induction of tumor-selective death signaling tnf-related apoptosis-inducing ligand (trail)
TRANSCRIPT
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Induction of tumor-selective death signaling
TNF-related apoptosis-inducing ligand (TRAIL)
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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
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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
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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
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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?
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RAR agonists induce NB4 APL cell apoptosis
(RAR selective)
(apoptotic particles <2n)
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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
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N°
Par
ticl
es
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
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Aberrant recruitment of HDACs in myeloid
leukemiaHDACi’s cooperate
with retinoic acid to induce differentiation of retinoid-resistant or insensitive
myeloid leukemia cells
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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
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Three HDACi’s induce differentiation, growth arrest and apoptosis with different
kinetics
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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
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ChromatinImmuno-precipitation
(ChIP) assay
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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
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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
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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
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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
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Few HDACs reside on the TRAIL promoter
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Ex vivo Cultures of AML Patients Blasts
• Differentiation
• Apoptosis
• Dramatically reduced colony formation
HDACi’s target the clonogenic blasts of patients
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Induction of TRAIL in AML patients’ blasts
• RPA
• ELISA
• Immunohisto-chemistry
• Blasts of more than 50 patients tested
• response >98%
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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
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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)
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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)
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Induction of TRAIL and DR5 expression in AML blasts
Immunohistochemistry
DR5 TRAIL
Expression only in differentiated blasts
Western DR5Multiplex-RPA
DR5
TRAIL
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Towards Therapy: PDEis
… and inhibit clonogenic growthOf AML patients’ blasts
LG1069 and PDEi’s induce differentiation of PLB985 cells(CD11c and NBT staining) …
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DR5
TRAIL
Altucci et al. Cancer Res 2005
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Seconda Università degli Studi di Napoli
Dipartimento di Patologia generale
A NebbiosoA ScognamiglioC AmbrosinoF ManzoG SavaresePP De RosaMR ConteC ScafoglioA WeiszF Bresciani
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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.