Acute myeloid leukemia: Therapeutic impact of epigenetic drugs

The International Journal of Biochemistry & Cell Biology 37 (2005) 1752–1762

Medicine in focus

Acute myeloid leukemia: Therapeutic impact of epigenetic drugs Lucia Altucci a,b,∗ , Nicole Clarke c , Angela Nebbioso a , Annamaria Scognamiglio a , Hinrich Gronemeyer c,∗∗ a

Dipartimento di Patologia generale, Seconda Universit`a degli Studi di Napoli (S.U.N.), vico L. De Crecchio 7, 80138 Naples, Italy b Centro di Oncogenomica AIRC, CEINGE Biotecnologia avanzata, Napoli, Italy c Department of Cell Biology and Signal Transduction, IGBMC, B.P. 10142, F-67404 Illkirch, Cedex C.U. de Strasbourg, France

Received 1 February 2005; received in revised form 20 April 2005; accepted 26 April 2005

Abstract Acute myeloid leukemia (AML) is not a single disease but a group of malignancies in which the clonal expansion of various types of hematopoietic precursor cells in the bone marrow leads to perturbation of the delicate balance between self-renewal and differentiation that is characteristic of normal hematopoiesis. An increasing number of genetic aberrations, such as chromosomal translocations that alter the function of transcription regulatory factors, has been identified as the cause of AML and shown to act by deregulating gene programming at both the genetic and epigenetic level. While the genetic aberrations occurring in acute myeloid leukemia are fairly well understood, we have only recently become aware of the epigenetic deregulation associated with leukemia, in particular with myeloid leukemias. The deposition of epigenetic “marks” on chromatin – post-translational modifications of nucleosomal proteins and methylation of particular DNA sequences – is accomplished by enzymes, which are often embedded in multi-subunit “machineries” that have acquired aberrant functionalities during leukemogenesis. These enzymes are targets for so-called “epi-drugs”. Indeed, recent results indicate that epi-drugs may constitute an entirely novel type of anti-cancer drugs with unanticipated potential. Proof-of-principle comes from studies with histone deacetylase inhibitors, promising novel anti-cancer drugs. In this review we focus on the epigenetic mechanisms associated with acute myeloid leukemogenesis and discuss the therapeutic potential of epigenetic modulators such as histone deacetylase and DNA methyltransferase inhibitors. © 2005 Elsevier Ltd. All rights reserved. Keywords: Leukemia; Epigenetics; Therapy; Deacetylases inhibitors

1. Introduction ∗

Corresponding author. Tel.: +39 081 5667569/5667564; fax: +39 081 450169. ∗∗ Corresponding author. Tel.: +33 388 653473/653212; fax: +33 388 653437. E-mail addresses: [email protected] (L. Altucci), [email protected] (H. Gronemeyer). 1357-2725/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2005.04.019

Leukemia is the manifestation of the inappropriate expansion of hematopoietic progenitor cells, often due to a block of cell maturation at early stages in the cell lineages that give rise to the various cell types that

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Table 1 Significance

Age

Causes

Clinical manifestations

Principles of treatment

Acute myeloid leukemias (AMLs) Hematologic urgency, usually fatal within weeks–months without chemotherapy. High mortality due to disease or treatment-related complications

Mainly in adults M > F curable in minority of adults

Idiopathic, underlying hematologic disorders, chemicals, drugs, ionizing radiation, viruses (HTLV I), hereditary or genetic conditions

Marrow failure, tissue infiltration, leukostasis, constitutional symptoms, disseminated intravascular coagulation

Induction and consolidation; combination chemotherapy: first goal is complete remission; supportive medical care: transfusions, antibiotics, nutrition; psychosocial support: patient and family

t(15; 17)(q22; q12–21) chromosomal translocation

As above

Combined treatment with all-trans retinoic acid and chemotherapy

PML–RAR␣-positive AML (FAB M3) Paradigm for cancer Mainly in differentiation therapy; cure adults, curable rates above 75% in 75%

