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“de novo” Acute Leukemias Defect in the differentiation process

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. BONE MARROW. °. 1. 2°. Expansion of a stem cell

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“de novo” Acute Leukemias Defect in the differentiation process

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  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. 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 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. 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 -7 +8 Normal -7 +8 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

  15. 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

  16. Singal et al. Blood

  17. DNA methylation and cancer

  18. S. Belinsky. Nature reviews 2004

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

  20. 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

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

  22. Clark S. et al Oncogene 2002

  23. Clark S. et al Oncogene 2002

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

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

  26. 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

  27. 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

  28. 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

  29. 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

  30. 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

  31. MDS with 3q26translocations CtBPs Sin3 N-cor HDAC AML1/EVI-1 fusion protein CtBPs hampered expression of target genes Koji Izutsu et al, Oncogene 2002

  32. CANDIDATE TARGETS FOR EPIGENETIC THERAPY DNMTs HDACs HATs

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

  34. Valproic acid • Used in the treatment of epilepsy • At the dosage used : 10 mg/kg (serum levels 40-100 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

  35. p<0.01 VPA & ATRAserum level (40-100 mcg/ml) 45 mg/m2 • Patients (10 AML, 7 RAEB) 17 • Patients with <1 month of therapy 7 • Patients evaluable 10 • Patients responsive 6 • 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: 6-8 months Responses in approximately 30% of the enrolled patients (ITT basis) and in 60% of those who were really treated

  36. Patient G.L. female, 63 year old, M4 AML WBC x 103/µl PLTS x 103/µl 250 5 4 200 150 3 RETINOICACID 100 2 VALPROICACID (VPA) 50 VPA target serum level 1 VPA suboptimal level 1 2 3 4 5 6 7 8 9 10 11 12 13 14 WEEKS

  37. 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

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

  39. Acethylation of H4 histone

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