epigenetics - slide 1

““Epigenetics-Epigenetics-

A Science and Novel Treatment Modality A Science and Novel Treatment Modality

in Haem-Oncologyin Haem-Oncology””

Dr Anthony Dear

Thursday March 15th 2007 ACBD, Department of Medicine, 6th Floor Burnet Tower,

Monash University, Prahran, Melbourne

Epigenetic InheritanceEpigenetic Inheritance

Definition:Definition:

Cellular information, other than DNA sequence itself, Cellular information, other than DNA sequence itself, that is heritable during cell division.that is heritable during cell division.

3 Main Types of Epigenetic Information3 Main Types of Epigenetic Information 1. Cytosine DNA methylation- methyl group is 1. Cytosine DNA methylation- methyl group is

transferred from transferred from SS-adenosylmethionine to the C-5 -adenosylmethionine to the C-5 position of cytosine by a family of cytosine (DNA-5)-position of cytosine by a family of cytosine (DNA-5)-methyltransferases (DNMT’s). methyltransferases (DNMT’s).

DNA methylation occurs almost exclusively at CpG DNA methylation occurs almost exclusively at CpG nucleotides and has an important contributing role in the nucleotides and has an important contributing role in the regulation/inhibition of gene expression.regulation/inhibition of gene expression.

Epigenetic MechanismsEpigenetic Mechanisms

CpG IslandCpG Island

Short region of DNA in which the frequency of the Short region of DNA in which the frequency of the CG sequence is higher than in other regions. "p" CG sequence is higher than in other regions. "p" indicates that "C" and "G" are connected by a indicates that "C" and "G" are connected by a phosphodiester bond.phosphodiester bond.

CpG islands are often located around the promoters CpG islands are often located around the promoters of housekeeping genes (which are essential for of housekeeping genes (which are essential for general cell functions) or other genes frequently general cell functions) or other genes frequently expressed in a cell.expressed in a cell.

Epigenetic Mechanisms (Cont.)Epigenetic Mechanisms (Cont.)

2. Genomic imprinting is parent-of-origin-specific 2. Genomic imprinting is parent-of-origin-specific allele silencing. It is maintained, in part, by allele silencing. It is maintained, in part, by differentially methylated regions and it is normally differentially methylated regions and it is normally reprogrammed in the germline.reprogrammed in the germline.

3. Histone modifications — including acetylation, 3. Histone modifications — including acetylation, methylation and phosphorylation — are important in methylation and phosphorylation — are important in transcriptional regulation and many are stably transcriptional regulation and many are stably maintained during cell division, although the maintained during cell division, although the mechanism for this epigenetic inheritance is not yet mechanism for this epigenetic inheritance is not yet well understood. well understood.

Histone AcetylationHistone Acetylation

Histone Acety/Deacety-lationHistone Acety/Deacety-lation

HATHAT -↑histone acetylation -↑histone acetylation (hyperacetylation)(hyperacetylation)

↑ ↑ transcriptional activity transcriptional activity

HDACHDAC -↓histone acetylation -↓histone acetylation

(hypoacetylation)(hypoacetylation)↓↓transcriptional transcriptional

activityactivity

The acetylation of histones results from the balance between histone acetyltransferases (HAT) and histone deacetylases (HDAC)

(Marks 2001)

Epigenetic MechanismsEpigenetic Mechanisms A connection between epigenetic gene silencing and A connection between epigenetic gene silencing and

chromatin modifications, was highlighted in studies of chromatin modifications, was highlighted in studies of X inactivation —X inactivation —

Mohandas Mohandas et alet al. . ScienceScience, 1981. 5-aza-2'-, 1981. 5-aza-2'-deoxycytidine (5-azaCdR), an inhibitor of DNA deoxycytidine (5-azaCdR), an inhibitor of DNA methylation could reactivate the inactive X methylation could reactivate the inactive X chromosome. chromosome.

45 human chromosomes intensely stained by an 45 human chromosomes intensely stained by an antibody specific for acetylated histone H4, with the antibody specific for acetylated histone H4, with the lone inactive X chromosome globally deacetylated lone inactive X chromosome globally deacetylated and unstained.and unstained.

