nuove mutazioni nella leucemia mielomonoci/ca cronica · nuove mutazioni nella leucemia...
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Nuovemutazioninellaleucemiamielomonoci/cacronica
Firenze31marzo2011
DANIEL A CILLONIUNIVERSITA’ DEGLI STUDI DI TORINODIPARTIMENTO DI SCIENZE CLINICHE EBIOLOGICHE
c‐Ros1c‐Ros1••HumanHumanorthologorthologofDrosophilaofDrosophilasevenlesssevenlesslocatedonlocatedonchromosome6,locusspanningover130Kbpchromosome6,locusspanningover130Kbp
••ExpressedinExpressedinfetal/ssuesfetal/ssues
••ExpressedinaExpressedinavarietyoftumorsvarietyoftumors(especiallyNSCLCand(especiallyNSCLCandglioblastoma)glioblastoma)
••ItencodesforanItencodesforanorphanReceptorTyrosinekinaseorphanReceptorTyrosinekinasestructurallystructurallyunrelatedtoanyotherRTKs.unrelatedtoanyotherRTKs.
FIG-ROS fusion geneIden/fiedinhumanglioblastomacelllineU118MG
Fusiongenedueto240kbdele/ononchr6
Fusion between exon 7 ofFIGandex36ofROS
CharestA.etal;Genes,Chromosomes&Cancer2003
Analysisoftheexpressionlevelsof96TKsindifferenttypesofMPNusingmicrofluidiccards
ROS1 is highly expressed in the 4 CMML cases included inthe study
p<0.001
p<0.001
ROS1 expression in CMML cells by RQ-PCR
CD34+
ROS1 protein in CMML cells by RQ-PCR
SW1088CMMLCTRLNEGCTRLBM PB BM PB
c‐ros260kDa→
45kDa→ Actin
ROS1 protein in CD34+ and monocytes
CD34+ Monocytes
CTRL CTRL
CMML CMML
Why is ROS1 overexpressed?
Which are the consequences of ROS1overexpression?
Which pathways are activated by ROS1?
May ROS1 represent a druggable target?
Whatleadsderegulatedc‐Ros1expression?
mutaPon/swithinthepromoterregion?
Sequencing800bpSequencing800bpupstreamATGupstreamATG
Nomuta/onsinCMMLptsNomuta/onsinCMMLpts
46,XY,3q-,6q-,-11,der(22),+mRP1-92C8RP1-179P9
InterphasecellswerelabelledwithInterphasecellswerelabelledwithprobesfromPAC(cloneRP1‐92C8)andprobesfromPAC(cloneRP1‐92C8)andBAC(cloneRP1‐179P9)BAC(cloneRP1‐179P9)
SNPs analysis
Why is ROS1 overexpressed?
Which are the consequences of ROS1overexpression?
Which pathways are activated by ROS?
May ROS1 represent a druggable target?
EGFR-ROS chimera
Transmembrane (TM) TK Domain
COOH
1 625 1852 1863 1882 1940 2218
EC Domain
NH2
EGFR ROS
Egfr/c-Ros CHIMERAEgfr/c-Ros CHIMERA
pBSK.Egfr/c-Ros
pcDNA3.1HisC.Egfr/c-Ros
SW1088 pcDNA ER
ROS
42 kDa
∼150 kDa
Actin
pcDNA ER
FBS+ FBS- EGF FBS+ FBS- EGF
ROS1
p-ROS1
Proliferation of ER transfected cells
H3 tymidine incorporation
MTT assay
HEK 293T
ROS1 activation reduces cell adhesion
ER ER + EGF
ANNEXIN VI ANNEXIN V
Perc
enta
ge o
f apo
ptos
is
ROS1 activation reduces apoptosis
ROS1 activation induces
Incresed proliferation
Reduction of cell adhesion
Mild reduction of apoptosis
Which pathways are activated by ROS?
