epigenetics and new therapies in cancer - centro

EPIGENETICS AND NEW THERAPIES

IN CaNCer29-30 November 2007

Madrid, Spain

chair Manel Esteller

co-ordinators Silvia MarsoniMiguel A. Piris

EPIGENETICS AND NEW THERAPIES

IN CaNCerchair

Manel Estellerco-ordinators

Silvia MarsoniMiguel A. Piris

EPIGENETICS AND NEW THERAPIES IN CaNCer

Dear Colleagues and Friends,

It is a pleasure for me to welcome you to the “Epigenetics and New Therapies in Cancer” Conference, a meeting whose aim is to present the recent advances in new antitumoral drugs and new cellular targets in a friendly and stimulating atmosphere. The approval by the Food and Drug Administration of four compounds that target DNA methylation and histone acetylation has opened a new area of translational research in epigenetics. It is a brave new world in which new therapies will benefit greatly from a better understanding of the role of non-coding RNAs and the full completion of the human epigenome projects.

This conference tries to cover and update these topics with the aim of stimulating and discussing about what we are doing and what should be done.

Thank you for coming to our place and we hope you have a pleasant and interesting conference.

Manel Esteller

CHaIrS’ MESSAGE

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EPIGENETICS AND NEW THERAPIES IN CANCERINDEx

General Information 7

Program 11

Speakers Abstract 17

Participants Posters 45

Faculty And Authors’ Index 97


GeNeraL INFORMATION


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GeNeraL INFORMATION

Conference VenueCentro Nacional de Investigaciones Oncológicas (CNIO)Melchor Fernández Almagro, 3E-28029 Madrid - Spain

Organising SecretariatSara Bertrand, PhDResponsable de Eventos Científicos / Scientific Events ManagerCentro Nacional de Investigaciones Oncológicas- CNIO(Spanish National Cancer Research Centre) Melchor Fernández Almagro, 3 - E-28029 Madrid - SpainTel.: +34 91224 69 00; Fax:+34 91 224 69 81E-mail: [email protected]

ESO SecretariatDaniela Mengato European School of OncologyVia del Bollo 4 -20123 Milan -Italy Tel: +39 02 85 46 45 23 Fax: +39 02 85 46 45 45 E-mail: [email protected]

Official LanguageThe official language of the meeting will be English.

CertificatesA certificate of participation will be issued to all participants.

BadgeThe badge is the only official evidence of registration and should be worn at all times during the course.

Lost and FoundFor lost and found objects please go to the registration desk.

InsuranceThe organisers bear no responsibility for untoward events in connection with, before, during and after the course. Participants are strongly advised to take out their own personal and travel insurance coverage.

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EPIGENETICS AND NEW THERAPIES IN CaNCerGeNeraL INFORMATION

Message CentrePeople can leave messages and other information for participants at the registration desk.

Registration FeeOn-site 300 EURO.

Registration PackageThe full registration package includes:

Attendance to the scientific sessionsProgram bookLunchesCoffee breaks

PostersPosters may be displayed in the morning of Thursday 29 November before the beginning of the conference and should be removed in the evening of Friday 30 November at the end of the conference.All posters will be on display during the entire conference. Coffee breaks will be served in the Hall of the building by the poster exhibition area in order to allow sufficient time for viewing and discussing.Each poster will be identified by a number (for position). The author must be present for discussion during breaks throughout the conference.

SlidesAll presenters should take their slides to the technicians in the conference room not less than one hour before the start of their session. All the presentations will be loaded in our auditorium computers (PC or Mac) so we will appreciate it if you could bring it on a CD or pen-drive at the beginning of your corresponding presentation day.

Smoking PolicyThere will be a strict no smoking policy in all areas of the conference.

StaffStaff members wearing the ESO badge will be pleased to assist you with any further information you may require.

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PrOGraM

PrOGraM

9:00 Welcome Opening Session

9:15 Therapeutic exploitation of the DNA repair defects in tumours A. Ashworth, UK

DNA METHYLATION INHIBITORS

PreclinicalM. Esteller, ES

9:45 Molecular modes of actions of DNA methyltransferase inhibitors F. Lyko, DE

10:15 The design of mechanism based, small-molecule inhibitors of DNA methyltransferases: nucleosides, nucleotide prodrugs and oligodeoxynucleotides V. E. Marquez, US

10:45 Coffee break and poster viewing

ClinicalM.A. Piris, ES

11:15 Demethylating approaches in the biological and therapeutic continuum of myelodysplasia and AML of the elderly M. Lubbert, DE

11:45 Epigenetic targets in acute myeloid leukaemia C. Nervi, IT

12:15 Use of Epi-drugs against cancer roles of class I and II HDACs in cell proliferation, differentiation and apoptosis L. Altucci, IT

12:45 Round table discussion

13:15 Lunch break

THURSDAY29 NOVEMBER


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EPIGENETICS AND NEW THERAPIES IN CaNCerPrOGraM

HDAC INHIBITORS

PreclinicalV. Fantin, US

14:30 Histone deacetylases and the development of epigenetic and non-epigenetic therapies S. Minucci, IT

15:00 Combination studies of DNMT and HDAC inhibitors in overcoming cytotoxic drug resistance R. Brown, UK

15:30 HDAC9 in cancer and epigenetic silencing of RAR signalling in AML A. Zelent, UK

16:00 Clinical applications of HDAC inhibitors N. B. La Thangue, UK


ClinicalR. Brown, UK

17:00 HDAC as a therapeutic target for cancer: development of Vorinostat V. Fantin, US

17:30 Epigenetic alterations in leukaemia G. Garcia-Manero, US

18:00 B-cell lymphoma. Therapy driven by molecular signatures M. A. Piris, ES

THURSDAY29 NOVEMBER

PrOGraM

PreclinicalA. Zelent, UK

9:00 A histone methyltransferase inhibitor A. Imhof, DE

9:30 Intervention in epigenetics, a tool to interven in tumor angiogenesis A. W. Griffioen, NL

10:00 Sirtuin, histone acetyltransferase and methyltransferase inhibitors A. Mai, IT

10:30 Functional genetic approaches identify cancerous miRNAs R. Agami, NL


ClinicalM.A. Piris, ES

11:30 Retinoids and arsenic trioxide in the treatment of APL F. Lo Coco, IT

12:00 Lessons to be learnt from the clinical development of HDAC inhibitors S. Marsoni, IT

12:30 Round table discussion

Closing SessionM. Esteller, ES

13:00 Cancer epigenomes and epigenetic drugs M. Esteller, ES

13:30 Closing remarks

FRIDAY30 NOVEMBER


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NOTeSEPIGENETICS AND NEW THERAPIES IN CaNCer


speakers AbstrAct

speakers AbstrActEPIGENEtIcs AND NEW tHErAPIEs IN CaNCer

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Alan Ashworth

Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK

About one in nine women in the Western world develop cancer of the breast and at least

5% of these cases are thought to result from a hereditary predisposition to the disease. Two

breast cancer susceptibility (BRCA) genes have been identified and mutations in these genes

account for most families with four or more cases of breast cancer diagnosed before the age

of 60. Women who inherit loss-of-function mutations in either of these genes have an up to

85% risk of breast cancer by age 70. As well as breast cancer, carriers of mutations in BRCA1

and BRCA2 are at elevated risk of cancer of the ovary, prostate and pancreas. The genes are

thought to be tumour suppressor genes as the wild-type allele of the gene is observed to be

lost in tumours of heterozygous carriers. Both BRCA1 and BRCA2 have significant roles in

the maintenance of genome integrity via roles in the repair of DNA damage via homologous

recombination. The specific DNA repair defect in BRCA-mutant cells provides opportunities

for novel therapeutic approaches based on selective inhibition of functionally interacting repair

pathways, in particular by inhibition of the enzyme PARP. Here I will describe recent work

defining determinants of sensitivity and resistance to PARP inhibitors, as well as the application

of the synthetic lethal approach to other cancer types.

Selected publications

Turner N, Tutt A, Ashworth A (2004) Hallmarks of ‘BRCAness’ in sporadic cancers. Nat Rev Cancer 4: 814-819

Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A (2005) Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434: 917-921

Lord, C.J., Garrett, M.D. and Ashworth, A. (2006) Targeting the double-strand DNA break repair pathway as a therapeutic strategy. Clin Cancer Res, 12, 4463-4468.

Iorns, E., Lord, C.J., Turner, N. and Ashworth, A. (2007) Utilizing RNA interference to enhance cancer drug development. Nat Rev Drug Disc, 6, 556-568.

1Therapeutic exploitation of the DNA repair defects in tumours

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EPIGENEtIcs AND NEW tHErAPIEs IN CaNCerspeakers AbstrAct

The DNA methyltransferase inhibitors 5-azacytidine (azacytidine) and 2’-deoxy-5-azacytidine

(decitabine) play a central role in epigenetic cancer therapies. Several studies have shown that

decitabine is able to induce DNA demethylation in patients, but the situation is much less clear

for azacytidine. We have now characterized the DNA methylation changes in myelodysplastic

syndrome patients undergoing azacytidine therapy. Our data demonstrate significant drug-

dependent methylation changes in blood and bone marrow samples from patients. Importantly,

demethylation was transient and methylation levels were restored to baseline within individual

treatment cycles. Importantly, these data also indicate that demethylation responses towards

azanucleosides are patient-specific. In order to identify the molecular basis for differential patient

responses, we are characterizing the molecular modes of action of azanucleosides in human

cancer cell lines. Our results show that azacytidine and decitabine utilize similar mechanisms for

their cellular uptake. However, the drugs showed different toxicity and demethylation profiles,

suggesting the involvement of different intracellular pathways. The identification of pathways

mediating the drug effects of azanucleosides will be an important aspect for the further

development of epigenetic therapies.

Frank Lyko

Division of epigenetics, German Cancer Research Center, Heidelberg, De

Molecular modes of actions of DNA methyltransferase inhibitors2

EPIGENEtIcs AND NEW tHErAPIEs IN CaNCer

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Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer

progression. Replacement of cytosine in CpG sites with 5-azacytosine results in potent inhibition of

DNA cytosine methyltransferases and reactivation of silenced genes. Yet, the use of 5-azacytidine

or 5-aza-2’-deoxcytidine as chemotherapeutic drugs is restricted by cytotoxicity and limited stability.

The 2-(1H)-pyrimidinone ribonucleoside (zebularine) is a more stable, non-toxic cytidine analog that

can inhibit DNA methylation in vivo with prolonged administration. To determine the mechanistic basis

for the difference in these inhibitors, we have carried out a detailed comparisons of the interaction

between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either

5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation.

Because of its chemical stability and oral activity in mice zebularine was considered for clinical

trials; however, an unexpected toxicity in primates has temporarily halted its progress. In view of the

tortuous metabolic pathway of zebularine, prior to DNA incorporation, efforts to either circumvent

its inefficient activation and to bypass first liver passage (a suspected cause of toxicity) are being

explored with two approaches: (1) use of synthetic nucleotide prodrugs that are able to deliver the key

metabolite, 2’-deoxyzebularine monophoshate, once they enter the cell; or (2) use synthetic ODNs

containing 2(1H)pyrimidinone at the target site. The crystal structure of bacterial M.HhaI methylase in

complex with zebularine-modifed ODNs shows that the nucleotide is rotated out of the double helix.

In forming a tight complex with the enzyme, the rotating motion of the nucleotide is perhaps more

critical than the nature of the nucleobase itself. Moreover, strong inhibition of DNA methylation can

be achieved with ODNs containing simple abasic sites. While exploring the activity of a 13-mer ODN

containing a conformationally restricted abasic pseudosugar at the recognition site of HhaI methylase,

we were able to capture the DNA-protein complex at the mid-point of the flipping trajectory. These

ODNs are some of the most potent inhibitors of DNA methylase known to date. Our approaches to

optimize modified nucleotide prodrugs and ODNs as potential clinical candidates will be highlighted.

The design of mechanism-based, small-molecule inhibitors of DNA methyltransferase: nucleosides, nucleotide prodrugs and oligodeoxynucleotides

Victor E. Marquez

Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, US

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4Epigenetic targets in acute myeloid leukaemia

Clara Nervi

Department of Histology & Medical embryology, University of Rome “La Sapienza” & San Raffaele Bio-medical Park Foundation, Rome, IT

Hematopoiesis is highly controlled by lineage-specific transcription factors that, by interacting

with specific DNA sequences directly activate or repress specific gene expression. These

transcription factors can be mutated or altered by chromosomal translocations associated to

leukemias, indicating their role in the pathogenesis of the differentiation block characterizing

these malignancies. The post genomic era however, has shown that transcription factors

are not the unique key regulators of gene expression. epigenetic mechanisms such as DNA

methylation, post-translational modifications of histone proteins, remodeling of nucleosomes

and expression of small regulatory RNAs all contribute to regulation of gene expression and

determination of tissue and cell specificity. epigenetic deregulations cooperate with genetic

alterations to the establishment and progression of tumors. In acute myeloid leukemia (AML),

which is the clonal expansion of myeloid precusors blocked at different stages of differentiation,

the oligomerization capabilities of chromosomal translocation-generated AML fusion proteins

(PML/RARα and AML1/eTO) results in the aberrant recruitment of histone deacetylase (HDAC)

and DNA-methyltransferase (DNMT) activities on genes that are relevant to differentiation and

transformation process. These enzymes remodel chromatin in a gene specific fashion and affect

proper cell function, differentiation and proliferation. However, in APL patients, the differentiating

agent retinoic acid (RA) epigenetically activates these chromatin sites and represents a

paradigmatic example of a highly effective transcriptional/differentiation therapy. We have shown

that the in vitro and in vivo inhibition of HDAC activities also restores the response of primary

AML blasts to RA, regardless of their underlying genetic lesion. Thus, therapeutic targeting

chromatin remodelling enzymes might represent a potentially novel treatment strategy for AML.

Recently, we demonstrated that the epigenetic regulation of the microRNA 223 (miR-223) levels

correlate with the differentiation fate of myeloid precursors. MiRNAs are negative regulators of

the expression of genes involved in development, differentiation, proliferation and apoptosis.

