Epigenetic Regulation of Hematological Malignancies

EPIGENETIC REGULATION OF HEMATOLOGICAL MALIGNANCIES

Elke De Bruyne, PhD Department of Hematology and Immunology-Myeloma Center Brussels, Vrije Universiteit Brussel

No conflicts of intrest to disclose

14-2-2013 Pag. 1 EPIGENOMICS

. Both genetic and epigenetic alterations play a fundamental role in tumor pathogenesis

Epigenetic changes = all heritable modifications of the genome which are not caused by changes in the primary DNA sequence and that result in phenotypic changes

Pag. EPIGENOMICS

Post-translational histone modifications

DNA methylation

Pag. Arrowsmith C et al, Nature Reviews Drug Discovery, 2012, vol 11. HISTONE POST-TRANSLATIONAL MODIFICATIONS

De Bruyne E et al, Springer Science, 2013. Pag. ALTERNATIVE TRANSCRIPTIONAL OUTCOMES

. DNA methylation and histone deacetylation: repressive

. Histone acetylation: active

. Histone methylation: both depending on site and degree of methylation - H3K27me3, H3K9 me2/3, H4K20me3 are repressive marks - H3K4me2/3, H3K36me2/3 and H3K79me are active marks

Co-operate intensively to organize the chromatin

structure and control gene expression

Pag. CHROMATINE MODIFYING PROTEINS

Acetylases (HATs) Deacetylases (HDACs) Bromodomain Methylases (DNMTs, HMTs) Demethylases (HDMs) Chromodomain Kinases Phosphatases PHD finger WD40 repeat

Pag. Gardner K et al, Journal of Molecular biology, 2011, Vol 409. EPIGENETIC LANDSCAPE IS DISTURBED IN CANCER CELLS:

Global hypomethylation: Genomic instability

Site specific hypermethylation: Loss-of-function of genes

Pag. McCabe M et al, Clinical Cancer Research 2009, Vol 15. RECURRENT EPIGENETIC SILENCED GENES

. Cell cycle progression (e.g., Rb, p14, p15, p16, p57, p73, …) . Survival (e.g., caspase-8, DAPK1, BIM, TMS-1, …) . Growth factor signaling (e.g., SOCS1, SOCS3, CRBP1, RAR- beta2,…) . DNA damage/ repair (e.g., Fancomi anemia-BRCA pathway members, GSTπ, hMLH1, MGMT …) . Metastatic potential (e.g., TIMP3, E-cadherin, CD9, …) . Angiogenesis (e.g., VHL, BNIP3, BNIP3L, IGFBP3, EGLN2, …)

…………

adverse prognosis

Pag. CAUSE OF GLOBAL SHIFTS IN CHROMATINE MODIFICATION, STRUCTURE AND FUNCTION?

. Mutations of chromatin modifying proteins

Malignancy writers erasers readers

AML Dnmt3a, EZH2, MLL, UTX, TET ASXL1, PHF6 NSD family, CBP, p300, Jak2

MDS Dnmt3a, EZH2, MLL, UTX, TET ASXL1 Jak2

CML EZH2, Jak2 UTX, TET ASXL1 ALL Dnmt3a, EZH2, MLL, UTX, TET ASXL, PHF6 CBP, Jak2 MM MMSET UTX Lymphoma Dnmt3a, EZH2, MLL, TET CBP, p300, Jak2

Pag. EPIGENETIC REPROGRAMMING

. Epigenetic modifications are reversible

. Plethora of epigenetic-modulating agents have been designed to target the epigenetic modifiying proteins

. Most advanced in terms of clinical applications: - DNA methyltransfersases inhibitors (DNMTi) - Histone deacetylase inhibitors (HDACi) mostly in the field of hematological malignancies

Pag. HYPOMETHYLATING CYTOSINE ANALOGUES

. Azacytidine (Vidaza) and Decitabine (Dacogen) . Dual mode of action: - low dose: hypomethylation (DNMTi) - high dose: cytotoxicity (DNA damage, cell cyle arrest and apoptosis) . FDA approved in high-risk MDS (standard of care) RD up to 18 months

