““Epigenetics-Epigenetics-
A Science and Novel Treatment ModalityA Science and Novel Treatment Modality
in Haem-Oncologyin...
Epigenetic InheritanceEpigenetic Inheritance
 Definition:Definition:
Cellular information, other than DNA sequence itself...
3 Main Types of Epigenetic Information3 Main Types of Epigenetic Information
 1. Cytosine DNA methylation- methyl group i...
CpG IslandCpG Island
 Short region of DNA in which the frequency of theShort region of DNA in which the frequency of the
...
Epigenetic Mechanisms (Cont.)Epigenetic Mechanisms (Cont.)
 2. Genomic imprinting is parent-of-origin-specific2. Genomic ...
Histone AcetylationHistone Acetylation
Histone Acety/Deacety-lationHistone Acety/Deacety-lation
HATHAT -↑histone acetylation-↑histone acetylation
(hyperacetylati...
Epigenetic MechanismsEpigenetic Mechanisms
 A connection between epigenetic gene silencing andA connection between epigen...
HDAC ClassesHDAC Classes
Epigenetics and OncogenesisEpigenetics and Oncogenesis
Epigenetics in HaematologicalEpigenetics in Haematological
Malignancies-MDSMalignancies-MDS
MyelodysplasiaMyelodysplasia
 Myelodysplastic syndrome (MDS) is one of the mostMyelodysplastic syndrome (MDS) is one of t...
CMML. Peripheral blood showing 5 over-large
mature monocytes with poorly formed nuclei and 2
agranular poorly segmented ne...
Methylation in HaematologicalMethylation in Haematological
Malignancies-MDSMalignancies-MDS
 CpG Island DNACpG Island DNA...
Methylation in HaematologicalMethylation in Haematological
Malignancies-MDS (cont.)Malignancies-MDS (cont.)
 Cyclin Depen...
 p15p15INK4BINK4B
identified as a candidate tumour suppressoridentified as a candidate tumour suppressor
genegene (Nobori...
Methylation in HaematologicalMethylation in Haematological
Malignancies-MDS (cont.)Malignancies-MDS (cont.)
 Aberrant p15...
Therapeutic Demethylating AgentsTherapeutic Demethylating Agents
Therapeutic Demethylating AgentsTherapeutic Demethylating Agents
 5-Azacytidine and 5-aza-2’-deoxycytidine5-Azacytidine a...
Therapeutic Demethylating AgentsTherapeutic Demethylating Agents..
 Cytidine analogues modified at position 5 of theCytid...
Therapeutic Demethylating AgentsTherapeutic Demethylating Agents
 Hypo-methylating effect restricted to C5 positionHypo-m...
Therapeutic Demethylating Agents.Therapeutic Demethylating Agents.
 Azacytidine incorporates into RNA whilst decitabineAz...
Clinical Trials with AzacytidineClinical Trials with Azacytidine
 CALGB phase III trial 191 patients with MDS treatedCALG...
Clinical Trials with Azacytidine (Cont.)Clinical Trials with Azacytidine (Cont.)
 Transformation to AML as 1Transformatio...
Survival from landmark date by cross-over
status (Kaplan-Meier method). Patients were
sub grouped as supportive care patie...
Clinical Trials Azacytidine (Cont).Clinical Trials Azacytidine (Cont).
 QOL follow up study demonstrated significantQOL f...
Clinical Trials with Demethylating AgentsClinical Trials with Demethylating Agents
 In MDS patients treated with Decitabi...
Therapeutic Histone Deacetylase InhibitorsTherapeutic Histone Deacetylase Inhibitors
(HDACi)(HDACi)
 HDACi cause growth a...
Therapeutic Histone DeacetylaseTherapeutic Histone Deacetylase
Inhibitors (HDACi)Inhibitors (HDACi) Cont…Cont…
(Marks 2001)
HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders
HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders
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 DeacetylaseTherapeutic Histone Deacetylase
Inhibitors (HDACi)Inhibitors (HDACi)
 DNMT also recruits H...
Cancer Res. 2006 Jun 15;66(12):6361-9
Combined DNA methyltransferase and histone
deacetylase inhibition in the treatment o...