Their expression is tissue-specific and highly regulated according to the cell’s developmental


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speakers AbstrAct

lineage and stage. Interestingly, miRNAs expressed in hematopoietic cells have been found

mutated or altered by chromosomal translocations associated to leukemias, indicating their role

in the pathogenesis of these malignancies. MiRNAs and their target genes are now regarded

as a potential new class of tumor suppressors or oncogenes. We found the activation of both

pathways of transcriptional regulation by the myeloid lineage-specific transcription factor

C/eBPα, and post-transcriptional regulation by miR-223 essential for granulocytic differentiation

and clinical response of acute promyelocytic leukemia blasts to RA. Results of our ongoing

research activity provide further evidence linking the epigenetic silencing of a miRNA locus to

AMLs. Indeed, miRNAs silencing follows aberrant chromatin remodeling events resulting from

a deregulated activity of myeloid transcription factors and results in the differentiation block of

hematopoietic progenitors and AML. This evidence underlies transcription factors, chromatin

remodeling and miRNAs as ultimate determinants for the correct organization of cell-type

specific gene arrays and hematopoietic differentiation, thus providing new targets for the

diagnosis and treatment of leukemias.

Epigenetic targets in acute myeloid leukaemia

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Chromatin is epigenetically modified to regulate gene expression. Upstream signals induce

complex patterns of enzyme-catalyzed modifications of DNA and histones, key protein

components of chromatin. emerging evidence causally links altered epigenetic functions to

oncogenesis, and suggests that chromatin regulators and upstream pathways are critical

targets for developing novel anti-cancer drugs (epi-drugs). Indeed, the demonstration that the in

vivo anti-cancer activity of histone deacetylase inhibitors (HDACi’s) is causally linked to induction

of cancer cell-selective apoptosis has provided proof-of-principle for the potency of epi-drugs.

The concept of “epigenetic cancer treatment” from the mechanism to animal models will be

discussed as well as applications of epi-drugs to other non cancer human diseases.

Angela Nebbioso, Marco Miceli, Floriana De Bellis, Mariarosaria Conte, Fabio Manzo, Vincenzo Carafa, FrancescoPaolo Tambaro, GianLuigi Franci, Ilaria Lepore, Vincenzo Di Cerbo, Lucia Altucci

Dipartimento di Patologia generale, Seconda Università degli Studi di Napoli, Napoli, IT

5Use of Epi-drugs against cancer roles of class I and II HDACs in cell proliferation, differentiation and apoptosis


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epigenetic changes occurs both during tumour development and during the acquisition

of drug resistance of tumours. Unlike genetic changes, epigenetic modifications require

active maintenance so they can be manipulated by small molecules and provide a potential

therapeutic target. Although epigenetic therapies such as DNA methyltransferase (DNMT)

inhibitors have shown considerable promise in the treatment of haematological malignancies,

their ability to inhibit growth of solid tumours is less clear. Clinical trials of DNMT inhibitors have

been ongoing for over 30 years ago, but initial studies in solid tumours showed little clinical

activity. However, clinical trials of such epigenetic therapies are now being re-evaluated as a

result of our increased understanding of the mechanisms of action of these agents and using

rational approaches to drug combinations based on preclinical animal model studies.

DNMT and HDAC inhibitors alone and in combination have shown additive or synergistic

activity in pre-clinical models in combination with a number of agents including cytotoxics.

The combination of DNMT and HDAC inhibitor can be more effective at reversing epigenetic

silencing than either agent alone. An increase in histone acetylation or decrease in CpG

methylation produces a more open DNA conformation and it has been proposed that this might

result in greater DNA damage following ionising radiation or platinum based chemotherapy.

There is also evidence that HDAC and DNMT inhibitors alter cellular responses to DNA damage

resulting in greater cell death. This may be mediated by altering expression of genes involved

in apoptosis, including re-expression of pro-apoptotic genes that are epigenetically silenced

during the acquisition of drug resistance.

Biological endpoints as trial objectives that assess whether a pharmacodynamic target is being

affected, such as demethylation in peripheral blood or histone deacetylation in PBMC, are

important and provide a first step in evaluating a potential epigenetic therapy. However markers

of biological effect such as markers of gene re-expression or induction of cell death may be

Combination studies of DNMT and HDAC inhibitors in overcoming cytotoxic drug resistance

Robert Brown

Imperial College, London, UK

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Combination studies of DNMT and HDAC inhibitors in overcoming cytotoxic drug resistance

more informative about efficacy and therefore be more useful in determining optimal schedules

and drug combinations. Identification of markers that predict for response to treatment is also

a high priority, but their sensitivity, specificity and reliability need to be carefully evaluated before

they should be used to determine future trial design. As these pharmacodynamic and biomarker

endpoints are more widely used, it will be vital to standardise and harmonise the methodology

in a quality assured manner. This will allow more sophisticated comparison of different treatment

regimens at an earlier stage and more confidence about the design of the eventual definitive

randomised Phase III trial. Randomised Phase II trials with biological endpoints included as trial

objectives, which assess biological as well as clinical efficacy and begin to evaluate the potential

of predictive markers, will become increasingly important.


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speakers AbstrAct

7DNA methylation-independent silencing of rara gene expression in acute myeloid leukemia

Annegret Glasow1,2, Angela Barrett1, Kevin Petrie1, Rajeev Gupta3, Da-Cheng Zhou4, David Grimwade5, Robert Gallagher4, Marieke von Lindern6, Samuel Waxman7, Tariq enver3, Arthur Zelent1

1. Section of Haemato-Oncology, Institute of Cancer Research, Sutton, UK2. University of Leipzig, De3. MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK4. Albert einstein Cancer Centre, New York, US5. King’s College, London, UK6. erasmus Medical Centre, Rotterdam, NL7. Mount Sinai School of Medicine, New York, US

Acute myeloid leukemia (AML) is characterized by an accumulation of undifferentiated progenitors

in the bone marrow and was originally classified into different subtypes on the basis of blast

morphology. More recently, however, AML is increasingly categorized according to recognized

cytogenetic and molecular characteristics that are likely to represent primary events in

leukemogenesis. Despite dramatic progress in the understanding of the molecular basis of AML

over the past three decades, overall survival rates remain low (http://seer.cancer.gov), particularly

for older individuals (40% for patients over 65 years of age), reinforcing the need for more effective

therapies. Among the subtypes of AML, acute promyelocytic leukemia (APL), which in the majority

of cases is associated with the translocation between the retinoic acid receptor alpha (RARA) and

PML genes leading to expression of the PML-RARα fusion oncoprotein (other much less common

rearrangements involving RARA have been describe), responds uniquely to differentiation therapy with

all-trans-retinoic acid (ATRA). Given that PML-RARα acts to inhibit the positive effects of physiological

ATRA on regulation of gene expression and myelomonocytic differentiation, these dramatic

therapeutic effects seemed at first paradoxical. However, progress in understanding the molecular

mechanisms through which RARs and other nuclear receptors regulate gene expression, which

involves ligand-mediated exchange of co-repressor for co-activator and post-activation receptor

degradation, provided a platform for understanding how administration of pharmacological levels of

ATRA can restore RARα signalling and differentiation in APL. Nevertheless, the molecular basis for the

general lack of response of non-APL AML to ATRA remains poorly understood.

RARA encodes two major isoforms and mediates positive effects of all-trans-retinoic acid

(ATRA) on myelomonocytic differentiation. We have previously demonstrated that expression

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of the ATRA-inducible RARα2 isoform increases with differentiation of murine hematopoietic

progenitors along the myelomonocytic lineage and AML cell lines including ATRA resistant

APL cells do not express this isoform effectively. This lack of RARα2 expression and ATRA

response may, at least in part, be a reflection of a general impairment of ATRA signaling, which

may involve genetic factors and/or epigenetic mechanisms. To examine this hypothesis we

have evaluated in parallel expression of the RARA gene and DNA methylation of its promoters

in normal hematopoietic stem/progenitor cells and primary AML blasts. We have determined

that relative to normal myeloid stem/progenitor cells, RARα2 expression is dramatically

reduced in primary AML blasts. expression of the RARα1 isoform is also significantly reduced

in primary AML cells, but not in AML cell lines. Although the promoters directing expression

of RARα1 and RARα2 are unmethylated and methylated, respectively, in AML cell lines, these

regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover,

in primary AML cells histones associated with the RARα2 promoter showed diminished levels

of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the

mechanisms responsible for silencing of gene expression in AML and support the notion that

diminished RARα expression contributes to leukemogenesis.

DNA methylation-independent silencing of RaRa gene expression in acute myeloid leukemia


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speakers AbstrAct

Aberrant acetylation has been strongly linked to tumourigenesis and the modulation of

acetylation through targeting histone deacetylase (HDAC) is gathering increasing pace as a

viable therapeutic strategy. However, the mechanism through which HDAC inhibitors kill cells is

not known, although it is widely believed to involve altered chromatin control. Using a genome-

wide loss-of-function screen, we have identified genes that influence the sensitivity of tumour

cells to HDAC inhibition. The results have important implications for understanding how HDAC

inhibitors kill cells, and provide informative biomarkers that might allow patient stratification into

favourable response groups.

Genome-wide loss-of-function screen reveals an unexpected mechanism for HDAC-inhibitor-induced apoptosis8

Susan Fotheringham1, Lindsay Stimson1, Omar Khan1, Victoria Wood1, Mirjam T. epping2, Rene Bernards2, Nicholas B. La Thangue1

1. Laboratory of Cancer Biology, Department of Clinical Pharmacology, Medical Sciences Division, John Radcliffe Hospital, University of Oxford, UK2. Division of Molecular Carcinogenesis and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, NL

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9 B-Cell receptor signaling and TCl1a expression delineate molecular and clinical variability in B-Cell non hodgkin lymphoma

Mohit Aggarwal1, Nerea Martínez1, Raquel Villuendas1, Margarita Sanchez-Beato1, Antonia Rodriguez1, Fatima Al-Sharour3, Gonzalo Gomez5, Abel Sanchez-Aguilera1, elena Ruiz-Ballesteros2, Francisca I. Camacho1, Alberto Pérez1, Paloma de la Cueva1, María Jesús Artiga1, Jose A. Garcia-Marco4, David G. Pisano5, eva Kimby6, Joaquín Dopazo3, Miguel A. Piris1

1. Molecular Pathology Programme, Centro Nacional de Investigaciones Oncológicas, Madrid, eS2. Genetics and Pathology Departments, Hospital Virgen de Salud, Toledo, eS3. Prince Felipe Research Centre, Valencia, eS4. Hematology Department, Hospital Universitario Puerta de Hierro, Madrid, eS5. Bioinformatics Unit, Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncológicas, Madrid, eS6. Department of Hematology, Huddinge Hospital, Karolinska Institutet, Stockholm, Se

B-cell lymphomas are presently diagnosed according to the WHO criteria based on the integration of

morphologic, immunophenotype and cytogenetic findings. However, the precise distinction among

common lymphoma types is frequently a difficult task, and there are areas of overlapping between

different lymphoma types, and heterogeneity within specific tumor types. More importantly, the

knowledge so far accumulated on B-Cell Lymphoma pathogenesis has not facilitated enough the

development of targeted therapies. Here we have analyzed whether gene expression profiling (GeP)

data, solely considered, could be used to validate the currently used B-cell lymphoma classification,

or proposing new lymphoma types, and for identifying functional signatures or eventual therapeutic

targets. To this aim, we have analyzed Gene expression Profiling (GeP) for 170 cases of B-cell NHL,

including the most frequent histological types.

The functional signatures overruling lymphoma classification were defining cell cycle, apoptosis,

cytokine-cytokine receptor interaction, T-cell receptor, B-cell receptor, cell adhesion, and

NF-kB activation. Moreover these signatures reveal sub-classes for diagnosed lymphoma

types, suggesting the existence of a distinct functional heterogeneity among CLL, MCL

and DLBCL. Multiple pathways define this molecular heterogeneity, but among them, BCR

signaling emerged as a decisive pathway, whose expression was found to be related with TCL1

expression. Validation studies allow to confirm the clinical and functional relevance of both BCR

signaling and TCL1 expression in different types of B-cell NHL.


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Histone modifications play a crucial role in the establishment and maintenance of gene

expression patterns. In order to faithfully maintain a particular modification pattern mechanisms

have to exist that allow the replication of histone modification patterns at defined loci. We

employ an efficient chromatin assembly system made from Drosophila embryonic extracts to

study the kinetic of particular histone modification in the test tube. We find a slow deacetylation

of the initially diacetylated isoform of histone H4 that occurs randomly and non-processively

at positions 5 and 12. In contrast to the observed deacetylation of H4, H3 is undergoing an

equilibrium shuffling between acetylated and non-acetylated states. Despite the fact that

histone H3 methyltransferases are present in the extract we do not observe methylation of

H3 indicating that methyltransferases are catalytically impaired in embryonic extracts. We will

describe the use of histone methyltransferase inhibitors to study the activity and the functional

interplay of these enzymes in setting up defined chromosome structures during and after

cellular division.

A histone methyltransferase inhibitor10Axel Imhof

Ludwig-maximilians University of Munich, De

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Epigenetic regulation of tumor angiogenesis; suppression of ICAM-1 in tumor endothelial cells and escape from anti-tumor immunity

Arjan W. Griffioen

Angiogenesis Laboratory (www.angiogenesis.nl), Research Institute for Growth and Development (GROW), Dept. of Pathology, Maastricht University & University Hospital, Maastricht, NL

In order to grow efficiently and metastasize to distant sites, tumors require angiogenesis to occur.

Tumor angiogenesis is an intricately regulated process requiring a strictly sequenced regulation of

gene expression in endothelial cells (eC). We recently showed that DNA methyltransferase (DNMT)-

and histone deacetylase (HDAC) inhibitors directly repress eC growth and tumor angiogenesis,

suggesting an epigenetic level of regulation. To investigate the background of this, we performed

microarray analysis to find genes downregulated in tumor-conditioned versus quiescent eC, and

identify the ones that are re-expressed by 5-aza-2’-deoxycytidine and trichostatin A. This approach

identified novel angiogenesis suppressing genes that are silenced in tumor eC and become

reactivated by DNMT- and HDAC inhibitors. We found that ICAM-1, a molecule that is suppressed

on tumor eC, is re-expressed after treatment with DNMT inhibitors 5-aza-2’-deoxycytidine and

zebularine, as well as HDAC inhibitor trichostatin A. This resulted in restored ICAM-1 expression and

leukocyte eC interactions both in vitro as well as in mouse tumor models, as measured by intravital

microscopy (see figure). Also an enhanced leukocyte infiltration was observed. In conclusion, our data

show that ICAM-1 is epigenetically silenced in tumor eC by promoter histone modifications, leading

to tumor escape from immunity. This problem can be overcome by treatment with DNMT- and HDAC

inhibitors, directly suggesting a new therapeutic strategy for cancer.

