1. Hybrid phatogenesis of MDS
Myeloproliferative diseases
Defect in the proliferative process
“de novo”
Acute Leukemias Defect in the differentiation process
MDS:
Defect in the proliferative and differentiative processes

2. CYTOPENIAS LEUKEMIA BONE MARROW ° 1 2° Expansion of a stem cell
clone showing an impaired
differentiation, that leads
to a premature cell death
Complete differentiation
block:
blast increase
PERIPHERAL BLOOD
CYTOPENIAS
LEUKEMIA

3. Genetic lesions in AMLs MDS Simple genetic Complex genetic Lesion
Single translocation
es. t(8;21)
Involvement of a single
Transcription Factor
Complex genetic
Lesion
Multiple translocations
and delitions
es. t(3;3) and del 7
Involvement of multiple
Transcription Factor
and deletion of genes

4. Relative percentage of various cytogenetic
The molecular and cytogenitic analysis is able to detect the presence of genetic lesions in about 60% of MDS patients
Relative percentage of various cytogenetic
abnormalities in de novo myelodysplastic syndrome
(MDS).
List. et al. ASH 2004

5. Gene expression profiling in healthy subjects and MDS patients
Normal Normal
Chen et al. Blood 2004

6. Chen et al. Blood 2004

7. Physiopathology of MDS
Genetic Abnormalities
-Chromosomal (+8, -7,-5,5q-, 7q-, 20q-)
-Point Mutations (RAS)
Epigenetic Modifications
-Aberrant DNA methylation
-Aberrant acetylation
Dysregulation of cytokine production
-Abnormal rate of apoptosis & proliferation

8. Epigenetic modifications
Potentially reversible DNA and chromatin modifications transmissed from a cell to its progeny, able to induce altered gene expression without changing DNA sequence and without any “new” genetic information

9. Epigenetic modifications
DNA Methylation: embryogenesis, differentiation, imprinting, X inactivation, cancer
Post translational modifications of proteins with subsequent chromatin alteration:
Histone acetylation
H. Methylation
H. Phosphorylation
H. Ubiquitination

10. Nucleasome structure Acetylation of lysines
Methylation of lysines and arginines
Phosphorylation of serines and threonines
Ubiquitination and sumulyation of lysines

11. Covalent modification of the N terminal tail of the core histones

12. Genetic and epigenetic changes that inactivate tumor suppressor genes
The number of cancer-related genes affected by epigenetic inactivation equals or exceeds the number that are inactivated by mutation

13. DNA Methylation
DNA methylation is a covalent chemical modification, adding a CH3 group at the carbon 5 position of cytosine situated in the sequence contest 5’CG3’. Its frequence is lower than predictable by sequence.
CpG dinucleotide clustered in promoter regions of 50% of human genes (CpG Islands 0.5 – 5kb every 100kb).
CpG islands are generally unmethylated in normal cells
Methylation of CpG islands determines gene silencing

14. Epigenetic alterations in MDS

16. Methylation of CpG island in gene promoter regions is associated with aberrant silencing of transcription and is a mechanism for inactivation of tumor suppressor genes

19. Singal et al. Blood

20. DNA methylation and cancer

22. S. Belinsky.
Nature reviews 2004

24. Hypermethylated genes in MDS
P15ink4a (65%)
DAPkinase (47%)
SOCS1
E-cadherin
calcitonin

25. p15ink4a Negatively regulates cell cycle (G1-S phase)
Inhibits cyclin dependent kinase 4 and 5
Gene hypermethylation correlates with
shorter MDS survival and disease progression

26. Survival of MDS patients according to the p15INK4b gene methylation status
Methylated
Unmethylated
T (months)
P survival
Quesnel, et al. Blood. 1998;91:2985

27. Clark S. et al Oncogene 2002

28. Clark S. et al Oncogene 2002

30. HAT
Sin3
N-cor
HDAC
histon acetylation -> chromatin relaxation -> transcription of target genes

31. La cromatina è accessibile ai fattori trascrizionali,
HAT complex
HDAC
complex
La cromatina è accessibile
ai fattori trascrizionali,
è possibile l’espressione di geni
necessari per la differenziazione

32. La cromatina non è accessibile ai fattori trascrizionali,
HAT complex
HDAC
complex
La cromatina non è accessibile
ai fattori trascrizionali,
non è possibile l’espressione di geni
indispensabile per la differenziazione.
Blocco differenziativo

33. recruitment of HDAC complex=
AML/MDS with t(8;21)
CBP/p300
CBFb
recruitment of HDAC complex=
transcription block
of target genes
AML1
AML1
HAT
AML1
Sin3
N-cor
ETO
HDAC
Wang J et al., PNAS 1998

34. PML RA In APL with PML-RAR, higher (phamacological)
concentrations of RA are needed
to detach the HDAC complex
(variant translocations are insensitive) RA RA RA RA
Sin3
N-cor
RAR RA
HDAC
DNMT
PML
transcription block
Grignani F. et al., Nature 1998

35. Inactivation of tumour suppressor genes
HDAC
Inactivation of genes regulating proliferation, differentiation and apoptosis
Crucial to prepare the histone template for methyltransferases removing acethyl groups

36. SWI/SNF AML with t(9;11) AML with t(6;11) AML with t(11;19)
MLL/partner gene
fusion protein
loss of the MLL C-terminal SET domain
hampered expression
of target genes

37. MDS with 3q26 translocations
CtBPs
Sin3
N-cor
AML1/EVI-1
fusion protein
CtBPs
HDAC
hampered expression
of target genes
Koji Izutsu et al, Oncogene 2002

39. FOR EPIGENETIC THERAPY
CANDIDATE TARGETS
FOR EPIGENETIC THERAPY
DNMTs
HDACs HATs

40. CANDIDATE DRUGS FOR EPIGENETIC THERAPY
DNMT inhibitors azacitidine, decitabine, zebularine, procainamide
DNMT antisense and siRNA
HDAC inhibitors

44. Valproic acid Used in the treatment of epilepsy
At the dosage used : 10 mg/kg (serum levels mcg/ml) it acts as a potent HDAC inhibitor
Used in clinical trials for the treatment of AML and MDS patients
Refractory to conventional chemotherapy
Not candidate for conventional chemotherapy

45. VPA & ATRA serum level (40-100 mcg/ml) 45 mg/m2
Patients (10 AML, 7 RAEB) 17
Patients with <1 month of therapy 7
Patients evaluable
Patients responsive
Plts before therapy: median 23 x 106/l
range ( 9-39)
Plts after therapy: median 157 x 106/l
range (76-271)
Median duration of response: months
p<0.01
Responses in approximately 30% of the enrolled patients (ITT basis)
and in 60% of those who were really treated

46. Patient G.L. female, 63 year old, M4 AML
WBC x 103/µl
PLTS x 103/µl
250 5
200 4
150
RETINOIC ACID 3
100 2
VALPROIC ACID (VPA) 50
VPA target serum level 1
VPA suboptimal level
WEEKS

48. Pazient G.L., BM at diagnosis
Dr. U. Familiari, Anatomia Patologica, Ospedale S.Luigi
Colorazione: Ematossilina-Eosina, ingrandimento 20 X
Pilatrino et al. Cancer 2005

49. Pazient G.L., BM post therapy
Dr. U. Familiari, Anatomia Patologica, Ospedale S.Luigi
Colorazione: Ematossilina-Eosina, ingrandimento 20 X

50. Acethylation of H4 histone

51. New therapeutical strategies
Association of HDAC inhibitors and demethylating agents.