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.
 ...
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
Epigenetics - Slide 1
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Epigenetics - Slide 1

  1. 1. ““Epigenetics-Epigenetics- A Science and Novel Treatment ModalityA 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
  2. 2. 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. 3. 3 Main Types of Epigenetic Information3 Main Types of Epigenetic Information  1. Cytosine DNA methylation- methyl group is1. Cytosine DNA methylation- methyl group is transferred fromtransferred 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 CpGDNA methylation occurs almost exclusively at CpG nucleotides and has an important contributing role in thenucleotides and has an important contributing role in the regulation/inhibition of gene expression.regulation/inhibition of gene expression. Epigenetic MechanismsEpigenetic Mechanisms
  4. 4. CpG IslandCpG Island  Short region of DNA in which the frequency of theShort 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 aindicates that "C" and "G" are connected by a phosphodiester bond.phosphodiester bond.  CpG islands are often located around the promotersCpG islands are often located around the promoters of housekeeping genes (which are essential forof housekeeping genes (which are essential for general cell functions) or other genes frequentlygeneral cell functions) or other genes frequently expressed in a cell.expressed in a cell.
  5. 5. Epigenetic Mechanisms (Cont.)Epigenetic Mechanisms (Cont.)  2. Genomic imprinting is parent-of-origin-specific2. Genomic imprinting is parent-of-origin-specific allele silencing. It is maintained, in part, byallele silencing. It is maintained, in part, by differentially methylated regions and it is normallydifferentially 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 inmethylation and phosphorylation — are important in transcriptional regulation and many are stablytranscriptional regulation and many are stably maintained during cell division, although themaintained during cell division, although the mechanism for this epigenetic inheritance is not yetmechanism for this epigenetic inheritance is not yet well understood.well understood.
  6. 6. Histone AcetylationHistone Acetylation
  7. 7. Histone Acety/Deacety-lationHistone Acety/Deacety-lation HATHAT -↑histone acetylation-↑histone acetylation (hyperacetylation)(hyperacetylation) ↑↑ transcriptional activitytranscriptional activity HDACHDAC -↓histone acetylation-↓histone acetylation (hypoacetylation)(hypoacetylation) ↓↓transcriptional activitytranscriptional activity The acetylation of histones results from the balance between histone acetyltransferases (HAT) and histone deacetylases (HDAC) (Marks 2001)
  8. 8. Epigenetic MechanismsEpigenetic Mechanisms  A connection between epigenetic gene silencing andA connection between epigenetic gene silencing and chromatin modifications, was highlighted in studies ofchromatin modifications, was highlighted in studies of X inactivation —X inactivation —  MohandasMohandas et alet al.. ScienceScience, 1981. 5-aza-2'-, 1981. 5-aza-2'- deoxycytidine (5-azaCdR), an inhibitor of DNAdeoxycytidine (5-azaCdR), an inhibitor of DNA methylation could reactivate the inactive Xmethylation could reactivate the inactive X chromosome.chromosome.  45 human chromosomes intensely stained by an45 human chromosomes intensely stained by an antibody specific for acetylated histone H4, with theantibody specific for acetylated histone H4, with the lone inactive X chromosome globally deacetylatedlone inactive X chromosome globally deacetylated and unstained.and unstained.
  9. 9. HDAC ClassesHDAC Classes
  10. 10. Epigenetics and OncogenesisEpigenetics and Oncogenesis
  11. 11. Epigenetics in HaematologicalEpigenetics in Haematological Malignancies-MDSMalignancies-MDS
  12. 12. MyelodysplasiaMyelodysplasia  Myelodysplastic syndrome (MDS) is one of the mostMyelodysplastic syndrome (MDS) is one of the most common haematological malignancies affecting adults.common haematological malignancies affecting adults.  The incidence of MDS is increasing and isThe incidence of MDS is increasing and is approximately 15-50/100,000 per year, primarily inapproximately 15-50/100,000 per year, primarily in patients over 60.patients over 60.  30-40% of cases transform to acute myeloid leukaemia30-40% 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, theSupportive care has remained, until recently, the standard of carestandard of care
  13. 13. CMML. Peripheral blood showing 5 over-large mature monocytes with poorly formed nuclei and 2 agranular poorly segmented neutrophils.
