Corso Nazionale di AggiornamentoIn Ematologia Clinica

Milano 28-29 Maggio 2007

Il rimodellamento cromatinico:agenti inibitori dell’istone deacetilasi e demetilanti

PG Pelicci

Upstream signals:

Enzyme-catalyzed (HDAC, HAT, HMT, DNMT)chromatin modifications(permanent or transient)

Regulation of transcription, replication,

Genome stability

Chromatin regulates all genome functions

Chromatin alterations

in cancer cells

• aberrant chromatin recruitment of HDACs byoncogenic fusion proteins (PML-RAR, PLZF-RAR,

AML1-ETO, TEL-AML1, Bcl6)

• missense mutations, rearrangements orinactivation of CBP or p300 (HATs) byoncogenic viral proteins

• DNA methylation at promoters of tumorsuppressors

• Over-expression or mutations of HMTs

Consequences: Abnormal transcriptional regulation oftarget genes that are relevant to the transformation process.

Chromatin “enzymes”

might represent

targets for the development of novel

anti-cancer drugs

Ongoing: HDAC-inhibitorsDemethylating agents

Pipeline: HMT-inhibitors/agonistsHist. demethylase inhib./agonists

Relevant features of HDAC-inhibitors

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+ HDACi

Breast cr.

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Lung cr.

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Colon cr.

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Broad anti-tumoractivity

Poor systemictoxicity

Adaptedfrom Blood,2002

Phase I-II clinical trialswith HDAC inhibitors:• modest systemic toxicity• clinical efficacy

Minucci S and Pelicci PG (2006)

Summary of the main clinical studies

using histone deacetylase inhibitors

Understand the molecular mechanismsunderlying the anti-tumor activities of HDACi

PatientStratification

Better drugs

• PML-RAR binds DNA and recruits

chromatin enzymes to target promoters

(HDAC, HMT, DNMT)

• Complex formation is critical

to all PML-RAR biological activities

• HDACs, HMTs, DNMTs all contribute

to PML-RAR functions

• PML-RAR induces heterochromatin

at target promoters

and transcriptional silencing

HDAC

Dnmt

PML

RAR

R A R E

SUVR

MBDs

HP1HP1

Dnmt

HDAC

HMT

MBDs

N-CoR

HDAC

Dnmt

PML

RAR

R A R E

MBDs

The APL model:

Epigenetic drugs(HDAC-inhibitors [VPA] anddemethylating agents [DAC])

enhance survival of APL mice

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Alive

Mic

e

10 15 20 25 30 35 40 45 50

Days after leukemia injection

PBS

DAC

VPA

VPA+DAC

p = 0,0045

p < 0,0045

p < 0,0001

Effects of HDAC-inhibitors (VPA):Placebo

VPA

TUNELH-E

2. Massive apoptosis only in the APL mice

1. Histone acetylation in both APL and Healthy mice

Ctrl

acetyl-H3

Healthy APL

H3

VPA

TSA

VPA

Ctrl

TSA

BM Spleen

Placebo

VPA

TUNEL H-E

Tumour-selective action of HDACi in acute promyelocytic leukaemia.

HDAC-inhibitors do not alleviatethe inhibitory effect of PML-RAR on transcription

and differentiation

Normalized RAR-expression

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1

0

Basal

+ PML-RAR

+VPA

+RA

+ Deaza

% Differentiation

80

20

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Ctrl + PML-RAR

+VPA

+RA

+ Deaza40

60

Cyt- + - + ++ +

CBP/

p300

PML

p53

PML-RAR

HDAC

Alternative mechanisms: p53 activation:

p53 acetylation (and activity)is inhibited by PML-RAR

HDACi do not target p53 in APLs

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m.

Su

rviv

al

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VPA

p<.0001

VPAPlacebo

Mechanisms of HDACi-induced apoptosis in APLs

IFOM-IEO campus

Milan, Italy

High-order chromatin changesinduced by PML-RAR

Ivan Dellino

Martin FalkMatteo CesaroniLucilla Luzi

Gabriele Bucci Stanislav Kozubek

Emily Lukasova

Saverio Minucci

IEO,Milan

Institute of BiophysicsAcademy of Science, Brno

U937 cells:

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U937 - Mt

U937- PR

U937-A1E

U937-PZR

CD34+

t(15;17)

t(8;21)

Inv(16)

expression

Cell Cell lineslines

Patients Patients (pools of 10 (pools of 10 individuals)individuals)

{{

+5

+4

+3

+2

1

-2

-3

-4

-5

U937 - Mt

U937- PR

U937-A1E

U937-PZR

CD34+

t(15;17)

t(8;21)

Inv(16)

expression

Cell Cell lineslines

Patients Patients (pools of 10 (pools of 10 individuals)individuals)

{{

Patients:

Analysis of PML/RAR transcriptional targets

using gene chip technology

- 1206 gene down-regulated by PML-RAR- Validation: approx. 90%- Only a minority of regulated genes contains bona fide RAREs in their promoter region

#1-2 #3,4 #5 #6 #9-10#7,8 #11 #13#12 #14 #16,17#15

Chr. 19

Gene Clusters

Chr. 12

#1 #2 #7#3-6 #8#1 #2 #7#3-6 #8

Gene Clusters

#1

Chr. 13

Gene Clusters

Clusters of PML-RAR - down-regulated genes(Chromosomes 19, 12 and 13)

#1-2 #3,4 #5 #6 #9-10#7,8 #11 #13#12 #14 #16,17#15

Chr. 19

Gene Clusters

#2#1 #3

Gene Clusters

Chr. 19p13.3 Clusters #1-3 (700-3000 Kb)

