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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