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Molecular Pathogenesis of Leukemia
PML/RAR
MLL translocations
PLZF/RARetc.AML1/ETO
CBF/SMMHCTEL/AML1
Other transloc.
BCR-ABL
Flt3 ITDor mutation
N/K-RASmutation
C-KIT mutation
PDGFR
Mutations that affect proliferation/survival
Type I mutationsType I mutations
Mutations that impair differentiation
Type II mutationsType II mutations
Trisomy 85q-, 7q-, 20q-
Complex Caryotypes
Oncogene addiction of human cancers
Weinstein, Science 2002
*
* C-myc in experimental modelsBcr/abl, PML/RAR in human (?)
Molecular Lesions Start in a Stem Molecular Lesions Start in a Stem Cell PopulationCell Population
Current Treatment approaches :Kill Cancer BulkKill Cancer Stem cells
Cancer stem cells undergo limited differentiationCancer stem cells undergo limited differentiation
Therapeutic Therapeutic Approach:Approach:
Induce Cancer Stem Induce Cancer Stem Cells DifferentiationCells Differentiation
Retinoic acid induces differentiation of APL blastsRetinoic acid induces differentiation of APL blastsExpressing PML/RARExpressing PML/RAR
1010-6-6 M M(or RA 10-9 M)
Acute Promyelocytic Leukemia (APL) and the Acute Promyelocytic Leukemia (APL) and the 15;17 translocation15;17 translocation
Grignani et al, Blood 1994
Oncogenic Biological Effects of PML/RAR Expression in Human Cells
Grignani et al. Cell 1993, Blood 2000
Commitment induction
PML/RAR expression
Block ofApoptosis
Self-renewal
Further mutations
XXXXX
Differentiationblock
LEUKEMIALEUKEMIA
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
RASelf-renewal
Differentiation
Commitment
Normal Hematopoiesis
Apoptosis
RARE RARE
RAR
RARE
RAR
PMLPML
RARE
CH3CH3
Dnmt1Sin3A
MeCP2
Ac AcAcAc
N-CoR
Transcriptional Repression by PML/RARTranscriptional Repression by PML/RARInvolves DNA Methyl-TransferasesInvolves DNA Methyl-Transferases
HDACHDAC
Ac Ac
Ac
Francesco Grignani
HMTHMT
RARE RARE
RAR
RARE
RAR
PMLPML
RARE
Dnmt1Sin3A
MeCP2N-CoR HDACHDAC
pASepharose
DNA+Protein
Immunoprecipitationw/anti-X antibody
RARE RARE
DNA extraction
PCR
Histone Methyl-transferases participate in the PML/RAR transcriptional complex
HMTHMT
RARE RARE
RAR
RARE
RAR
PMLPML
RARE
CH3CH3
Dnmt1Sin3A
MeCP2
Ac AcAcAc
N-CoR
Transcriptional Repression by PML/RARTranscriptional Repression by PML/RAR
HDACHDAC
Ac Ac
Ac
Francesco Grignani
RARE RARE
RAR
RARE
RAR
PMLPML
RARE
Ac AcAcAc
Transcriptional Repression by PML/RARTranscriptional Repression by PML/RAR
RA RA10-6 M
Francesco Grignani
CH3CH3
Dnmt1Sin3A
MeCP2N-CoR
HDAC
Ac Ac
Ac
HMTHMT
CH3CH3
Transcriptional Activation by Retinoic AcidTranscriptional Activation by Retinoic Acid
MeCP2
Sin3A
HDAC
Dnmt1
N-CoR
RAR
RARE
RAR
PMLPML
RARE
RA RA10-6 M
Ac AcAcAc
Ac AcAcAcCBP
pCAF
pCAF complex
N-CoA
PRMT1PRMT1BTG2BTG2
CH3CH3
PML/RARPML/RAR degradation by Retinoic Acid degradation by Retinoic Acid
MeCP2
Sin3A
HDAC
Dnmt1
N-CoR
RARE RARE
CBP
pCAF
pCAF complex
N-CoA
PRMT1PRMT1BTG2BTG2
90-95%90-95%
3-4%3-4%
1%1%
1%1%
1%1%
APL Chromosomal Translocations Always Involve APL Chromosomal Translocations Always Involve RARRARBut not all APLs are the SameBut not all APLs are the Same
RA-sensitiveRA-sensitive
RA-resistantRA-resistant
??
??
??
