ANOMALIE DELLA TRASDUZIONE DEL SEGNALE DEI FATTORI DI CRESCITA EMOOIETICI NELLE MDS ED EFFETTO MODULATORE DELLA LENALIDOMIDE Elena Spinelli, UF Ematologia Università degli Studi di Firenze
ALTERAZIONI DELLA TRASDUZIONE DEL SEGNALE ATTIVAZIONE di STAT5 JAK 2 Stat5 Stat5 EO, IL-3 G-CSF Stat5 JAK 2 Stat5 ISRE transcription In MDS: Fas in GLY A+ cells EO Stat5 Hoetsloot LH et al. Blood, 1997 Altered BFU-E/CFU-E Stat5 EO Fontenay-Roupie M. et al. B. J. of Hematology, 1999
ERK1/2 ACTIVATION UNREGULATED ACTIVATION OF STAT-5 AND ERK1/2 MAY CONTRIBUTE TO THE HENOTYIC TRANSFORMATION FROM MYEOLODYSLASTIC SYNDROME TO ACUTE LEUKAEMIA IL-3 EO cytochine receptors GM-CSF FL FLT3 M-CSF c-fms SCF c-kit LIGANDS TYR-CHINASE JAK-2 RAS Cellular membrane Stat5 Stat5 B-RAF MEK RAS/ B-RAF SIGN. ATHWAY ERK1/2 GENE EXRESSION Nuclear membrane ROLIFERATION Kolonics A. Haematologia 2001
ERK1/2 ACTIVATION and EO RESONSE -ERK1/2 IS SIGNIFICANTLY LOWER IN CD71+CD45-GA- CELLS FROM NON-RESONDERS TO EO COMARED TO RESONDERS OR CONTROLS AND IT IS A REDICTIVE FACTOR OF RESONSE TO ESA IN LOW/INT-1 MDS Fontenay M. et al. Haematologica, 2010
p38 ACTIVATION p38 ACTIVATION CORRELATES WITH AOTOSIS IN LOW-GRADE MDS Navas T.A. Blood, 2006.
caspase-3 ACTIVATION a) CLASSICAL CASASE-ACTIVATION ATHWAYS b) CASASE-ACTIVATION ATHWAYS IN EARLY-STAGE MDS Over-expression of CD95 and TRAILR1 and their ligands Corey. Nat Rev Cancer. 2007 In RARS the mitochondrial pathway might be stimulated by increased iron deposition, leading to increased production of reactive oxygen species (ROS). In other forms of MDS, ROS might be generated by other mechanisms. AOTOSIS INCREASE IN MDS
AIM OF STUDY In separated CD45+, CD34+ and CD71+CD45- BONE MARROW MDS CELLS BASAL ACTIVATION CYTOKINE STIMULATION (EO 100U/ml 15 G-CSF 100ng/ml 10 ) ERK1/2 STAT-5 p38 CASASE 3 ROLIFERATIVE SIGNALLING DIFFERENTIATION/ AOTOTIC SIGNALLING INTRACELLULAR HOSHO-SECIFIC FLOW CYTOMETRY
EXERIMENTAL ROCEDURES MDS cell subpopulations prepared using a simple density gradient separation of marrow blood sample (Ficoll-Hypaque; density 1.077) G-CSF 100ng/ml 10 or EO 100U/ml 15 stimulation at 37 C Fixation (BD Cytofix buffer) 10 at 37 C ermeabilization with ice cold methanol (BD erm BufferIII) 30 on ice Intracellular phospho-specific Flow Cytometry: Alexa-Fluor488 anti-stat5 (py694) AC anti-human CD34 E anti-human CD71 erc anti-human CD45 Alexa-Fluor488 anti-erk1/2 (pt202/py204) Alexa-Fluor488 anti-p38 (pt180/py182) Alexa-Fluor488 anti-cleaved CASASE-3 (D175) Alexa-Fluor488 Isotype control Analysis on FacsCanto cytometer (BD) with 6 colour beams G.. Nolan, Cell 2004
MFI (MEDIAN FLUORESCENCES INTENSITY) RATIO OF EACH SAMLE S MFI DIVIDED BY THE MFI OF ISOTYE CONTROL Cut-off=1.2
