Nuove mutazioni nella leucemia mielomonoci/ca cronica
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1 Nuove mutazioni nella leucemia mielomonoci/ca cronica Firenze 31 marzo 2011 DANIEL A CILLONI UNIVERSITA DEGLI STUDI DI TORINO DIPARTIMENTO DI SCIENZE CLINICHE E BIOLOGICHE
2 c Ros1 Human Human ortholog of Drosophila of Drosophila sevenless located on chromosome 6, locus spanning over 130 Kbp Expressed in Expressed in fetal /ssues Expressed in a Expressed in a variety of tumors variety of tumors (especially NSCLC and glioblastoma) It encodes for an It encodes for an orphan Receptor Tyrosine kinase orphan Receptor Tyrosine kinase structurallystructurally unrelated to any other RTKs.
3 FIG-ROS fusion gene Iden/fied in human glioblastoma cell line U118 MG Fusion gene due to 240 kb dele/on on chr 6 Charest A. et al; Genes, Chromosomes & Cancer 2003 Fusion between exon 7 of FIG and ex 36 of ROS
4 Analysis of the expression levels of 96 TKs in different types of MPN using micro fluidic cards ROS1 is highly expressed in the 4 CMML cases included in the study
5 ROS1 expression in CMML cells by RQ-PCR p<0.001 CD34+ p<0.001
6 ROS1 protein in CMML cells by RQ-PCR SW1088 CMML CTRL NEG CTRL BM PB BM PB 260 kda c ros 45 kda Actin
7 ROS1 protein in CD34+ and monocytes CTRL CTRL CMML CMML CD34+ Monocytes
8 Why is ROS1 overexpressed? Which are the consequences of ROS1 overexpression? Which pathways are activated by ROS1? May ROS1 represent a druggable target?
9 What leads deregulated c Ros1 expression? mutapon/s within the promoter region? Sequencing 800 bp upstream ATG No muta/ons in CMML pts
10 46,XY,3q-,6q-,-11,der(22),+m RP1-92C8 RP1-179P9 Interphase cells were labelled with probes from PAC (clonerp1 92C8) and BAC (clone RP1 179P9)
11 SNPs analysis
12 Why is ROS1 overexpressed? Which are the consequences of ROS1 overexpression? Which pathways are activated by ROS? May ROS1 represent a druggable target?
13 EGFR-ROS chimera
14 Egfr/c-Ros CHIMERA EGFR ROS NH2 EC Domain Transmembrane (TM) TK Domain COOH pbsk.egfr/c-ros pcdna3.1hisc.egfr/c-ros
15 SW1088 pcdna ER ROS 150 kda Actin 42 kda pcdna ER FBS+ FBS- EGF FBS+ FBS- EGF ROS1 p-ros1
16 Proliferation of ER transfected cells H3 tymidine incorporation MTT assay
17 ROS1 activation reduces cell adhesion HEK 293T
18 ROS1 activation reduces apoptosis ER ER + EGF Percentage of apoptosis ANNEXIN VI ANNEXIN V
19 ROS1 activation induces Incresed proliferation Reduction of cell adhesion Mild reduction of apoptosis
20 Which pathways are activated by ROS?
21 IP (GRB2 and ROS/SOS) SW1088 pcdna ER ROS ROS SOS
22 ERK and AKT pathways are activated by ROS1 ER+FBS ER-FBS ER+EGF pros perk/erk pakt/akt p-erk 1/2 Erk1/2 p-akt Akt actin
23 The same pathways are activated in CMML patients overexpressing ROS1 CTRL CMML perk/erk pakt/akt pros perk 1/2 Erk 1/2 pakt Akt Actin
24 Why is ROS1 overexpressed? Which are the consequences of ROS1 overexpression? Which pathways are activated by ROS? May ROS1 represent a druggable target?
25 ROS1 inhibitors ( Nippon-Shinyaku-Japan)
26 APOPTOSYS in HCC 78 cell line HCC-78-CTRL HCC-78-COMPOUND A 1 µm HCC-78-COMPOUND D 1 µm PROPIDIUM PE- A 93.6% 2.5% 3.9% PROPIDIUM PE- A 90.9% 3.7% 5.4% PROPIDIUM PE- A 94.1% 2% 3.9% ANNEXIN FITC-A HCC-78-COMPOUND E 1 µm ANNEXIN FITC-A ANNEXIN FITC-A HCC-78-COMPOUND F 1 µm PROPIDIUM PE- A 70.6% 13.5% 15.8% PROPIDIUM PE- A 72.6% 18.8% 8.5% ANNEXIN FITC-A ANNEXIN FITC-A
27 CMML patient ROS + RQ PCR expression = 15393
28 APOPTOSYS (CD34+ pz CMML ROS+) PROPIDIUM PE- A pz CMML-CTRL 6.1% 48.6% 44.6% pz CMML-COMPOUND A 300 nm 2.7% PROPIDIUM PE- A 37.1% 60.1% pz CMML-COMPOUND D 300 nm PROPIDIUM PE- A 5.1% 48.4% 46.5% ANNEXIN FITC-A ANNEXIN FITC-A ANNEXIN FITC-A pz CMML-COMPOUND E 200 nm PROPIDIUM PE- A 5.8% 34.6% 59.2% pz CMML-COMPOUND F 300 nm PROPIDIUM PE- A 43.2% 8.4% 48.3% ANNEXIN FITC-A ANNEXIN FITC-A
29 APOPTOSYS in PMF patient ROS1 negative pz PMF-CTRL pz PMF-COMPOUND A 300 nm pz PMF-COMPOUND D 300 nm PROPIDIUM PE- A 2% 83.8% 14.1% PROPIDIUM PE- A 78.7% 1.7% 19.4% PROPIDIUM PE- A 2.4% 74.3% 23.1% ANNEXIN FITC-A ANNEXIN FITC-A ANNEXIN FITC-A pz PMF-COMPOUND E 200 nm pz PMF-COMPOUND F 300 nm PROPIDIUM PE- A 3.4% 77.5% 19.1% PROPIDIUM PE- A 77.4% 1.5% 20.9% ANNEXIN FITC-A ANNEXIN FITC-A
30 University of Turin Department of clinical and biological sciences Enrico Bracco Sonia Carturan Valentina Campia ROS inhibitors Nippon-Shinyaku-Japan) Masaki Nogawa SNPs array University of Bologna Ilaria Iacobucci Giovanni Martinelli
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