of arsenite on VEGF expression coincided with its ability to inhibit phosphorylation of eIF2alpha and ATF4 protein expression. Thus, arsenite-induced up-regulation of VEGF gene transcription occurs by an ATF4-dependent mechanism.
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http://dx.doi.org/10.1074/jbc.M411275200DOI Listing
May 2005

Expression of angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 is highly responsive to ambient glutamine availability: role of nuclear factor-kappaB and activating protein-1.

Cancer Res 2004 Jul;64(14):4858-69

Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, 915 Camino de Salud NE, Albuquerque, NM 87131, USA.

Vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8) are prominent pro-angiogenic and pro-metastatic proteins that represent negative prognostic factors in many types of cancer. Hypoxia is thought to be the primary environmental cause of VEGF and IL-8 expression in solid tumors. We hypothesized that a lack of nutrients other than oxygen could stimulate the expression of these factors and previously demonstrated that expression of VEGF and IL-8 is responsive to amino acid deprivation. In the present study, we examined the effect of glutamine availability on the expression of these factors as well as the role of transcription factors NFkappaB and activating protein-1 (AP-1) in the response of TSE human breast carcinoma cells to glutamine deprivation. VEGF and IL-8 secretion and mRNA levels were dramatically induced by glutamine deprivation. mRNA stabilization contributed to this response. Glutamine deprivation increased NFkappaB (p65/p50) and AP-1 (Fra-1/c-Jun+JunD) DNA-binding activities. Blocking NFkappaB and AP-1 activation with curcumin as well as expression of dominant inhibitors, inhibitor of nuclear factor-kappaB (IkappaB) super repressor (IkappaBM), and a mutant form of c-Fos (A-Fos) demonstrated that the activation of NFkappaB and AP-1 transcription factors was necessary for the induction of IL-8 expression but dispensable for the induction of VEGF expression. A macro-array containing 111 NFkappaB target genes identified a total of 17 that were up-regulated 2-fold or more in response to glutamine deprivation. These included growth regulated oncogene alpha (GROalpha/GRO1/CXCL1), another neutrophil chemoattractant implicated in tumor angiogenesis and metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-0682DOI Listing
July 2004