HDAC Classes HDAC Classes

Epigenetics and OncogenesisEpigenetics and Oncogenesis

Epigenetics in Haematological Epigenetics in Haematological

Malignancies-MDSMalignancies-MDS

MyelodysplasiaMyelodysplasia

Myelodysplastic syndrome (MDS) is one of the most Myelodysplastic syndrome (MDS) is one of the most common haematological malignancies affecting adults.common haematological malignancies affecting adults.

The incidence of MDS is increasing and is The incidence of MDS is increasing and is approximately 15-50/100,000 per year, primarily in approximately 15-50/100,000 per year, primarily in patients over 60.patients over 60.

30-40% of cases transform to acute myeloid leukaemia 30-40% of cases transform to acute myeloid leukaemia (AML) and most patients die from infection or bleeding.(AML) and most patients die from infection or bleeding.

Supportive care has remained, until recently, the Supportive care has remained, until recently, the

standard of carestandard of care

CMML. Peripheral blood showing 5 over-large mature monocytes with poorly formed nuclei and 2 agranular poorly segmented neutrophils.

Methylation in Haematological Methylation in Haematological Malignancies-MDSMalignancies-MDS

CpG Island DNA CpG Island DNA hyper-hyper-methylation identified in methylation identified in AML and MDS at global (genome) and individual AML and MDS at global (genome) and individual gene levelgene level. . (Rush et al (Rush et al Blood Blood 2001, Melki et al 2001, Melki et al Cancer ResCancer Res 1999). 1999).

DNA DNA hypo-hypo-methylation in T cell lymphomas methylation in T cell lymphomas identified in transgenic mice with 90% deficiency in identified in transgenic mice with 90% deficiency in

DNMTDNMT (Gaudet et al (Gaudet et al ScienceScience 2003). 2003).

Methylation in Haematological Methylation in Haematological Malignancies-MDS (cont.)Malignancies-MDS (cont.)

Cyclin Dependent Kinase Inhibitors (CDKI’s), Cyclin Dependent Kinase Inhibitors (CDKI’s), particularly, p15particularly, p15INK4BINK4B, are up regulated during normal , are up regulated during normal CD34+ differentiation, and negatively regulate CDK CD34+ differentiation, and negatively regulate CDK during the cell cycle.during the cell cycle.

p15p15INK4B INK4B identified as a candidate tumour suppressor identified as a candidate tumour suppressor

genegene (Nobori et al (Nobori et al NatureNature 1994). 1994).

Excessive, aberrant DNA methylation in the p15Excessive, aberrant DNA methylation in the p15INK4BINK4B gene promoter identified in some MDS patients gene promoter identified in some MDS patients particularly RAEB and RAEBt and those with xs particularly RAEB and RAEBt and those with xs methylation at Dx have a shorter survival cf normal methylation at Dx have a shorter survival cf normal methylation pattern and higher chance to AML methylation pattern and higher chance to AML

progressionprogression (Quesnel et al (Quesnel et al Blood Blood 1998, Tien et al 1998, Tien et al Br J HaemBr J Haem 2001). 2001).



Aberrant p15Aberrant p15INK4B INK4B hypermethylation also identified in hypermethylation also identified in de-novo AML and CML.de-novo AML and CML.

Hyper-methylation may be a consequence of excessive Hyper-methylation may be a consequence of excessive DNMT activity in these malignanciesDNMT activity in these malignancies (Mizuno et al (Mizuno et al Blood Blood 2001).2001).

Inhibition of DNMT results in reactivation of silenced Inhibition of DNMT results in reactivation of silenced genesgenes (Bender et al (Bender et al Pharm ResPharm Res 1998). 1998).

Therapeutic Demethylating AgentsTherapeutic Demethylating Agents


5-Azacytidine and 5-aza-2’-deoxycytidine 5-Azacytidine and 5-aza-2’-deoxycytidine (Decitabine) restore normal methylation pattern in (Decitabine) restore normal methylation pattern in vitro and in vivo to several genes including p15vitro and in vivo to several genes including p15 INK4BINK4B in multiple haematological malignanciesin multiple haematological malignancies (Daskalakis et al (Daskalakis et al Blood Blood 2002).2002).

