www.sciencetranslationalmedicine.org/cgi/content/full/4/120/120ra17/DC1

Supplementary Materials for

Mechanisms of Acquired Crizotinib Resistance in ALK-Rearranged Lung Cancers

Ryohei Katayama, Alice T. Shaw, Tahsin M. Khan, Mari Mino-Kenudson, Benjamin J.

Solomon, Balazs Halmos, Nicholas A. Jessop, John C. Wain, Alan Tien Yeo, Cyril Benes, Lisa Drew, Jamal Carlos Saeh, Katherine Crosby, Lecia V. Sequist, A. John

Iafrate, Jeffrey A. Engelman*

*To whom correspondence should be addressed. E-mail: jengelman@partners.org

Published 8 February 2012, Sci. Transl. Med. 4, 120ra17 (2012) DOI: 10.1126/scitranslmed.3003316

The PDF file includes:

Fig. S1. Identification of secondary resistance mutations within the ALK TK domain. Fig. S2. Characterization of secondary resistance mutations within the ALK TK domain. Fig. S3. Sensitivity of wild-type and mutated EML4-ALK to next-generation ALK inhibitors and the hsp90 inhibitor 17-AAG. Fig. S4. The secondary ALK 1151Tins mutation mediates crizotinib resistance in H3122 CR2 cells. Fig. S5. Sensitivity of crizotinib-resistant H3122 cell lines to next-generation ALK inhibitors and the hsp90 inhibitor 17-AAG. Fig. S6. EGFR activation in H3122 CR3 cells is due to up-regulation of EGFR, amphiregulin, and NRG1 mRNA. Fig. S7. Failure to up-regulate BIM mRNA in resistant H3122 CR3 cells. Fig. S8. EGFR activation in crizotinib-naïve, ALK-positive NSCLC. Fig. S9. Structural models of wild-type and mutated ALK kinase domain with ALK inhibitors. Tables S1 and S2. In vitro kinase assay data of ASP-3026.

1

Supplementary Figure Legends

Supplementary Figure S1. Identification of secondary resistance mutations within the

ALK TK domain. (A) Electrophoretograms of ALK gDNA (exon23) from 3 different

crizotinib-resistant tumors (upper panels). Each mutation was confirmed by subcloning into

bacteria and sequencing individual clones (lower panels). (B) Electrophoretogram of ALK

cDNA from a wild-type control sample (upper panel) and from a crizotinib-resistant tumor

(lower panel) showing an 1151T insertion mutation. （C） Absence of the L1196M mutation

prior to crizotinib therapy using a highly sensitive, allele-specific PCR assay. PCRs were

performed in 50µL reactions containing 30ng genomic DNA purified from H3122 and H3122

CR1 cells. Genomic DNAs purified from each cell line were mixed in the indicated ratios to

determine the detection limit of the assay (left panel). L1196M allele-specific PCR was then

performed using total nucleic acid from the resistant specimen MGH020 and the

corresponding sensitive (i.e., pre-crizotinib) sample (right panel). As a control, GAPDH

exon8 was PCR amplified.

Supplementary Figure S2. Characterization of secondary resistance mutations within

the ALK TK domain.

(A) Expression of wild-type and mutant forms of EML4-ALK in Ba/F3 cells. Ba/F3 cells were

2

transformed by stably expressing wild-type EML4-ALK or EML4-ALK harboring one of the

four identified mutations (L1196M, G1202R, S1206Y or 1151Tins). Cell lysates were

probed with ALK-specific antibodies as indicated. (B-E) Sensitivity of each ALK resistance

mutation to the indicated ALK inhibitors and the hsp90 inhibitor 17-AAG, as assessed in

Ba/F3 cells. Parental, IL-3 dependent Ba/F3 cells or transformed (IL-3 independent) Ba/F3

cells expressing wild-type or mutated EML4-ALK were treated with NVP-TAE684 (B),

CH5424802 (C), ASP3026 (D), or 17-AAG (E) at the indicated doses for 48 hrs. Cell

survival was measured using CellTiter-Glo. Each concentration was measured in sixplicate,

and the average and standard deviation are shown. (F) The average IC50s of each drug

across 6 different Ba/F3 cell lines, including parental, IL-3 dependent Ba/F3 cells as well as

transformed Ba/F3 cells expressing the indicated EML4-ALK constructs. Values shown are

the average of three independent experiments. For each drug tested, the number in

parentheses represents the ratio of the IC50 value shown relative to the IC50 in wild-type

