Molecular Pathogenesis of Leukemia

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

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

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/RARa 10-6 M (or RA 10-9 M)

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

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

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

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

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

11. Transcriptional Activation by Retinoic Acid PML/RARa degradation by Retinoic Acid HMT Dnmt1 PRMT1 PRMT1 Dnmt1 Dnmt1 BTG2 BTG2 Sin3A pCAF complex N-CoR N-CoR pCAF complex N-CoR Sin3A Sin3A MeCP2 MeCP2 MeCP2 pCAF PML PML PML PML pCAF N-CoA N-CoA CBP CBP HDAC HDAC HDAC RARE RARE RARE RARE 10-6 M 10-6 M RARa RARa RARa RARa CH3 CH3 RA RA RA RA RARE RARE RARE RARE Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac CH3 CH3 CH3 CH3 Transcriptional Repression by PML/RARa Francesco Grignani

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

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

14. Altered regulation of chromatin structure is common in AML

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

16. As2O3 induces degradation of PML/RARa Sin3A HDAC Dnmt1 Dnmt1 As As As As HDAC N-CoR N-CoR As As MeCP2 MeCP2 PML PML As As RARa RARa CH3 CH3 CH3 CH3 RARE RARE Ac Ac Ac As As Ac As As Ac Ac As As As2O3 induces degradation of PML/RARa Sin3A

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

18. HDAC inhibitors in AML and MDS treatment 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. 2006 Feb;20(2):212-7. 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. 2006 Jan 1;106(1):112-9. 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. 2005 Dec;84 Suppl 13:61-6. McMullin MF, Nugent E, Thompson A, Hull D, Jones FG, Grimwade D.Prolonged molecular remission in PML-RARa-positive acute promyelocytic leukemia treated with minimal chemotherapy followed by maintenanceincluding the histone deacetylase inhibitor sodium valproate. Leukemia. 2005 Sep;19(9):1676-7.

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

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

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

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

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

24. C E PML A/B D F N-CoR/SMRT IDN IDC RD3 Fusion Proteins / Co-Repressors Interaction Domains PML/RARa AML1 ETO ZnF AML1/ETO

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

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

27. Disruption of the Corepressor Complex Increases the Expression of Fusion Proteins Target Genes NB4/ NB4R4 SKNO1 AML1/ETO-regulated PML/RARa-regulated

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

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

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

31. IDC/IDN Expression Induces PML/RARa Protein Degradation The Fusion RNA is Expressed The Protein is Undetectable

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

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

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

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

36. BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas. 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

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

39. IDC Expression Allowes Histone Acetylation on RARa Target Genes by Low RA Concentrations NB4 NB4 IDC NB4 IDC NB4 C+ C+ RA hrs 0 15 30 60 180 0 15 30 60 180 0 30 60 180 0 30 60 180 RAR-b Actin ChIP a-acetyl H3 ChIP a-acetyl H4 5x10-8 M RA

40. Transcriptional Activation by Retinoic Acid Sin3A HDAC N-CoR pCAF complex N-CoR pCAF RXRa RARa N-CoA RXRa RARa CBP HDAC RARE RARE RARE RARE RARE RARE 10-9 M 10-9 M RA RA RA RA Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Inactive RARa Sin3A Francesco Grignani

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

42. IDC-induced PML/RARa degrdation reconstitute PML-nuclear bodies NB4-IDC NB4 NB4-R4 NB4-R4-IDC

43. Many HDAC Inhibitors are entering clinical trials

44. HL60 Cells NB4 Cells NB4 Cells +RA 10-6 M (PML/RARa) (NO PML/RARa) Expression of the PML/RARa Fusion Protein Alters PML Nuclear Bodies. RA Restores Them a-PML Mo-Ab