11


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12

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) are key enzymes involved in

determining the histone acetylation, which play an important role in epigenetic regulation of

gene expression. Class I/II HDAC inhibitors can reactivate gene expression and result potent

inducers of growth arrest, differentiation, or apoptotic cell death in a variety of transformed cells

in culture and in tumor bearing animals. Class III HDACs (sirtuins, SIRT1-7) are less validated

as therapeutic targets. In yeast, Sir2 is critical for transcriptional silencing at the telomeres, the

ribosomal DNA, and the silent mating type loci. Sir2-like proteins also deacetylate non-histone

proteins, including the tumor suppressor p53 and α-tubulin. The human Sir2 ortholog, SIRT1,

has been implicated in a variety of important disease-related processes including silencing of

p53, inflammatory responses, cell defence and survival, and fatty acid metabolism, and finding

molecules that modulate these enzymes is considered a possible route for disease treatment.

In a search for potent sirtuin inhibitors as apoptotic and/or cytodifferentiating agents, we

prepared a series of sirtinol analogues, and two of them (meta- and para-sirtinol) were from

2- to 10-fold more potent than sirtinol against human SIRT1 and SIRT2 enzymes. Moreover,

a lot of them showed interesting pro-apoptotic and/or cytodifferentiating properties against

the human leukaemia U937 cell line. Afterwards, using the SIRTi cambinol as a template,

we prepared some benzooxadeazaflavines as potent, SIRT1-selective inhibitors active at

nanomolar concentration.

HATs misregulation also is invariably associated to human pathologies such as tumors. We

identified the quinoline MC1626 as a novel, cell permeable Gcn5p inhibitor able to inhibit yeast

cell growth, the Gcn5-dependent transcription, and HAT acetylation in vivo. Such compound

was a useful starting point for the further development of new, more potent molecules that can

be applied to study the expression profile of genes regulated by histone H3 acetylation.

Sirtuin, histone acetyltransferase and methyltranferase inhibitors

Antonello Mai

Dipartimento di Studi Farmaceutici, La Sapienza Università di Roma, Roma, IT

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In addition to acetylation, histone and non-histone proteins can undergo methylation reactions

at both Lys and Arg residues. Protein arginine methyltransferases (PRMTs) and histone lysine

methyltransferases (HKMTs) are epigenetic enzymes involved in regulation of gene expression

and cellular processes such as cell proliferation, signal transduction, mRNA splicing, RNA

transport, and protein-protein interactions. PRMTs are known coactivators for nuclear

receptors, so they may represent likely candidates to be overexpressed in the hormone-

dependent prostate and breast cancers. Among HKMTs, SeT7/9 has been reported to act

on histones as well as on p53 leading to gene silencing. In an effort to find small molecules

that could represent lead compound MT inhibitors, we designed and synthesize a series of

compounds as simplified analogues of eosin, recently reported as HMTi. A number among

such compounds behaved as epigenetic multiple ligands (epi-MLs), by inhibiting at the same

time not only PRMTs and HKMTs but also HAT and SIRTs. Such epi-MLs, tested on U937 cells,

showed interesting pro-apoptotic and cytodifferentiating properties, differently from the related

single-target inhibitors.

Sirtuin, histone acetyltransferase and methyltranferase inhibitors


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speakers AbstrAct

Functional genetic approaches identify cancerous miRNAs

microRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes.

Patterns of mis-expression of miRNAs in cancer suggest key functions of miRNAs in

tumorigenesis. However, current bioinformatics tools do not fully support the identification

and characterization of the mode of action of such miRNAs. To perform genetic screens for

novel functions of miRNAs we developed a library of vectors expressing the majority of cloned

human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that

cooperate with oncogenes in cellular transformation we identified miR-372 and miR-373, each

permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic

RAS and active wild type p53. We provided evidence that these miRNAs are potential novel

oncogenes participating in the development of human testicular germ cell tumors by numbing

the p53 pathway, thus allowing tumorigenic growth in the presence of wild type p53. Recently,

we have used a novel functional genetic approach and identified miR-221 and miR-222

(miR-221&222) as potent regulators of p27Kip1, a cell cycle inhibitor and tumor suppressor.

Interestingly, high miR-221&222 levels appear in signatures of poor prognosis cancers. Using

miRNA-inhibitors we demonstrated that certain cancer cell lines require high activity of miR-

221&222 for the maintenance of low p27Kip1 levels and continuous proliferation. Thus, high

levels of miR-221&222 promote cancerous growth by inhibiting the expression of p27Kip1.

Reuven Agami

Department of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, Ne

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14Treatment of acute promyelocytic leukemia with ATRA and arsenic trioxide

As reported in several large multicenter trials, front-line use of combined ATRA and

anthracycline chemotherapy results in long-term remission and potential cure in >70%

of newly diagnosed APL patients. The Italian multicenter Group GIMeMA reported in

1997 high rates of molecular remission in newly diagnosed and genetically confirmed APL

using a simultaneous Atra plus IDArubicin (AIDA) combination for induction treatment,

followed by 3 courses of intensive chemotherapy as consolidation. This protocol, with slight

modifications, was subsequently adopted by other groups including the Spanish PeTHeMA

cooperative group who reported similar antileukemic efficacy by omitting Ara-C and other

non-intercalating agents from the original AIDA, with the advantage of sparing toxicity and

increasing compliance to treatment. Despite the dramatic progress achieved in front-line

therapy with the ATRA/chemotherapy combination, relapses still occur in approximately 20%

of APL patients. Moreover, these regimens are associated with significant toxicity due to

severe myelosuppression frequently resulting in life-threatening infections, and with serious,

though infrequent late complications such as cardiomyopathy and the occurrence of secondary

myelodysplastic syndromes and/or acute myeloid leukemias. Several means are available to

decrease toxicity in the treatment of newly diagnosed APL, including the availability of less toxic

and highly effective agents such as arsenic trioxide (ATO). The mechanism of action of ATO in

APL is complex and not yet known in detail. At a high concentration (0.5 to 2.0 µmol/L) ATO

induces apoptosis in vitro, through induction of caspases 2 and 3, while at lower concentrations

(0.1 to 0.5 µmol/L) it induces partial differentiation of leukemic promyelocytes through PML/

RARa degradation; furthermore, ATO is known to inhibit angiogenesis via down- regulation of

vascular endothelial growth factor (VeGF). According to original clinical trials reported in China,

ATO was able to induce hematologic CR in >85% patients who relapsed after front-line ATRA.

These results were subsequently reproduced in the USA and europe. Significantly, unlike ATRA,

ATO as a single agent was able to induce durable molecular remission after two cycles in the

majority of patients treated for disease recurrence. In addition to trials in which ATO was used

Francesco Lo-Coco

Department of Biopathology, University Tor Vergata, Rome, IT


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a single agent, some studies investigated its efficacy and toxicity profile in combination with

other agents including ATRA. Synergism with ATRA and increased antileukemic efficacy in APL

was demonstrated in a Chinese randomised study comparing ATO+ATRA vs. either ATO or

ATRA used as single agents. Following the experience in relapsed patients and based on the

favourable toxicity profile, several investigators have more recently explored the effect of ATO in

newly diagnosed APL patients and reported very encouraging preliminary findings in front-line

therapy. Although the results of such studies need to be strengthened by studies in larger

series and with more prolonged observation, they strongly suggest that at least non-high risk

APL patients may be cured without chemotherapy. This relevant issue will be addressed in the

next trial of the Italian Gimema group.

Treatment of acute promyelocytic leukemia with ATRA and arsenic trioxide

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15 Epigenetic Alterations in Cancer and Therapy

Manel Esteller

Cancer epigenetics Group, Spanish National Cancer Research Centre (CNIO), Madrid, eS

epigenetic alterations have moved to a prominent place in cancer research. Key breakthroughs

in the epigenetics include the identification of a code of histone modifications associated

with specific functions, the finding of mechanistic links between DNA methylation and

histone modifications or the recognition that epigenetics not only defines cell identity but

also constitutes a dynamic readout of environment. In addition, the identification of profound

alterations in epigenetic regulation in cancer has atracted the interest of many reserachers and

pharmaceutical companies, since these alterations are potentially reversible.

The discovery that drastic changes in DNA methylation and histone modifications are commonly

found in human tumors has inspired various laboratories and pharmaceutical companies to

develop and study epigenetic drugs. One of two most promising groups of agents are the

inhibitors of histone modification and DNA methylation enzymes.

We are at the dawn of an era when epigenetic drugs will be an important weapon in our arsenal

in the war against cancer. Hematological malignancies have provided a promising starting point,

but studies will surely extend to all solid tumors. However, we need to continue our research

to develop more specific DNA demethylating agents as well as specific inhibitors for histone

modification enzymes, to understand their biologic effects, and to determine whether they may

be successfully combined with other therapeutic compounds.

NoTesEPIGENEtIcs AND NEW tHErAPIEs IN CaNCer

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40


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parTICIpaNTs POstErs

1parTICIpaNTs POstErsEPIGENEtIcs AND NEW tHErAPIEs IN CaNCer

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Glucocorticoid resistance in childhood acute lymphoblastic leukaemia associated with epigenetic silencing of the pro-apoptotic BH3-only gene Bim

P. S. Bachmann, R. Piazza, V. Magistroni, C. Gambacorti-Passerini, R. C Gorman, R. B.Lock

Leukaemia Biology Program, Children’s Cancer Institute Australia for Medical Research, Sydney, AUSchool of Women’s and Children’s Health, University of New South Wales, Sydney, AUDepartment of ‘Medicina Clinica, Prevenzione e Biotecnologie Sanitarie’, University of Milano-Bicocca, Monza, IT

Glucocorticoids play a central role in all protocols for the treatment of human lymphoid

malignancies, including childhood acute lymphoblastic leukaemia (ALL) in which initial response

to glucocorticoid therapy is a critical prognostic factor. However, the mechanisms by which

glucocorticoids induce apoptosis of leukaemia cells, and the lesions underlying resistance,

remain poorly understood. An in vivo model of childhood ALL has been developed in our

laboratory, using patient biopsies established as xenografts in immune-deficient (NOD/SCID)

mice. The responses of these xenografts to the glucocorticoid dexamethasone range from

sensitivity to high-level resistance both in vivo and in vitro. We have previously shown that

response to glucocorticoids in this model system is associated with: (i) ligand-induced

nuclear translocation of the glucocorticoid receptor (GR); (ii) GR-mediated changes in gene

transcription; and (iii) induction of the pro-apoptotic Bcl-2 family member Bim resulting in

activation of the intrinsic apoptotic pathway and cell death. The difference between sensitive

and resistant xenografts was evident in the failure of 5/6 highly resistant xenografts to induce

mRNA and protein levels of Bim (Bachmann et al, 2005, Blood, 105:2519-26; Bachmann et al,

2007, Cancer Research, 67:4482-90). The bim promoter has been shown to be encompassed

by a CpG island that is actively methylated in lymphoid cell lines, and treatment with

demethylating agents caused induction of bim and apoptosis (Piazza et al, 2005, Blood, 106:

Abstract 2625). Analysis of the bim genomic locus by chromatin immunoprecipitation in this

panel of xenografts revealed that failure to induce bim was significantly associated with histone-

H3 deacetylation, and the extent of Bim protein induced by dexamethasone (1 mM, 16h)

correlated with basal acetylation levels (r=0.9091, p<0.001). Furthermore, bisulfite sequencing

and methylated DNA immunoprecipitation assay showed 2/3 highly resistant xenografts

also exhibited DNA methylation of this region, in contrast with 3/3 sensitive xenografts that

were demethylated. When treated with the histone deacetylase inhibitor vorinostat, a dose-

dependent increase in acetyl-histone H3 associated with the bim promoter was observed,

and, when combined with dexamethasone in vitro, an increased response to dexamethasone

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parTICIpaNTs POstErsEPIGENEtIcs AND NEW tHErAPIEs IN CaNCer

was observed in the highly resistant xenograft that did not also exhibit DNA methylation

at this genomic region. These results indicate that pharmacological modulation of histone

acetylation and DNA methylation at the bim locus may provide a novel therapeutic strategy for

circumventing glucocorticoid resistance in childhood ALL.

Keywords: leukaemia, glucocorticoid, resistance, bim, vorinostat

Glucocorticoid resistance in childhood acute lymphoblastic leukaemia associated with epigenetic silencing of the pro-apoptotic BH3-only gene Bim


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2 Epigenetic diagnostic method of brain tumors

A. M. Barciszewska, S. Nowak, R. Zukiel, I. Gawronska and M. Z. Barciszewska

Chair and Clinic of Neurosurgery and Neurotraumatology, Karol Marcinkowski University of Medical Sciences, Przybyszewskiego 49, Poznan, PLInstitute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego, Poznan, PL

.

´´

Background

Human brain tumours remain to be a diagnostic and therapeutic challenge for contemporary

medicine. Despite many advanced tools and drugs their detection is still too late and the

treatment uneffective. We describe a new, simple and reliable method for diagnosis of brain

tumours. It is based on thin layer chromatography (TLC) quantitative determination of 5-

methylcytosine (m5C) in relation to its some damage products of DNA from tumour tissue and

blood from brain tumour patients. Currently there are many evidences that oxidative stress

through reactive oxygen species (ROS) play an important role in the etiology and progression of

a number of human diseases. m5C along with other basic components of DNA are the targets

for ROS what results in the appearance of modified nucleic acid bases. Therefore the analysis

of global m5C amount in DNA, can put a new light on neoplasia.

Aim

The aim of our work was to check the global m5C content in tissues taken from patients with

brain tumors.

Methods

We present the results of the analysis of over 400 DNA samples taken from brain tumour

tissues, 100 of them combined with blood samples from the same patients. DNA was isolated,

hydrolysed into nucleotides and separated on TLC after labelling with 32P. Chromatograms

were evaluated using phosphoimager and the amounts of m5C calculated as a ratio (R) of spot

intensities of m5C to m5C+C+T.

Results/Discussion/Conclusion

The R value decreases as the malignancy of brain tumour increases, so it could be a good

diagnostic marker not only for brain tumours but also differentiate low and high grade gliomas.

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Therefore DNA methylation pattern might be a useful tool to give primary diagnosis of brain

tumour or could be a marker for early detection of relapse of disease, especially because the R

values for the same patients are similar in their blood and brain tumour tissues. The method has

several advantages over those existing now.

Keywords: DNA methylation, m5C content, epigenetics, brain tumor, diagnostics, TLC analysis

Epigenetic diagnostic method of brain tumors


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3 The role of the histone 9 methyltransferase in epigenetic regulation of facultative heterochromatinisation during embryonic development

S. Bongiorni, B. Pasqualini, S. Volpi, F. Avanopoulou, A. Imhof, P. B Singh and G. Prantera

University of Tuscia, Viterbo, IT

Facultative heterochromatin denotes those euchromatic regions that adopt all the cytological and

functional features of heterochromatin in a developmentally, and/or tissue-specific regulated manner. As

such, facultative heterochromatinisation represents a paradigmatic example of epigenetic regulation.