EML4-ALK-expressing Ba/F3 cells.

generation ALK inhibitors and the hsp90 inhibitor 17-AAG. (A) Impact of next

generation ALK inhibitors on ALK phosphorylation in Ba/F3 cells expressing wild-type or

mutant EML4-ALK. Ba/F3 cells transformed by wildtype or mutant EML4-ALK were treated

Supplementary Figure S3. Sensitivity of wild-type and mutated EML4-ALK to next-

3

with NVP-TAE684, CH5424802 or ASP-3026 at the indicated concentrations for 1 hr.

Lysates were probed with the anti-ALK specific antibodies as shown. (B) IC50s of different

ALK inhibitors with either wild-type or mutant ALK, as determined by in vitro kinase assays

(see Methods). Shown are IC50 values for each drug across different ALK proteins (wild-

type, L1196M, G1202R, and S1206Y). The ratio compares the IC50 values of mutant

versus wild-type ALK for each drug. (C) Ba/F3 cells expressing wildtype or mutant EML4-

ALK were treated with 17-AAG at the indicated concentrations for 24 hrs. Lysates were

probed with the anti-ALK specific antibodies as shown.

Supplementary Figure S4. The secondary ALK 1151Tins mutation mediates crizotinib

resistance in H3122 CR2 cells. (A) Persistence of ALK activation and downstream

signaling in crizotinib-treated H3122 CR2 cells. Parental H3122, H3122 CR1, and H3122

CR2 cells were treated with crizotinib at the indicated doses for 6 hrs. Cell extracts were

immunoblotted to detect the proteins shown. (B) Quantitative PCR measuring Exon23 of

ALK were performed in triplicate using 30ng genomic DNA purified from H3122, H3122

CR1, CR2 and CR3 cells. (C) Electrophoretogram of EML4-ALK cDNA from H3122 CR2

cells showing the presence of a threonine insertion at residue 1151.

Supplementary Figure S5. Sensitivity of crizotinib-resistant H3122 cell lines to next-

4

crizotinib-resistant H3122 cells (CR1, CR2, and CR3), and control cell lines expressing

wild-type ALK (HCC827, PC9 and A549) were treated with NVP-TAE684 (A), CH5424802

(B), ASP-3026 (C), or 17-AAG (D) at the indicated doses for 72 hrs. Cell survival was

determined using CellTiter-Glo. Each concentration was measured in sixplicate, and the

average and standard deviation are shown.

of EGFR, amphiregulin, and NRG1 mRNA. (A-E) Quantitative RT-PCR comparing

parental H3122 and crizotinib-resistant H3122 CR3 cells. Cells were treated in the absence

or presence of 1 µM crizotinib for 6 hrs. Total RNA was extracted for cDNA synthesis

according to standard protocols. Quantitative PCR was performed to determine transcript

levels of EGFR (A), EGF (B), amphiregulin (C), ERBB3 (D), and NRG-1 (E). Measurements

were performed in triplicate. Gene expression is shown relative to actin mRNA.

cells. H3122 and H3122 CR3 cells were treated with vehicle control, 1 µM crizotinib, 2 µM

gefitinib, or the combination for 24 hrs. BIM and actin mRNA levels were measured by

quantitative PCR performed in triplicate. Shown are expression levels of BIM mRNA

generation ALK inhibitors and the hsp90 inhibitor 17-AAG. (A-D) Parental H3122 cells,

Supplementary Figure S6. EGFR activation in H3122 CR3 cells is due to up-regulation

Supplementary Figure S7. Failure to up-regulate BIM mRNA in resistant H3122 CR3

5

normalized to actin mRNA.

Supplementary Figure S8. EGFR activation in crizotinib-naïve, ALK-positive NSCLC.