Fenaux P et al, Lancet Oncology, 2009, Vol 10. Pag. HYPOMETHYLATING CYTOSINE ANALOGUES

. Promising activity in elderly AML patients: trend for improved overall survival

. Remaining hematological malignancies: only modest single agent activity

. Main side-effects: myelosuppression, injection site side-effects, nausea, vomiting, diarrhea

Pag. HDACi

Class Compound Study phase

Hydroxamic acid TSA Voronistat (SAHA) FDA approved Panobinostat (LBH589) III Belinostat (PXD101) II LAQ824 I IFT2357 II Short-chain fatty Sodium butyrate acids Valproic acid I AN-9 I II

Benzamidines Entinostat (MS275) II Mocetinostat II (MGCD0103)

Cyclic peptides Romidepsin (FK228) FDA approved Aplicidin

Pag. HDACi

. Potent preclinical anti-tumor activity and remarkable tumor specificity . Mechanisms of action are pleiotropic: - cell cycle arrest - apoptosis - immuno modulation - angiostatic . Clinical efficacy for CTCL, PTCL, HL, AML . MM, CLL, CML, ALL, MDS: response unpredictable and rather poor . Main side-effects: fatigue, diarrhea, nausea, anemia and thrombocytopenia

Pag. EPIGENETIC MODULATING DRUGS

. Single agent activity is only modest . Therapy is not curative and resistance develops . Ascribed to: 1. Response depends on the compound, concentration and time of exposure and cellular context → most relevant target(s) responsible for the anti-cancer activity remain undefined 2. No validated biomarkers available to identify patients whom would benefit from epigenetic targeted treatment or predict for clinical response

Pag.

NEW AVENUES FOR (PRE)CLINICAL RESEARCH

. Test new dosing schedules and compare routes of administration (SGI-110) . Design (new) drug combinations: with conventional and/or other epigenetic agents . Unravel underlying mechanisms, especially in vivo . Develop new agents with higher specificity (e.g. HTMi, HDMi, specific HDACi, ….) . Identify patients who would benefit from epigenetic targeted treatment (= personalized treatment)

Pag. MULTIPLE MYELOMA (MM)= MODEL FOR EPIGENETIC DYSREGULATION

– Different 5TMM models

moderate growth (12w) 5T2 osteolytic lesions rapid growth (3-4w) 5T33 no osteolytic lesions In vitro cell line (5T33vt)

– Similar characteristics as human disease

Pag. JNJ-26481585 (JNJ-85) IN 5TMM MODEL (Johnson&Johnson)

. Novel, second generation hydroxamate based pan- HDACi, with significantly improved pharmacodynamic parameters.

. Potent anti-tumor activity in preclinical solid and hematological tumor models.

. Currently in phase II clinical trial (CTCL).

Pag. EFFECT ON HISTONE ACETYLATION AND PROLIFERATION

Histone acetylation Proliferation

Deleu S et al, Leukemia, 2009, Vol 23.

Pag. EFFECT ON CELL CYCLE

* *

5T33MMvt STR-10

HDACi in MM 14/02/2013 | pag. 20

Pag. EFFECT ON APOPTOSIS

Pag. JNJ-85 IN THE 5TMM MODEL – PROPHYLATIC AND THERAPEUTIC SETTING

5T33MM Week 4 Day 0

•Vehicle •JNJ-85 (20mg/kg, Tumorload every other day,s.c.) Bone disease

5T2MM

Day 0 Week 8 Week 12 M-protein

Pag. IN VIVO ANTI-MM ACTIVITY (I)

BM plasmacytosis Serum M-protein 5T33MM 80 * * 4 60 * * 3

40 2

20 1 Paraprotein (g/dl) Paraprotein 0 0 % MM cells in the bonecells %MM marrow Naive Vehicle 20mg/kg Naive Vehicle 20mg/kg *P<0.0001 *P<0.0001 JNJ-26481585 JNJ-26481585