constitute normal blood. Through recurrent specific genetic aberrations, such as chromosome breaks, rearrangements that transfer genes into distinct genetic contexts or generate fusion proteins with altered functionalities, genes have been identified whose altered functionality was critically or causally linked to leukemogenesis. In particular, acute myeloid leukemia (AML) is a malignant tumour of hematopoietic precursor cells of non-lymphoid lineage, with an annual incidence of 1/10,000 and a frequency that increases with age (Table 1). While AMLs were previously sub-divided following the French–American–British (FAB) classification according to blast morphology, immunophenotype, cytogenetics and clinical features, the WHO classification incorporates in addition to those parameters more recent discoveries regarding the genetics and clinical features of AML in an attempt to define entities that are biologically homogeneous and that have prognostic and therapeutic relevance (WHO; Table 2). The most studied AML subtype is acute promyelocytic leukemia (APL; FAB M3) of which more than 90% display the chromosomal translocation t(15;17) that generates the PML–RAR␣ fusion protein comprising parts of the PML and retinoic acid receptor alpha (RAR␣) genes. In the past years, the functions that have been altered by the presence of different oncogenic fusion proteins such as PML–RAR␣ in APL or AML–ETO in some AML are increasingly well understood. However, only recently, we have begun to unravel the epigenetic (and thus in principle, reversible) phenomena that contribute, together with genetic aberrations, to the development of leukemia, and malig-

nancies in general, by the recognition and dissection of multi-subunit chromatin-modifying machineries. Epigenetic phenomena refer to ‘mitotically and meiotically heritable changes in gene expression that are not coded in the DNA sequence itself’ (Egger, Liang, Aparicio, & Jones, 2004). But often the term ‘epigenetic’ is in general applied to describe events originated from the post-translational modification of the main chromatin components, histones and DNA, or even transcription factors. Moreover, an ‘histone code’ hypothesis has been formulated (Strahl & Allis, 2000) to propose that the panel of modifications such as acetylation, methylation, phosphorylation, ubiquitination, sumoylation or ADP ribosylation deposit epigenetically relevant information at defined genetic Table 2 French–American–British (FAB) AML classification M0 Undifferentiated M1

Early myeloblastic

M2 M3

Late myeloblastic Promyelocytic

M4 M5

Myelomonocytic Monoblastic

M6

Erythroleukaemic

M7

Megakaryoblastic

WHO AML with recurrent genetic abnormalities AML with multilineage dysplasia AML and myelodysplastic syndromes; therapy related AML not otherwise categorised Acute leukemias of ambiguous lineage

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Fig. 1. Illustration of the nucleosomal histone octamer with the N- and C-terminal regions of histones extending from the core. Partial sequences of the terminal are given; numbers refer to positions in the amino acid sequences. Colored dots above residues indicate known post-translational modifications and may constitute signal transduction readouts or even constitute an “epigenetic code”.

loci to regulate most, if not all, chromatin-templated processes (see Fig. 1 for epigenetic modifications of the histone tails). Theoretically, the complexity of such a ‘histone code’ would by far exceed that of the classical genetic code. However, an alternative view is that histone modifications may just correspond to steps in a complex signalling process (Kurdistani, Tavazoie, & Grunstein, 2004). Importantly, modifications of the DNA and histone modification patterns have both been associated with cancer and epigenetic deregulations are increasingly recognized as mechanisms exploited by tumors to silence gene programs that regulate growth, DNA repair and apoptosis (Ayton, Chen, & Cleary, 2004; Egger et al., 2004; Esteller, 2005; Feinberg &

Tycko, 2004; Laird, 2003; Villa et al., 2004). Notably, in contrast to genetic modifications, epigenetic alterations are transient and can be reversed, at least partially, by treatment with epigenetic drugs (Brueckner & Lyko, 2004; Somech, Izraeli, & Simon, 2004). Pioneering work has unraveled the link between oncogenic fusion proteins (such as PML–RAR␣, PLZF–RAR␣, AML–ETO, etc.) – that cause acute myeloid leukaemia – with the aberrant recruitment of histone deacetylases and DNA methyltransferase, enzymes involved in gene silencing. Various types of inhibitors (HDAC-Is and DNMT-Is) have been generated and we are now witnessing the elucidation of structure–activity relationships of HDAC-Is (Marks, Richon, Miller, &

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Kelly, 2004) and the mechanism(s) of their anti-cancer action (Insinga et al., 2005; Nebbioso et al., 2005). Moreover, therapies are being explored in which two epigenetic (e.g. an HDAC and a DNA methyltransferase inhibitor) drugs or an epigenetic and a signalling drug (e.g. HDAC-I and retinoic acid) are combined to specify action, decrease effective concentration due to drug synergy and thus, reduce side effects.

