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/DR5 TRAIL Trimerization DD FADD DED Autocatalytic activation intitiator procasp-8 or-10 DISC Activation of effector casp-3, -6, -7 APOPTOSIS Substrate cleavage DNA fragmentation APOPTOSIS SIGNALING: 2 pathways Bid tBid Bax/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 NB4NB4-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° Particles 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 leukemia HDACi’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 TRAIL p21 WAF1/CIP1 Multiplex RPA TRAIL ELISA p21 Western Nebbioso et al., Nat Med 2005 Insinga et al., Nat Med 2005

Chromatin Immuno-precipitation (ChIP) assay

RNA interference Short 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 TRAIL si Control 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 EMBO J 18, ) 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 Apoptosis Differentiation 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 growth Of 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 Nebbioso A Scognamiglio C Ambrosino F Manzo G Savarese PP De Rosa MR Conte C Scafoglio A Weisz F 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 Grymwade Division of Medical and Molecular Genetics, GKT School of Medicine, London, UK.