5T2MM 2,5 80 2 60 * * * * 1,5

40 1

20 Paraprotein (g/dl)Paraprotein

% MM cells in the BM the in cells MM % 0,5

0 0 Naive Vehicle 20mg/kg Naive Vehicle 20mg/kg *p<0,001 JNJ-26481585 *p<0,001 JNJ-26481585

Pag. IN VIVO ANTI-MM ACTIVITY (II)

Pag. JNJ-85 IN COMBINATION WITH BORTEZOMIB (Bz)

Week 0 Week 8 Week 12

M-protein i.v. injection of Sacrifice 5T2MM cells Treatment of the mice (s.c.) • Vehicle • Bortezomib (0.8mg/kg, twice weekly) • Bortezomib (0.6mg/kg, twice weekly) • JNJ-85 (1.25mg/kg, every other day,s.c.) • Combo (low)

Deleu S et al, Cancer Reserach, 2009, Vol 69.

Pag. IN VIVO ANTI-MM ACTIVITY (I)

Sarah Deleu, Fig.1 A BMTumor plasmacytosisload in BM B Serum MParaprotein-protein

60 2,5 * * * 50 * * * 2,0 * * *

40 (g/dl)

in the BM the in 1,5 30

cells 1,0

Paraprotein %MM %MM 10 0,5

Bz 5T2MM + 5T2MM +

Bz Bz

0,0 Bz

5T2MM +

JNJ JNJ

Naive

Vehicle

5T2MM + + JNJ

5T2MM + 5T2MM +

+ JNJ

5T2MM +

5T2MM + 5T2MM +

(0.6mg/kg)

(0.8mg/kg) (0.6mg/kg)

- -

85 85

C Microvessel density (MVD)

* 30 * * * *

25 staining 20

Pag.

CD31 bloodvessels

by 15 of

Nr 10 counted 5

Bz Bz

5T2MM +

0 Naive Bz

5T2MM +

JNJ

Vehicle

+ JNJ

5T2MM + 5T2MM + 5T2MM +

(0.6mg/kg)

(0.8mg/kg) (0.6mg/kg)

85 Sarah Deleu, Fig.2

A B Bone Volume

* 6 *

5 * *

1. 2. 3. 4 *

Trabecular *

3 Volume/ Volume/

2 Bone

4.IN VIVO5. ANTI6. -MM ACTIVITY % 1 (III)

Bz Bz Bz

5T2MM+

Naive

Vehicle

+ + JNJ

JNJ 5T2MM+

5T2MM+ 5T2MM+

(0.6mg/kg)

(0.8mg/kg) (0.6mg/kg)

- Sarah Deleu, Fig.2 85 C TrabecularTrabecular number number Bone Volume A B Bone Volume 1,2 * * * 6 * * * 5 * *

* nr 0,8 *

1. 2. 3. 4 * Trabecular

* nr

Trabecular

0,4 Volume/

Bone

Trabecular

% Bone Bone Volume/ % Trabecular 4. 5. 6. % 1

0 0 5T2MM +

Bz Bz

Bz Bz Bz

5T2MM+ Naive

5T2MM+

Vehicle

(0.6mg/kg) Bz + JNJ

Naive Naive

Naive

5T2MM + (0.8mg/kg)

Vehicle

CD31 staining5T2MM

JNJ Bz

+ + JNJ

JNJ 5T2MM+

JNJ + JNJ 5T2MM +

5T2MM+ Bz

5T2MM+ 5T2MM+

5T2MM + 5T2MM +

(0.6mg/kg)

Bz 5T2MM +

(0.8mg/kg) (0.6mg/kg)

Vehicle

5T2MM +

JNJ

5T2MM +

(0.8mg/kg)

(0.6mg/kg) Bz Bz

- + JNJ

Bz Bz

85 85

+ JNJ85 1.25

C Trabecular number

1,2 *

* *

* nr 0,8 *

Pag. Trabecular

0,4

Bz Bz

5T2MM+

Naive

Vehicle

JNJ + JNJ

5T2MM+

5T2MM+ 5T2MM+

(0.6mg/kg)

(0.8mg/kg) (0.6mg/kg)

- +85 JNJ85 1.25 IN VIVO ANTI-MM ACTIVITY (III)