  14. 14. Methylation in HaematologicalMethylation in Haematological Malignancies-MDSMalignancies-MDS  CpG Island DNACpG Island DNA hyper-hyper-methylation identified inmethylation identified in AML and MDS at global (genome) and individualAML and MDS at global (genome) and individual gene levelgene level.. (Rush et al(Rush et al BloodBlood 2001, Melki et al2001, Melki et al Cancer ResCancer Res 1999).1999).  DNADNA hypo-hypo-methylation in T cell lymphomasmethylation in T cell lymphomas identified in transgenic mice with 90% deficiency inidentified in transgenic mice with 90% deficiency in DNMTDNMT (Gaudet et al(Gaudet et al ScienceScience 2003).2003).
  15. 15. Methylation in HaematologicalMethylation 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 CDKCD34+ differentiation, and negatively regulate CDK during the cell cycle.during the cell cycle.
  16. 16.  p15p15INK4BINK4B identified as a candidate tumour suppressoridentified 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 patientsgene promoter identified in some MDS patients particularly RAEB and RAEBt and those with xsparticularly RAEB and RAEBt and those with xs methylation at Dx have a shorter survival cf normalmethylation at Dx have a shorter survival cf normal methylation pattern and higher chance to AMLmethylation pattern and higher chance to AML progressionprogression (Quesnel et al(Quesnel et al BloodBlood 1998, Tien et al1998, Tien et al Br J HaemBr J Haem 2001).2001). Methylation in HaematologicalMethylation in Haematological Malignancies-MDS (cont.)Malignancies-MDS (cont.)
  17. 17. Methylation in HaematologicalMethylation in Haematological Malignancies-MDS (cont.)Malignancies-MDS (cont.)  Aberrant p15Aberrant p15INK4BINK4B hypermethylation also identified in de-hypermethylation also identified in de- novo AML and CML.novo AML and CML.  Hyper-methylation may be a consequence of excessiveHyper-methylation may be a consequence of excessive DNMT activity in these malignanciesDNMT activity in these malignancies (Mizuno et al(Mizuno et al BloodBlood 2001).2001).  Inhibition of DNMT results in reactivation of silencedInhibition of DNMT results in reactivation of silenced genesgenes (Bender et al(Bender et al Pharm ResPharm Res 1998).1998).
  18. 18. Therapeutic Demethylating AgentsTherapeutic Demethylating Agents
  19. 19. Therapeutic Demethylating AgentsTherapeutic Demethylating Agents  5-Azacytidine and 5-aza-2’-deoxycytidine5-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 p15INK4BINK4B inin multiple haematological malignanciesmultiple haematological malignancies (Daskalakis et al(Daskalakis et al BloodBlood 2002).2002).
  20. 20. Therapeutic Demethylating AgentsTherapeutic Demethylating Agents..  Cytidine analogues modified at position 5 of theCytidine analogues modified at position 5 of the pyrimidine ring are potent inhibitors of DNApyrimidine ring are potent inhibitors of DNA methylation, with Decitabine having 30x higher potency.methylation, with Decitabine having 30x higher potency.
  21. 21. Therapeutic Demethylating AgentsTherapeutic Demethylating Agents  Hypo-methylating effect restricted to C5 positionHypo-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-methylationAzacytidine and gemcitabine have no de-methylation activity.activity.  Sorm et al synthesised both Azacytidine and DecitabineSorm et al synthesised both Azacytidine and Decitabine in 1964 .in 1964 .  Bi-modal action noted in vitro and in vivo, cytotoxic atBi-modal action noted in vitro and in vivo, cytotoxic at high [ ] and demethylation at low [ ].high [ ] and demethylation at low [ ].
  22. 22. Therapeutic Demethylating Agents.Therapeutic Demethylating Agents.  Azacytidine incorporates into RNA whilst decitabineAzacytidine 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 byEither as a result of incorporation into nucleic acid or by direct inhibition of DNMT demethylation is achieveddirect 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.