(A) MGH006 cells were treated with increasing doses of crizotinib in the presence or

absence of 2 µM gefitinib or 1 µM erlotinib for 72 hrs. Cell survival was assayed using

CellTiter-Glo. Each concentration was measured in sixplicate, and the average and

standard deviation are shown. (B) MGH006 cells were treated with vehicle control, crizotinib

(1 µM), or with crizotinib and gefitinib (2 µM) for 6 hrs. Cell extracts were immunoblotted to

detect the proteins shown. (C) Enhanced apoptosis of MGH006 cells with combined

crizotinib and gefitinib treatment. MGH006 cells were treated with vehicle control, 1 µM

crizotinib, 2 µM gefitinib, or the combination. After 72 hrs, cells were stained with Alexa-633

labeled Annexin-V and PI, and analyzed by flow cytometry. The percentage of cells

undergoing apoptosis is shown for each treatment condition.

domain with ALK inhibitors.

(A) An overlay of three ALK co-crystal structures: crizotinib (2XP2, yellow stick), NVP-

TAE684 (2XB7, green stick) and CH5424802 (3AOX PBD, in blue stick). The sidechains

for the mutations discussed in this manuscript are highlighted in CPK representation. (B)

Supplementary Figure S9. Structural models of wild-type and mutated ALK kinase

6

Ribbon diagram of ALK kinase domain with CH5424802 (3AOX, PDB). Analysis of the

crystal structure of ALK reveals a critical hydrogen bonding interaction between the

backbone N-H of T1151 and the backbone carbonyl of E1129. The location of E1129 on the

P-Loop, and adjacent to the catalytic Lys1150, led Bossi et al (ref 42) to hypothesize that a

mutation around this amino-acid residue may de-stabilize the hydrogen bonding interaction

and may lead to changes in the affinity of ALK for ATP. (C) The co-crystal structure of

CH5424802 and ALK (3AOX, PDB) was used to generate the accessible surface (left). The

color reflects the electrostatic potential of the protein and was calculated using Vida 4.1.1.

The molecular surface of the minimized G1202R mutant is represented on the right.

Starting with the published structure of CH5424802 (PDB 3AOX), G1202R was modeled by

mutating G1202 and performing a minimization of the mutant using Prime Ver.3.0 using

default settings. The surface is colored by the electrostatic potential of the surface using

Vida 4.1.1 where blue is positively charged and red is negatively charged. The reduction in

potency of CH5424802 can be rationalized structurally by noting that G1202 is near the

solvent front. The mutation of a small glycine to a bulkier and highly basic arginine is

hypothesized to lead to steric clash with the inhibitors. Additionally, the presence of basic

functionalities in each of the ligands, including CH5424802, leads to non-complementary

electrostatic interactions.

7

ASP3026 was profiled against 451 kinases by KINOME scan (SanDiego, CA) using the

KINOMEscan screening, which utilizes DNA tagged recombinant kinase, ligand conjugated

beads, and three inhibitor concentrations of 10 nM, 100nM and 1 µM. After incubation,

wash and elution, the kinase concentration was measured by qPCR.

(http://www. kinomescan.com)

Supplementary Table S1 and S2. In vitro kinase assay data of ASP-3026.

Katayama  et  al,  Supplementary  Figure  S1

S1206Y

Exon

PC

R fr

om

geno

mic

DN

A

Subc

lone

d D

NA

Forward Reverse

G1202R

Forward Reverse

L1196M

Forward Reverse

A

mt

WT

Exon 22 Exon 21

1151 T insertion

B

C

Pre-

Criz

Post

Criz

wat

er

99.9% 99.7% H3122 pt 99% 99.97% 90%

H3122 CR1

0%

0.1% 0.3% 1% 0.03% 10% 100%

100%

0%

MGH020

L1196M specific

GAPDH (exon8)

0

50

100

0 10 100 1000 10000concentration of 17-AAG (nM)

Rela

tive

cell n

umbe

r (%

of c

ontro

l)

E WT 1151-T-ins L1196M G1202R S1206Y Ba/F3 + IL-3

B

0

50

100

0 1 10 100 1000 10000concentration of TAE684 (nM)

Relat

ive

cell n

umbe

r (%

of c

ontro

l)