Physical map of Chrom. 19 Cluster #1 of PML-RAR - down-regulated genes

Chr.19 Cluster #1 (535 kb; 21 genes)

PML/RAR binds to TSSs of all regulated Cluster #1 genes

RARE clusters coincide with clusters of Alu repeat sequences

Each DR2-RARE sequence is located within one Alu repeat elements

DR2-RARE

Cluster #1 genes are flanked by clusters of RAREs

Alu-RARE

Clusters

Alu-RARE Clusters

#1-2 #3,4 #5 #6 #9-10#7,8 #11 #13#12 #14 #16,17#15

Chr. 19Gene

Clusters

Chr. 12

#1 #2 #7#3-6 #8#1 #2 #7#3-6 #8

Gene Clusters

Alu-RARE

Clusters

#1

Chr. 13Gene

Clusters

Alu-RARE Clusters

Alu-RARE Clusters

Alu-RAREs are distributed as clusters that are located in the proximity of clusters of PML-RAR regulated genes

PML-RAR binds Alu-RAREs

PML-RAR binding toThe Ya5 Alu-subfamily

PML-RAR binding toIndividual Alu-RAREswithin Chr.19 Cluster #1

1. 50% of PML-RAR - down-regulated genes are clustered in the genome

Regulated genes

Conclusions (1):

3. Alu-RAREs bind PML/RAR

2. Clustered genes:

- do not contain RAREs in their proximal promoter

Alu-RARE cluster Alu-RARE cluster

- are flanked by clustered Alu-RAREs

- bind PML/RAR at their TSS

• How is PML-RAR recruited to TSS?• Is PML-RAR first recruited to Alu-RAREs, and, from there, to TSSs (RAR interacts with TFIIH)• Does PML/RAR expression triggers juxtaposition of Alu-RAREs and the TSS regionsof regulated genes?

ELA2 gene - A1 Alu-RARE (370 Kb)

5’ HA-1 gene - A1 Alu-RARE (150 kb)

A1/A2 Alu-RARE (130 Kb)

3’ HA-1 gene - A1 Alu-RARE (145 kb)

ELA2 gene - A2 Alu-RARE (500Kb)

5’ HA-1 gene - A2 Alu-RARE (280 kb)

3’ HA-1 gene - A2 Alu-RARE (275 kb)

ELA2 gene - 5’ H1 gene (210 Kb)

3C-capturedfragments

3C-negativefragments

Analysis of long-range intrachromosomal interactionsby the chromosome conformation capture (3C) technology

Chr. 19

Chr. 13

Chr. 12

#1 #2 #7#3-6 #8

#1

#1-2 #3,4 #5 #6 #9-10#7,8 #11 #13#12 #14 #16,17#15

Gene Clusters

Gene Clusters

Gene Clusters

3D-FISH DNA probes: 1-2 Mb Probe BProbe A

PML-RAR induces shortening (up to 25%) of the distance between Two DNA probes flanking Regulated Gene Alu-RARE Clusters

- + PR - + PR + PR

Control + PML-RAR + RA

Chr. 19 Chr. 12Chr. 12

Expression of PML-RAR induces:

• changes in higher-order chromatin structure (juxtaposition of Alu-RAREs and the TSS regions of regulated genes)

• transcriptional repression of neighbouring clustered genes

Alu cluster

Alu cluster

Conclusions (2):

TSA revert the effectsOf PML-RAR on high order-chromatin structure

Chr. 19 Chr. 12Chr. 12

PML-RAR + RA + TSA

TSA induces acetylation at TSSs of clustered genesAnd increases Pol II recruitment

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HA-1 Alu 1135 STK11 ATP5D MIDN Int K7

Polymerase II

H3 AcK14

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AZU1 HA-1 Alu K0 STK11 ATP5D MIDN CIRBP Int K7 RARbeta1135

Understand the molecular mechanismsunderlying the anti-tumor activities of HDACi

PatientStratification

Better drugs

Class A:“Hypo-acetylated”

Breast cancer

Class B:“Hyperacetylated”

Breast cancer

“Hyper-acetylated”

Breast normaltissue

N

T

T

N

A fraction of Breast Cancer Samples show “global hypo-acetylation” of chromatin

class A:

“hypoacetylated” tumors

class B:

“hyperacetylated tumors”

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VPA TSAC

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“Hypoacetylated” tumors

are sensitive to HDAC-inhibitors

MSCV

MTA1

HDAC2

HDAC1

H4-Ac

1 week

TSA

MSCV MTA1 HDAC2 HDAC1TSA: - + + +- + - -

Overexpression of HDACs or MTA1 in the resistant cellsinduces histone-hypoacetylation and sensitivityto the apoptogenic effect of HDACi

Understand the molecular mechanismsunderlying the anti-tumor activities of HDACi

PatientStratification

Better drugs

Different in vitro potency of different HDACi

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DAC-60 1 μM

DAC-60 3 μM

% sub G1 population in K562 celll line

SAHA 3 μM

Hours

%S

ub

G1

po

pu

lati

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% Sub G1 population in HCT-116 cell line

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DAC-60 1μM

DAC-60 3μM

SAHA 3μM

Hours

% S

ub

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lati

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Company confidential

In vivo efficacy in papilloma induced mouse model

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In vivo efficay in papilloma induced mouse model

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VPA

Dac-60

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men

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VehicleVPA DAC-60

Company confidential

Different in vivo potency of different HDACi