The male-specific facultative heterochromatinisation in mealybugs offers thus a very sharp

tool to investigate this phenomenon, since the heterochromatinisation onset can be carefully

monitored during male embryo development; and we can observe a unique phenomenon of de-

heterochromatinisation in some adult male tissues.

We showed that the developmental timing of two heterochromatin proteins, HP1-like (named

PCHeT2) and HP2-like, is strictly correlated to the nucleation of the male-specific facultative

heterochromatin. Both proteins mark the chromatin before it acquires the full cytological

characteristics of the male-specific heterochromatin. We found that tri-methylated lysine 9 of

histone H3 and tri-methylated lysine 20 of histone H4 are a distinctive feature of male-specific

heterochromatin. Using RNAi we have conducted a functional analysis of the Hp1-like chromobox

gene pchet2 during embryogenesis. extinguishing pchet2 expression results in decondensation of

the male-specific heterochromatin. Along with the disappearance of the chromocenter the levels of

the two associated histone modifications (Me(3)K9H3, Me(3)K20H4) are reduced to undetectable.

Strikingly, HP2-like is not affected by HP1-like depletion.

Further, we verified the interplay of di- and tri-MeK9H3 with HP1 during facultative

heterochromatinisation by using the mealybug Su(var)3-9-like dsRNA in RNAi experiments and the

histone methyltransferase inhibitor, chaetocin.

As a whole, our results strongly argue that HP1 and SU(VAR)3-9 play a central role in facultative

hetrochromatinisation and that they are likely involved also in chromosome condensation and in

chromosome mitotic behaviour.

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Chromatin modifying proteins have received increasing attention during recent years.

Histone tails can be enzymatically modified in several ways. In addition to acetylation, a main

regulatory mode is through methylation. Histone methylation of certain lysine residues, such

as lysine 9 on histone 3 (H3K9), is generally viewed as repressive for transcription, while

methylation of other residues, such as lysine 4 on histone 3 (H3K4) exerts positive effects

on transcription. We have previously showed that H3K9 is normally hypoacetylated in neural

stem cells (NSC) in vitro, and increased acetylation of H3K9 (but not other residues) at a

subset of genes is specifically associated with neuronal differentiation. A pre-requirement

for this acetylation control mechanism is that H3K9 is demethylated. Historically, histone

methylation has been considered a stable modification, but recently it has been shown that a

certain family of proteins containing the so-called Jumonji domain (JmjC) can act as histone

demethylases.

In this study, we have investigated the role of H3K9-demethylating enzymes, in particular

JMJD1 and JMJD2 Jumonji families, in neural stem cells (NSCs) derived from embryonic

rodent cortices and expanded with basic fibroblast growth factor (FGF2) as monolayers.

experiments using RT-PCR and RT-qPCR revealed that the seven members of JMJD1 and

JMJD2 families investigated were expressed in the NSCs.

We subsequently treated the NSCs with two different extrinsic factors that can induce

neuronal and oligodendrocyte differentiation: valproic acid (VPA) and thyroid hormone (T3),

respectively. NSCs treated with VPA, an inhibitor of histone deacetylases (HDACs), showed

an increased transcription of JMJD2C that correlated with a decrease in the methylation

state of H3K9. Furthermore, preliminary results showed that the overexpression of JMJD2C

in these cells led to a decrease in the methylation pattern of the same lysine. Our results

further suggest that T3 treatment causes not only a significant reduction in the methylation

4A. Cascante and O. Hermanson

Department of Neuroscience and Center of excellence in Developmental Biology, Karolinska Institutet, Stockholm, Se

Epigenetic regulation of neural stem cell state and fate by jumonji histone demethylases


52

state of the H3K9, but also maintenance of the expression levels of JMJD2D in NSCs.

Moreover, we observed an upregulation of JMJD2D during oligodendrocyte differentiation in

an oligodendrocyte precursor cell line. Thus, our results suggest that different Jumonji histone

demethylases play distinct roles during neural stem cell differentiation.

Key words: Jumonji histone demethylases, neural stem cells, valproic acid and thyroid hormone

Epigenetic regulation of neural stem cell state and fate by jumonji histone demethylases

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Dendritic cells constitute extremely attractive vectors for immunotherapy, due to their unique

potential to induce primary immune responses in naïve CD4+ and CD8+ T cells using their

ability to efficiently uptake, process, and present the antigens .

The present research was aimed to optimize the immune response by studying alternative

methods of dendritic cells’ antigenic loading.

Results demonstrated that CD8+ T cell cross-priming strategy using monoclonal antibody-

coated tumor cells is superior both quantitatively and qualitatively to the standard antigen-

loading procedures currently used.

By generating an increased number of tumor-specific T cells with a broad range multivalent

repertoire, this novel method offers new strategies for dendritic cell-based vaccines and

adoptive T cell therapy.

Novel strategies for modulation of dendritic cells’ antigen-presenting activity

D. P. Cioca, D. A. Rusu, S. Anghel, C. Gluhovschi

Timisoara University of Medicine and Pharmacy, Timisoara, RO

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6 Structure, cytotoxic properties and mechanism of several gold and silver tiolates compounds

Mª A. Roy1, e. Sinués1, S. emperador1, F. de la Iglesia1, C. Alcaine1, A. Gascón1, Á. Alcalá1, A. Laguna2, C. Gimeno3, B. Conde1

1. Departamento de Anatomía e Histología Humanas, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, eS2. Departamento de Química Inorgánica, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, eS3. Instituto de Ciencias de los Materiales de Aragón (Universidad de Zaragoza y CSIC), Zaragoza, eS

Although cisplatin (CDDC) is de most important quimiotherapic agent, it has undesirable

side effects and in some cases the tumor shows resistance. Besides, it is no effective for the

treatment of certain tumors like lung carcinoma and colon adenocarcinoma. Coordination

compounds with transition metals are expected to have antitumor activity. We have studied

several compounds with thiol ligands and different metallic centre (gold and silver, binuclear

or mononuclear). Some of them show interesting cytotoxic properties when tested against

two breast cancer cell lines, MDA-MB-231 and MCF-7. As expected, ligands alone also have

toxic effects. The study of cell cycle shows an important percentage of apoptotic cells and

decreased number of cells in phase G1. Gold thiolates are used in the treatment of arthritis and

reduce inflammatory response. Tetraacetatethioglucose or Auranofin is active against tumor

cells as HeLa and P388. Thus gold coordination compounds may be suitable also for the

treatment of myeloma.

The mechanism of cytotoxicity is still unknown. Like other inorganic drugs, there are several

possibilities. Due to differences of structure when compare with cisplatin, direct union to DNA is

unlikely to be the main way. Thus, these compounds might be potential agents in the treatment

of CDDC-resistant tumors. As arsenic trioxide, interactions with proteins is possible by binding

to –SH. There is an evident effect on lysosome and mitochondrial, possibly due to ROS

generation. We also investigated the role of some apoptotic key molecules, such as caspases,

AIF, Bax and Bcl-2.

keywords: gold-tiolate compounds, breast adenocarcinoma, apoptosis

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Aging ADN cancer induced chromatin perturbation through DNA methylation and histone acetylation/deacetylation dynamics7

N. Cucu1, G. Anton2, L. Burlibasa1, M. Matei3, C. Birsan4, G.L. Radu4, T. elena5, A. Botezatu2

1. University of Bucharest, Faculty of Biology, Dept. of Genetics, epigenetic Lab, Bucharest-Romania, RO2. National Institute of Virology “Stefan Nicolau”, Bucharest, Dept of Molecular Biology, Romania, RO3. CFR2 General Hospital, Dept. of Genetics, Bucharest-Romania, RO4. National Institute of Biological Sciences, Bio-Analysis Dept. Bucharest-Romania, RO5. Ana Aslan National Institute of Gerontology and Geriatrics, Bucharest-Romania, RO

epigenetic factors such as DNA hypo-and hypermethylation, as well as histone acetylation/

deacetylation play an important role in both aging and cancer induced genome instability. However,

the two cell types characteristic for both processes differ by their regenerative capacity and their

cell cycle activity. Our study involved six - elderly (60 plus years), adult (30-45 years), and young

(18-30 years) groups, as follows: one young group with controlled sportive life, one young group with

uncontrolled lifestyle, two adult groups with and without health problems, and two elderly groups,

with vs. without significant pathology. This experimental model was chosen to prove the similarity of

DNA methylation dynamics both throughout the whole genome and, locally, in specific, eR alpha and

RAR beta, genes, during both processes. Also, the inhibition of both important epigenetic effector

enzymes, DNMT and HDAC, have been performed in the genome of the lymphocytes derived from

the young group, in order to demonstrate their role in senescence induction through both DNA and

histone modification dynamics. Moreover, the cell division processes have been studied through

karyotyping which showed frequent anneuploydies and premature chromatide separations, due to

altered pericentromeric heterochromatin structures.

The study has been completed with methyl-metabolomic results which showed correspondingly

low SAM/SAH ratios to the DNA hypomethylated state and deacetylated histones, inspite

normal mthfr polymorphism.

A conclusion has been raised regarding the importance of knowledge of the molecular

mechanisms of the heterochromatin formation and maintenance in both cancer and aging

processes for desciphering the ability of cellular senescence for suppressing tumorigenesis,

which may be further used for new anticancer therapeutical interventions.

Keyword: DNA hypomethylation, DNA hypermethylation, histone deacetylase inhibitor, cancer, aging, senescence, pericentromeric heterochromatin


56

lineage specific response of pediatric acute lymphoblastic leukemia xenografts to vorinostat

C. Davies, P. S. Bachmann, R. Papa, R. B. Lock

Children’s Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, NSW, AU

Advances in chemotherapy protocols have improved the cure rate of pediatric acute

lymphoblastic leukemia (ALL) which now approaches 80%. However, a significant number of

patients relapse, and new drugs are required to treat drug-resistant and aggressive cases of

the disease. The histone deacetylase inhibitor (HDACi), vorinostat, is currently in clinical trials for

a variety of malignancies, though its exact mechanism of action remains unclear. The purpose

of this study was to evaluate the efficacy of vorinostat in experimental models of pediatric

ALL. A model of pediatric ALL has been developed in our laboratory from a heterogeneous

panel of patient biopsies established as xenografts in immune-deficient (NOD/SCID) mice. The

efficacy of vorinostat to induce death in the xenograft cells was assessed by in vitro cytotoxicity

assays. Cells from T-lineage (T-ALL) xenografts were more sensitive to the effects of vorinostat

(IC50 318nM-750nM, n=5), in comparison to B-cell precursor (BCP) xenograft cells (IC50

580-2200nM, n=4). The increased in vitro efficacy was reflected in vivo, with vorinostat (100

mg/kg i.p., 28 days) causing a leukaemia growth delay (LGD) in T-ALL samples of 4.0-13.7

days (n=4), compared to a minimal response in BCP samples (LGD 2.5-4.2 days, n=4). A

combination of vorinostat and etoposide (3mg/kg i.p., Mon-Fri, 4 weeks) was also evaluated,

having a greater than additive effect against 4/8 xenografts. Molecular markers of HDAC

inhibition were used to define the lineage specific response to vorinostat. Both BCP and T-ALL

xenografts showed increased histone H4 acetylation and total acetylated lysine after vorinostat

treatment. Transcription of p21WAF1 is regulated in part by histone acetylation and CpG island

methylation, and is used as a marker for the transcriptional effects of HDACi. expression of

p21WAF1 was determined in xenograft cells and cell lines after exposure to vorinostat and

etoposide, a drug that induces p21WAF1 transcription through the p53 pathway. exposure

of xenograft cells to both agents caused induction of p21WAF1 protein in BCP samples (up

to 6-fold that of controls), yet failed to induce p21WAF1 at the protein and mRNA level in four

T-ALL samples. The inability to induce p21WAF1 mRNA corresponded with a 9-fold decrease

in acetylated histone H3 associated with the p21WAF1 promoter of the T-lineage xenograft,

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ALL-16, compared to a BCP xenograft (ALL-19), signifying a less transcriptionally active region

of DNA. Xenografts with defective p21WAF1 induction were, in general, more sensitive in vitro

to etoposide and vorinostat, which was also reflected through increased phosphatidylserine

externalization and caspase-3/-7 activity. As dysfunctional p21WAF1 induction following

treatment correlated with enhanced apoptotic response in xenografts, this finding suggests

that p21WAF1 alters the mechanism by which vorinostat kills tumor cells and could bestow an

apoptosis resistant phenotype on leukemia cells.

Keywords: leukaemia, vorinostat, apoptosis, p21WAF1, in vivo.

Address of corresponding author: Children’s Cancer Institute Australia, PO Box 81, High Street, Randwick, NSW 2031, Australia; Telephone: 61+ 293821843; Fax Number: +61 293821850; email: [email protected].

All authors approve of this abstract being published

Lineage specific response of pediatric acute lymphoblastic leukemia xenografts to vorinostat


58

Neuroblastoma cell differentiation by the histone deacetylase inhibitor helminthosporium carbonum (HC)-toxin

H. E. Deubzer1,2, V. ehemann3, A. e. Kulozik2, F. Westermann4, M. Schwab4, O. Witt1,2

1. Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), Heidelberg2. Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg3. Cytometry Facility, Institute for Pathology, University of Heidelberg4. Department of Tumor Genetics, German Cancer Research Center (DKFZ), Heidelberg, De

The survival rate of children with high-risk neuroblastoma (NB) is unsatisfactory despite intensive

multimodal therapy. Limited efficacy and serious side effects of currently used therapeutic

regimens necessitate new, less toxic treatments.

This study shows that the histone deacetylase inhibitor (HDACI) Helminthosporium carbonum

(HC)-toxin suppresses the malignant phenotype of both established NB cell lines and primary

cultures with and without amplified MYCN oncogene more efficiently than retinoids (RAs) and

all other HDACIs tested (MS-275, n-butyric acid, suberoylanilide hydroxamic acid, trichostatin

A, valproic acid). HC-toxin induces cell cycle arrest and apoptosis as well as neuronal

differentiation and diminishes both colony formation and invasive growth. The shift to a benign

phenotype is associated with an activation of the retinoblastoma (RB) tumor suppressor

network. This includes the transcriptional repression of cell cycle regulators found at high levels

in NBs associated with poor prognosis, like e2F-1, N-myc, Skp2, Mad2 and survivin proteins.

