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Molecular Pathogenesis of Leukemia PML/RAR  MLL translocations PLZF/RAR  etc. AML1/ETO CBF  /SMMHC TEL/AML1 Other transloc. BCR-ABL Flt3 ITD or mutation.

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Presentation on theme: "Molecular Pathogenesis of Leukemia PML/RAR  MLL translocations PLZF/RAR  etc. AML1/ETO CBF  /SMMHC TEL/AML1 Other transloc. BCR-ABL Flt3 ITD or mutation."— Presentation transcript:

1 Molecular Pathogenesis of Leukemia PML/RAR  MLL translocations PLZF/RAR  etc. AML1/ETO CBF  /SMMHC TEL/AML1 Other transloc. BCR-ABL Flt3 ITD or mutation N/K-RAS mutation C-KIT mutation PDGFR  Mutations that affect proliferation/survival Type I mutations Mutations that impair differentiation Type II mutations Trisomy 8 5q-, 7q-, 20q- Complex Caryotypes

2 Oncogene addiction of human cancers Weinstein, Science 2002 * * C-myc in experimental models Bcr/abl, PML/RAR  in human (?)

3 Molecular Lesions Start in a Stem Cell Population Current Treatment approaches :  Kill Cancer Bulk  Kill Cancer Stem cells

4 Cancer stem cells undergo limited differentiation Therapeutic Approach: Induce Cancer Stem Cells Differentiation

5 Retinoic acid induces differentiation of APL blasts Expressing PML/RAR  M (or RA M)

6 Acute Promyelocytic Leukemia (APL) and the 15;17 translocation Grignani et al, Blood 1994

7 Oncogenic Biological Effects of PML/RAR  Expression in Human Cells Grignani et al. Cell 1993, Blood 2000 Commitment induction PML/RAR  expression Block of Apoptosis Self-renewal Further mutations XXXXX Differentiation block LEUKEMIA Self-renewal Differentiation Commitment Normal Hematopoiesis Apoptosis Biological Effects of Retinoic Acid in PML/RAR  Expressing Human Cells PML/RAR  expression Grignani et al. Cell 1993, Blood 2000 Self-renewal Commitment induction Differentiation Apoptosis RA Self-renewal Differentiation Commitment Normal Hematopoiesis Apoptosis

8 RARE RAR  RARE RAR  PML RARE CH3 Dnmt1 Sin3A MeCP2 Ac N- CoR Transcriptional Repression by PML/RAR  Involves DNA Methyl-Transferases HDAC Ac Francesco Grignani

9 HMT RARE RAR  RARE RAR  PML RARE Dnmt1 Sin3A MeCP2 N-CoR HDAC pA Sepharose DNA+Protein Immunoprecipitation w/anti-X antibody RARE DNA extraction PCR Histone Methyl-transferases participate in the PML/RAR  transcriptional complex

10 HMT RARE RAR  RARE RAR  PML RARE CH3 Dnmt1 Sin3A MeCP2 Ac N- CoR Transcriptional Repression by PML/RAR  HDAC Ac Francesco Grignani

11 RARE RAR  RARE RAR  PML RARE Ac Transcriptional Repression by PML/RAR  RA M Francesco Grignani CH3 Dnmt1 Sin3A MeCP2 N- CoR HDAC Ac HMT CH3 Transcriptional Activation by Retinoic Acid MeCP2 Sin3A HDAC Dnmt1 N-CoR RAR  RARE RAR  PML RARE RA M Ac CBP pCAF pCAF complex N-CoA PRMT1 BTG2 CH3 PML/RAR  degradation by Retinoic Acid MeCP2 Sin3A HDAC Dnmt1 N-CoR RARE CBP pCAF pCAF complex N-CoA PRMT1 BTG2

12 90-95% 3-4% 1% 1% 1% APL Chromosomal Translocations Always Involve RAR  But not all APLs are the Same RA-sensitive RA-resistant ? ? ?