WT 1151-T-ins L1196M G1202R S1206Y Ba/F3 + IL-3

0

50

100

0 1 10 100 1000 10000concentration of CH5424802 (nM)

Rel

ativ

e ce

ll nu

mbe

r (%

of c

ontro

l)

C WT 1151-T-ins L1196M G1202R S1206Y Ba/F3 + IL-3

D

0

50

100

0 10 100 1000 10000concentration of ASP3026 (nM)

Rela

tive

cell n

umbe

r (%

of c

ontro

l)

WT 1151-T-ins L1196M G1202R S1206Y Ba/F3 + IL-3

Katayama  et  al,  Supplementary  Figure  S2

F IC50 (nM)(fold > WT)

WT 1151T-ins L1196M G1202R S1206Y Ba/F3+IL3

26.5 431.8 399.7 242.4 120.8 755.7

(1) ( 16 x ) ( 15 x ) ( 9 x ) ( 5 x ) ( 29 x )

1.4 28.3 5.4 15.0 5.9 2389.0

(1) ( 20 x ) ( 4 x ) ( 11 x ) ( 4 x ) ( 1700 x )

10.4 165.7 101.8 596.7 28.0 715.7

(1) ( 16 x ) (10 x ) ( 57 x ) ( 3 x ) ( 69 x )

89.8 1755.0 584.2 390.3 205.5 5743.0

(1) ( 20 x ) ( 7 x ) ( 4 x ) ( 2 x ) ( 64 x )

98.9 44.5 92.1 33.4 64.9 183.7

(1) ( 0.4 x ) ( 0.9 x ) ( 0.3 x ) ( 0.7 x ) ( 1.9 x )17-AAG

ASP3026

CH5425802

TAE684

Crizotinib

A

WT

L119

6M

G12

02R

S120

6Y

1151

-T-in

s

Ba/

F3

pALK

ALK

Actin

Katayama  et  al,  Supplementary  Figure  S3

A

pALK ALK

pALK ALK

pALK ALK

pALK ALK

TAE684 (nM)

0 10

100

1000

CH5424802 (nM)

0 10

100

1000

pALK ALK 1151-T-ins.

WT

L1196M

G1202R

S1206Y

0 10

100

1000

ASP3026 (nM)

pALK ALK

pALK ALK WT

L1196M

pALK ALK

pALK ALK

G1202R

S1206Y

17AAG (nM)

0 10

100

1000

C

1151-T-ins. pALK

ALK Actin

Actin

Actin

Actin

Actin

B IC50 WT L1196M Ratio G1202R Ratio S1206Y Ratio

Crizotinib 0.003 0.019 7.440 0.010 4.160 0.009 3.480 CH5424802 0.002 0.008 3.435 0.018 7.826 0.013 5.696 ASP3026 0.009 0.030 3.471 0.028 3.306 0.026 3.012

Katayama  et  al,  Supplementary  Figure  S4

pALK

ALK

pERK pAKT

ERK AKT

1000

300

30

0 100

H3122 H3122 CR1

1000

300

30

0 100

1000

300

30

0 100

H3122 CR2 A

C

C G T

B

1

2

3

0

Mal

e no

rmal

H31

22

H31

22 C

R1

H31

22 C

R2

H31

22 C

R3

Rel

ativ

e PC

R p

rodu

cts

(ALK

/LIN

E-1)

0

50

100

0 1 10 100 1000 10000

TAE684 (H3122)

TAE684 (H3122 CR2)TAE684 (H3122 CR3)

TAE684 (H3122 CR1)

TAE684 (HCC827)TAE684 (A549)TAE684 (PC9)

concentration of TAE684 (nM)

rela

tive

cell

num

ber (

% o

f con

trol)

Katayama  et  al,  Supplementary  Figure  S5

A

0

50

100

0 1 10 100 1000 10000

CH5424802 (H3122)CH5424802 (H3122 CR1)CH5424802 (H3122 CR2)CH5424802 (H3122 CR3)CH5424802 (HCC827)CH5424802 (A549)CH5424802 (PC9)

concentration of CH5424802 (nM)

rela

tive

cell

num

ber (

% o

f con

trol)