Sin3A
The AML1/ETO Fusion Protein Recruits a The AML1/ETO Fusion Protein Recruits a Repressor ComplexRepressor Complex
HDACHDAC
Ac Ac
Ac
AML1AML1 AML1AML1
ETOETOETOETON-CoRN-CoR
Altered regulation of chromatin structure is common in AML
Beyond Retinoic Acid and APLBeyond Retinoic Acid and APL
1- Some PML/RAR1- Some PML/RAR APL relapse and are ATRA resistant APL relapse and are ATRA resistant
2- PLZF/RAR2- PLZF/RAR APLs are ATRA resistant APLs are ATRA resistant
3- The AML1/ETO fusion protein of M2/4 AMLs3- The AML1/ETO fusion protein of M2/4 AMLs fuctions by recruiting a co-repressor/HDAC complexfuctions by recruiting a co-repressor/HDAC complex
4- Other leukemias are associated to altered chromatin 4- Other leukemias are associated to altered chromatin structure regulationstructure regulation
Sin3A
N-CoR
Dnmt1
MeCP2HDAC
Ac Ac
Ac
RAR
RARE
RAR
PMLPML
RARE
AsAs22OO3 3 induces degradation of PML/RARinduces degradation of PML/RAR
CH3CH3
As
As
As
As
As
As
As
Sin3A
N-CoR
Dnmt1
MeCP2
HDAC
Ac AcAc
CH3CH3
As
As
As
As
As
As
As
AsAs22OO3 3 induces degradation of PML/RARinduces degradation of PML/RAR
Riadattata da: Esteller M., J Pathol 2005
HDACinhibitors
(TSA,VPA,etc)5-azacytidine
Terapia Epigenetica
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-RAR-positive acute promyelocytic leukemia treated with minimal chemotherapy
followed by maintenance including the histone deacetylase inhibitor sodium valproate. Leukemia. 2005 Sep;19(9):1676-7.
HDAC inhibitors in AML and MDS treatment
E2F
RbHDAC 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
HDAC inhibition: potential effects on EBV regulated pathways
HDAC inhibition may have “EBV-like” effects
RARE RARE
RAR
RARE
RAR
PMLPML
RARE
CH3CH3
Dnmt1Sin3A
MeCP2N-CoR
PML/RARPML/RARActivity Depends on its Activity Depends on its Corepressors Interaction InterfaceCorepressors Interaction Interface
HDAC
Ac Ac
Ac
Sin3A
The AML1/The AML1/ETO Activity Depends on its Activity Depends on its Corepressors Interaction InterfaceCorepressors Interaction Interface
HDACHDAC
Ac Ac
Ac
AML1AML1 AML1AML1
ETOETOETOETON-CoR/N-CoR/SMRTSMRT
Sin3A
N-CoR
CH3CH3
Dnmt1
MeCP2RAR
RARE
RAR
PMLPML
RARE
Block of N-CoR/PML-RARBlock of N-CoR/PML-RAR interactions interactions
HDAC
Ac Ac
Ac
RAR
RARE
RAR
PMLPML
RARE
CH3CH3
A/BA/B CC DD EEPMLPML FF
PML/RARPML/RAR
IDNIDN IDCIDCN-CoR/SMRTN-CoR/SMRT
RD3RD3
AML1/ETOAML1/ETO
ZnFZnFAML1AML1
Fusion Proteins / Co-Repressors Interaction DomainsFusion Proteins / Co-Repressors Interaction Domains
ETOETO
Sin3A
N-CoR
CH3CH3
Dnmt1
MeCP2RAR
RARE
RAR
PMLPML
RARE
Transcriptional Repression by PML/RARTranscriptional Repression by PML/RAR
HDAC
Ac Ac
Ac
RAR
RARE
RAR
PMLPML
RARE
CH3CH3
IDC Expression impairs N-CoR/RARIDC Expression impairs N-CoR/RAR binding binding
CH3CH3
PMLPML
RAR
RARE
RAR
RARE
Sin3A
HDAC
Dnmt1
N-CoR
MeCP2
The AML1/The AML1/ETO Fusion Protein Recruits a Fusion Protein Recruits a Repressor ComplexRepressor Complex
AML1AML1 AML1AML1
ETOETOETOETO
Sin3A
HDACHDACN-CoR/N-CoR/SMRTSMRT
PML/RAR-regulated AML1/ETO-regulated
Disruption of the Corepressor Complex Increases the Expression of Fusion Proteins Target Genes
NB4/ NB4R4 SKNO1
Block of Fusion Proteins-Corepressors Interaction Block of Fusion Proteins-Corepressors Interaction Restores D3-Induced Differentiation ResponseRestores D3-Induced Differentiation Response
PML/RAR AML1/ETO
NB4R4+RANB4R4+RA NB4R4-IDC+RANB4R4-IDC+RA
Block of PML/RARBlock of PML/RAR-Corepressors Interaction Restores -Corepressors Interaction Restores RA-Induced Terminal Differentiation in RA-Resistant CellsRA-Induced Terminal Differentiation in RA-Resistant Cells
NBT Surface Markers Growth
Block of AML1/ETO-Corepressors Interaction Increases Block of AML1/ETO-Corepressors Interaction Increases RA-Induced Differentiation ResponseRA-Induced Differentiation Response
IDC/IDN Expression Induces PML/RARIDC/IDN Expression Induces PML/RARProtein DegradationProtein Degradation
The Fusion RNA is Expressed
The Protein is Undetectable
Sin3A
N-CoR
CH3CH3
Dnmt1
MeCP2RAR
RARE
RAR
PMLPML
RARE
Transcriptional Repression by PML/RARTranscriptional Repression by PML/RAR
HDAC
Ac Ac
Ac
RAR
RARE
RAR
PMLPML
RARE
CH3CH3
IDC Expression impairs N-CoR/RARIDC Expression impairs N-CoR/RAR binding binding
CH3CH3
PMLPML
RAR
RARE
RAR
RARE
Sin3A
HDAC
Dnmt1
N-CoR
MeCP2
CH3CH3
Sin3A
HDAC
Dnmt1
N-CoR
MeCP2
RARE RARE
IDC Expression induces PML/RARIDC Expression induces PML/RAR degradation degradation
Protein transfer strategiesProtein transfer strategies
Protein production in bacteria
Addition to cell culture medium
Lysis and 6-H-based
purification
TATHA IDC6H
TAT6H HA IDC
6H IDC
6H IDC
6H IDC
PEP1
Coupling with “cargo peptide”
