Meccanismi di resistenza all’inibizione della via di
segnale PI3K/Akt/mTOR nella LAM
University of Modena and Reggio
Emilia
Cell Signaling Unit, ChiMOMo
Sandra Marmiroli
Jessika Bertacchini
Laura Mediani
Hematology Department
Mario Luppi
Fabio Forghieri
Patrizia Barozzi
University of Padova
Oncohematology Laboratory
Giuseppe Basso
Benedetta Accordi
George Mason University
Manassass Virginia USA
Center for Applied Proteomics
and Molecular Medicine
Lance Liotta
Emmanuel Petricoin
RTKs!
GPCR
Ras!
PTEN!
PIP2!
PIP3!
PIP2!
IRS! p85! p110α/β/δ
PTEN!
PDK-1!
Gβγ
p87/101! p85!
p110β
p110γ
P
Akt1/2/3!
P
mTORC2!
mTORC1!
S6K!
Bad!
IKK! MDM2! FOXO! GSK3! TSC2! PFK2! ACL!
Cell!
Survival!
Cell!
Cycle!
Cell!
Growth!
Glycolysis!
Metabolism!
Signalling of the PI3K/AKT/mTOR pathway and
relevant drugs that target each of the components of
the pathway
Rodon, J. et al. (2013) Development of PI3K inhibitors: lessons learned from early clinical trials
Nat. Rev. Clin. Oncol. doi:10.1038/nrclinonc.2013.10
Growth factor
Receptor Tyr kinase
PTEN
Energy stress
PI3K
Ras
PI-3,4,5P3
Raf
AKT
PDK1
MEK
AMP
LKB1
A.A.
Tuberin
Erk
Rheb
Rsk
AMPK
RagB/RagD
Raptor/mTOR
4EBP1
S6-kinase
S6
Protein synthesis
NF1
Bertacchini et al. Leukemia 2014
Bertacchini et al. Leukemia 2014
Bertacchini et al. Leukemia 2014
Bertacchini et al. Leukemia 2014
AKT inhibitors
mTOR
inhibitors
Perifosine, Akti 1/2
TCN
Torin 1
Rapamycin
Bertacchini et al. Leukemia 2014
Bertacchini et al. Leukemia 2014
  Activation
of PI3K/Akt pathway in AML is
modulated by feedback
  Akt inhibitors reactivate PI3K/Akt through
stabilization of IRS-1 and FOXO, which in turn
increases IR or other RTKs
  The results highlight the limitations of these drugs
if used as monotherapy
  Association of Akt and RTKs inhibitors prevents
re-induction of Akt
Vitalità %/CTRL
THP1 (PF-04691502)
μM
100
80
60
40
20
0
Dose (μM)
Vitalità %/CTRL
CEM (PF-04691502)
100
80
60
40
20
0
Dose (μM)
  IC50 1,1 µM in cellule THP1
 
1,3 nM in cellule CEM.
μM
Etoposide + Citarabina + GDC-0068
100
80
60
40
20
DMSO
GSK
E+C
E+C+GSK
Vitalità %/CTRL
Etoposide + Citarabina + PF04691502
100
80
60
40
20
Trattamenti GSK 60
ETO + + AraC
40
DMSO
PF
E+C
E+C+PF
+ GDC ETO + + AraC
+ PF   .
DMSO
GDC
E+C
E+C+GDC
INDICI DI COMBINAZIONE ETO + AraC +
80
0
100
0
0
20
Vitalità %/CTRL
Vitalità %/CTRL
Etoposide + Citarabina + GSK690693
ED50 ED75 ED90 0,047 0,013 0,045 0,051 0,011 0,0037 0,0062 0,052 0,055 Bertacchini et al. Leukemia 2014
  Our
data support the development of targeted
treatment paradigms for PI3K/Akt/mTOR-altered
adult leukemias and also demonstrate that
therapies must be tailored to the specific RTKs and
phosphorylome context.
fold change upon treatment
3
2
1
rapamycin
0
Akt T308 Akt S473
-1
-2
-3
PRAS40
T246
GSK3 S
21/9
P70 S6K
T389
4EBP1
S65
IRS-1
Torin1
Phosphorylation profile of receptor tyrosine
kinases in human primary blast cells
RTKs!
GPCR
Ras!
PTEN!
PIP2!
PIP3!
PIP2!
IRS! p85! p110α/β/δ
PTEN!
PDK-1!
Gβγ
p87/101! p85!
p110β
p110γ
P
Akt1/2/3!
P
mTORC2!
mTORC1!
S6K!
Bad!
IKK! MDM2! FOXO! GSK3! TSC2! PFK2! ACL!
Cell!
Survival!
Cell!
Cycle!
Cell!
Growth!
Glycolysis!
Metabolism!
sample collection
84 samples with >80% blast cells analyzed by
RPPA, then validated by western blotting
Fresh peripheral blood and bone marrow
specimens with newly diagnosed AML.
Patients are diagnosed according to blast
content, FAB classification and cytogenetic
analysis.
Overlapping profiles of peripheral blood (PB) or bone
marrow (BM) derived blast cells from the same patients
.
18 blood and bone marrow specimens from the same
patient were available.
Overlapping profiles of fresh and cryopreserved
samples from the same patient
samples derived from the same patient
before and after cryopreservation in
10% DMSO were analyzed, with similar
results
blast
Reverse Phase Protein Array
Automated sample printing on
nitrocellulose-coated glass
Statistical analysis
Sypro Ruby staining for
protein quantification
Detection and quantification
of specific endpoints
Through RPPA technology 90 endpoints were analyzed,
involved in different signaling pathways: survival,
apoptosis, oxidative-stress and metabolism.
Areas with pathways hyper-activated in limited clusters of
patients are highlighted.
Correlation of protein profiles with FAB groups :
 p-Akt, p-PKA, p-PKC, p-mTOR, p-GSK, p-p70S6K, p-p90RSK and
 p-p38 cluster preferentially in M4/M5 FAB subtype;
 pro-apoptotic proteins cluster preferentially in M1/M2 FAB
subtype.
M1
M2
M4
C-Kit receptor expression correlates with
hyperphosphorylation of the PI3K/Akt pathaway
90
50
LY294002
40
PERIFOSINE
100
0
DM
5
10
20
50
60
% SURVIVAL
70
TORIN
TRICIRIBINE
20
10
0
5
10
AKT INHIBITORS +
TORIN 1
20
[µM]
% SURVIVAL
DMSO
50
90
80
70
60
50
40
30
20
10
0
RAPAMYCI
N
[µM]
VIII
30
% SURVIVAL
% SURVIVAL
RAPAMYCIN
AKT INHIBITORS
80
100
50
0
TORIN
pAkt S473
2,5
fold change upon treatment
2
1,5
1
0,5
0
-0,5
-1
-1,5
-2
-2,5
Akti VIII 20 hr
3
2,5
2
1,5
1
0,5
0
Akt S473 FOXO3a IRS1 S612 PRAS40 Akt T308 4EBP1
p70S6K CC9
Perifosine 20 hr
3
2,5
2
1,5
1
0,5
0
The Akt inhibitor paradox:
 sustained apoptosis
 sustained Akt activation
Fold change upon treatment
0
-0,5
-1
-1,5
-2
-2,5
pAkt S473
4 fold change upon treatment
3 Perifosine
Akti VIII
2 1 0 -­‐1 -­‐2 hr
-­‐3 2
4
20
2
4
20
Bertacchini et al. Leukemia 2014
Etoposide + Citarabina
120
Vitalità %/CTRL
100
80
60
40
20
0
0,5+5
1+10
Dose (μM)
2+20