2. Genetic aberrations causing acute myeloid leukemia affect epigenetic gene regulation Acute myeloid leukemia (AML), the collective description of a variety of myeloid leukemias characterized by a block of differentiation at various early stages in the myeloid lineage, is frequently associated with the common karyotype abnormalities that affect a number of specific genes believed to regulate critical steps in normal myelopoiesis. Common abnormalities cluster often, but not always, with common AML subtypes. In some cases, such as the t(15;17) translocation generating the PML–RAR␣ onco-fusion protein that causes acute promyelocytic leukaemia (APL), the affected genes and abnormalities have been characterised in detail and mouse models have confirmed the leukemogenic potential of the abnormal gene product, albeit secondary mutations may be necessary for expression of the acute phenotype (for review and references see Altucci & Gronemeyer, 2001; Gilliland, 2002). Clues to understand the altered functionality of PML–RAR␣ came from the mechanistic studies of nuclear receptor-mediated gene activation (in presence of an agonistic ligand) and silencing (in the absence of ligand), which revealed that gene expression is regulated at the level of chromatin by the action of histone acetyltransferases (HATs) and deacetylases (HDACs). Indeed, histone acetylation is dynamically regulated and is directly involved in the gene expression regulation: hyper-acetylated histones are frequently found at transcriptionally active loci, while the chromatin of inactive genes and heterochromatin contains generally hypo-acetylated histones. Transcription activation involves the recruitment of multi-subunit machineries containing co-activators, which display HAT activity such as P/CAF, p300 or CBP, by DNA binding transcription factors to target gene promoters. Mechanistically, HATs tethered to a promoter acetylate

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histones thus inducing a decondensation, resulting in de-represssion of chromatin-mediated gene silencing. This gene silencing can be established by multicomponent machineries, which contain enzymes with HDAC activity. Their recruitment results in hypoacetylation and generates a repressive chromatin conformation. In addition to inducing silencing HDACs contribute to epigenetic stabilization of the repressive pattern by recruiting DNA-methyl transferases (DNMTs) and leading to a stable methylation of silenced gene program(s). The interdependency of histone hypo-acetylation and DNA methylation is further strengthened by the existence of methylated DNAbinding proteins (MBDs) that bind methyl-C residues and recruit HDACs. Initial evidence that alterations of the acetylation/deacetylation equilibrium might lead to cancer came from studies on the molecular pathogenesis of APL. In more than 95% of the cases APL is associated with chromosomal translocations generating the PML–RAR␣ fusion protein. Pharmacological doses of retinoic acid (RA) induce disease remission by triggering terminal differentiation and apoptosis of APL blasts. From a mechanistic perspective, PML–RAR␣ acts like a retinoic acid receptor that has gone awry. Normally, in the absence of its natural ligand all-trans retinoic acid (RA), RAR␣ represses target gene transcription by recruiting the HDAC complex through direct binding of the HDAC-associated nuclear co-repressors N-CoR or SMRT. Physiological concentrations of RA lead to dissociation of the HDAC complex and recruitment of HAT/coactivator complexes. In contrast to RAR␣, physiological concentrations of RA cannot dissociate the co-repressor-HDAC complex from PML–RAR␣ resulting in permanent silencing of the RA-regulated gene program(s), such as those triggering cell maturation and death. Strikingly, at pharmacologically high doses of RA the co-repressorHDAC complex does dissociate from PML–RAR␣ allowing RA signalling to proceed. It is believed that this phenomenon, together with RA induction of the death ligand TRAIL (Clarke, Jimenez-Lara, Voltz, & Gronemeyer, 2004) (Fig. 2), accounts for the successful “differentiation therapy” of APL patients with RA (for review and references see Altucci & Gronemeyer, 2004). A number of points have, however, still to be clarified. These concern (i) the question whether RA-bound

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Fig. 2. The TRAIL promoter can be activated by retinoids, interferons and HDAC-Is. (a) The TRAIL promoter contains several consensus sequences, two GC-boxes, an ISRE and an IRF-E. In un-stimulated cells, SP1 and SP3 are bound to the GC-boxes and the IRF-E and/or ISRE are bound by interferon regulatory factor-2, IRF-2. Treatment of cells with INF␥ and/or RA induces the expression of interferon regulatory factor-1, IRF-1, which is recruited to the IRF-E and ISRE of the promoter. (b) HDAC-Is require the GC-boxes for TRAIL induction and induce recruitment of additional SP3 protein to the promoter. (c) In the above two cases, CREB-binding protein (CBP), a co-activator with HAT activity and RNA polymerase II (PolII) are subsequently recruited to the promoter, histones are hyper-acetylated and the gene is transcribed.

