Università degli Studi di Torino Ruolo del fattore trascrizionale FoxO-3 nei progenitori CD34+ Ph positivi nella leucemia mieloide cronica (LMC) Francesca Messa Forkhead family CBP?? Ub Ub CBP Ub Ac Ac Ac UbUb AcAc? Ub Ac Forkhead family of transcription factors (39 members, 19 subgroups) Highly conserved across species (C.elegans, Drosophila, Mammals) Vary degrees of expression in all mammals tissue (FoxO1: adipose tissue;foxo3/6 nervouse tissue; FoxO4:developing brain) Wide range of roles during development (from organogenesis to language acquisition) 1
FoxO regulation Greer, 2005 FoxO target genes and cellular roles Differentiation Metabolism Apoptosis Cell Cycle inhibition Oxidative-stress resistance Modified from Greer, 2005 2
FoxO in cancer development Reagan-Shaw, 2007 Tumor suppressor FoxO inactivation in many cancer type (breast cancer, prostate cancer, glioblastoma) Involved in chromosomal translocations in tumors LOF of FoxOs in conditional KO models results in tumorigenesis in a highly tissue dependent and selective manner FoxO in leukemia Greer, 2005 3
FoxO and the HSC compartment FoxO and the HSC compartment 4
FoxO and Malignant Stem Cells Aim of the study To clarify the role of FoxO3 in Bcr-Abl induced apoptotic arrest and cell growth To investigate the role of FoxO3 and the effects of Imatinib treatment in CML progenitor cells 5
FoxO3 is delocalyzed in the cytoplasm in CML patients CML CP CML CCyR CTRL CML#1 CTRL CML#2 FoxO-3 C N C N C N α-tubulin lamin Abnormal cytoplasmatic localization of FoxO3 in CML patients leads to the abrogation of FoxO3 functions CTRL#1 CTRL#2 CML#1 2.00 100000 d e r p S t!c! - 2 1.75 1.50 1.25 1.00 0.75 0.50 0.25 BCR-ABL copies/ 10 4 ABL copies 2 -ΔΔct SPRED 10000 1000 100 10 1 0,6 0,5 0,4 0,3 0,2 0,1 0.00 0 ES MCyR CCyR MR m m 6
% APOPTOSIS 20 10 0 Re-localization of FoxO-3 reduces proliferation and induces apoptosis in K562 cell line *** ** *** ** K562 CTRL K562 FOXO WT K562 FOXO TM 12500 0 % INHIBITION PROLIFERATION 10000 20 40 7500 60 5000 80 2500 1000 ** *** *** FoxO3 K562 CTRL K562 CTRL+IM K562 FOXO WT K562 FOXO WT+IM K562 FOXO TM K562 FOXO TM+IM Lamin Actin FoxOs signalling is highly conserved across species Drosophila InR Mammals dpten dp110 dakt1 P P P dfoxo PTEN FoxO1 FoxO3 FoxO4 FoxO6 7
To demonstrate that BCR-ABL is responsible for FoxO inactivation The UAS-GAL4 binary system X Tissue specific enhancer TATA GAL4 UUUUUUUUUU AAAAAAAAAA Human Bcr-Abl p210 Tissue specific enhancer TATA GAL4 UUUUUUUUUU AAAAAAAAAA Human Bcr-Abl p210 8
FoxOgmrG4/ is directly uas bcr-abl 1M inactivated by hbcr-abl gmrg4, uas dfoxo/uas lacz gmrg4, uas dfoxo/uas bcr-abl 1M CD47 expression, a marker of proliferant cells, decreased in CD34+ cells from CML patients CD34 progenitor cells Untreated Imatinib 1 µm Imatinib 83% Ctrl 99% ARA-C 80% CML primary cells FoxO3 re-activation induces quiescence of progenitor cells compartment CD47 expression Imatinib 1µM CML CD34+ cells 9
In quiescent LSC FoxO-3 is localyzed within the nucleus Lyn+ CD34+ CD38+ Lyn- CD34+ CD38- There is a LSC pool in which quiescence is maintained by active form of FoxO3 FoxO and LSC IMATINIB Therapeutic resistance? 10
To further investigate if FoxO3 could be responsable for therapeutic resistance in Bcr-Abl transformed LSC The UAS-GAL4 binary system X Tissue specific enhancer TATA GAL4 UUUUUUUUUU AAAAAAAAAA Human Bcr-Abl p210 Tissue specific enhancer TATA GAL4 UUUUUUUUUU AAAAAAAAAA Human Bcr-Abl p210 11
Drosophila Lymph Gland CORTICAL ZONE, containing maturing hemocytes POSTERIOR SIGNALING CENTER, containing small cluster of signaling cells MEDULLARY ZONE, containing prohemocytes Dorsal vessel Pericardial cells, separarting each lobe Primary lobe Secondary lobes Bcr-Abl expression into fly lymph gland induced the formation of melanotic tumors SrpG4/UAS Bcr-Abl 4M 3 rd larval instar CgG4/UAS Bcr-Abl 3M 0-5 days adult fly 12
Next studies To analyze the role of FoxO3 in the quiescence and in the mainteinance of LSC using the Drosophila lymph gland as model system ACKNOWLEDGMENTS San Luigi Hospital University of Turin Daniela Cilloni Giuseppe Saglio Cristina Panuzzo Francesca Arruga Monica Pradotto Emanuela Messa Enrico Bracco Elisabetta Greco Chiara Maffè Sonia Carturan Antonia Rotolo University of Bologna Roberto Bernardoni Seragnoli Institute University of Bologna Michele Baccarani Giovanni Martinelli Ilaria Iacobucci 13