Therapeutic Demethylating AgentsTherapeutic Demethylating Agents..

Cytidine analogues modified at position 5 of the Cytidine analogues modified at position 5 of the pyrimidine ring are potent inhibitors of DNA pyrimidine ring are potent inhibitors of DNA methylation, with Decitabine having 30x higher potency.methylation, with Decitabine having 30x higher potency.


Hypo-methylating effect restricted to C5 position Hypo-methylating effect restricted to C5 position alterations as other cytidine analogues eg ara-C, 6-alterations as other cytidine analogues eg ara-C, 6-Azacytidine and gemcitabine have no de-methylation Azacytidine and gemcitabine have no de-methylation activity.activity.

Sorm et al synthesised both Azacytidine and Decitabine Sorm et al synthesised both Azacytidine and Decitabine in 1964 .in 1964 .

Bi-modal action noted in vitro and in vivo, cytotoxic at Bi-modal action noted in vitro and in vivo, cytotoxic at high [ ] and demethylation at low [ ].high [ ] and demethylation at low [ ].

Therapeutic Demethylating Agents.Therapeutic Demethylating Agents.

Azacytidine incorporates into RNA whilst decitabine Azacytidine incorporates into RNA whilst decitabine incorporates into DNA. Neither are orally available.incorporates into DNA. Neither are orally available.

Either as a result of incorporation into nucleic acid or by Either as a result of incorporation into nucleic acid or by direct inhibition of DNMT demethylation is achieved direct inhibition of DNMT demethylation is achieved after several rounds of DNA replication.after several rounds of DNA replication.

zebularine [1-(beta-D-ribofuranosyl)-1,2-zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one]. Orally available analogue.dihydropyrimidin-2-one]. Orally available analogue.

Clinical Trials with AzacytidineClinical Trials with Azacytidine

CALGB phase III trial 191 patients with MDS treated CALGB phase III trial 191 patients with MDS treated with 75mg/mwith 75mg/m22/day AZA s.c for 7 days, no pre-/day AZA s.c for 7 days, no pre-treatment vs. supportive care only treatment vs. supportive care only (Silverman et al 2002

J Clin Oncol).

Supportive arm able to cross over to Aza arm in the Supportive arm able to cross over to Aza arm in the case of disease progression.case of disease progression.

Median time to leukemic transformation or death was Median time to leukemic transformation or death was 21 months for Aza vs. 13 months for supportive care 21 months for Aza vs. 13 months for supportive care p<0.007.p<0.007.

Clinical Trials with Azacytidine (Cont.)Clinical Trials with Azacytidine (Cont.)

Transformation to AML as 1Transformation to AML as 1stst event in 15% Aza vs. event in 15% Aza vs. 38% supportive care (p<0.001) suggesting Aza may 38% supportive care (p<0.001) suggesting Aza may prevent leukemic transformation. Aza acts as a prevent leukemic transformation. Aza acts as a BRM??BRM??

After removal of cross over confounding effect After removal of cross over confounding effect analysis at 6 months demonstrated 18 month for Aza analysis at 6 months demonstrated 18 month for Aza vs. 11 month survival for supportive care (P<0.03).vs. 11 month survival for supportive care (P<0.03).

Survival from landmark date by cross-over status (Kaplan-Meier method). Patients were sub grouped as supportive care patients who either never crossed over or crossed over after 6 months, supportive care patients who crossed over before 6 months, and patients who were initially randomized to Aza C.

Clinical Trials Azacytidine (Cont).Clinical Trials Azacytidine (Cont).

QOL follow up study demonstrated significant QOL follow up study demonstrated significant

improvement cf supportive care onlyimprovement cf supportive care only (Kornblith et al (Kornblith et al J Clin J Clin OncolOncol 2002). 2002).

Studies demonstrate for the first time potential to Studies demonstrate for the first time potential to modify the natural history of MDS by a non- modify the natural history of MDS by a non-

intensive drug regimeintensive drug regime (confirmed using low dose (confirmed using low dose Decitabine with no significant chromosomal destabilisation Decitabine with no significant chromosomal destabilisation Lubbert et al Lubbert et al Br J HaemBr J Haem 2001). 2001).