B

0

50

100

0 1 10 100 1000 10000

17-AAG (H3122)17-AAG (H3122 CR1)17-AAG (H3122 CR2)17-AAG (H3122 CR3)17-AAG (HCC827)17-AAG (A549)17-AAG (PC9)

concentration of 17AAG (nM)

rela

tive

cell

num

ber (

% o

f con

trol)

D

0

50

100

0 10 100 1000 10000

ASP3026 (H3122)ASP3026 (H3122 CR1)ASP3026 (H3122 CR2)ASP3026 (H3122 CR3)ASP3026 (HCC827)ASP3026 (A549)ASP3026 (PC9)

concentration of ASP3026 (nM)

rela

tive

cell

num

ber (

% o

f con

trol)

C

Katayama  et  al,  Supplementary  Figure  S6

D

A

Crizotinib - + - +

Rel

ativ

e G

ene

Expr

essi

on

(NR

G1/

Act

in)

1

2

3

4

- + - +

1

2

3

4

E

Rel

ativ

e G

ene

Expr

essi

on

(EG

FR/A

ctin

) B

Rel

ativ

e G

ene

Expr

essi

on

(ER

BB

3/A

ctin

)

EGFR

Crizotinib

Rel

ativ

e G

ene

Expr

essi

on

(EG

F/A

ctin

)

H3122 H3122 CR3

H3122 H3122 CR3

1

2

3

Crizotinib - + - + H3122 H3122 CR3

1

2

3

- + - + Crizotinib H3122 H3122 CR3

Rel

ativ

e G

ene

Expr

essi

on

(Am

phre

gulin

/Act

in)

1

2

3

- + - + Crizotinib H3122 H3122 CR3

CEGF Amphiregulin

ERBB3 NRG1

0

1

2

3

4

Katayama  et  al,  Supplementary  Figure  S7

H3122 H3122 CR3

Rel

ativ

e m

RN

A ex

pres

sion

(B

im/A

ctin

)

Gefitinib Crizotinib + + -

+ + - - -

+ + - + + -

- -

Katayama  et  al,  Supplementary  Figure  S8

pALK

ALK

pERK

pEGFR

300

1000

30

0 100

gefitinib (1µM)

Crizotinib (nM): 300

1000

30

0 100

AKT

B A

0

50

100Crizotinib + erlotinib

0 10 100 1000 10000

CrizotinibCrizo+Gefitinib

concentration of crizotinib (nM)

Rel

ativ

e ce

ll nu

mbe

r (%

of c

ontr

ol)

pAKT

EGFR

ERK

Actin

C control Crizotinib Crizo + Gef Gefitinib

5% 42% 6% 68% PI

Annexin-V

Katayama  et  al,  Supplementary  Figure  S9

T1151

E1129

B A

S1206 G1202

T1151

L1196

S1206

G1202

C

E1210 S1206 D1203

L1197

Sol

vent

Hinge M1199

G1202 G1202R

E1210

S1206 D1203

L1197

Sol

vent

Hinge M1199

ALK (WT) with CH5424802 ALK (G1202R) with CH5424802

Kinase Target 10 nM 100 nM 1000 nM

ALK 12 0.7 0.95MKNK2 20 0.25 0BMPR1B 25 11 1.6TNK2 35 4 1.7ROS1 51 19 6ABL1(F317L)-phosphorylated 63 10 1.9FLT3(D835Y) 63 14 2.2ABL1(F317I)-phosphorylated 58 26 11LTK 83 13 0.55FLT3(D835H) 72 25 7.4SIK2 74 27 5.2TNK1 85 18 2.1SBK1 79 33 9.1EGFR(E746-A750del) 62 48 18FLT3(N841I) 76 39 12TEC 62 48 30EGFR(T790M) 90 37 5.3EGFR(L747-S752del, P753S) 68 52 18FRK 80 48 6.4BTK 99 35 1.2ABL1(F317L)-nonphosphorylated 100 38 14PLK4 95 43 4SRC 82 51 2.2DCAMKL1 69 56 25PYK2 94 53 10

Percent Control

% control0-11-910-3435-5960-100

Katayama  et  al,  Supplementary  Table  S1

In Vitro Kinase Assay Data of ASP3026*

*This data shows the top 25 kinase targets. The full data is shown in Supplementary Table S2.