Caratteristiche dei 60 casi di MDS casi WHO 27 RA +8 12 RCMD 7 RAEB-1 5 RAEB-2 1 MDS/ML 1 complex 3 complex 1 del 11q 1 del 7q and 5q +8 t(9;21) 1 5q- 5q- 2 RARS +8 3 CMML Anomalie citogenetiche 2 CMML-2 +8 1 complex ISS 29 LOW casi 16 INT-1 9 INT-2 1 HIGH +6 MIDOLLI di CONTROLLO
BASAL ACTIVATION OF CD45+ CELLS Cluster analysis 45 MDS 6 NORMAL N N N NN N NOT CLUSTERING FOR WHO CLASSIFICATION STAT5 is significantly activated in MDS (p=0.0007 ) Wilcoxon-test RA (p=0.0002) RAEB-1 (p= 0.001) Kruskal-Wallis test Statistical analysis Caspase-3 is significantly activated in MDS (p=0.0027 ) Wilcoxon-test
BASAL ACTIVATION OF CD45+ CELLS CASE #38 ISOTYE CONTROL -ERK 1/2 MFI=1 - STAT 5 CD45+ - p38 CASASE 3 cliv. MFI=5,1 MFI=1 MFI=3,6
BASAL ACTIVATION OF CD34+ CELLS Cluster analysis 54 MDS 6 NORMAL N N N NNN NOT CLUSTERING FOR WHO CLASSIFICATION STAT5 is significantly activated in MDS (p=0.0006 ) Wilcoxon-test Statistical analysis Caspase-3 is significantly activated in MDS (p=0.0024) Wilcoxon-test RA (p=0.0003) RAEB-1 (p= 0.0005) Kruskal-Wallis test p38 is activated primarily in RA
BASAL ACTIVATION OF CD34+ CELLS CASE #38 ISOTYE CONTROL -ERK1/2 -STAT 5 CD34+ -p38 MFI=1 CASASE 3 cliv. MFI=4,6 MFI=2,4 MFI=4
BASAL ACTIVATION OF CD71+CD45- CELLS Cluster analysis 43 MDS 6 NORMAL N N N N NN NOT CLUSTERING FOR WHO CLASSIFICATION Statistical analysis only STAT5 is significantly activated in MDS (p=0.0059 ) Wilcoxon-test RA (p=0.001) RAEB-1 (p= 0.001) Kruskal-Wallis test
CASE #38 BASAL ACTIVATION OF CD71+CD45- CELLS CD71+ CD45- ISOTYE CONTROL -ERK 1/2 MFI=1 - STAT5 -p38 CASASI 3 cliv. MFI=6,2 MFI=1 MFI=2,2
CYTOKINE STIMULATION G-CSF 100ng/ml 10 EOETIN 100U/ml 15 MFI stimulated/ MFI unstimulated Ratio of median fluorescence intensity of stimulated versus unstimolated cell populations Cut-off=1.2
CHANGES IN ROTEIN ACTIVATION FOLLOWING G-CSF STIMULATION G-CSF G-CSF ERK1/2 p38 CD34+ CD45+ CD71+CD45- G-CSF CAS. 3 cliv
CHANGES IN ROTEIN ACTIVATION FOLLOWING G-CSF STIMULATION CD45+ G-CSF STAT-5 IN CD71+CD45- G-CSF STAT-5 IN CD34+
STAT5 ACTIVATION by G-CSF in MDS CD34+ cells Cluster analysis 34 MDS 6 NORMAL N N N N N N MFI stim / 9.9 8.4 MFI unstim 6.1 6.8 4.7 4.4 4 Statistical analysis NO SIGNIFICANT DIFFERENCES (Wilcoxon test: p = 0.49) BETWEEN NORMAL AND MDS
STAT5 ACTIVATION by G-CSF in MDS CD34+ cells CASE #45 ISOTYE CONTROL -STAT 5 CD34+ BASAL ACTIVATION ISOTYE CONTROL -STAT 5 G-CSF STIMULATION MFI stim./ MFI unst.=1,6
CHANGES IN ROTEIN ACTIVATION FOLLOWING EO STIMULATION EO EO EO ERK1/2 p38 CAS3 cliv CD34+ CD45+ CD71+CD45-
CHANGES IN ROTEIN ACTIVATION FOLLOWING EO STIMULATION CD34+ EO STAT-5 IN CD45+ EO STAT-5 IN CD71+CD45-
STAT5 ACTIVATION by EO in MDS CD71+CD45- cells Cluster analysis 36 MDS 6 NORMAL CLUSTER 2 CLUSTER 3 CLUSTER 1 N NNN NN 11 MDS: 1.4<MFI stim/ MFI unstim<1.2 2 23 MDS: MFI stim/ MFI unstim<1.2 3 1 Statistical analysis SIGNIFICANT DIFFERENCES (Wilcoxon test: p = 0.0001) BETWEEN NORMAL AND MDS