Quick urea removal
A. B.
Vitamin D3-induced differentiation
Corepressors interaction peptides are effective when transduced via TAT-mediated protein transfer
GSH
GST PMLRAR
35S N-CoRGSH
GSHGST PMLRAR
IDC 20-mer competes for PML/RAR binding to N-CoR in GST pull-down experiments
35 S N-C
oR
GSTGST-P
ML/R
AR
- 1:5 1:50IDC 20-mer - -
+ IDC 20-mer
Specific peptide interference reveals BCL6 transcriptionaland oncogenic mechanisms in B-cell lymphoma cellsJose 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.
ConclusionsConclusions
1- The search for the best molecular target in 1- The search for the best molecular target in leukemia therapy points again at fusion proteinsleukemia therapy points again at fusion proteins
2- Fusion proteins alter epigenetic regulation of 2- Fusion proteins alter epigenetic regulation of target genes by recruiting enzymestarget genes by recruiting enzymes
3- Enzyme inhibitors are being tested in clinical 3- Enzyme inhibitors are being tested in clinical trialstrials
4- The use of blocking peptides could contribute to 4- The use of blocking peptides could contribute to neutralize oncogenic proteins neutralize oncogenic proteins
Francesco GrignaniFrancesco Grignani
Serena Racanicchi, Serena Racanicchi, Monia Billi,Monia Billi,Maddalena PanigadaMaddalena Panigada,Chiara Maccherani,Concetta LiberatoreGiovanni Rizzo
Clara Nervi Clara Nervi
Vania GelmettiVania 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
0 30 60 180 0 30 60 180
5x10-8 M RA
RAR-
Actin
C+ C+
ChIP -acetyl H3 ChIP -acetyl H4
0 30 60 18015 0 30 60 18015
NB4 NB4 IDC NB4 NB4 IDC
IDC Expression Allowes Histone Acetylation on RAR Target Genes by Low RA Concentrations
RA hrs
RAR
RARE
RXR
RARERARE RARE
Sin3A
Ac AcAcAc
N-CoR
Inactive RARInactive RAR
HDAC
Ac Ac
Ac
RA RA10-9 M
Francesco Grignani
Transcriptional Activation by Retinoic AcidTranscriptional Activation by Retinoic Acid
Sin3A
HDACN-CoR
CBP
pCAF
pCAF complex
N-CoA
RAR
RARE
RXR
RARE
RA RA10-9 M
Ac AcAcAc
Ac AcAcAc
RARGST GSHGST PMLRAR
+
35S N-CoR
+/- RAGSH
GSH
GST PMLRAR
PML/RAR binds N-CoR with high affinity
Grignani et al. Nature 1998
NB4-R4NB4-R4 NB4-R4-IDCNB4-R4-IDC
NB4NB4 NB4-IDCNB4-IDC
IDC-induced PML/RARIDC-induced PML/RAR degrdation reconstitute degrdation reconstitute PML-nuclear bodiesPML-nuclear bodies
Many HDAC InhibitorsMany HDAC Inhibitorsare entering are entering clinical trialsclinical trials
-PML Mo-Ab-PML Mo-Ab
Expression of the PML/RARExpression of the PML/RAR Fusion Protein Fusion Protein Alters PML Nuclear Bodies. RA Restores ThemAlters PML Nuclear Bodies. RA Restores Them
HL60 CellsHL60 Cells(NO PML/RAR(NO PML/RAR))
NB4 CellsNB4 Cells(PML/RAR(PML/RAR))
NB4 Cells NB4 Cells +RA 10+RA 10-6-6 M M