13 Sin3A The AML1/ETO Fusion Protein Recruits a Repressor Complex HDAC Ac AML1AML1 ETO ETO N-CoR

14 Altered regulation of chromatin structure is common in AML

15 Beyond Retinoic Acid and APL 1- Some PML/RAR  APL relapse and are ATRA resistant 2- PLZF/RAR  APLs are ATRA resistant 3- The AML1/ETO fusion protein of M2/4 AMLs fuctions by recruiting a co-repressor/HDAC complex fuctions by recruiting a co-repressor/HDAC complex 4- Other leukemias are associated to altered chromatin structure regulation structure regulation

16 Sin3A N- CoR Dnmt1 MeCP2 HDAC Ac RAR  RARE RAR  PML RARE As 2 O 3 induces degradation of PML/RAR  CH3 As Sin3A N- CoR Dnmt1 MeCP2 HDAC Ac CH3 As As 2 O 3 induces degradation of PML/RAR 

17 Riadattata da: Esteller M., J Pathol 2005 HDAC inhibitors (TSA,VPA,etc) 5-azacytidine Terapia Epigenetica

18 Maslak P, Chanel S, Camacho LH, Soignet S, Pandolfi PP, Guernah I, Warrell R, Nimer S. Pilot study of combination transcriptional modulation therapy with sodium phenylbutyrate and 5- azacytidine in patients with acute myeloid leukemia or myelodysplastic syndrome. Leukemia Feb;20(2): Kuendgen A, Schmid M, Schlenk R, Knipp S, Hildebrandt B, Steidl C, Germing U, Haas R, Dohner H, Gattermann N. The histone deacetylase (HDAC) inhibitor valproic acid as monotherapy or in combination with all-trans retinoic acid in patients with acute myeloid leukemia. Cancer Jan 1;106(1): Kuendgen A, Knipp S, Fox F, Strupp C, Hildebrandt B, Steidl C, Germing U, Haas R, Gattermann N. Results of a phase 2 study of valproic acid alone or in combination with all-trans retinoic acid in 75 patients with myelodysplastic syndrome and relapsed or refractory acute myeloid leukemia. Ann Hematol Dec;84 Suppl 13:61-6. McMullin MF, Nugent E, Thompson A, Hull D, Jones FG, Grimwade D. Prolonged molecular remission in PML-RAR  -positive acute promyelocytic leukemia treated with minimal chemotherapy followed by maintenance including the histone deacetylase inhibitor sodium valproate. Leukemia Sep;19(9): HDAC inhibitors in AML and MDS treatment

19 E2F Rb HDAC X A. B. C. HDAC inhibition: potential effects on cell growth Activation of myc-target genes Suppression of Rb activity Increased activity of “HAT” fusion proteins

20 HDAC inhibition: potential effects on EBV regulated pathways HDAC inhibition may have “EBV-like” effects

21 RARE RAR  RARE RAR  PML RARE CH3 Dnmt1 Sin3A MeCP2 N- CoR PML/RAR  Activity Depends on its Corepressors Interaction Interface HDAC Ac

22 Sin3A The AML1/ Activity Depends on its Corepressors Interaction Interface The AML1/ETO Activity Depends on its Corepressors Interaction Interface HDAC Ac AML1AML1 ETO ETO N-CoR/SMRT

23 Sin3A N- CoR CH3 Dnmt1 MeCP2 RAR  RARE RAR  PML RARE Block of N-CoR/PML-RAR  interactions HDAC Ac RAR  RARE RAR  PML RARE CH3

24 A/B C D E PML F PML/RAR  IDN IDCN-CoR/SMRTRD3 AML1/ETO ZnF AML1 Fusion Proteins / Co-Repressors Interaction Domains ETO

25 Sin3A N- CoR CH3 Dnmt1 MeCP2 RAR  RARE RAR  PML RARE Transcriptional Repression by PML/RAR  HDAC Ac RAR  RARE RAR  PML RARE CH3 IDC Expression impairs N-CoR/RAR  binding CH3 PML RAR  RARE RAR  RARE Sin3A HDAC Dnmt1 N-CoR MeCP2

26 The AML1/ Fusion Protein Recruits a The AML1/ETO Fusion Protein Recruits a Repressor Complex AML1AML1 ETO ETO Sin3A HDAC N-CoR/SMRT

27 PML/RAR  -regulated AML1/ETO-regulated Disruption of the Corepressor Complex Increases the Expression of Fusion Proteins Target Genes NB4/ NB4R4SKNO1