Whole genome expression analysis followed by the functional characterization of 100 candidate

genes with medium-throughput RNA-interference (RNAi) identified those early regulated genes

that contribute to the initiation of neuroblastoma cell differentiation. HC-toxin’s epoxy group is

essential for inhibiting HDACs and promoting anti-NB activity. Without this functional group,

those cellular effects are not observed. In conclusion, the anti-NB activity of HC-toxin is superior

to that of RAs and that of all other HDACIs tested.

Key words: epigenetic therapy, cell cycle arrest, differentiation, E2F-1 regulated genes, RB tumor suppressor network

Supported by the Bundesministerium für Bildung und Forschung (BMBF) through the National Genome Research Network 2 (NGFN2)

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Genome-wide functional knockdown reveals an unexpected mechanism for HDAC inhibitor-induced apoptosis10

S. Fotheringham1, M. T. epping2, R. Bernards2, N. B. La Thangue1

1. Laboratory of Cancer Biology, Medical Sciences Division, University of Oxford, The John Radcliffe Hospital, Oxford, UK2. Division of Molecular Carcinogenesis and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, NL

Many cancer drugs exhibit limited clinical efficacy, reflecting the poor therapeutic window

between effects on tumour cells relative to their normal cellular counterparts. Biomarkers that

inform on the tumour response to a therapeutic modality might help overcome this limitation.

Aberrant acetylation has been strongly linked to tumourigenesis, and the modulation of

acetylation through targeting histone deacetylase (HDAC) is gathering increasing pace as a

viable therapeutic strategy. However, the mechanism through which HDAC inhibitors kill cells

is not known, although it is widely believed to involve chromatin control. Using a genome-wide

functional knockdown screen, we have identified genes that influence the sensitivity of tumour

cells to HDAC inhibition. The results have important implications for our understanding of HDAC

inhibitors as new cancer agents, and provide informative response biomarkers that might allow

patients to be stratified into groups that undergo favourable responses to drugs that target

HDACs.

Keywords: HDAC inhibitor, screen, biomarkers


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Clonal heritability of cta expression and promoter methylation patterns in melanoma cells: bases for epigenetic therapy?11

E. Fratta1, L. Sigalotti1, F. Colizzi1, A. Covre1, S. Coral1, H. Nicolay1,2, e. Cortini2, L. Calabrò2, e. Fonsatti2, M. Altomonte2, M. Maio1,2

1. Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, IT 2. Division of Medical Oncology and Immunotherapy, Department of Oncology, University Hospital of Siena, Siena, IT

Cancer-testis antigens (CTA), expressed by neoplastic cells of different histotypes, represent

ideal targets for tumor immunotherapy due to their limited expression in normal tissues and

their in vivo immunogenicity. However, their heterogeneous intratumoral expression may

limit their clinical usefulness since the elicited anti-CTA immune reactions will eventually

target only a portion of the tumor mass. Using a model of intratumor heterogeneity of CTA

expression, represented by 14 single cell clones expressing different constitutive levels of

the therapeutic CTA MAGe-A3, we have recently demonstrated promoter methylation as the

molecular mechanism directly responsible for the heterogeneous intratumoral expression

of CTA in melanoma. In this scenario, it remained to be addressed whether, in the context

of a constitutive intratumor heterogeneity, CTA expression and promoter methylation are

stable throughout cellular division at single cell level or evolve/modify leading to pleiomorphic

neoplastic populations hardly targetable by CTA-directed immunotherapy. To this end, Mel

313 clones 5 and 14, previously characterized for having weak and strong expression of the

CTA MAGe-A3, were sub-cloned by limiting dilution. RT-PCR and quantitative RT-PCR assays

demonstrated that both presence and levels of CTA expression were highly maintained in

sub-clones derived from the same parental clone, while concomitantly preserving the original

difference between the two parental clones. Noteworthy, the observed maintenance of the

levels of MAGe-A3 expression correlated with the single cell-level heritability of the methylation

status of its promoter, as assessed by sequence analysis of genomic DNA treated with sodium

bisulfite. Consistent with the heritability of CTA promoter methylation status throughout cellular

divisions, treatment of melanoma cell clones with the DNA hypomethylating agent 5-aza-

2’-deoxycytidine (5-AZA-CdR) resulted in demethylation of CTA promoters that ended up in

a stable co-expression of different CTA at single cell level. In fact, 60 days after the end of

treatment, a de novo expression of at least one CTA was still present in 78% of sub-clones

obtained from clone 5 previously treated with 5-AZA-CdR (5-AZA sub-clones), and 11%

of these displayed a de novo expression of all CTA analyzed. Noteworthy, all 5-AZA sub-

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clones were still sensitive to 5-AZA-CdR-treatment, which consistently resulted in a de novo

expression or up-regulation of all CTA analyzed, leading to a homogeneous expression of CTA

among 5-AZA sub-clones. Altogether, these findings support the notion that CTA expression is

stable throughout cellular divisions, therefore granting for a successful immunological targeting

of melanoma cells in the course of the disease. Furthermore, the reported single cell-level

heritability of CTA promoter methylation patterns and enduring sensitivity to the demethylating

activity of 5-AZA-CdR suggest DNA hypomethylating drugs as effective modifiers of melanoma

antigenic phenotype that may be utilized to design novel chemo-immunotherapeutic regimens

that may improve the therapeutic efficacy of multivalent CTA-directed vaccines in metastatic

melanoma patients.

Clonal heritability of cta expression and promoter methylation patterns in melanoma cells: bases for epigenetic therapy?


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MAGE-A1 expression is associated with good prognosis in neuroblastoma12

E. Grau1,2, S. Oltra1, F. Martínez1, C. Orellana1, A. Cañete2, J. M. Fernández2, M. Hernández-Martí3, V. Castel2

1. Unidad de Genética y Diagnóstico Prenatal, Hospital Universitario La Fe, Valencia, eS2. Unidad de Oncología Pediátrica, Hospital Universitario La Fe, Valencia, eS3. Unidad de Anatomía Patológica, Hospital Universitario La Fe, Valencia, eS

Advanced neuroblastoma has a dismal prognosis, with frequent bone marrow disease.

Minimal Residual Disease (MRD) studies are becoming very important to evaluate response to

therapy. MAGe-A1 gene has been used as MRD marker but its utility is limited due to a more

frequent expression in localized tumours than in advanced ones. MAGe-A1 gene is regulated

by epigenetic mechanisms and this could explain why its expression does not show a good

correlation with NB stage.

Material and methods

MAGe-A1 gene methylation pattern was studied in a series of 47 primary NB tumours by

methylation specific PCR (MSP)1, in order to assess the role of epigenetic mechanisms in its

regulation. Microsatellite instability (MSI) analysis was performed by amplification of BAT-26

molecular marker. P-values < 0,05 were considered significant.

Results

Nineteen (42.6%) tumours showed an abnormal demethylated pattern. Methylation status of

MAGe-A1 gene showed a significant correlation with its expression (P-value= 0.0006). There

were tumours from advanced cases that failed in showing MAGe-A1 expression in spite of

its hypomethylated sequence, suggesting that they might share other silencing mechanism.

Given the association between genome-wide hypomethylation and MSI, we analyzed the MSI

status of tumor samples, observing that there was a significant correlation between MAGe-

A1 expression and MSI (P-value= 0.028) This last observation appears to be more specifically

associated to the unmethylated pattern of the MAGe-A1 gene (P-value = 0.007). There was a

significant correlation between the epigenetic status of MAGe-A1 and MSI in localized tumors

(P-value = 0.0192).

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Conclusions

1- Almost half of our cases showed an abnormal demethylated pattern,

2- Methylation status of MAGe-A1 gene was correlated with its ectopic expression.

3- MSI analysis showed that there might be different mechanisms repressing MAGe-1

expression in advanced neuroblastoma, pointing at a protective effect against progression.

MAGE-A1 expression is associated with good prognosis in neuroblastoma


64

Development of small modulators of gene activation and repression by targeting epigenetic regulators: introduction of a new research network13

e. Ballestar., S. Cuthill, M. esteller, J. Hochstatter, T. Kouzarides, D. Schuebeler, A. Imhof

Histone Modification Group, Adolf-Butenandt-Institut, LMU, Munich, De

Fundamental aspects of chromatin function are more and more recognized as important factor

in the development of many severe and often untreatable diseases. Therefore many proteins

that are involved in the regulation of chromatin structure are potential drug targets and small

molecules directed against these factors will play an increasingly important role in treating

patients that are affected by one of these maladies.

Here we would like to introduce the newly founded SMARTeR Network consisting of

four research groups with high expertise in different chromatin techniques and Chroma

Therapeutics, a company focused on the generation of cancer and inflammation treatments.

The SMARTeR project aims at the development and improvement of compounds targeting

epigenetic regulators. These compounds will be tested in various assays making it possible

to collect data sets of several parameters as histone modifications, chromatin states, gene

expression patterns and physiological characteristics in an integrative manner.

This cooperation between leading european chromatin labs and Chroma Therapeutics is

expected to strengthen the company’s knowledge base, thus having a strong impact on

its ability to enter drug candidates in clinical trials. Our network will thereby promote the

development and improvement of a new branch of cancer drugs and as well support validation

of new potential drug target enzymes. Importantly, tools will be generated which allow new

insights in fundamental mechanisms of gene regulation by epigenetic modification.

Key words: histone modification, small molecule inhibitors, mass spectrometry, expression profiling, mouse model

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14Microsattelit instability on APC and PlA2G2A genes in colorectal cancer

Introduction: Colorectal cancer is one of the most frequent causes of cancer deaths in the world. The

chromosomal instability pathway involves the activation of oncogenes (e.g. K-ras), the inactivation of

tumour suppressor genes such as APC, p53, DCC and the microsatellite instability (MSI) caused by a

defective mismatch repair (MMR) system. In colorectal cancer, instability at the level of chromosomes

results in losses or gains of whole chromosomes or large portions of it.

Aims: to compare the significance of APC and PLA2G2A proteins expression with the investigated

rate of allelic imbalance (microsatellite instability – MSI /loss of heterozygosity – LOH) of APC and

PLA2G2A microsatellite region loci on chromosomes 5q and 1p, respectively.

Material and methods: We evaluated the MSI/LOH on chromosome 1p (1p35-36.2) and 5q

(5q14-q23) with the ABI PRISM 310 Genetic Analyzer by using a panel of 9 microsatellites loci from

human chromosomes 1p and 5q. We analyzed DNA and tissue samples from blood and tumors of

patients with colorectal cancer. Samples were obtained with the consent of 27 patients, consisting of

histopatologically confirmed colorectal adenomas. The median age was 60 (range 55-74 years old)

and the most frequent symptoms were intestinal occlusion, bleeding, the macroscopic once usually,

and anemia because of occult gastrointestinal bleeding.

Results: Allelic imbalance analyses revealed that the most frequent alterations appeared to the

microsatellite markers D1S2644, D1S2843, D1S199, D1SPLA2 loci mapped in 1p35-36.2 and D5S82,

D5S421 loci mapped in 5q14-q23. At 6 patients out of 27 (22,22%) LOH/ MSI appears to the PLA2G2A

locus and/or in the surrounding microsatellites loci. At 5 patients aut of these 6, AI appear at the D1S234

locus. At 12 patients out of 27 pacienti (44,44%) LOH/MSI appears at different microsatelittes loci on

chromosome 5q. At 9 patients aut of these 12, AI appear at the D5S421 locus. These results confirm our

previous results on APC and PLA2 type IIA expression by immunohistochemistry.

Conclusions: Taking togheder all these results we conclude that only 44,44% of patiens have AI/

LOH and for half of theme (18,5%) we observed an association with MLPA (data not show).

A. Mihalcea, V. Tica, M. Serban, C. Tesio, M. Costache, E. Ionica

University of Bucharest, Molecular Biology Center, RO


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15 New indolone inhibitors of NAD+-dependent histone deacetylases

Histone deacetylases (HDACs) are enzymes that are able to deacetylate lysine side chains in

histones and other proteins which lead to altered states of conformation and activity for the

proteins in question. Three classes of HDACs have been recognized in humans: class I and

II are zinc- dependent amidohydrolases and a first hydroxamic acid derivative as selective

inhibitor of classes I and II enzymes has obtained FDA approval.

Much less is known about class III proteins. The so called sirtuins depend in their deacetylase

mechanism on NAD+ and O-acetyl ADP ribose and nicotinamide are formed as a consequence

of the acetyl transfer. Seven members of class III proteins are found in humans and called

SIRT1-7 due to their homology to the yeast histone deacetylase Sir2p. Sirtuins are implicated in

the regulation of apoptosis, gene silencing, metabolism and HIV Tat transactivation and might

therefore present a promising target for new antiviral and cancer drugs.

Here we present new indolone containing inhibitors of sirtuins. They show activity in the low

micromolar region against both GST-hSirt1 and His-hSirt2.

K. Huber1, J. Schemies2, F. Bracher1, M. Jung2

1. LMU University of Munich, De2. University of Freiburg, De

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Methylation of tumor related genes in neoadjuvant treated gastric cancer: relation to therapy response, clinico-pathological and molecular features16

R. Napieralski1, K. Ott2, M. Kremer1, K. Becker1, A.-L. Boulesteix3, F. Lordick2, 4, J. R. Siewert2, H. Höfler1, 5, G. Keller1

1. Department of Pathology2. Department of Surgery3. Department of Medical Statistics and epidemiology4. 3rd Department of Internal Medicine, Technische Universität München, Munich, De 5. Institute of Pathology, National Research Center for environment and Health, GSF-Forschungszentrum, Oberschleissheim, De

Aims: The objective of this study was to analyze the hypermethylation of tumor related

gene promoters for an association with therapy response and clinicopathological features

of neoadjuvant treated gastric cancer patients. Furthermore, we analyzed the relationship

of promoter hypermethylation with other molecular features of the tumors as microsatellite

instability (MSI) and loss of heterozygosity (LOH).

Methods: Pretherapeutic biopsies of 61 patients, subsequently treated with cisplatin and 5FU,

were studied. Using MethyLight technology, promotor methylation analysis of the following six

genes was performed: MGMT, LOX, p16, e-cadherin, 14-3-3 sigma, and HPP1. Microsatellite

analysis was performed with 11 microsatellite markers using standard procedures.

