induction of tumor-selective death signaling tnf-related apoptosis-inducing ligand (trail)
TRANSCRIPT
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
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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.