Sarah Deleu, Fig.3 Sarah Deleu, Fig.3 Sarah Deleu, Fig.3 Nr Osteoclasts A NrNr osteoclasts Osteoblastsper mm B Nr osteoblasts per mm A Nr osteoclasts per mm B Nr osteoblasts per mm A Nr osteoclasts per mm B Nr osteoblasts per mm 5 * * * 5 * 30 5 * * * * * * * 30 * * 304 * * * * * * * * * 4 * 25 * /mm * 4 * * * 25

/mm * * 25 /mm

/mm *

3 /mm * 20 3 /mm * 3 20 20

osteoclasts 15

osteoclasts 2 15

osteoclasts 15 osteoblasts

2 2 Nr

osteoblasts

Nr osteoblasts Nr 10 10 Nr

10 Nr Nr 1 1 1 5 5 5 CD31 staining 0 0

Bz Bz 5T2MM+

0 Bz 0 Bz 0

Bz Bz 5T2MM +

5T2MM+ 5T2MM+

Bz 0

JNJ

Naive Naive

5T2MM+ 5T2MM+

JNJ

Bz Bz 5T2MM+

Naive Naive

Bz Bz 5T2MM+

Vehicle Vehicle

JNJ

Naive Naive

Vehicle Vehicle

5T2MM+ 5T2MM+ + JNJ

Bz 5T2MM +

5T2MM+

Vehicle Vehicle

5T2MM+ 5T2MM+ + JNJ

5T2MM+

JNJ

(0.6mg/kg)

+ JNJ +

5T2MM + 5T2MM + 5T2MM + 5T2MM +

JNJ

(0.8mg/kg) (0.6mg/kg)

(0.6mg/kg)

JNJ

5T2MM+ 5T2MM+ + JNJ

(0.8mg/kg) (0.6mg/kg)

5T2MM+

(0.6mg/kg)

(0.8mg/kg) (0.6mg/kg)

5T2MM+ 5T2MM+ + JNJ

5T2MM+

(0.6mg/kg)

+ JNJ +

5T2MM + 5T2MM + 5T2MM +

(0.8mg/kg) (0.6mg/kg)

5T2MM + 5T2MM +

(0.6mg/kg)

(0.8mg/kg) (0.6mg/kg)

(0.6mg/kg)

(0.8mg/kg)

(0.6mg/kg)

Pag. CONCLUSION- JNJ-85 IN MM

. Potent anti-MM drug affecting survival at low nM range . Selectivity towards malignant cells . Overcomes the protective signals of BM niche . Decreases MM associated bone disease . Bz + low dose JNJ-85: ↑ anabolic bone properties of Bz

. Promising novel anti-MM agent . Demonstrate importance for further clinical evaluation in MM

Pag. PANOBINOSTAT (LBH589) AND THE IGF-1 RECEPTOR INHIBITOR PPP

. IGF-1: one of 2 major growth and survival factors for MM cells . IGF-1 receptor (IGF-1R) is highly expressed in MM cells and its expression is correlated with poor prognosis

Sprynski et al, Blood, 2009, Vol 113.

Pag. IGF-1R TARGETING: PICROPODOPHYLLIN (PPP)

. PPP is a potent inhibitor of IGF-1R signaling

HOWEVER: mice eventually relapsed

Strömberg T et al, Blood, 2006, 107. Menu E et al, Blood, 2006, 107. Pag. Menu E et al, Int J Cancer, 2007, 121. EFFECT ON VIABILITY

CI: Combination index calculated according to Chou-Talalay

CI > 1 Antagonism CI = 1 additive effect CI < 1 Synergy

Pag. Lemaire et al, Clin Cancer Res, 2012, Vol 18. EFFECT ON CELL CYCLE AND APOPTOSIS

RPMI8226

Pag. LBH589 + PPP IN THE 5T33MM MODEL – PROPHYLATIC SETTING

n weeks . naive (n=10) . vehicle (n=10) Day 0: . 1.5 mg PPP/5 g FOOD(n=10) Inoculation of mice Survival . 2.5 mg/kg LBH589 (i.p. daily, n=10) . Combo (n=10)

Pag. CANDIDATE GENE APPROACH: BIM

. IGF-1 down-regulates BIM expression in MM

Karpas 707 OPM-2 24 h 48 h - + IGF-1 - + - + IGF-1 BimEL BimEL

BimL BimS BimL Actin BimS

Actin

De Bruyne E et al, Blood, 2010, Vol 115.