PML–RAR␣ recruits the same or distinct coactivator/HAT complexes as RAR␣, (ii) the altered functionality of PML in the onco-fusion protein (Lin, Bergmann, & Pandolfi, 2004; Salomoni & Pandolfi, 2002), (iii) novel functionalities generated by fusing two distinct signalling factors (Insinga et al., 2004), (iv) the impact of site-specific sumoylation on PML–RAR␣ function (Zhu et al., 2005) and (v) the role of the reciprocal fusion product (Rego & Pandolfi, 2002). For more detailed information, which is beyond the scope of this

review, the reader is referred to the indicated publications. A minority of APL cases exhibits variant translocations; although these translocations also generate fusion proteins with RAR␣ the patients are RA resistant. It has been demonstrated that in the case of the PLZF–RAR␣ binding of the co-repressor-HDAC complex does not only involve RA-sensitive interaction with RAR but also with the PLZF part of the fusion protein (for review and references see Altucci &

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Gronemeyer, 2004). Notably, however, the combination of RA and HDAC inhibitors (HDAC-Is) relieves the differentiation block by PLZF–RAR␣ in vitro, suggesting that HDAC-Is might be effective in the treatment of RA-resistant APL patients (Lin et al., 1998). There is increasing evidence indicating that aberrant recruitment of HDACs is a recurrent theme in multiple AML subtypes. For example, in the AML1ETO fusion protein, one of the most common fusion proteins in AMLs, the DNA binding domain of a transcription factor (AML1) is fused to nearly the entire coding sequence of ETO, which interacts with the HDAC complex (Amann et al., 2001). Additionally, the HATs CBP (Borrow et al., 1996; Petrij et al., 1995) and p300 (Chaffanet et al., 2000; Ida et al., 1997), and the nuclear receptor co-activator TIF2 that normally interacts with CBP (Voegel et al., 1998) causes myeloid leukaemia when fused to a distinct HAT, MOZ upon chromosomal translocation (Deguchi et al., 2003; Huntly et al., 2004). Interestingly, monoallelic loss of CBP, but not of p300, determines multi-lineage defects in hematopoiesis and haematological malignancies in mice (Kung et al., 2000). These findings indicate that alterations in the equilibrium of HAT/HDAC activity are frequent in leukemias.

3. Differentiation therapy of myeloid leukaemia Cancer ‘differentiation therapy’ refers to a novel type of treatment by which a (signaling, epigenetic) drug forces a tumor cell to adapt a more, preferentially terminally, differentiated state that leads to apoptosis. Such drugs do not necessarily display the toxicity commonly associated with chemotherapy. Presently, several compounds are in clinical use but the most successful example of differentiation therapy is APL with the use of all-trans retinoic acid which combined with chemotherapy leads to a complete remission in more than 75% of patients that carry that characteristic t(15;17) translocation. The introduction of RA therapy for APL has changed a cancer with a poor outcome into one of the most treatable forms of myeloid leukemia. Unfortunately, variant APL translocations are insensitive to RA (see below), as are the great number of other AMLs, which frequently originate from several chromosomal aberrations. Given the alteration in DNA methylation patterns in cancers, including leukemias,