SW1088 pcDNA ER
IP (GRB2 and ROS/SOS)
SOS
ROS
ROS
p-Erk 1/2
ER+FBS ER-FBS ER+EGF
Erk1/2
p-Akt
Akt
actin
pROS pErk/Erk pAkt/Akt
ERK and AKT pathways are activated by ROS1
pAkt
pErk 1/2
Erk 1/2
Akt
Actin
CTRL CMML
pROS
pErk/Erk pAkt/Akt
The same pathways are activated in CMML patientsoverexpressing ROS1
Why is ROS1 overexpressed?
Which are the consequences of ROS1overexpression?
Which pathways are activated by ROS?
May ROS1 represent a druggable target?
ROS1 inhibitors ( Nippon-Shinyaku-Japan)
ROS JAK2 JAK3 Src TRKA ALK INSR Syk FLT-3 EGFRcompond E 0.88 47 300 33 16 440 3500 3700 2100 > 10000compound F 6.0 690 > 10000 1300 18 1900 > 10000 > 10000 > 10000 > 10000
kinase inhibition (IC50, nM)
ROS JAK2 JAK3 Src TRKA ALK INSR Syk FLT-3 EGFRcompound A 6.8 1.8 65 20 52 110 > 10000 1300 560 > 10000compound B 8.4 15 390 100 10 730 6700 9600 740 > 10000compound C 2.6 130 790 110 51 1700 > 10000 > 10000 6900 > 10000compound D > 10000 0.8 32 > 10000 n.d. n.d. n.d. n.d. n.d. n.d.
kinase inhibition (IC50, nM)
APOPTOSYS in HCC 78 cell line
HCC-78-CTRL
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
2.5%
3.9%93.6%
HCC-78-COMPOUND A 1 µM
PR
OP
IDIU
M P
E-
AANNEXIN FITC-A
90.9%5.4%
3.7%
HCC-78-COMPOUND D 1 µM
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
94.1%3.9%
2%
HCC-78-COMPOUND E 1 µM
ANNEXIN FITC-A
PR
OP
IDIU
M P
E-
A
70.6%15.8%
13.5%
HCC-78-COMPOUND F 1 µM
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
72.6%8.5%
18.8%
CMMLpatientROS+
RQ‐PCRexpression=15393
APOPTOSYS (CD34+ pz CMML ROS+)
pz CMML-CTRL
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
48.6% 44.6%
6.1%
pz CMML-COMPOUND A 300nM
ANNEXIN FITC-A
37.1%
2.7%
60.1%P
RO
PID
IUM
PE
-A
pz CMML-COMPOUND D 300 nM
ANNEXIN FITC-A
PR
OP
IDIU
M P
E-
A
48.4% 46.5%
5.1%
pz CMML-COMPOUND E 200 nM
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
34.6% 59.2%
5.8%pz CMML-COMPOUND F 300 nM
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
43.2%
8.4%
48.3%
APOPTOSYS in PMF patient ROS1 negative
pz PMF-CTRL
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
83.8% 14.1%
2%
pz PMF-COMPOUND A 300 nM
ANNEXIN FITC-A
78.7%
1.7%
19.4%P
RO
PID
IUM
PE
-A
pz PMF-COMPOUND D 300 nM
ANNEXIN FITC-A
PR
OP
IDIU
M P
E-
A 74.3% 23.1%
2.4%
pz PMF-COMPOUND E 200 nM
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
77.5% 19.1%
3.4%
pz PMF-COMPOUND F 300 nM
PR
OP
IDIU
M P
E-
A
ANNEXIN FITC-A
77.4%
1.5%
20.9%
UniversityofTurin
Departmentofclinicalandbiologicalsciences
Enrico BraccoSonia CarturanValentina Campia
ROS inhibitors
Nippon-Shinyaku-Japan)Masaki Nogawa
SNPs array
University of BolognaIlaria IacobucciGiovanni Martinelli