Clinical Trials with Demethylating AgentsClinical Trials with Demethylating Agents

In MDS patients treated with Decitabine, over time, In MDS patients treated with Decitabine, over time, demonstrate demethylated p15 alleles and reversion demonstrate demethylated p15 alleles and reversion to normal karyotype, indicating suppression of clonal to normal karyotype, indicating suppression of clonal

growthgrowth (Wijermans et al (Wijermans et al BloodBlood 2002). 2002).

Ongoing trials: randomised, open label, phase 3. cf Ongoing trials: randomised, open label, phase 3. cf s.c Aza+ best supportive care vs. conventional s.c Aza+ best supportive care vs. conventional regime+ best supportive care in MDS.regime+ best supportive care in MDS.

Therapeutic Histone Deacetylase Inhibitors Therapeutic Histone Deacetylase Inhibitors (HDACi)(HDACi)

HDACi cause growth arrest, differentiation, HDACi cause growth arrest, differentiation, cytotoxicity, and induction cytotoxicity, and induction of apoptosis by inducing specificof apoptosis by inducing specificchanges in gene expression.changes in gene expression.

Therapeutic Histone Deacetylase Therapeutic Histone Deacetylase Inhibitors (HDACi)Inhibitors (HDACi) Cont… Cont…

(Marks 2001)

HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders

HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders

Br J Cancer. 2006 Dec;95 Suppl 1:S7-S12

Therapeutic Histone Deacetylase Therapeutic Histone Deacetylase Inhibitors (HDACi)Inhibitors (HDACi)

DNMT also recruits HDAC thus HDACi could reduce DNMT also recruits HDAC thus HDACi could reduce DNMT activity.DNMT activity.

Combination of demethylating Combination of demethylating

agents and HDACi may resultagents and HDACi may result

in augmentation of therapeutic in augmentation of therapeutic

response.response.

Cancer Res. 2006 Jun 15;66(12):6361-9

Combined DNA methyltransferase and histone deacetylase inhibition in the treatment of myeloid neoplasms

Gore SD, Baylin S, Sugar E, Carraway H, Miller CB, Carducci M, Grever M, Galm O, Dauses T, Karp JE, Rudek MA, Zhao M, Smith BD, Manning J, Jiemjit A, Dover G, Mays A, Zwiebel J, Murgo A, Weng LJ, Herman JG.

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland 21287, USA. [email protected]

Patients with myelodysplastic syndrome or acute myeloid leukemia (AML) were treated with the methyltransferase inhibitor 5-azacitidine (aza-CR) followed by the histone deacetylase inhibitor sodium phenylbutyrate. Major responses associated with cytogenetic complete response developed in patients receiving prolonged dosing schedules of aza-CR. Six of six responding patients with pretreatment methylation of p15 or CDH-1 promoters reversed methylation during the first cycle of therapy (methylation-specific PCR), whereas none of six nonresponders showed any demethylation. Administration of both drugs was associated with induction of acetylation of histones H3 and H4. This study provides the first demonstration that molecular mechanisms responsible for responses to DNA methyltransferase/histone deacetylase inhibitor combinations may include reversal of aberrant epigenetic gene silencing. The promising percentage of major hematologic responses justifies the testing of such combinations in prospective randomized trials.

HDACi in Combination TherapyHDACi in Combination Therapy

Br J Cancer. 2006 Dec;95 Suppl 1:S7-S12

SummarySummary

Epigenetic mechanisms are important in oncogenesis.Epigenetic mechanisms are important in oncogenesis.

Epigenetic mechanisms are important in the Epigenetic mechanisms are important in the pathogenesis of Haematological malignancies.pathogenesis of Haematological malignancies.

Demethylation and Histone Acetylation represent two Demethylation and Histone Acetylation represent two epigenetic mechanisms with relevance to oncogenesis.epigenetic mechanisms with relevance to oncogenesis.

Therapeutic modulation of these mechanisms may Therapeutic modulation of these mechanisms may afford novel treatment strategies in Haem-oncology, afford novel treatment strategies in Haem-oncology, particularly in pre-treated patients and in combination.particularly in pre-treated patients and in combination.

epigenetics - slide 1

Documents