Dissecting the
the Molecular Vulnerabilities
Vulnerabilities
Of Myeloma using High Throughput RNAi
Screening
A. Keith Stewart
Scottsdale, Arizona
Rochester,
Rochester Minnesota
Jacksonville, Florida
RNA interf
te erence
>90% transfection and high reproducibility
U)L
(R
iabilityV
r
r
e
A
A
e
e
A
A
e
Buff
siRN
siRN
Buff
siRN
siRN
ontrol
Lethal
ontrol
Lethal
C
C
RNAi HTS ­ Kinome (1,278) and Druggable Genome (14,000)
Geneti
tic Di
Diversit
ity f
o Cell
ll Li
Lines (KMS11
(KMS1 , IAN6
IAN6, JJN3)
Incubate 96h
Day 0
Day 1
Day 4
Cell line
+/- Bortezomib
Vi
Viability
+ Kinome or DG2 siRNA library
(CellTitre Glo)
+ Transfection reagent
+ fresh media
siRNA screening of druggable genome in myeloma cells
Lethal
Control
siRNA
e.g. UBB
Vulnerable genes (in green)
siRNA lethality screening of kinome genes in KMS11
reagent control
NS -ve control
GFP -ve control
Vi
V able
Kinase
siRNA
B-score
Dead
UbB +ve controls
Primary screening: kinase vulnerabilities
1+ siRNA hits
KMS11
INA6
14% of Kinases
9% of Kinases
CLK3
ITPKC
CNKSR1
JAK3
4%
AKAP3
KIP2
CSNK1D
KDR
AKAP6
MAP4K1
DAPK2
LOC91807
ACVR1B
MAP3K1
ALS2CR2
MARCKS
AAK1
DGKB
MAP3K9
ANKRD3
MAP3K11
ALS2CR7
MARK1
AATK
DGKQ
MAPK12
ATM
MAPK11
BMPR2
NJMU-R1
ABL1
EPHA7
MERTK
NME3
BMX
PDGFRA
ADCK5
EPHB2
MVK
NME3
AURKA
CAMK1D
PI4KII
AK3
EPHB6
NEK9
PKN1
AURKB
CAMKK2
PIP5K2A
AKAP7
FASTK
NRBP
CKB
PLK1
CLK2
PLK4
AKAP9
FES
NTRK3
DYRK1B
PRKAB1
AKT1
FGFR3
PCTK3
EPHA5
CSNK1A1L
PRKAR1A
PTK2
AKT2
FLT3LG
PFKM
FGFR4
ROR2
DGKA
PTK9L
ROR2
APEG1
FLT4
PIK3CD
DYRK4
PXK
FLT1
RYK
CAMK1G
GAK
RIOK3
EPHA1
RPS6KA5
FYN
TK2
CDC2L1
GRK4
RIPK1
ERBB3
STK16
HK1
TNK1
CDK2
GRK6
SLK
FGFR2
STK25
VRK3
CDK5
GUK1
SMG1
IBTK
STK29
CDK5R2
HIPK3
TAOK3
IKBKAP
HUNK
TNK2
JAK1
ILK
TSKS
ITPKA
TYRO3
ITPKB
YES1
R Tiedemann et al.
Myeloma kinome vulnerability map | shortest paths network
PKN1
Validation of survival kinases: 4 siRNA / kinase
NS -ve controls
non-lethal
re)
2t
2 st.dev th
thresh l
o d
ld
sco
(B
ityilba
Vi
UbB +ve controls
Kinases
Many MM survival kinases are not universally vulnerable
Non silencing siRNA -ve controls
Myeloma:
Viable
JJN3
2 St Dev
lethal
Embryonic
viable
kidney: 293
lethal
2 St Dev
PLK1
UBB
kinases
13 Kinases Vulnerable and Validated
Only 2% of Kinases were lethal in all 3 MM cell
lines using multiple siRNA, relatively
gy non lethal in
epithelial cell lines and plausibly expressed in
myeloma cell lines and patient samples
AKT1
AK3L1,
AURKA
AURKB,
CDC2L1
CDK5R2
FES
FLT4
GAK
GRK6
PKN1
SMG1
TNK2
Therapeutic Window: Myeloma survival kinases
preferentially expressed in MM
Normal plasma cells
Somatic tissues
Myeloma tumors
GRK6
PKN1
CDC2L1
mRNA level (MAS5)
not expressed
p
GRK6 is selectively vulnerable in myeloma cells
myeloma
non myeloma
Control
GRK6 shRNA
Control
GRK6 shRNA
3.13
14.2
18.5
52.4
2.99
12.2 1.32
9.41
KMS11
MCF7
(breast Ca)
75.9
21.8
