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.

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