  23. 23. Clinical Trials with AzacytidineClinical Trials with Azacytidine  CALGB phase III trial 191 patients with MDS treatedCALGB 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 onlytreatment vs. supportive care only (Silverman et al 2002 J Clin Oncol).  Supportive arm able to cross over to Aza arm in theSupportive 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 wasMedian time to leukemic transformation or death was 21 months for Aza vs. 13 months for supportive care21 months for Aza vs. 13 months for supportive care p<0.007.p<0.007.
  24. 24. 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 may38% supportive care (p<0.001) suggesting Aza may prevent leukemic transformation. Aza acts as aprevent leukemic transformation. Aza acts as a BRM??BRM??  After removal of cross over confounding effectAfter removal of cross over confounding effect analysis at 6 months demonstrated 18 month for Azaanalysis 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).
  25. 25. 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.
  26. 26. Clinical Trials Azacytidine (Cont).Clinical Trials Azacytidine (Cont).  QOL follow up study demonstrated significantQOL follow up study demonstrated significant improvement cf supportive care onlyimprovement cf supportive care only (Kornblith et al(Kornblith et al J ClinJ Clin OncolOncol 2002).2002).  Studies demonstrate for the first time potential toStudies 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 destabilisationDecitabine with no significant chromosomal destabilisation Lubbert et alLubbert et al Br J HaemBr J Haem 2001).2001).
  27. 27. 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 reversiondemonstrate demethylated p15 alleles and reversion to normal karyotype, indicating suppression of clonalto normal karyotype, indicating suppression of clonal growthgrowth (Wijermans et al(Wijermans et al BloodBlood 2002).2002).  Ongoing trials: randomised, open label, phase 3. cfOngoing trials: randomised, open label, phase 3. cf s.c Aza+ best supportive care vs. conventionals.c Aza+ best supportive care vs. conventional regime+ best supportive care in MDS.regime+ best supportive care in MDS.
  28. 28. Therapeutic Histone Deacetylase InhibitorsTherapeutic Histone Deacetylase Inhibitors (HDACi)(HDACi)  HDACi cause growth arrest, differentiation,HDACi cause growth arrest, differentiation, cytotoxicity, and inductioncytotoxicity, and induction of apoptosis by inducing specificof apoptosis by inducing specific changes in gene expression.changes in gene expression.
  29. 29. Therapeutic Histone DeacetylaseTherapeutic Histone Deacetylase Inhibitors (HDACi)Inhibitors (HDACi) Cont…Cont… (Marks 2001)
  30. 30. HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders
  31. 31. HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders
  32. 32. HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders
  33. 33. HDACi in Lymphoproliferative DisordersHDACi in Lymphoproliferative Disorders Br J Cancer. 2006 Dec;95 Suppl 1:S7-S12
  34. 34. Therapeutic Histone DeacetylaseTherapeutic Histone Deacetylase Inhibitors (HDACi)Inhibitors (HDACi)  DNMT also recruits HDAC thus HDACi could reduceDNMT also recruits HDAC thus HDACi could reduce DNMT activity.DNMT activity.  Combination of demethylatingCombination of demethylating agents and HDACi may resultagents and HDACi may result in augmentation of therapeuticin augmentation of therapeutic response.response.
  35. 35. 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. gorest@jhmi.edu 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.
  36. 36. HDACi in Combination TherapyHDACi in Combination Therapy Br J Cancer. 2006 Dec;95 Suppl 1:S7-S12
  37. 37. SummarySummary  Epigenetic mechanisms are important in oncogenesis.Epigenetic mechanisms are important in oncogenesis.  Epigenetic mechanisms are important in theEpigenetic mechanisms are important in the pathogenesis of Haematological malignancies.pathogenesis of Haematological malignancies.  Demethylation and Histone Acetylation represent twoDemethylation and Histone Acetylation represent two epigenetic mechanisms with relevance to oncogenesis.epigenetic mechanisms with relevance to oncogenesis.  Therapeutic modulation of these mechanisms mayTherapeutic 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.

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