nuove mutazioni nella leucemia mielomonoci/ca cronica · nuove mutazioni nella leucemia...

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