Induction of tumor-selective death signaling

TNF-related apoptosis-inducing ligand (TRAIL)

Cancer Therapy - Two Options

•Define the basis of the oncogenic event or critical markers of the specific neoplastic disease and derive selective drugs

•Kill the tumor cells

– Activate endogenous defense system that kills tumor cells without affecting normal cells

TRAIL kills cancer cells• TRAIL or activating TRAIL-R bodies kill tumor

cells in culture cell-automomously

• Antitumor activity in xenograft experiments

• NK and NKT cells use TRAIL (receptor) signaling required for tumor surveillance

• TRAIL ko: increased tumor incidence and sensitivity to chemical carcinogenesis

• Stepwise tumorigenesis cell model - TRAIL acts cancer cell-selective

TRAIL does not kill normal cells

Extrinsic Death Signaling Pathway

Intrinsic Death Signaling Pathway

DR4/DR5TRAIL

Trimerization

DDFADD

DEDAutocatalytic

activation intitiator procasp-8

or-10

DISC

Activation of effector

casp-3, -6, -7

APOPTOSIS

Substrate cleavageDNA fragmentation

APOPTOSISSIGNALING:2 pathways

Bid

tBidBax/Bak

Apaf-1

Procasp-9

Apoptosome

Bcl-2/Bcl-XL

Casp-9 activation

Cytochrome c

c-IAP

FLIP

Starting point - Ret(x)inoids are powerful anti-cancer

agents• Ret(x)inoids are proven cancer therapeutic (eg

APL; cutaneous T cell lymphoma) and cancer preventive (eg leukoplakia) agents

• APL is prototype of a “cancer differentiation therapy” - more than 75% of patients are cured by combination of ATRA and CT

Are there any activities of retinoids beyond the induction of differentiation which could

account for their cancer therapeutic & preventive action?

RAR agonists induce NB4 APL cell apoptosis

(RAR selective)

(apoptotic particles <2n)

ATRA induces the death ligand TRAIL, member of the TNF family in

APL cells

NB4 NB4-R2

Multiplex RNAse Mapping

Activation of TRAIL and caspase-8 expression in APL patients’ blasts by ATRA

Altucci et al (2001) Nat Med 7 : 680 Altucci & Gronemeyer (2001) Nat. Rev. Cancer 1 : 181

N°

Par

ticl

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Annexin V (marker of apoptosis)

Induction of Tumor-Selective TRAIL is the Cause of Retinoid-Induced Apoptosis in APL cells

Apoptotic cells

Living cells

TRAIL/R-Fc chimeras sequester TRAIL and make it unavailable for its receptors

Aberrant recruitment of HDACs in myeloid

leukemiaHDACi’s cooperate

with retinoic acid to induce differentiation of retinoid-resistant or insensitive

myeloid leukemia cells

Reactivation of growth control programs by HDACi

• HAT/HDAC balance is altered in cancer (ex.:APL) resulting in silencing of growth control programs

• Epigenetic silencing is not irreversible (like DNA mutation) - the involved enzymes are drugable

• Is it possible to re-activate programs that are silenced during tumorigenesis?

• Start: Define action of HDACi’s

Three HDACi’s induce differentiation, growth arrest and apoptosis with different

kinetics

HDACi’s induce p21 and TRAIL

TRAILp21WAF1/CIP1

Multiplex RPA TRAIL ELISA

p21 Western

Nebbioso et al., Nat Med 2005Insinga et al., Nat Med 2005

ChromatinImmuno-precipitation

(ChIP) assay

RNA interferenc

eShort dsRNA (21-23bp) homologous to a given gene can be used “knock down” expression from this gene by destruction of its mRNA

Permanent RNA interference

Efficient knockdown of TRAIL, p21 or both together

• TRAIL and p21 knockdown blunt apoptosis and G1 arrest, respectively

• TRAIL-mediated apoptosis, p21-induced growth arrest and differentiation along the granulocyte lineage are separable activities of MS275

TRAIL induction is the cause of death by HDACi’s

• TRAIL induction is dose dependent and correlates with the extent apoptosis (in myeloid cell lines and AML patient blasts)

• ALL 3 HDACi’s - MS275, SAHA and VPA - induce TRAIL but not class 2 selective HDAC inhibitors

TRAIL knock-down:

No activation of initiator caspase 8

No tBid-mediated activation of the intrinsic death pathway

TRAILsiControl

MS275

Mechanistic analysis of HDACi induction of TRAIL

RA(IRF1)

Promoter mapping

one GC boxes is an HDACi-RE

ChIP assays

Acetylation of chromatin and recruitment and acetylation of SP1 family members at TRAIL promoter cause induction

Few HDACs reside on the TRAIL promoter

Ex vivo Cultures of AML Patients Blasts

• Differentiation

• Apoptosis

• Dramatically reduced colony formation

HDACi’s target the clonogenic blasts of patients

Induction of TRAIL in AML patients’ blasts

• RPA

• ELISA

• Immunohisto-chemistry

• Blasts of more than 50 patients tested

• response >98%

HDACi-Induced Apoptosis is Leukemia Cell Specific

• In all AML blasts TRAIL gets induced and blasts die (>65 cases) independent of karyotype, immunophenotype, FAB status.

• In all cases blast apoptosis correlated with TRAIL protein induction

• CD34+ cells in culture (>10) are heterogenous for TRAIL expression/induction but cells expressing TRAIL do not die

Starting observations(Benoit et al. 1999 EMBO J 18, 7011-7018)

• NB4 APL cells do not respond to pure rexinoids• Combining rexinoids with PKA agonists leads to NB4 cell

differentiation• Even ATRA-resistant NB4 cell can be differentiated with

rexinoids in presence of elevated cAMP levels

Rexinoids are powerful differentiation and apoptosis-inducing agents when the cAMP level is increased

• ALL AML cells respond despite their vast heterogeneity in immunophenotype, karyotype, FAB status

• Also patients blasts enter into apoptosis

• Possible novel anti-AML therapy option

• Mechanism of x-talk and apoptosis understood (took 6 years)

AML patient blasts ex vivo cultures

• Several established AML cell lines are responsive

• All (>50) tested blast cultures from AML patients responded ex vivo

ApoptosisDifferentiation

Undifferentiated blasts(ATRA insensitive)

Induction of TRAIL and DR5 expression in AML blasts

Immunohistochemistry

DR5 TRAIL

Expression only in differentiated blasts

Western DR5Multiplex-RPA

DR5

TRAIL

Towards Therapy: PDEis

… and inhibit clonogenic growthOf AML patients’ blasts

LG1069 and PDEi’s induce differentiation of PLB985 cells(CD11c and NBT staining) …

DR5

TRAIL

Altucci et al. Cancer Res 2005

Seconda Università degli Studi di Napoli

Dipartimento di Patologia generale

A NebbiosoA ScognamiglioC AmbrosinoF ManzoG SavaresePP De RosaMR ConteC ScafoglioA WeiszF Bresciani

External Collaborators• Hinrich Gronemeyer,

IGBMC, SXB, FRANCE

• EM Schiavone & F Ferrara Hospital Cardarelli, Division of Hematology, Naples, Italy

• Angel de Lera Dept of Organic Chemistry, University of Vigo, Vigo, Spain

• Hugues de The 4CNRS UMR 7151, Paris, France

• Arthur Zelent (PLZF-RAR)Institute of Cancer Research, London, UK

• David GrymwadeDivision of Medical and Molecular Genetics, GKT School of Medicine, London, UK.