28 Block of Fusion Proteins-Corepressors Interaction Restores D3-Induced Differentiation Response PML/RAR  AML1/ETO

29 NB4R4+RA NB4R4-IDC+RA Block of PML/RAR  -Corepressors Interaction Restores RA-Induced Terminal Differentiation in RA-Resistant Cells

30 NBT Surface Markers Growth Block of AML1/ETO-Corepressors Interaction Increases RA-Induced Differentiation Response

31 IDC/IDN Expression Induces PML/RAR  Protein Degradation The Fusion RNA is Expressed The Protein is Undetectable

32 Sin3A N- CoR CH3 Dnmt1 MeCP2 RAR  RARE RAR  PML RARE Transcriptional Repression by PML/RAR  HDAC Ac RAR  RARE RAR  PML RARE CH3 IDC Expression impairs N-CoR/RAR  binding CH3 PML RAR  RARE RAR  RARE Sin3A HDAC Dnmt1 N-CoR MeCP2 CH3 Sin3A HDAC Dnmt1 N-CoR MeCP2 RARE IDC Expression induces PML/RAR  degradation

33 Protein transfer strategies Protein production in bacteria Addition to cell culture medium Lysis and 6-H-based purification TAT HAIDC 6H TAT 6H HA IDC 6H IDC 6H IDC 6H IDC PEP1 Coupling with “cargo peptide” Quick urea removal A. B.

34 Vitamin D3-induced differentiation Corepressors interaction peptides are effective when transduced via TAT-mediated protein transfer

35 GSH GST PML RAR  35 S N-CoR GSH GST PML RAR  IDC 20-mer competes for PML/RAR  binding to N-CoR in GST pull-down experiments 35 S N-CoR GST GST-PML/RAR  - 1:5 1:50 IDC 20-mer -- + IDC 20-mer

36 Specific peptide interference reveals BCL6 transcriptional and oncogenic mechanisms in B-cell lymphoma cells Jose M Polo, Tania Dell’Oso, Stella Maris Ranuncolo, Leandro Cerchietti, David Beck, Gustavo F Da Silva,Gilbert G Prive, Jonathan D Licht & Ari Melnick Nature Medicine 10:1329, 2004 BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas.

37 Conclusions 1- The search for the best molecular target in leukemia therapy points again at fusion proteins 2- Fusion proteins alter epigenetic regulation of target genes by recruiting enzymes 3- Enzyme inhibitors are being tested in clinical trials 4- The use of blocking peptides could contribute to neutralize oncogenic proteins

38 Francesco Grignani Serena Racanicchi, Monia Billi, Maddalena Panigada Maddalena Panigada, Chiara Maccherani, Concetta Liberatore Giovanni Rizzo Clara Nervi Vania Gelmetti Vania Gelmetti, Patologia Generale and MISO, Dept. of Clin. and Experimental Medicine, Perugia University, Italy Dept. of Histology and Embriology, Universita` di Roma ‘La Sapienza’, Parco Bio-Medico Scientifico San Raffaele, Roma, Italy

39 x10 -8 M RA RAR-  Actin C+ ChIP  -acetyl H3 ChIP  -acetyl H NB4 NB4 IDC NB4 NB4 IDC IDC Expression Allowes Histone Acetylation on RAR  Target Genes by Low RA Concentrations RA hrs

40 RAR  RARE RXR  RARE Sin3A Ac N- CoR Inactive RAR  HDAC Ac RA M Francesco Grignani Transcriptional Activation by Retinoic Acid Sin3A HDAC N-CoR CBP pCAF pCAF complex N-CoA RAR  RARE RXR  RARE RA M Ac

41 RAR  GST GSH GST PML RAR  + 35 S N-CoR +/- RA GSH GST PML RAR  PML/RAR  binds N-CoR with high affinity Grignani et al. Nature 1998

42 NB4-R4NB4-R4-IDC NB4 NB4-IDC IDC-induced PML/RAR  degrdation reconstitute PML-nuclear bodies

43 Many HDAC Inhibitors are entering clinical trials

44  -PML Mo-Ab Expression of the PML/RAR  Fusion Protein Alters PML Nuclear Bodies. RA Restores Them HL60 Cells (NO PML/RAR  ) NB4 Cells (PML/RAR  ) NB4 Cells +RA M


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