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and the link between HDACs and DNMTs, it is tempting to speculate that alterations in histone acetylation levels may affect the methylation of specific genes. AML fusion proteins may directly target, through the HDAC/DNMT association, DNMTs to those genes, leading to their methylation (Di Croce et al., 2002). The alteration in DNA methylation patterns may in turn lead to a complex profile of altered gene expression (epigenetically inherited) in cancer cells. Mixed lineage leukaemia (MLL) fusion proteins may become oncogenic due to a related epigenetic phenomenon. MLL, the homologue of Drosophila trithorax, encodes a methyltransferase that methylates histone H3 on lysine 4. MLL is an epigenetic modulator that is required for the maintenance but not the induction of its targets, such as Hox genes. Recent results indicate that MLLassociated oncogenesis may be linked to the (altered) recognition of CpG islands in target promoters (Ayton et al., 2004; Milne et al., 2002; Nakamura et al., 2002). Therapeutic concepts based on inclusion of epigenetic targets, such as the interaction of AML fusion proteins with HDAC–DNMT complexes may represent a novel anti-leukemic strategy. Indeed, initial evidence comes from an example of RA-resistant APL, where the combination RA and HDAC-Is treatment led to in vitro differentiation (Ferrara et al., 2001). Thus the combination of signalling drugs and epigenetic modulators may be a promising approach in leukemias. Furthermore, novel signalling pathways (regulated by ligand-bound retinoid X receptors and activated protein kinase A) leading to differentiation have been identified in APL-derived cells, thus adding to the list of potentially useful compounds (Benoit et al., 1999; Guillemin et al., 2002). Combination paradigms established for leukemias may in turn pave the way to the still largely unexplored area dealing with the use of these agents in the treatment of other cancers.

4. Coming of age: Epigenetic therapy of leukemia Solid evidence demonstrates that in addition to genetic alteration, aberrant epigenetic regulation, such as silencing of tumor suppressors, is used by cancer cells to escape growth and death control mechanisms (for review and references see Esteller, 2005). Thus, compounds able to influence the epigenetic status of

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Table 3 Molecule

Structure

Haematological cancer type

Combination

Phase

Histone-deacetylase inhibitors in clinical trialsa

SAHA (suberoylanilide hydroxamic acid)

Leukemia, lymphoma, multiple myeloma

I

Cutaneous T-cell lymphoma, sezary syndrome, mycosis fungoides Diffuse large B-cell lymphoma

II/III

VPA (valproic acid)

AML, CLL, SLL CML, MDS, acute leukemia

MS-275

Acute leukemia, CML, chronic leukemia, plasma cell neoplasm, MDS Leukemia, lymphoma AML, CML, MDS, secondary AML

Depsipeptide (FR901228)

Phenylbutyrate

Leukemia, T-cell lymphoma, non-Hodgkins lymphoma, ALL, cutaneous T-cell lymphoma Non-Hodgkins lymphoma

AML, CML, chronic myelomonocytic leukemia, MDS Recurrent adult AML

II/III

Decitabine

I

I

Isotretinoin Azacitidine

I I

II

Fludaribine, rituximab

I/II

Tretinoin

I

Azacitidine Dexamethasone & sargramostim (GM-CSF)

I/II II

a Information regarding these clinical trials can be found at http://www.clinicaltrials.gov/ AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; CLL, chronic lymphoblastic leukemia; SLL, small lymphocytic leukaemia.

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Table 4 Molecule Methyltransferase inhibitors in clinical trialsa Decitabine (5-aza-2 -deoxycitidine)

Azacitidine

Haematological cancer type

Combination

Phase

CML, MDS AML, ALL, MDS, CLL, CML, SLL CML

Valproic acid Imatinib mesylate (Gleevec)

II I/II II

Phenylbutyrate

I/II

Amifostine MS-275

II I

AML, atypical CML, MDS, non-Hodgkins lymphoma MDS MDS, CML, AML

a Information regarding these clinical trials can be found at http://www.clinicaltrials.gov/ AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; CLL, chronic lymphoblastic leukemia; SLL, small lymphocytic lymphoma.

a cell have promise for cancer treatment. DNMT-Is have been already largely tested in cancers. One of the most recent results obtained 30–60% response rates in leukemias (Issa et al., 2004; Silverman et al., 2002). Some concerns to the use of DNMT-Is have been raised for the finding of genome instability in mice with reduced DNA methylation levels due to prolonged demethylating treatment (Gaudet et al., 2003), even if those results are not directly comparable to the transient DNMT inhibition applied in cancer patients (Yang, Estecio, Garcia-Manero, Kantarjian, & Issa, 2003). Moreover, the question has been raised whether the effect of azacytidine nucleotides might be mediated by its own cytotoxicity (Eden, Gaudet, Waghmare, & Jaenisch, 2003). Recently, additional compounds such as the local anesthetic procaine (and its derivative procainamide) (Villar-Garea, Fraga, Espada, & Esteller, 2003) and the main polyphenol compound in the green tea EGCG ((−)-epigallocatechin-3-gallate) (Fang et al., 2003) have been shown to inhibit DNMTs and some of them are currently in phase I trials. Moreover, the psammaplins class has shown both DNMT and HDAC activity inhibition which might enable a combinatorial inhibition (Pina et al., 2003). Regarding acetylation and deacetylation processes, several HDAC-Is exhibit impressive anti-tumor activity potentiated by little toxicity in in vitro, ex vivo and in vivo models (Insinga et al., 2005; Nebbioso et al., 2005) and are now in clinical trials as monotherapy as well as in combination with other drugs (see Tables 3 and 4). Several classes of HDAC-Is have been identified including short-fatty acid (such as butyric acid), hydroxamic acids (such as suberoy-