Results: The methylation frequencies for the analyzed genes were: MGMT, 44%; LOX, 53%;

p16, 46%, e-cadherin, 30%; 14-3-3 sigma, 69%; HPP1, 82%. Concordant methylation of more

than 3 genes was found in 46% of the tumors and was inversely correlated with the LOH-rate

(p=9x10-5), was associated with female gender (p=0.049), nonintestinal type tumors (p=0.04)

and a nonproximal tumor location (p=0.003). No statistically significant association between the

methylation of a single gene was found with response or survival. Considering the concordant

methylation of multiple genes, patients with tumors demonstrating methylation at more than 3

genes were found more frequently among the nonresponding group (49% versus 27%), but this

difference was statistically not significant (p=0.20). In respect to survival, patients with none or

only one methylated gene demonstrated a trend for an increase in survival (5 year survival rate

83% versus 35%, p=0.067).

Conclusion: The highly significant inverse correlation of promoter methylation and LOH-

rate suggests that epigenetic instability, reflected by the concordant methylation of multiple


68

genes and chromosomal instability, reflected by a high LOH-rate, represent major, alternative

molecular pathways in gastric carcinogenesis. Methylation was not statistically significantly

associated with the response to cisplatin/5FU based therapy. However, a concordant

methylation of more than 3 genes defines subgroups of gastric cancer with distinct biological

and genetic characteristics. This may be of clinical relevance for a potential application of an

epigenetic-based chemotherapy in gastric cancer.

Published: Clinical Cancer Research 2007; 13(17) September 1

Key words: neoadjuvant chemotherapy, gastric cancer, methylation, loss of heterozygosity, chromosomal instability

Address for correspondence:

PD Dr. Gisela Keller

Institute of Pathology

Klinikum rechts der Isar

Technische Universität München

Trogerstr. 18

D-81675 Munich

Germany

Tel ++49 89 4140 4592

e-mail: [email protected]

Methylation of tumor related genes in neoadjuvant treated gastric cancer: relation to therapy response, clinico-pathological and molecular features

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17Aberrant promoter hypermethylation of multiples tumor suppressor genes in diffuse large B-cell lymphomas correlates with the presence of simian virus 40 DNA sequences

A. Khaled1, M.Trimeche1, S. Ziadi1, A. Laatiri2, M Hachana1, B. Sriha1, M. MoknI1, S Korbi1

1. Department of Pathology, CHU Farhat Hached, Sousse, TN2. Department of Clinical Hematology, CHU Farhat Hached, Sousse, TN

Diffuse large B-cell lymphomas (DLBCLs) represent the most common category of non-

Hodgkin’s lymphomas (NHLs) and are characterized by heterogeneous biological, clinical,

immunophenotypic, and molecular features. Several studies have reported the presence of

simian virus 40 (SV40) DNA sequences in 15% to 55% of diffuse large B-cell lymphomas

(DLBCLs) cases. Hypermethylation in promoter regions is a frequent process of silencing tumor

suppressor genes (TSGs) in cancers. In this study, we investigate the presence of SV40 DNA

sequences in 108 cases diagnosed at the laboratory of Pathology at Farhat-Hached University

Hospital of Sousse (Tunisia), and correlate the data with methylation status of a panel of 15

TSGs known or suspected to be altered by hypermethylation in several cancers including

lymphomas, comprising P14, P15, P16, GSTP1, DAPK, SHP1, P53, RB1, APC, CDH1,

TIMP3, RASSF1A, P33, BLU, and VHL. SV40 DNA presence was investigated by PCR assays

targeting the large T-antigen. Hypermethylation was carried out by methylation-specific PCR.

To compare the overall extent of methylation for the panel of genes examined, we calculated

the Methylation Index, defined as the total number of genes methylated divided by the total

number of genes analyzed, for each case and the mean of methylation index of different groups

was compared using the Mann-Whitney U nonparametric test. SV40 DNA was detected in

63/108 (56%) of DLBCL cases. The frequency of hypermethylation of the tested genes varied

notably and ranged from 3 to 74%. The hypermethylation of SHP1, DAPK, CDH1, GSTP1, and

P16 genes were significantly higher in SV40-positive DLBCLs than in SV40-negative cases (p

values ranging from 0.0006 to 0.0001). Interestingly, the Methylation Index was significantly

higher in SV40-postive DLBCLs compared to SV40-negative cases (0.530 ± 0.019 versus

0.324 ± 0.022; p=0.001). In summary, our results support a role of SV40 in DNA methylation in

a significant proportion of diffuse large B-cell lymphomas cases. However, further studies will

be necessary to elucidate the mechanisms by which the virus interferes with the methylation in

those lymphomas and to determine its role in lymphomagenesis.


70

Aberrant promoter hypermethylation of multiples tumor suppressor genes in diffuse large B-cell lymphomas correlates with the presence of simian virus 40 DNA sequences

Key words: diffuse large B-cell lymphomas; hypermethylation; tumor suppressor genes; simian virus 40; Tunisia

corresponding author:

AMARA KHaled

CHU Frahat Hached

Department of Pathology

4000 Sousse

Tunisia

Tel: 0026198386265

Fax: 0021673210355

email: [email protected]

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18Prognostic value of aberrant promoter hypermethylation of multiple tumor supprossor genes in diffuse large B-cell lymphomas in tunisia

A. Khaled1, M.Trimeche1, S. Ziadi1, A. Laatiri2, A. Khelif2, S. Korbi1

1. Department of Pathology, CHU Farhat Hached, Sousse, TN2. Department of Clinical Hematology, CHU Farhat Hached, Sousse, TN

DNA hypermethylation of the promoter region of genes has emerged as a common mechanism

of inactivation of tumor suppressor genes (TSGs) in human cancers including lymphomas.

Diffuse large B-cell lymphomas (DLBCLs) represent the most common category of non-

Hodgkin’s lymphomas (NHLs) and are characterized by heterogeneous biological, clinical,

immunophenotypic, and molecular features. It constitutes approximately 30–40% of all NHLs

diagnosed in the Western countries, and accounts for an even higher proportion in developing

countries, including Tunisia. The aim of the study was to assess the prognostic significance

of the hypermethylation of the promoter regions of DAPK, GSTP1, P14, P15, P16, APC,

CDH1, BLU, TIMP3, RASSF1A, P33, and VHL and their in DLBCLs in Tunisian patients. The

methylation status of these TSGs was investigated by methylation-specific polymerase chain

reaction in 46 DLBCL cases selected on the basis of the availability of complete fellow-up

information. The extent of each gene methylation status on patient’s overall survival was

assessed using the Kaplan-Meier methods and compared with the log-rank test. All samples

showed at least one gene methylated and 78% of cases had 3 or more genes methylated.

Overall, the frequency of hypermethylation of each gene varied notably. There was no significant

correlation between methylation status of those genes and patient’s age, gender, or clinical

stage. Regarding the clinical follow-up data, promoter hypermethylation of DAPK (17.03 versus

34.75 months; p=0.01), p16 (13.79 versus 33.41; p=0.001), and VHL (19.22 versus 30.19;

p=0.018) were significantly associated with shortened overall survival. Taken in consideration

the international prognostic index (IPI), hypermethylation of DAPK and P16 remains significantly

associated with shortened overall survival among patients with low IPI scores (p =0.036 and

p=0.006, respectively). In summary, our results indicate that hypermethylation of DAPK and P16

could be a potential prognostic marker for long-term outcome prediction in patients with diffuse

large B-cell lymphomas and may be useful for determining appropriate treatment.


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Establishment of a high throughput screening platform for inhibitors of human DNMT1

D. Kuck and F. Lyko

German Cancer Research Center, Division of epigenetics, Heidelberg, De

The inhibition of the DNA methyltransferase activity has been shown to reverse epimutations

and to reactivate epigenetically silenced genes. Clinical use has been established for two

compounds, 5-azacytidine and 2’-deoxy-5-azacytidine, for the treatment of leukemias

and other tumors. However, novel inhibitors need to be developed that result in a more

direct and specific inhibition of DNA methyltransferase activity. Our aim is to set up a high

throughput screening platform for inhibitors of DNMT1. Therefore, a biochemical benchtop

assay was established based on a recombinant human DNMT1. DNMT1 was expressed in

insect cells and purified by Ni2+ immobilized metal affinity chromatography and subsequent

gel filtration to obtain protein of high purity. Several important features i.e dose and time

kinetics, stability during storage, enzymatic activity at 25°C and 37°C as well as the stability

of the enzymatic activity were investigated. For standardization purposes the IC50 value of

S-Adenosylhomocysteine was determined. In addition, several published inhibitors of DNMT

like RG108, procaine, procainamide, hydralazine and S-adenosylhomocysteine were tested

in the assay. In conclusion, the recombinant human DNMT1 revealed a robust performance in

terms of storage stability and enzymatic activity in the assay. Notably, most of the published

compounds failed to inhibit DNMT1.

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20Differential methylation profile of ovarian cancer: a possible detection biomarker

A. A. Melnikov, D. Scholtens, A. K. Godwin, V. V. Levenson

Robert H. Lurie Comprehensive Cancer Center and Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago; Fox Chase Cancer Center, Department of Medical Oncology, Philadelphia, US

early detection through regular screening can reduce ovarian cancer-related mortality. Increased

accuracy of cancer detection is paramount for early detection, so further improvements to

existing biomarkers are urgently needed. We evaluated the potential of ovarian cancer detection

by methylation profiling with a panel of 56 potentially methylated promoters. In the first part of

the project we compared profiles of tumor sections (n=30) of serous papillary adenocarcinoma

and profiles of uninvolved ovaries (n=30) from women of a high-risk group. A composite

biomarker consistently included promoters of ten genes (BRCA1, eP300, NR3C1 (GR), MLH1,

DNAJC15 (MCJ), CDKN1C (p57kip2), TP73, PGR (proximal promoter), PYCARD (TMS1),

THBS1). Stratified five-fold cross-validation of this biomarker showed 70% accuracy of ovarian

cancer detection. Methylation profiles of circulating DNA from blood were used in the second

part of the project. These profiles were compared between patients with serous papillary

adenocarcinoma (n=33) and healthy controls (n=33), and five genes (BRCA1, HIC1, PAX5,

PGR (proximal promoter), THBS1) were found to be informative for a composite biomarker.

This biomarker identified cancer patients with 85% sensitivity and 61% specificity as estimated

by stratified five-fold cross-validation. Thus, differential methylation profiling in heterogeneous

samples is possible with either whole sections of ovarian tissues or circulating DNA from

blood. While the accuracy of resulting biomarkers has to be further improved, even now the

blood-based biomarker is sufficiently sensitive and can be tested as a first-line screening tool in

combination with imaging techniques.


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21 Molecular biological patterns of A2780 cells upon formation of anticancer drug resistance

N. Y. Lukianova1, D.O. Mykytenko1, S.I. Shpileva1, I.P. Pogribny2, V.F. Chekhun1

1. Institute of experimental Pathology, Oncology and Radiobiology, Kiev, UA2. National Center for Toxicological Research, Arkansas, US

It is well known that successful therapy of malignant tumors requires clear understanding of interactions

between multiple mechanisms involved in formation of drug resistance. The detailed comparative

study of biological patterns of the sensitive cells and their resistant variants allows understanding the

mechanisms of formation of drug resistance and prediction the efficacy of anticancer therapy.

The aim of our work was to study the molecular patterns of the human A2780 ovarian carcinoma

cells sensitive and resistant to anticancer chemotherapeutic agents.

The results of the study showed that formation of the resistance of A2780 cancer cells to cisplatin

occurs via disruption of cellular apoptotic programme characterized by altered methylation and elevated

expression of Bcl-2 gene. This was evidenced by the flow cytometry data showing the increase in the

index of fluorescence peak (IFP) from 1.1 in sensitive cells to 3.4 in their cisplatin-resistant variants.

Additionally, we detected promoter hypomethylation of the gluthathione-S-transferase (GSTp) gene and

accompanied by increased level of GSTp protein (IFP increased from 1.9 to 3.4). At the same time, we

did not detect changes in promoter methylation and expression of mdr1 gene.

In contrast, the resistance to doxorubicin in A2780 cells occurs by ATP-dependent mechanism via

development of hypomethylation of promoter of mdr1 gene and appearance of hyperexpression of

P-glycoprotein (IPF was 1.9 and 2.8 in sensitive and doxorubicin resistant cells, respectively).

In conclusion, acquisition of the cancer drug resistance in the human A2780 ovarian carcinoma cells

to cisplatin and doxorubicin occurs via involvement of different genetic and epigenetic mechanisms.

Specifically, formation of the cancer drug-resistant phenotype is associated with altered methylation

patterns of genes associated with drug resistance subsequently leading to activation of cellular

antiapoptotic defense programme, detoxicating and ATP-dependent mechanisms.

Key words: drug resistance, ovarian cancer, cisplatin, doxorubicin.

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22Anti-estrogenic actions of histone deacetylase inhibitors in MCF-7 breast cancer cells

M. De los Santos, O. A. Martinez-Iglesias, A. Aranda

Instituto de Investigaciones Biomédicas Alberto Sols. CSIC-UAM, Madrid, eS

Anti-estrogens are the current endocrine therapy of choice in the treatment of estrogen receptor

(eR)-positive breast cancers. Histone deacetylase inhibitors (HDACi) also constitute a promising

treatment for therapy, and combination of anti-estrogens with HDACi may improve efficacy

while reducing side effects. We have examined the effect of the HDACi sodium butyrate and

suberoylanilide hydroxamic acid (SAHA), alone and in combination with 17β-estradiol (e2)

and the pure anti-estrogen ICI 182.780 (ICI) in human MCF-7 breast cancer cells. HDACi

caused a sustained increase of histone H3 acetylation and caused cell death as shown by flow

cytometry analysis. In surviving cells both inhibitors were even stronger than ICI in depleting

cyclin D1 levels, inducing expression of the cyclin kinase inhibitor p21Waf1/Cip1, blocking

phosphorylation of the retinoblastoma protein (pRb) or inhibiting cell growth. No additive

effects of ICI with either butyrate or SAHA were found. In addition, these drugs we able to

antagonize the effects of e2 on expression of cell cycle proteins, cell growth and transcription

of eR-dependent genes. The anti-estrogenic effects of HDACi appear to be related to a strong

down-regulation of the expresión of eRα that appears to be secondary to both transcriptional

and post-transcriptional regulation. eRα phosphorylation is involved in estrogen signalling,

and HDACi also prevented receptor phosphorylation in serine-118 both in the absence and

presence of eR ligands. These results provide further support for the use of deacetylase

inhibitors as chemotherapeutic agents in the treatment of breast cancer tumors.

