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Discovery Of Potent And Selective Inhibitors Of USP7 With Anti-Tumor Activity In Vitro And In VivoYamini Ohol, Michael Sun, Gene Cutler, Paul Leger, Dennis Hu, Berenger Biannic, Payal Rana, Cynthia Cho, Scott Jacobson, Steve Wong, Jerick Sanchez, Xinping Han, Kyle Young, Akinori Okano, Nick
Shah, Betty Abraham, Deepa Pookot, Jack Maung, Delia Bradford, Nathan Kozon, Silpa Suthram, Lavanya Adusumilli, Deepika Kaveri, Oezcan Talay, Christophe Colas, Andrea Kim, Jacob Schwarz, David Wustrow, Dirk Brockstedt, Paul Kassner; FLX Bio, Inc. South San Francisco, CA
Abstract
USP7 is a deubiquitinase that regulates the levels of multiple downstream targets with roles in cancer progression and immune response. Inhibitors of USP7 (USP7i) may decrease oncogene function, increase tumor suppressor function, enhance immune function and sensitize tumor cells to DNA damaging agents. We have developed USP7i that are potent and selective in biochemical and cellular assays. Our USP7i reduce the viability of multiple p53-wild type cell lines, including blood cancer and neuroblastoma cell lines, as well as a subset of p53-mutant tumor cell lines in vitro. Further, oral administration of our USP7i inhibits MM.1S (multiple myeloma; p53-wild type) and H526 (small cell lung cancer; p53-mutant) tumor growth in vivo.
Conclusions
§ We have developed a novel series of potent, highly selective, orally available small molecule USP7 inhibitors with anti-tumor activity in vitro and in vivo.
§ FLX USP7 inhibitors are effective against multiple blood cancer and MYCN-amplified neuroblastoma cell lines with p53-WT status in vitro.
§ FLX USP7 inhibitors are effective against a subset of p53-mutant tumor cell lines and synergize with DNA-damaging agents, PIM and PI3K inhibitors.
§ Oral delivery of FLX USP inhibitors reduces growth of MM.1S (multiple myeloma; p53-wild type) and H526 (small cell lung cancer; p53-mutant) tumors and prolongs survival in vivo.
2
USP7i
Oncogenes + Tumor suppressors
•MDM2 p53•FOXO•PTEN
DNA damage response•RAD18•Claspin•CHFR
•PHF8, etc.
Immune response•FOXP3•TIP60•NFkB•IKK
Ø Increased sensitivity to DNA damaging agents
Ø Decreased oncogene activity
Ø Increased tumor suppressor activity
Ø Direct anti-tumor effect
Ø Decreased Tregfunction
Ø Increased anti-tumor immunity
Multiple Beneficial Mechanisms of USP7 Inhibitors
USP7i USP7 IC50
FLX-A 10 nM
FLX-B < 1 nM
FLX-C < 1 nM
Active enantiomer < 1 nM
Inactive enantiomer
< 100 nM
FLX-D (in vivo tool) < 1 nM
FLX-E (in vivo tool) < 1 nM
USP7 Inhibitors Inhibit p53-Wild Type Tumors
In Vitro and In Vivo
(A) p53 wild type status is a predictor of USP7i sensitivity. (B) MM.1S cells were treated with USP7 inhibitors for 5 days, after which viability was measured by CellTiterGlo assay. (C) MM.1S cells were treated with USP7 inhibitor for 4h, after which USP7 engagement was measured using Ub-PA probe. (D) MM.1S cells were treated with USP7 inhibitor or Idasanutlin for 6h, after which p53 and p21 levels were measured by Western blot. (E) NOD-scid mice engrafted with multiple myeloma MM.1S tumors were treated with USP7 inhibitor for the indicated time, and tumor volume and survival were monitored.
(C) (D)
(A) (B)
(E)
Wild typeMutantp53 Status
Nor
mali
zed
signa
l at 1
µM
1.0
0.5
0.0
USP7-UbUSP7
Probe:FLX-C (nM):
- + + + + + + + + + + + + 0 0 500 200 85 35 15 6 2 1 0.4 0.2 0.06
FLX-D (nM):Idasanutlin (nM):
- 100 500 -
- - - 125
1 0 - 8 1 0 - 7 1 0 - 60
2 0
4 0
6 0
8 0
1 0 0
[ C o m p o u n d ] M
% L
um
ine
sc
en
ce
(A
TP
)
A c t i v e e n a n t i o m e r
I n a c t i v e e n a n t i o m e r
1 0 - 8 1 0 - 7 1 0 - 6 1 0 - 5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
[ C o m p o u n d ] ( M )
% L
um
ine
sc
en
ce
(A
TP
)
F L X - D
0 1 0 2 0 3 00
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
D a y s o f T r e a t m e n t
Tu
mo
r V
olu
me
(m
m3
) V e h i c l e c o n t r o l
F L X - D 1 0 0 m p k
F L X - D 5 0 m p k
T u m o r V o lu m e
0 1 0 2 0 3 00
2 0
4 0
6 0
8 0
1 0 0
D a y s o f T r e a t m e n t
Pe
rce
nt
Su
rviv
al
(Tu
mo
r R
ea
ch
ing
15
00
mm
3)
V e h i c l e c o n t r o l
F L X - D 1 0 0 m p k
F L X - D 5 0 m p k
S u r v i v a l
USP7 Inhibitors are Highly Selective and Potent
in Biochemical and Cellular Assays
(A) USP7 inhibitor inhibits USP7 in biochemical assay using rhodamine-labeled ubiquitin. (B) USP7 inhibitor elevates p53 in a luciferase reporter gene cell-based assay. (C) USP7 inhibitor IC50 is <10 nM for USP7 but >10 µM for all other DUBs tested.