lanilide hydroxamic acid, SAHA and trichostatin A TSA), cyclic tetrapeptides and benzamides (such as MS-275) (see Table 3 for a selection of HDAC-Is that are enrolled in clinical trials). While it was well established that an important component of the actions spectrum of HDAC-Is is the induction of the cyclindependent kinase inhibitor p21WAF1/CIP1 , very recent data point to an exciting potential of HDAC-Is that may explain the selective anti-cancer activity of these compounds, which are known to be more toxic for tumor cells than for normal cells. These data derived from two studies of the anti-tumor activity of several HDACIs in AML models, which led to the discovery of the induction of TRAIL/Apo2L and its death receptors. Activation of the TRAIL death pathway is well known to kill cancer cells preferentially (for more details see Litwack, 2004). TRAIL inducibility is elicited by HDAC-Is inhibition, partial HDAC displacement from the TRAIL promoter plus SP1/SP3 recruitment and acetylation to the TRAIL promoter; notable is the poor toxicity of HDAC-Is (Guo et al., 2004; Insinga et al., 2005; Nebbioso et al., 2005) (Fig. 1). The possibility to create second and third generation compounds with a higher specificity for a single enzyme or a selected class of DNMTs or HDACs (Mai et al., 2003) will pave the way for new discovery and specific applications in leukemia treatment.

5. Conclusions and perspectives The progresses on the functional characterization of DNMTs and HDACs will enable a more rational

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approach to drug design. Targeted design of epigenetic modulators together with the development of molecular models for screening purposes will facilitate the identification of more specific and selective molecules. Furthermore, defining the action spectra of HDACs, using genetics, antisense oligonucleotides, siRNAs and inhibitors, will provide the basis for targeted clinical use of potentially very powerful, isotype-selective, inhibitors. By combining these with retinoids or other differentiating agents, it might be possible to induce growth inhibitory (p21WAF1 ), differentiative and apoptogenic (TRAIL and/or TRAIL receptor induction; unblocking of RAR␤ inducibility) programs. Notably, reversal of DNA hypermethylation by demethylating agents has already been shown to restore RA-mediated signalling in some tumors. Studies on the molecular basis that modulate epigenetic events during tumorigenesis, and their effect on differentiation and apoptosis pathways, alone or in combination with other drugs, will provide new tools to fight cancer. While research on leukemia has spearheaded the recognition of the role of epigenetic phenomena in leukemogenesis and the potential of epi-drugs, similar data are being reported for solid tumor. For example, dysregulated expression of the polycomb group protein enhancer of zeste homolog 2 (EZH2) may be involved in prostate cancer progression and breast cancer aggressiveness, and its overexpression in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion (Kleer et al., 2003; Varambally et al., 2002). Notably EZH2 functions as transcriptional repressor, displays histone H3 Lysine 27 methyltransferase activity and forms complexes with proteins that interact directly with type 1 HDACs. Thus, much of what we learn about the epigenetics of leukemia and the use of epi-drugs may be important for tumorigenesis and cancer therapy in general.

Acknowledgements Supported by the European Community (QLG1-CT2000-01935 and QLK3-CT-2002-02029), the Institut Nationale de la Sant`e et de la Recherche M´edicale, the Centre National de La Recherche Scientifique, the H`opital Universitaire de Strasbourg, the Association for International Cancer Research, the Association

pour la Recherche sur le Cancer, the Fondation de France, the Regione Campania Legge 5/2002, PRIN 2004-055579, the Ministero dell Salute R.F.02/184, the French-Italian GALILEO project.

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