Keywords: sodium butyrate, SAHA, breast cancer cell proliferation, estrogen receptor


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23 Opimization of chemotherapy of cancer with 5-aza-2’-deoxycytidine (decitabine, dacogen)

R. L. Momparler, M. Lemaire, G.G. Chabot, N. Raynal, M. L. Bernstein, L. F. Momparler, A. Hurtubise

Dept Pharmacology, University of Montreal and Centre de recherche, Service Hematology-Oncology, Hoptial Sainte-Justine, Montreal, Quebec, CA

The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an

important role in oncogenesis. Since this epigenetic change is reversible, it is an interesting

target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-

2’-deoxycytidine (decitabine, dacogen, DAC). Although clinical studies show that DAC has

activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent

still needs to be established. Using an in vitro assay on human HL-60 myeloid leukemic cells

and Calu-6 lung carcinoma cells, we observed that the loss of clonogenicity increased with

DAC concentration and duration of exposure. DAC at 100 ng/ml produced a greater loss of

clonogenicity and greater reactivation of the TSGs p57CDKN1C (HL-60 cells) and p16CDKN2A

(Calu-6 cells) than 10 ng/ml. In mice with murine L1210 leukemia, the antineoplastic action of

DAC increased with the dose. An 18 h i.v. infusion of DAC at total dose of 20 mg/kg produced

100% cures . The steady state plasma level of DAC during this infusion was estimated to be

greater than 500 ng/ml . In mice with murine eMT6 mammary tumor, the survival fraction of the

tumor cells also decreased with the DAC dose and duration of i.v. infusion. These preclinical

observations will be helpful in the design of dose-schedules for future clinical trials to fully

evaluate the potential of DAC in cancer therapy. Supported by grants from Canadian Cancer

Society and the Cancer Research Society.

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24Effect of homocysteine on the formation of drug-resistant phenotype of cancer cells

V.F. Chekhun1, D. O. Mykytenko1, N.Yu. Lukianova1, I.P. Pogribny2

1. Institute of experimental Pathology, Oncology and Radiobiology, Kiev, UA2. National Center for Toxicological Research, Arkansas, US

Homocysteine is a natural factor in affecting the process of cellular methylation and elevated level

of this amino acid has been registered in some cancers. It is known that epigenetic aberrations,

in particular altered methylation of DNA, play an important role in the acquisition of drug-resistant

phenotype tumors.

In view of these consideration, in the present study we investigated whether or not the increased level

of homocysteine will affect sensitivity of cancer cells to chemotherapeutic agents.

The maintenance of human ovarian A2780 cancer cells and breast MCF-7 cancer cells with

homocysteine at concentration of 100 µM/L decrease sensitivity to cisplatin and doxorubicin.

Immunophenotype patterns of A2780 cells cultured with homocysteine are characterized by

increased activity of mdr-dependent, detoxication (GSTp) and antiapoptotic (Bcl-2) systems

accompanied by promoter hypermethylation of apoptosis-associated genes (tp53, p73 and bcl-2),

hypomethylation of the genes of drug resistance (mdr1, GSTp) and e-cadherin gene. Maintenance of

MCF-7 breast cancer cells with homocysteine at the same concentration resulted in hypometylation

at mdr1 and GSTp genes and hypermethylation of tp53, p73, and bcl-2 genes accompanied by mdr-

dependent and detoxication pathways and inhibition of cellular apoptotic program, respectively.

The results of our study showed that homocysteine-induced alteration in the gene specific

methylation patterns is one of the primary events in initiation of signal cascades of drug resistance

and may be a leading mechanism of regulation of activity of defense systems of ovarian cancer cells

and human breast cancer cells upon elevated level of homocysteine in the system in vitro.

Key words: drug resistance, homocysteine, methylation, cisplatin, doxorubicin


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25 Epigenetic down-regulation of Snk/Plk2 is associated with clinical relapse and chemotherapy resistance in ovarian cancer

S. Nelofer1, I. McNeish2, T. Crook1

1. Breakthrough Breast Cancer Centre at The Institute for Cancer Research, London, UK.2. Department of Medical Oncology, St Bartholomew’s Hospital, London, UK

To seek genes regulating chemotherapy sensitivity, we derived new ovarian carcinoma cell

lines with acquired resistance to taxanes. We show that paclitaxel resistance is predominantly

epigenetic. Using methylation reversal/micro-array analysis, we have identified transcriptional

down-regulation of the Polo-Like Kinase Snk/Plk2 as a major contributor to paclitaxel

resistance. Bisulphite sequencing revealed aberrant methylation in the CpG island located

at the 5’ end of the Snk/Plk2 gene in each of the drug resistant cell lines compared to their

drug sensitive parental cell lines, implying that methylation-dependent transcriptional silencing

underlies the reduced expression of Snk/Plk2 mRNA. RNAi-dependent knock down of Snk/Plk2

resulted in decreased sensitivity to taxol and platinum, whereas ectopic expression conferred

resistance to both drugs. In clinical cases of epithelial ovarian cancer, aberrant methylation of

the Snk/Plk2 CpG island was significantly more common at relapse with drug-resistant disease

than at initial presentation (p= 0.021). Moreover, the presence of methylated Snk/Plk2 in serum

was strongly predictive of both disease recurrence and failure of second line chemotherapy.

Together, our results implicate transcriptional silencing of Snk/Plk2 as a contributory mechanism

to acquired drug resistance in epithelial ovarian cancer

Key words: Snk/Plk2; epithelial ovarian cancer; chemotherapy

Corresponding author: Nelofer Syed

Telephone: +44 20 7352 8133

Fax +44 20 7724 8586

Email: [email protected]

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26 Role of histone deacetylase 8 in neuroblastoma cells

I. Oehme1, H. e. Deubzer1, 2, D. Wegener1, D. Pickert1, 3, O. Witt1, 2

1. CCU Pediatric Oncology, DKFZ, Heidelberg, De2. Dpt of Pediatric Oncology, University of Heidelberg, De 3. University of Applied Sciences Lausitz, Senftenberg , De

The control of histone acetylation and deacetylation by histone acetyl transferases and histone

deacetylases (HDACs) plays an important role in regulating transcriptional programs in normal

cells, and deregulation of this epigenetic key process has been linked to human cancer. We

want to dissect the functional role of class I, II and IV HDAC family members for controlling

biological properties of malignancy in neuroblastoma cells.

Silencing of the class I family member HDAC8 with RNA interference revealed that this enzyme

plays a role in tumor cell proliferation, cell cycle progression and clonogenic growth. Additionally,

knockdown of HDAC8 induced neuronal differentiation with outgrowth of neurite-like structures

and upregulation of neuronal markers, like neurofilament (mRNA and protein), which is in

contrast to the knockdown of other class I family members. On the one hand our data show

that HDAC8 contributes to the malignancy of neuroblastoma cells and on the other hand that

distinct HDAC family members control specific cellular functions. Thus, HDAC8 could serve as

a specific drug target for novel targeted therapy strategies.

Keywords: histone deacetylases; neuroblastoma; HDAC8

To whom correspondence should be addressed:

Ina Oehme, Clinical Cooperation Unit Pediatric Oncology G340, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

Phone: 0049-6221-423388

Fax: 0049-6221-423277

E-mail: [email protected]


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27 Thyroid hormone regulates HP1b dissociation through a lSD1- and Aurora B-mediated mechanism

B. Gil de Araujo, D. Moreno, M. Tardáguila, M. e. Gonzalez Gugel, M. Lasa, A. Sánchez-Pacheco

Instituto de Investigaciones Biomedicas A. Sols (UAM-CSIC), Madrid, eS

epigenetic mechanisms, such as histone covalent modifications, are essential for the

regulation of gene expression in eukaryotes as they control chromatin structure and dynamics.

Perturbations of epigenetic balances may lead to alterations in gene expression, ultimately

resulting in cellular transformation and malignant outgrowth. Here we show that the thyroid

hormone (T3) causes a dynamic reorganization of chromatin in parallel with rapid epigenetic

changes that can precede transcription of target genes. We demonstrate that, in thyroid

hormone receptor (TR)-expressing cells, T3 produces in a few minutes heterochromatin protein

1b (HP1b) displacement to the nuclear periphery concomitant with a rapid increase in histone

H3K9 di-methylation (H3K9me2) and H3Ser10 phosphorylation (H3Ser10ph). HDACs inhibition

by TSA treatment abolished these effects. Our data show an in vivo interaction of TR with the

lysine-specific demethylase-1 (LSD1) and the Aurora Kinase B (AIM-1), as well as between

these proteins. Inhibition of expression or activity of these enzymes abolishes the effect of T3

on H3K9me2 and H3Ser10ph, blocks HP1b delocalization, and reduces significantly ligand-

dependent transactivation withβshowing a novel role for LSD1 together with Aurora-B on

association of HP1 chromatin. Since the effect of T3 occurs in GO/G1, our findings reveal a

novel role for Aurora-B different from the well-known mitotic function of this kinase. Aurora-

B and LSD1 are therefore part of a “methyl/phos switch” mechanism that displaces HP1b,

preparing chromatin for the initiation of transcriptional activation of T3 regulated genes.

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28Epigenetic Gene Silencing of Secreted Frizzled-Related Proteins (SFRPs) in Acute Myeloid leukemia

J. Schmid, S. Wilop, R. Osieka, e. Jost, O. Galm

Medizinische Klinik IV, Universitaetsklinikum Aachen, RWTH Aachen, De

Acute myeloid leukemia (AML) originates in the transformation of hematopoietic stem cells,

leading to clonal proliferation of immature progenitor cells and severe multilineage bone

marrow suppression. In addition to numerous genetic aberrations in AML, it has become

evident that epigenetic alterations play a significant role in the pathogenesis of AML. Silencing

of tumor suppressor gene expression by aberrant CpG methylation in promoter regions is an

established epigenetic principle in carcinogenesis. The Wnt signaling pathway plays a key role

in cell proliferation as well as differentiation and has been shown to be important for normal

hematopoiesis. The family of secreted Frizzled-related proteins (SFRPs), functioning as Wnt

signaling antagonists, has been found to be downregulated by promoter hypermethylation

in acute lymphoblastic leukemia (ALL) and solid tumors. Data suggest a correlation between

epigenetic silencing of SFRPs and poor prognosis in ALL. To demonstrate the relevance of

SFRP downregulation in AML, we examined the promoter-associated methylation status of

CpG islands of SFRP-1,-2,-4 and -5 in the AML cell lines KG1a and HL60 and the Burgkitt’s

lymphoma cell line Raji by methylation-specific polymerase chain reaction (MSP). Promoter

hypermethylation was found for all four SFRP genes in HL60 and Raji and for SFRP-1,-2 and

-5 in KG1a. Transcriptional gene silencing as a consequence of SFRP hypermethylation was

confirmed by real-time reverse transcriptase quantitative polymerase chain reaction (RT-

PCR) for SFRP-1 and SFRP-2 in both AML cell lines. Treatment of KG1a and HL60 with the

demethylating agent 5-aza-2’deoxycytidine led to a reexpression of SFRP-1 and SFRP-2 in

real-time RT-PCR analysis. By MSP, we analyzed the methylation status of SFRP-1,-2,-4 and

-5 in 97 specimens obtained from AML patients at diagnosis. The frequencies of aberrant

methylation among the primary patient samples were 28,9 % (28/97) for SFRP-1, 18,6 %

(18/97) for SFRP-2, 0 % (0/97) for SFRP-4 and 8,2 % (8/97) for SFRP-5.

In our AML patient cohort there was a clinical correlation between the methylation status

of SFRP-5 and decreased white blood cell counts at diagnosis (p=0,01). Patients with


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SFRP-2 hypermethylation tended to have lower hemoglobin values at diagnosis, but this

difference was not statistically significant (p=0,11). We could not find correlations between the

methylation status of any SFRP gene with the clinical parameters platelet count, serum lactate

dehydrogenase, age and gender. Furthermore, there was no impact of the SFRP methylation

patterns on overall survival.

Our results indicate that hypermethylation of the SFRP promoter region is a frequent epigenetic

event in AML, with the potential to cause aberrant Wnt signaling. The challenge in the future

will be to clarify the effect of SFRP gene silencing and subsequent Wnt signaling on AML

pathogenesis and to benefit from this knowledge to develop epigenetically active therapies for

AML.

Epigenetic Gene Silencing of Secreted Frizzled-Related Proteins (SFRPs) in Acute Myeloid Leukemia

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29 The NTA protein mediates cellular uptake of 5-azacytidine and plays a functional role in drug-mediated dna demethylation

C Stresemann1, M. Rius2, D. Keppler2, F Lyko1

1. Division of epigenetics, German Cancer Research Center, Heidelberg, De2. Division of Tumor Biochemistry, German Cancer Research Center, Heidelberg, De

5-azacytidine is a potent DNA methyltransferase inhibitor that is commonly used in laboratories

and has found increasing clinical use for the treatment of leukemias. Nevertheless the transport

proteins that mediate the uptake of 5-azacytidine into the cells have not been identified yet.

We show that nucleoside transporters, which are encoded by the SLC29 (eNT) and SLC28

(CNT) gene families, exhibit differential expression in leukemic cancer cell lines. After treatment

of these cells with 5-azacytidine, expression levels of transporters changed. We therefore

established stably transfected cell lines which ectopically express candidate nucleoside

transporters. With this model we could identify the NTA protein that mediates efficient uptake of

5-azacytidine with a Km of 63 µM. Additionally, ectopic expression of the nucleoside transporter

increased the growth inhibiting effects of 5-azacytidine when compared to controls. The

elevated expression of NTA facilitated DNMT1 depletion and global DNA demethylation, which

demonstrated a functional role of NTA in drug-mediated DNA demethylation. Together, our

results indicate that the potency of nucleoside DNA methyltransferase inhibitors is dependent

not only on their intracellular metabolism but also on the expression of specific transport

proteins, which mediate their uptake.

Keywords: nucleoside transporter, 5-azacytidine, DNA methylation


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30Epigenetic silencing of the dual specificity phosphatases (DUSPs) in human cancers

J. R. Taylor, N. Syed, P. Smith, T. Crook

The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK

Deregulated MAPK signalling is a hallmark of the transformed phenotype in many cancers, but

mechanisms mediating this are unclear. Using methylation reversal and mirco-array, we have

identified aberrant methylation in the CpG island of the dual specificity phosphatases (DUSP)

in a number of human cancers. In ovarian and breast cancer cells, DUSP2 (PAC-1) methylation

is distributed throughout the CpG island and efficiently silences basal transcription. In contrast,

methylation of the DUSP1 (MKP-1) CpG island occurs in a highly defined region of the

promoter and does not silence basal expression. Rather, the effect of methylation is to abrogate

transcriptional induction of DUSP1 mRNA by mitogens that activate MAPK. Methylation of the

DUSP1 CpG island therefore causes sustained MAPK signalling in cancer cell lines which is

independent of the expression of DUSP2. Furthermore in melanoma, methylation of the DUSP1

CpG island only occurs in the mutant B-Raf cell lines and not in cells which have wildtype B-Raf

including normal melanocytes. Therefore DUSP1 methylation may enhance the constitutive

activation of MAPK caused by mutant B-Raf.