81.2
86.8
6.86
7.29
3.67
2.49
ADA
AAD
A 3.42
18.9
50
5. 2
02
50 4
.
7 56
5. 3
63
82
8. 9
29 13
21 9
.
7
OPM1
SF767
71 8
.
13.7
(glioma) 84.4
61.5
5.83
30.8
1.73
3.53
Annexin V
Annexin V
Annexin V
Annexin V
V
Annexin V
Annexin
GRK6 knockdown is selectively lethal to myeloma cells
A non-selective GRK6 kinase inhibitor is selectively lethal to MM cells at
concentrations above IC50 for GRK6 inhibition (>100x IC50 for PKC)
(20uM)
inhibitor6K
GR
GRK6 inhibitor (uM)
GRK6 Binds to and is Regulated by Heat
Shock Protein 90
Overview of Druggable
gg
Genome Screen Results
Killer siRNA Control
Vehicle
Bortezomib 1.5nM
IC10
Bortezomib 2 nM
IC30
Bortezomib 2.5 nM IC60
Bortezomib 3 nM
nM
IC80
IC80
Secondary siRNA screen: confirmation of kinase targets that synergise with
bortezomib
Tiedemann et al. ASH 2007
Kinases That Synergize with Bortezomib
QuickTimeTM and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Aurora Kinase Inhibitor Cytotoxic to
1.1
All MM Cell Lines Tested
KMS11
1
KMS12PE
KMS18
0.9
OPM1
SKMM2
0.8
MM1R
MM1S
0.7
JJN3
MY5
H929
0.6
U266
8226
0.5
0.4
0.3
0.2
0.1
0
0
50nM
100
200
300
400
500
600
AURORA Inhibitor nM
Aurora Kinase A Inhibitor + Bortezomib
A Rational
Rational Combination
Combination for Trials
Trials
1.2
Synergy
1
0.8
0nM VELCADE
1nM
2nM
0.6
3nM
0.4
0.2
0
0nM
15nM
30nM
45nM
60nM
75nM
90nM
105nM
MLN 8237
CDK5 Silencing Increases Sensitivity to Proteasome Inhibitors
OPM1
1.2
1.1
GFP control
10
1.0
CDK5
CDK shRN
shR A
N 1
0.9
CDK5 shRNA 2
GFP ad control
CDK5 shRNA 3
0.8
yit 0.7
CDK5 shRNA 3
il 0.6
CDK5 shRNA 4
iab 0.5
V 04
CDK5 shRNA 5
0.4
CDK5 shRNA 1
0.3
OPM1
CDK5 shRNA 4
0.2
0.1
0.0
0
1
2
3
4
Bot
Bo ez
e om
o i
m b
i nM
nM
CDK5 shRNA 2
CDK5 shRNA 5
1.25
GFP control
1.00
CDK5 shRNA 1
CDK5 shR
sh N
R A
N 2
y
CDK5 shRNA
it
0.75
CDK5 shRNA 3
il
CDK5 shRNA 4
OPM1
1
2
3
4
5
GFP ad
iab
0.50
V
CDK5 shRNA 5
CDK5
OPM1
02
0. 5
25
-actin
0.00
0
10
20
30
40
PR047 nM
CDK5 and Primary Patient
CD13
46%
14%
8
Vehi
h cle
37%
3%
CD138
Vel 1.5nM 48h
30%
11%
48%
11%
11
CD138
Ros 10
10 M
u
48h
30%
15%
47%
8%
8
CD138
Vel + Ros 48h
17%
8%
67%
8%
Annexin V
MC0888 ­ Phase II trial of CDKSCH
727965 a novel, potent, small
molecule inhibitor of cyclin
dependent kinases (CDK5)
(CDK5) in
Multiple Myeloma
PI: Kumar
Kumar and Tiedemann
Tiedemann
Summary
1. Vulnerable Kinases Identified
GRK6
2. Vulnerable Targets in Druggable Genome identified
MCL-1, Proteasome (Tiedemann)
3. Bortezomib Sensitizers Identified
CDK5
Aurora A
Summary
Clinical Trials That Logically
gy Follow From Screening

Aurora Kinase +/- Proteasome

MCL 1(
-1 Ob
(Obat
l
oc ax) /
+ - Pt
Proteasome

CDK5 (SCH 727965) + Proteasome
For the Future
GRK6
CDC42SE
Questions, Comments or
or Insults
Insults ?
­ Rodger Tiedemann
­ Estaban Braggio and Jonathon Keats
­ Fonseca/Bergsagel/Mikhael/Reeder
­ Kumar et al.