ASP3026Entrez  Gene  Symbol

KINOMEscan  Gene  Symbol 10  nM 100  nM 1000  nM

AAK1 AAK1 83 74 36ABL1 ABL1(E255K)-­‐phosphorylated 100 80 27ABL1 ABL1(F317I)-­‐nonphosphorylated 92 58 13ABL1 ABL1(F317I)-­‐phosphorylated 58 26 11ABL1 ABL1(F317L)-­‐nonphosphorylated 100 38 14ABL1 ABL1(F317L)-­‐phosphorylated 63 10 1.9ABL1 ABL1(H396P)-­‐nonphosphorylated 100 100 15ABL1 ABL1(H396P)-­‐phosphorylated 100 78 14ABL1 ABL1(M351T)-­‐phosphorylated 100 100 11ABL1 ABL1(Q252H)-­‐nonphosphorylated 100 55 9.4ABL1 ABL1(Q252H)-­‐phosphorylated 100 70 13ABL1 ABL1(T315I)-­‐nonphosphorylated 100 100 68ABL1 ABL1(T315I)-­‐phosphorylated 90 76 26ABL1 ABL1(Y253F)-­‐phosphorylated 100 82 6.8ABL1 ABL1-­‐nonphosphorylated 92 73 12ABL1 ABL1-­‐phosphorylated 100 77 15ABL2 ABL2 100 100 45ACVR1 ACVR1 100 78 15ACVR1B ACVR1B 100 83 96ACVR2A ACVR2A 100 94 78ACVR2B ACVR2B 100 100 100ACVRL1 ACVRL1 100 83 34CABC1 ADCK3 100 90 100ADCK4 ADCK4 100 100 63AKT1 AKT1 84 100 81AKT2 AKT2 98 100 100AKT3 AKT3 100 100 100ALK ALK 12 0.7 0.95PRKAA1 AMPK-­‐alpha1 100 100 76PRKAA2 AMPK-­‐alpha2 98 100 100ANKK1 ANKK1 100 100 100NUAK1 ARK5 100 100 55MAP3K5 ASK1 92 100 100MAP3K6 ASK2 98 85 72AURKA AURKA 81 70 30AURKB AURKB 92 100 100AURKC AURKC 100 66 46AXL AXL 100 81 34BMP2K BIKE 100 84 89BLK BLK 87 71 6.2BMPR1A BMPR1A 78 47 58BMPR1B BMPR1B 25 11 1.6BMPR2 BMPR2 100 100 100BMX BMX 100 91 72BRAF BRAF 100 100 100BRAF BRAF(V600E) 100 100 78

Percent  Control

PTK6 BRK 89 78 16BRSK1 BRSK1 75 84 95BRSK2 BRSK2 98 50 94BTK BTK 99 35 1.2BUB1 BUB1 87 89 78CAMK1 CAMK1 78 83 70CAMK1D CAMK1D 97 100 95CAMK1G CAMK1G 98 100 96CAMK2A CAMK2A 98 100 70CAMK2B CAMK2B 100 100 76CAMK2D CAMK2D 93 96 47CAMK2G CAMK2G 100 100 80CAMK4 CAMK4 100 98 99CAMKK1 CAMKK1 100 100 79CAMKK2 CAMKK2 100 100 48CASK CASK 87 67 76CDK11B CDC2L1 96 79 100CDC2L2 CDC2L2 71 55 100CDK13 CDC2L5 100 79 87CDK19 CDK11 90 69 88CDK2 CDK2 100 100 100CDK3 CDK3 100 94 100CDK4 CDK4-­‐cyclinD1 80 91 94CDK4 CDK4-­‐cyclinD3 100 100 100CDK5 CDK5 76 79 100CDK7 CDK7 100 100 100CDK8 CDK8 93 100 88CDK9 CDK9 96 88 100CDKL1 CDKL1 90 100 100CDKL2 CDKL2 98 98 100CDKL3 CDKL3 100 97 100CDKL5 CDKL5 100 100 100CHEK1 CHEK1 91 78 89CHEK2 CHEK2 100 100 85CIT CIT 100 100 100CLK1 CLK1 100 100 81CLK2 CLK2 100 100 73CLK3 CLK3 100 100 100CLK4 CLK4 92 100 100CSF1R CSF1R 98 63 16CSF1R CSF1R-­‐autoinhibited 100 100 100CSK CSK 98 89 63CSNK1A1 CSNK1A1 86 95 92CSNK1A1L CSNK1A1L 100 93 100CSNK1D CSNK1D 59 56 100CSNK1E CSNK1E 100 100 100CSNK1G1 CSNK1G1 100 85 100CSNK1G2 CSNK1G2 92 92 100CSNK1G3 CSNK1G3 100 100 100