Key words: DUSP, MAPK, methylation.

Corresponding author: Jessica Taylor

The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB

Telephone: +44 (0)20 7153 5504 Fax: +44 (0)20 7153 5340


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31 Analysis of APC ,β-catenin and p53 mutations on CaCo2 cell line. In vitro chemotherapeutic investigation for the treatment of colorectal cancer

Introduction: Cancer of the colon and rectum is the third leading cause of death cancer

in males and the fourth leading cause of death cancer in females. The most mutations in

colorectal cancer occur at the level of adenomatous polyposis coli (APC) or β-catenin genes.

The APC gene is transcriptionally up-regulated by p53 in response to DNA damage. Material

and methods: Using immunofluorescence and Western-blot methods we investigated the

expression of APC, β-catenin and p53 proteins on Caco2 cell line established from human

colonic tumor. We also performed western blot and MTT to determine cytotoxic properties

of 5-fluorouracil by a 24 hours exposure of Caco2 cell line to FU. Results: We observed

the existence of an APC and p53 proteins mutation that lead to cytoplasmatic and nuclear

expression of β-catenin which heterodimerizes with Tcf-Lef transcription factor and actively

transcribes cell cycle related genes causing cellular proliferation. The results of Caco2 cell

line treatment with FU showed that, as a single agent, it is modestly active against advanced

colorectal cancer. Conclusion: Disruption of the functions of APC, β-catenin and p53 proteins

may have an important role in initiation and progression of colorectal tumors. Metastatic

colorectal cancer remains incurable with the available systemic therapeutic options. efforts

to ameliorate these poor results of 5-fluorouracil are likely to lead to a new era for patients

suffering from advanced colorectal carcinoma.

Keywords: CaCo2, APC, β-catenin, p53, colorectal cancer

A. Mihalcea, I. Stefan, V. Tica, M. Costache, e. Ionica

University of Bucharest, Molecular Biology Center, RO


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Aspects regarding the involvement of procaine in the methylome of living organisms

E. Turcu1, V. Lungu2, N. Cucu3, R. Pircalabu1, e. Lupeanu1, V. Andrei1, D. Chiper3

1. “Ana Aslan” National Institute of Gerontology and Geriatrics– Bucharest, RO2. “H.Hulubei” National Institute of Physics and Nuclear engineering – Bucharest-Magurele, RO3. University of Bucharest, Genetics chair – Bucuresti, RO

The paper includes the presentation of some original pharmacokinetc and pharmacodynamic

results regarding Procaine’s action mechanisms in the living mammalian organism, obtained

in a research program during over 20 years in Ana Aslan Institute from Bucharest. Aside

an extensive bibliographic overview, these data allowed certain inferences regarding the

mechanisms of Procaine’s involvement in the mammalin methylome, and consecutively, about

the anti-aging and potentially anti-carcinogenetic actions of that molecule.

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33Differential Control of H3K9 Acetylation and Neuronal Gene Expression by Class I Histone Deacetylases

K. Wallenborg1, D. Solum2, A. I. Teixeira1, J. K. Duckworth1, K. Jepsen2, P. ernfors3, M. G. Rosenfeld2, O. Hermanson1

1. Department of Neuroscience and Department of Cell and Molecular Biology (CMB), Center of excellence in Developmental Biology (CeDB), Karolinska Institutet, Stockholm, Se2. Howard Hughes Medical Institute, Department and School of Medicine, University of California, San Diego, La Jolla, CA, US3. Department of Medical Biochemistry and Biophysics (MBB), Center of excellence in Developmental Biology (CeDB), Karolinska Institutet, Stockholm, Se

epigenetic regulation of stem cell characteristics is a crucial issue in development and disease.

We show that a very rapid pro-neuronal gene program induced in embryonic neural stem cells

by inhibition of HDAC2 and HDAC3, is associated with a specific increase in H3K9 acetylation –

but not other investigated residues – at genes proven required for the enhanced neurogenesis,

such as BDNF. In contrast, astrocytic differentiation is repressed by a temporal DNA

methylation/N-CoR dependent mechanism. Inhibition of individual deacetylases is not sufficient

for increased neurogenesis, but although HDAC2 and HDAC3 are detected simultaneously

at target genes such as BDNF and Numb, inhibition of single HDACs surprisingly results in

enzyme-specific alterations in gene expression. Our findings reveal non-redundant gene-

specific requirements for H3K9-controlling HDACs regulating neuronal gene expression.


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34M. B. Wozniak1, P. L.. Ortiz-Romero2, P. de la Cueva1, Mª e. Rodriguez1, D. Martin-Perez1, M. A. Piris1, R. Villuendas1

1. Molecular Pathology Programme, CNIO, Madrid, eS2. Dermatology Department, Hospital 12 de Octubre, Madrid, eS

Suberoylanilide hydroxamic acid (SAHA, Vorinostat, ZolinzaTM), an orally administered inhibitor

of histone deacetylases, has recently been approved by the U.S. Food and Drug Administration

for the treatment of cutaneous T-cell lymphoma (CTCL). This drug is being used in patients

with progressive, persistent, or recurrent disease or after 2 systemic therapies. SAHA inhibits

the activity of histone deacetylases (HDACs), including all 11 known human class I and class

II HDACs. Treatment with SAHA was found to increase acetylation of histones, growth arrest

and death of transformed cells with no significant toxicity on normal cells. However, in spite of

emerging information on SAHA effect on many cancers, the exact mechanism, the kinetics of

gene expression and players involved in resistance to this drug are still unknown. In order to

adress these questions we performed oligonucleotide microarrays to investigate the changes

in gene expression profile in five CTCL cell lines after SAHA treatment over time. SAHA at

concentration 2.5, 5 and 10 uM for 24 and 48 hours induced apoptosis and growth arrest in

a concentration- and time-dependent manner. Based on these results, for gene expression

studies, five CTCL cell lines (Hut78, Myla, MJ, HH, SeAx) were treated with SAHA at 5 uM

concentration for 1, 2, 4, 8, 12 and 24 hours. Using short-time series expression miner (STeM)

we selected genes significantly down- or up-regulated by SAHA over different time periods

(2-fold change, FDR corrected p-value<0.05). Our results demonstrate a dramatic change in

gene expression occuring after 8-hours treatment in all cell lines, showing a higher proportion of

repressed genes (100) over activated genes (57). The functional analysis of all significant genes

suggests that SAHA alters the signaling of T cell receptor, MAPK, Wnt and JAK-STAT (STAT1,

STAT3) pathways as well as cell cycle (CDKN1A), apoptosis (TRAIL-R), purine and pyrimidine

metabolisms. Our data highligt the importance of TCR associated genes (CD3D, CD3G,

CD3e CD4, LAT, ZAP70) and Th2 cell cytokines - IL-5, IL-10 (downregulated by SAHA) in the

pathogenesis of CTCL. In summary, our results demonstrate the potential targets of SAHA and

underline the importance of TCR signaling in pathogenesis of CTCL.

Gene expression profiling reveals underlying molecular mechanisms of SAHA action in cutaneous T-cell lymphoma

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Key words: SAHA, gene expression, cutaneous T-cell lymphoma

Magdalena B. Wozniak

Laboratorio de Linfomas

Programa de Patología Molecular

Centro Nacional de Investigaciones Oncológicas (CNIO)

Melchor Fernandez Almagro 3

Madrid 28029

Tel: 34 91 224 69 61

Fax: 34 91 224 69 23

[email protected]

Gene expression profiling reveals underlying molecular mechanisms of SAHA action in cutaneous T-cell lymphoma


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FaCULTY aND aUTHOrS’ INDEX

FaCULTYEPIGENETICS AND NEW THERAPIES IN CaNCer

98

R. Agami, The Netherlands Cancer Institute, Amsterdam, NL

L. Altucci, Seconda Università degli Studi di Napoli, IT

A. Ashworth, Chester Beatty Laboratories, London, UK

R. Brown, Cancer Research UK Beatson Laboratories, Glasgow, UK

M. Esteller, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ES

V. Fantin, Merck Research Laboratories, Boston, US

G. Garcia-Manero, The University of Texas, Houston, US

A. W. Griffioen, University Hospital Maastricht, NL

A. Imhof, University of Munich, DE

N. B. La Thangue, University of Oxford, UK

F. Lo Coco, Tor Vergara University, Rome, IT

M. Lubbert, University of Freiburg Medical Center, Freiburg, DE

F. Lyko, German Cancer Research Center, Heidelberg, DE

A. Mai, University of Rome La Sapienza, Rome, IT

V. E. Marquez, Center for Cancer Research, Frederick, US

S. Marsoni, SENDO, Milan, IT

S. Minucci, IFOM, Milan, IT

C. Nervi, University of Rome La Sapienza, Rome, IT

M. A. Piris, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ES

A. Zelent, Leukaemia Research Fund Centre at the Institute of Cancer Research (LRFC at ICR), London, UK

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R. Agami, The Netherlands Cancer Institute, Amsterdam, NLpage 34

L. Altucci, Seconda Università degli Studi di Napoli, ITpage 23

A. Ashworth, Chester Beatty Laboratories, London, UKpage 18

P. S. Bachmann, Children’s Cancer Institute Australia for Medical Research, Sydney, AUpage 46

A. M. Barciszewska, Karol Marcinkowski University of Medical Sciences, Poznan, PLpage 48

S. Bongiorni, University of Tuscia, Viterbo, ITpage 50

R. Brown, Cancer Research UK Beatson Laboratories, Glasgow, UKpage 24

A. Cascante, Karolinska Institutet, Stockholm, SEpage 51

D. P. Cioca, Timisoara University of Medicine and Pharmacy, Timisoara, ROpage 53

B. Conde, Universidad de Zaragoza, ESpage 54

N. Cucu, University of Bucharest, Bucharest-Romania, ROpage 55

C. Davies, Children’s Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, AUpage 56

H. E. Deubzer, University of Heidelberg, Heidelberg, DEpage 58

´

aUTHOrS’ INDEX


100

M. Esteller, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ESpage 37

V. Fantin, Merck Research Laboratories, Boston, USAbstract not available

S. Fotheringham, University of Oxford, The John Radcliffe Hospital, Oxford, UKpage 59

E. Fratta, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, IT page 60

G. Garcia-Manero, The University of Texas, Houston, USAbstract not available

E. Grau, Hospital Universitario La Fe, Valencia, ESpage 62

A. W. Griffioen, University Hospital Maastricht, NLpage 31

J. Hochstatter, Adolf-Butenandt-Institut, LMU, Munich, DEpage 64

A. Imhof, University of Munich, DEpage 30

E. Ionica, University of Bucharest, Molecular Biology Center, ROpage 65

M. Jung, University of Freiburg, DEpage 66

G. Keller, Technische Universität München, Munich, DE page 67

A. Khaled, CHU Frahat Hached, Sousse, TNpage 69 - 71

D. I. Kuck, German Cancer Research Center, Heidelberg, DEpage 72

aUTHOrS’ INDEX

101

aUTHOrS’ INDEX

eP

IGe

Ne

TIC

S a

ND

Ne

W T

He

ra

PIe

S IN

Ca

NC

erM

adrid

Nov

embe

r 29·

30 2

007


N. B. La Thangue, University of Oxford, UKpage 28

V. V. Levenson, Feinberg School of Medicine, Northwestern University, Chicago, USpage 73

F. Lo Coco, Tor Vergara University, Rome, ITpage 35

M. Lubbert, University of Freiburg Medical Center, Freiburg, DEAbstract not available

N. Y. Lukianova, Institute of Experimental Pathology, Oncology and Radiobiology, Kiev, UApage 74

F. Lyko, German Cancer Research Center, Heidelberg, DEpage 19

A. Mai, University of Rome La Sapienza, Rome, ITpage 32

V. E. Marquez, Center for Cancer Research, Frederick, USpage 20

O. A. Martinez-Iglesias, Instituto de Investigaciones Biomédicas Alberto Sols. CSIC-UAM, Madrid, ESpage 75

S. Marsoni, SENDO, Milan, ITAbstract not available

S. Minucci, IFOM, Milan, ITAbstract not available

R.L. Momparler, Hoptial Sainte-Justine, Montreal, Quebec, CApage 76

D. O. Mykytenko, Institute of Experimental Pathology, Oncology and Radiobiology, Kiev, UApage 77


102

S. Nelofer, Breakthrough Breast Cancer Centre at The Institute for Cancer Research, London, UKpage 78

C. Nervi, University of Rome La Sapienza, Rome, ITpage 21

I. Oehme, CCU Pediatric Oncology, DKFZ, Heidelberg, DEpage 79

M. A. Piris, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ESpage 29

G. Prantera, University of Tuscia, Viterbo, ITpage 50

A. Sánchez-Pacheco, Instituto de Investigaciones Biomedicas A. Sols (UAM-CSIC), Madrid, ESpage 80

J. Schmid, Medizinische Klinik IV, Universitaetsklinikum Aachen, RWTH Aachen, DEpage 81

C. Stresemann, German Cancer Research Center, DEpage 83

J. R. Taylor, The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKpage 84

V. Tica, University of Bucharest, Molecular Biology Center, ROpage 85

E. Turcu, “Ana Aslan” National Institute of Gerontology and Geriatrics,Bucharest, ROpage 86

S. Volpi, University of Tuscia, Viterbo, ITpage 50

aUTHOrS’ INDEX

103

aUTHOrS’ INDEX

eP

IGe

Ne

TIC

S a

ND

Ne

W T

He

ra

PIe

S IN

Ca

NC

erM

adrid

Nov

embe

r 29·

30 2

007


K. Wallenborg, Karolinska Institutet, Stockholm, SEpage 87

M. B. Wozniak, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, ESpage 88

A. Zelent, Leukaemia Research Fund Centre at the Institute of Cancer Research, London, UKpage 26

eSO european School of Oncology Via del Bollo, 420123 Milan, Italywww.cancerworld.org

CNIO Centro Nacional de Investigaciones OncológicasMelchor Fernández almagro, 328029 Madrid, Spainwww.cnio.es

Coordination and edition Sara Bertrand and Daniela MengatoDirection of art and producction Bocetocolor SLPhotographic archive CNIO

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