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Protein
translation
Proliferation
Viability
Energy
Monitoring
MTOR
motility
Nutrient
Availability
Angiogenesis
Glucose
metabolism

IRS1
PI3KINASE
AKT & SGK
VIABILITY
PKC
ADHESION
& MOTILITY
mLST8
RAPTOR
mTOR
Rapalogs
pp242
Comp'd 2
mTOR
RICTOR
mLST8
TORC1
mSIN1
PROTOR
Feedback
P70 and 4EBP1
TORC2
Pathways
phosphorylation
ERK
Enhanced
CELL CYCLE TRANSIT
Translation
of cell cycle
proteins

Active site mTOR inhibitors
· Inhibit mTOR at ATPbinding site
· Pp242, torin 1, INK 128, OSI are examples; In trials currently
· More active than rapamycin against MM cells in preclinical
survival assays; Synergizes with bortezomib
· Inhibits TORC1 inhibits phosphorylation of p70 and 4EBP1
and, thus, inhibits protein translation
· Inhibits TORC2 inhibits AKT phosphorylation and SGK1
activity
· Improved activity vs rapalogs thought due to more intense
inhibition of 4EBP1 phosphorylation and decreased protein
translation
· However, rictor knock down (TORC2 paralysis) is deleterious
to MM cells suggesting inhibition of TORC2 plays a role in
enhanced efficacy

8226
MM1.S
rap
pp242
rap
pp242
0
100 250 500 1000100 250 500 1000
0 100 250 500 1000 100 250 500 1000 nM
erk-P
erk-total
P70-P
p70-total
10
8226
25
in
MM1S
0
100 250 500 pp242
8
20
p44
p42
ERK-P
6
15
increase
ERK-T
4
10
Fold
phospho-ERK
2
5
Patient #1
nM 100 250 500 1000
100 250 500 1000
0 1hr 3hr pp242
P-ERK
8226
MM1.S
T-ERK
Patient #2
DMSO
Rap
Pp242
IL-6
DMSO
Rap
pp242
IL-6
ERK
P-ELK

FIGURE 6
100
8226
U0126
A)
B) 100
8226
5000
80
80
1000
U0126
60
0
60
250
Y 40
250
40
0
VER 20
O
1000
20
SIS
C
O
5000
RE 100
100
U0126
MM1.S
MM1.S
U0126
APOPT
5000
80
%
VIABLE
80
1000
%
0
60
60
250
250
40
0
1000
40
20
5000
20
PP242 100 250 500 PP242 100 250 500

8226
A)
ric1
ric2
scr
0
250 1000
pp242(nM)
0
250 1000
0
250 1000
Erk-P
erk
rictor
rap1
rap2
scr
B)
0
250 1000
pp242(nM)
0
250 1000
0
250 1000
Erk-P
erk
raptor

HIGH THROUGHPUT SCREEN FOR INHIBITOR OF RICTORmTOR
INTERACTION
1) MAKE YEAST HYPERPERMEABLE TO SMALL INHIBITORS
2) CLONE mTOR AND RICTOR INTO YEAST VECTORS FOR
TWO-HYBRID ASSAY
small molecule
inhibitor
AD
protein Y
DBD
protein X
HIS3,
LacZ
Gal UAS

CLONE 1 CLONE 2
COMPONENT 1
COMPONENT 2
DBD
AD
RICTOR
AD
DBD
mTOR
DBD
mTOR
RICTOR
AD

C
1
4
COMP'D 2 (uM)
AKT-P
AKT-T
P70-P
P70-T
75
MM1.S
OPM-2
8226
SIS
M
M
O
DMSO
1
4
50
erk P
erk
APOPT
25
%
akt P
akt
Control
1 uM
4 uM
Control
1 uM
48 hrs
72 hrs

CONCLUSIONS
· Although active site TOR kinase inhibitors are better
than rapalogs against MM cells, they induce ERK
activation
· ERK activation functions as a mechanism of
resistance
· Identification of a selective TORC2 inhibitor, by a
high throughput yeasttwohybrid assay against
mTORRICTOR binding, demonstrates the induction
of MM apoptosis without activation of ERK

DEPTOR
· A 48 kDa protein that binds to mTOR within both TORC1 and
TORC2 complexes
· Negatively regulates both TORC1 and TORC2 activity
· Not significantly expressed in any malignancies other than
myeloma
· Overexpressed in 28% of myeloma specimens
· Overexpression specifically found in cases with Ig
translocations
· Is a MAF target, explaining especially high expression in
MAFtranslocated myeloma
· Its inhibition of TORC1 results in marked feedback activation
of PI3K/AKT
· Knockdown results in prevention of MM cell growth and
apoptosis

Ramifications of high DEPTOR expression
1. INHIBITED PROTEIN TRANSLATION
INDUCING PROTECTION AGAINST ER
STRESS
PI3K
AKT
2. CAUSE OF HIGH BASAL AKT
ACTIVATION
TORC 1
3. POSSIBLE MECHANISM OF ANTI-
RAPTOR
APOPTOTIC SIGNALING IN MYELOMA
CELLS
DEPTOR
mTOR
4. POTENTIAL INDUCER OF AKT
ADDICTION IN MYELOMA CELLS
P70S6K
4E-BP1
PROTEIN
TRANSLATION

WHO DID THE WORK
· Joseph Gera
· Bao Hoang
· Angie Benavides
· Yijiang Shi
· Yonghui Yang
· SUPPORTED BY
· 1) NIH
· 2) DOD
· 3) VA
· 4) MMRF