STAT-5 ACTIVATION by EO in MDS CD71+CD45- cells CASE #62 CD71+ CD45- ISOTYE CONTROL -STAT 5 BASAL ACTIVATION ISOTYE CONTROL -STAT 5 EO STIMULATION MFI stim. /MFI unstim.=2,5
CORRELAZIONE ROSETTICA RISOSTA IN VIVO/VITRO AD EO RISOSTA AD EO IN VIVO ERITROOIETINA ALHA almeno 40.000 U/sett. per 8 sett. RISOSTA AD EO IN VITRO EO ESAMINAZIONE ARAMETRI EMATOLOGICI dopo 8 sett Hb> 2 g/dl STAT-5 CD71+CD45- AUMENTO ATTIVAZIONE MFI stimol./mfi unstimol.>1.2
CORRELAZIONE ROSETTICA RISOSTA IN VIVO/VITRO AD EO 22/36 MDS sono state trattate con EO in vivo: 7/22 responsivi in vivo 5/22 responsivi in vitro 5/22 responsivi vitro/vivo 15/22 refrattari vitro/vivo CORRELAZIONE FRA RISOSTA AD EO IN VIVO ED IN VITRO 20/22 (90,9%) Spearman =0.62; p=0.002 (**)
Does Lenalidomide treatment restore EO response? EO STAT-5 LENALIDOMIDE IN MDS W/O del 5q- CD71+CD45- MFI stimol./mfi unstimol.>1,2 INCREASED ACTIVATION INTRACELLULAR HOSHO-SECIFIC FLOW CYTOMETRY
= basal STAT5 activation and no increase of -STAT5 by EO stim. = increase of -STAT5 by EO stim. CASE CD34+ CD34+ CD71+ CD71+ NT +EO NT +EO #1 baseline 1 1.3 1.1 1.3 #1 3 month 2 2 1.2 1.4 #1 6 month 1 1 1 1.2 #2 baseline 1.2 1.5 1.1 1.2 #2 3 month 1 1 1.3 1.5 #2 6 month 1.7 1.5 1.3 1.5 #3 baseline 1.1 1.1 1.2 1.4 #3 3 month 1.5 1.9 1.2 1.9 #3 6 month 1.1 1.5 1 1.4
CASE CD34+ CD34+ CD71+ CD71+ NT +EO NT +EO #4 baseline 1 1.4 1.2 1.2 #4 3 month 1.1 1.3 1.1 1.1 #4 6 month 1.2 1.5 1.2 1.2 #5 baseline 1.5 2.1 1.2 1.3 #5 3 month 1.1 1 1 1.3 #6 baseline 1.2 1.3 1.2 2 #6 3 month 1.4 2 1 1.2 #7 baseline 1.1 1 1 1.1 #7 3 month 1.6 1.4 1.3 1.3 #8 baseline 1.3 2.3 1.3 1.3 #8 3 month no cells no cells 1 1.1
CASE CD34+ CD34+ CD71+ CD71+ NT +EO NT +EO #9 baseline 1.1 1.4 1 1 #9 3 month 1 1 1 1.4 #9 6 month no cells no cells 1.1 1.2 #10 baseline 1 1.2 1.1 1.1 #10 3 month no cells no cells 1 1
STAT5 ACTIVATION by EO in MDS CD71+CD45- cells after Lenalidomide treatment CASE #60 BASAL ACTIVATION EO STIMULATION BASELINE MFI= 1.1 MFI= 1.2 BASAL ACTIVATION EO STIMULATION LENALIDOMIDE TREATMENT MFI= 1.3 MFI= 1.5
CONCLUSIONS BASAL ACTIVATION Baseline activation of MAK phospho-proteins, STAT5 and caspase-3 is variable among MDS cases and among distinct cellular subpopulations G-CSF STIMULATION Activates STAT5 only in CD34+ MDS and normal cells EO STIMULATION Induces STAT5 activation in 13/36 CD71+CD45- MDS cells, weaker than in normal cells EO response in vivo strongly correlates with in vitro EO dependent STAT5 activation
RINGRAZIAMENTI UF Ematologia, Firenze rof.ssa V. Santini Dott.ssa F. Buchi Dott. E. Masala Dott.ssa A. Valencia Dott.ssa A. Gozzini Dott. A. Sanna Dott.ssa F. Sassolini Dott.ssa B. Caciagli rof. A. Bosi Laboratorio di citometria Dott. R. Caporale Analisi statistica rof. V. Boddi Cluster Analysis Centro di Riferimento Oncologico (CRO), Aviano Dott. V. Gattei Dott.ssa A. Zucchetto