1
(C)USP7
FLX-A
(B)(A)
USP7 Inhibitors Inhibit p53-Mutant Tumors
In Vitro and In Vivo
3
(A) H526 small cell lung cancer cells were treated with USP7 inhibitors or idasanutlinfor 5 days, after which viability was measured by CellTiter Glo assay. (B) Nu/Nu mice engrafted with H526 tumors were treated with USP7 inhibitor for the indicated time, and tumor volume and survival were monitored.
(A)
(B)
1 0 - 8 1 0 - 7 1 0 - 6 1 0 - 50
2 0
4 0
6 0
8 0
1 0 0
1 2 0
[ C o m p o u n d ] M
% L
um
ine
sc
en
ce
(A
TP
)
I n a c t i v e e n a n t i o m e r
A c t i v e e n a n t i o m e r
1 0 - 8 1 0 - 7 1 0 - 6 1 0 - 50
2 0
4 0
6 0
8 0
1 0 0
1 2 0
[ C o m p o u n d ] M
% L
um
ine
sc
en
ce
(A
TP
)
I d a s a n u t l i n
1 0 - 8 1 0 - 7 1 0 - 6 1 0 - 50
2 0
4 0
6 0
8 0
1 0 0
1 2 0
[ C o m p o u n d ] M
% L
um
ine
sc
en
ce
(A
TP
)
F L X - E
0 1 0 2 0 3 00
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D a y s o f T r e a t m e n t
Tu
mo
r V
olu
me
(m
m3
)
V e h i c l e c o n t r o l
F L X - E
T m T
T u m o r V o l u m e
0 1 0 2 0 3 00
2 0
4 0
6 0
8 0
1 0 0
D a y s o f T r e a t m e n t
Pe
rce
nt
Su
rviv
al
(Tu
mo
r R
ea
ch
ing
15
00
mm
3)
V e h i c l e c o n t r o l
F L X - E
S u r v i v a l
5
(E)
USP7 Inhibitors Synergize With DNA-
Damaging Agents, PIM and PI3K Inhibitors
(A)
(B)
4
(A, B) H526 cells were treated with indicated drug (y-axis) and USP7 inhibitor (x-axis) at indicated dose (µM) for 5 days, after which viability was measured by CellTiter Glo assay and synergy was calculated by Bliss analysis. Combinations of USP7 inhibitors with DNA-damaging agents and PARP inhibitor were tested in (A), PIM and PI3K inhibitors in (B).
2
- 9 - 8 - 7 - 6 - 5 - 4
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
C a r b o p l a t i n + F L X - E
l o g [ c a r b o p l a t i n , ( M ) ]
% l
um
ine
sc
en
ce
(A
TP
)
- 1 0 - 9 - 8 - 7 - 6
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
D o x o r u b i c i n + F L X - E
l o g [ d o x o r u b i c i n , ( M ) ]
% l
um
ine
sc
en
ce
(A
TP
)
- 8 - 7 - 6 - 5 - 4 - 3
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
O l a p a r i b + F L X - E
l o g [ o l a p a r i b , ( M ) ]
% l
um
ine
sc
en
ce
(A
TP
)
2
0 . 5
0 . 1 2 5
0
0 . 1 2 5
0 . 5
2
C o m p o u n d + F L X - E ( µ M )
F L X - E a lo n e ( µ M )
- 9 - 8 - 7 - 6 - 5
0
2 0
4 0
6 0
8 0
1 0 0
A Z D 1 2 0 8 + F L X - E
l o g [ A Z D 1 2 0 8 , ( M ) ]
% l
um
ine
sc
en
ce
(A
TP
)
- 9 - 8 - 7 - 6
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
P i c t i l i s i b + F L X - E
l o g [ p i c t i l i s i b , ( M ) ]
% l
um
ine
sc
en
ce
(A
TP
)
- 9 - 8 - 7 - 6
0
2 0
4 0
6 0
8 0
1 0 0
T a s e l i s i b + F L X - E
l o g [ t a s e l i s i b , ( M ) ]
% l
um
ine
sc
en
ce
(A
TP
)
C o m p o u n d + F L X - E ( µ M )
0
0 . 0 6 2 5
0 . 2 5
1
0 . 0 6 2 5
0 . 2 5
1
F L X - E a lo n e ( µ M )
DNA-damaging agent DNA-damaging agent PARP inhibitor
pan-PIM kinase inhibitor PI3K-selective inhibitor PI3Kα-selective inhibitor
5.00E-09
5.00E-08
5.00E-07
5.00E-06
USP
1U
SP2
USP
4U
SP5
USP
6U
SP7
USP
8U
SP9
xU
SP1
1U
SP1
4U
SP1
5U
SP1
6U
SP1
9U
SP2
0U
SP2
1U
SP2
5U
SP2
7xU
SP2
8U
SP3
0U
SP3
5U
SP3
6U
SP4
5U
SP4
7C
YLD
UCH
L1U
CHL3
UCH
L5BA
P1O
TU1
OTU
B2O
TUD
1O
TUD
3O
TUD
5O
TUD
6AO
TUD
6BC
ezan
neV
CPIP
AM
SH-L
PA
taxin
3A
taxin
3L
JOSD
1JO
SD2
DUB
IC50
DUB Selectivity Profile
5 e-8
5 e-7
5 e-6
5 e-9
DU
B IC
50(M
)
3 e-7
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