Pag. BIM

. Member of the BH3-only group of the Bcl-2 protein family . Bim is a sensors of stress: apoptosis

. Bim is a potent tumor supressor . In B-cell lymphomas and AML: silencing by promoter (hyper)methylation

Pag. EPIGENETIC REGULATION OF BIM IN MM

Karpas707 - + - + LBH589 B - - + + DAC C A * - + - + LBH589 - - + + DAC BimEL 3 * * Acetyl-H3 2,5 * Acetyl-H4 2 1,5 BimL Actin

mRNA 1 BimS 0,5 Actin relative amount of relative Bim 0 control LBH589 DAC Combo D - + - + LBH589 E - + - + LBH589 - - + + DAC - - + + DAC

*p<0.05 OPM-2 BimEL Acetyl-H3 Acetyl-H4

Actin BimL BimS

Actin

Pag. EFFECT OF IGF-1 ON HISTONE MODIFICATIONS OF BIM PROMOTER

Chromatin immunoprecipitation (ChIP) analysis

Karpas707 OPM-2

MZS IGF-1 MZS IGF-1 1,4 * 2 1,2 * 1 1,5 0,8 * * 1 0,6 0,4 0,5

0,2 relative fold relative enrichment

0 fold enrichmentrelative 0 3M H3K4 2M H3K9 Ac H3K9 3M H3K4 2M H3K9 Ac H3K9

*p<0.05 Condensed chromatin

Pag. CONCLUSION

. PPP and LBH589 synergistically induced MM apoptosis

. PPP and LBH589 significantly prolonged overall survival compared to single agents.

. IGF-1 down-regulates Bim in MM cells by:

1. ↓ Ac H3K9 and 3M H3K4 and/ or 2. ↑ 2M H3K9 at the BIM promoter

Pag. CURRENT WORK AND FUTURE (I)

. Anti-MM effects of decitabine (DAC) alone and in combination with JNJ-85: – DAC induces caspase-mediated apoptosis – Depending on the cell line, a G1- or a G2/M-phase arrest is induced – DAC slows down MM progression and increases survival probability in 5T33MM model – JNJ-85 augments DAC-mediated anti-myeloma activity in vitro and in vivo . Unravel molecular mechanisms underlying these anti-MM effects

Poster P05 by Maes K et al Pag.

SUMMARY

. Although epigenetic therapy is clinically usefull in MDS (DNMTi) and CTCL and PTCL (HDACi) many questions remain . Therapy is not curative and resistance develops commonly . Combinations with conventional and/or epigenetic agents are under intense (pre)-clinical investigation . Identify patients who would benefit from epigenetic targeted treatment (= personalized treatment) (Moreaux J et al, Mol Cancer Ther, 2012, Vol 11. DM score to predict the efficacy of DAC in MM)

Pag. Myeloma Center Brussels Prof. Dr. Karin Vanderkerken Prof. Dr. Ivan Van Riet Prof. Dr. Rik Schots Prof. Dr. Els Van Valckenborgh Prof. Dr. Eline Menu Lemaire Miguel Ken Maes Haneen Nur Liesbeth Bieghs Susanne Lub Kim De Veirman Nathan De Beule Mérèdis Favreau Jinheng Wang Carine Seynaeve Collaborations Andrea Desmedt - Prof. H. Jernberg-Wiklund, Upssala University, Marie Joos De Ter Beerst Sweden. Nicole Arras - Prof. JM. Délaissé and Dr. T. Andersen, Southern

Denmark University, Denmark.

- Prof. B. Klein and Dr J. Moreaux , Inserm, Montpellier,

France.

- Prof P Croucher, Garvan Institute, Sydney, Austrelia.

Pag.