JAK mutations resistant to

ATP-competitive JAK inhibitors

L. Knoops

IL-9R signal transductionIL-9 R signal transduction

IL-9 R phe116 receptor

BaF3phe116

BaF3phe116

IL-9 responding

IL-9 IL-9

BaF3phe116

IL-9

IL-9 responding

IL-9

Autonomous

/ /

BaF3phe116

IL-9

IL-9 responding

IL-9

Autonomous

/ /

In vitro model of tumorigenesis

BaF3phe116

IL-9

IL-9 responding

IL-9

Autonomous

/ /

JAK1mutations

JAK1 overexpression

In vitro model of tumorigenesis

JH7 JH6 JH5 JH2 JH1

FERM pseudokinase kinase

K1026E

Y1035C

S1043I

F958V/C/S/L

L910Q

T901R

D895H

F635V

Y652H

V658I/L/F

S646F

S703I

P815S

L783F

A723D

E897KR724S

F734L

JH4 JH3

“SH2”

P960T/S

JAK1

Schematic modeling of JAK1 structure

JAK1 activating mutations

JH7 JH6 JH5 JH2 JH1

FERM pseudokinase kinase

K1026E

Y1035C

S1043I

F958V/C/S/L

L910Q

T901R

D895H

F635V

Y652H

V658I/L/F

S646F

S703I

P815S

L783F

A723D

E897KR724S

F734L

JH4 JH3

“SH2”

P960T/S

ALL-associated JAK1 mutations

JAK1

Schematic modeling of JAK1 structure

JAK1 activating mutations

JH7 JH6 JH5 JH2 JH1

FERM pseudokinase kinase

K1026E

Y1035C

S1043I

F958V/C/S/L

L910Q

T901R

D895H

F635V

Y652H

V658I/L/F

S646F

S703I

P815S

L783F

A723D

E897KR724S

F734L

JH4 JH3

“SH2”

P960T/S

ALL-associated JAK1 mutations

JAK1

JH2

domain

JH1

domain

E897K

D895HT901R

F958

V/C/S/L

K1026E

Y1035C

S1043I

L910Q

P815S

L783S

A723D

V658I/L/F F635V

Y652H

S646F

F734L

S703I

R724S

P960T/S

Schematic and 3D modeling of JAK1 structure

JAK1 activating mutations

T901RD895H

F958V/C/S/LL910Q

K1026E

Y1035C

S1043I

Hinge

G-loop

A- loop

13

2

C

4

5

7

8

D

E

K

J H

I

F

G

E897K

P960T/S

Crystal structure of JAK1 KD with CMP6

JAK1 activating mutations : kinase domain

Western Blot

JAK1 F958V is resistant to JAK inhibitor I

0

20

40

60

80

100

120

0.001 0.01 0.1 1

INCB018424 (M)

Pro

lifera

tion (

% o

f contr

ol)

M-RAS

Aut(V658F)

Aut(V658F)

M-RAS

H3-Thymidine incorporation

0

20

40

60

80

100

120

0.001 0.01 0.1 1

INCB018424 (M)

Pro

lifera

tion (

% o

f contr

ol)

M-RAS

Aut(V658F)

Aut(F958V)

Aut(F958V)

Aut(V658F)

M-RAS

H3-Thymidine incorporation

JAK1 F958V is resistant to INC424

P960T/ S F958V/ C/ S/ L ( JAK i nhi bi t or s)

JAK1 925 KLIMEFLPSGSLKEYLPK-NKN 973 .:|.||:..|:|.:||.: |:.

ABL1 312 YIITEFMTYGNLLDYLRECNRQ 333 T315I / A/ S ( I mat i ni b) E316D F317L/ I M318A G321V

Alignment of the human JAK1 and ABL protein sequences around hinge region

JAK1 and ABL1 hinge regions

JAK1, CP-690,500

(PDB 3EYG)JAK2, CP-690,500

(PDB 3FUP)

ABL1, Imatinib

(PDB 2HYY)

F958 Y931 F317

P960T/ S F958V/ C/ S/ L ( JAK i nhi bi t or s)

JAK1 925 KLIMEFLPSGSLKEYLPK-NKN 973 .:|.||:..|:|.:||.: |:.

ABL1 312 YIITEFMTYGNLLDYLRECNRQ 333 T315I / A/ S ( I mat i ni b) E316D F317L/ I M318A G321V

The hinge region in crystal structures of JAK1, JAK2, ABL1

JAK1 and ABL1 hinge regions

JAK1, CP-690,500

(PDB 3EYG)JAK2, CP-690,500

(PDB 3FUP)

ABL1, Imatinib

(PDB 2HYY)

F958 Y931 F317

P960T/ S F958V/ C/ S/ L ( JAK i nhi bi t or s)

JAK1 925 KLIMEFLPSGSLKEYLPK-NKN 973 .:|.||:..|:|.:||.: |:.

ABL1 312 YIITEFMTYGNLLDYLRECNRQ 333 T315I / A/ S ( I mat i ni b) E316D F317L/ I M318A G321V

The hinge region in crystal structures of JAK1, JAK2, ABL1

JAK1 and ABL1 hinge regions

BaF3 cells - H3-Thymidine incorporation

0

0.2

0.4

0.6

0.8

0 1 2 3 4

Days without IL-3

Auto

nom

ous p

rolif

era

tion (

cells

x10

6)

CT

WT

V617F

V617F

WT

The JAK2 Y931C mutation – autonomous proliferation

0

0.2

0.4

0.6

0.8

0 1 2 3 4

Days without IL-3

Auto

nom

ous p

rolif

era

tion (

cells

x10

6)

CT

WT

V617F

Y931CV617F

WT

Y931C

The JAK2 Y931C mutation – autonomous proliferation

BaF3 cells - H3-Thymidine incorporation

0

0.2

0.4

0.6

0.8

0 1 2 3 4

Days without IL-3

Auto

nom

ous p

rolif

era

tion (

cells

x10

6)

CT

WT

V617F

Y931C

V617F/Y931C

V617F

WT

Y931C

V617F/Y931

C

The JAK2 Y931C mutation – autonomous proliferation

BaF3 cells - H3-Thymidine incorporation

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10

INCB018424 (uM)

Pro

lifera

tion (

in %

of contr

ol)

V617F

The JAK2 Y931C mutation – INC424 response

BaF3 cells - H3-Thymidine incorporation

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10

INCB018424 (uM)

Pro

lifera

tion (

in %

of contr

ol)

V617F

Y931C

BaF3 cells - H3-Thymidine incorporation

The JAK2 Y931C mutation – INC424 response

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10

INCB018424 (uM)

Pro

lifera

tion (

in %

of contr

ol)

V617F/

Y931C

V617F

Y931C

BaF3 cells - H3-Thymidine incorporation

The JAK2 Y931C mutation – INC424 response

Perrine

Laurent

Muriel

Emilie

LaureMagali

Lorraine Thomas AstridIsabelle(s)

ReeceMonique

Jacques

Tekla

Jean-Christophe

+ SN Constantinescu