CSNK2A1 CSNK2A1 100 100 97CSNK2A2 CSNK2A2 86 100 100MATK CTK 100 93 85DAPK1 DAPK1 89 80 96DAPK2 DAPK2 100 100 100DAPK3 DAPK3 98 100 94DCLK1 DCAMKL1 69 56 25DCLK2 DCAMKL2 90 94 85DCLK3 DCAMKL3 100 80 40DDR1 DDR1 90 73 26DDR2 DDR2 100 89 54MAP3K12 DLK 88 66 50DMPK DMPK 100 100 100CDC42BPG DMPK2 93 87 100STK17A DRAK1 100 92 100STK17B DRAK2 100 98 100DYRK1A DYRK1A 100 100 100DYRK1B DYRK1B 100 96 100DYRK2 DYRK2 97 74 64EGFR EGFR 95 89 32EGFR EGFR(E746-­‐A750del) 62 48 18EGFR EGFR(G719C) 88 54 42EGFR EGFR(G719S) 83 76 43EGFR EGFR(L747-­‐E749del,  A750P) 90 74 18EGFR EGFR(L747-­‐S752del,  P753S) 68 52 18EGFR EGFR(L747-­‐T751del,Sins) 80 60 23EGFR EGFR(L858R) 93 74 26EGFR EGFR(L858R,T790M) 100 84 40EGFR EGFR(L861Q) 75 68 22EGFR EGFR(S752-­‐I759del) 81 81 47EGFR EGFR(T790M) 90 37 5.3EIF2AK1 EIF2AK1 91 94 100EPHA1 EPHA1 99 59 19EPHA2 EPHA2 100 100 74EPHA3 EPHA3 87 100 91EPHA4 EPHA4 75 68 58EPHA5 EPHA5 100 100 100EPHA6 EPHA6 95 98 100EPHA7 EPHA7 100 100 100EPHA8 EPHA8 100 100 100EPHB1 EPHB1 100 97 36EPHB2 EPHB2 100 92 100EPHB3 EPHB3 92 94 76EPHB4 EPHB4 100 100 75EPHB6 EPHB6 99 85 46ERBB2 ERBB2 100 100 79ERBB3 ERBB3 100 100 58ERBB4 ERBB4 100 100 40MAPK3 ERK1 100 100 100

MAPK1 ERK2 94 100 100MAPK6 ERK3 96 86 100MAPK4 ERK4 99 92 100MAPK7 ERK5 100 84 95MAPK15 ERK8 100 98 100ERN1 ERN1 100 100 78PTK2 FAK 94 67 3.8FER FER 94 84 20FES FES 98 96 44FGFR1 FGFR1 100 85 80FGFR2 FGFR2 100 97 93FGFR3 FGFR3 100 89 100FGFR3 FGFR3(G697C) 100 86 100FGFR4 FGFR4 100 100 100FGR FGR 91 92 32FLT1 FLT1 99 92 100FLT3 FLT3 100 76 23FLT3 FLT3(D835H) 72 25 7.4FLT3 FLT3(D835Y) 63 14 2.2FLT3 FLT3(ITD) 97 85 32FLT3 FLT3(K663Q) 100 84 25FLT3 FLT3(N841I) 76 39 12FLT3 FLT3(R834Q) 93 80 49FLT3 FLT3-­‐autoinhibited 100 100 100FLT4 FLT4 92 91 90FRK FRK 80 48 6.4FYN FYN 93 96 68GAK GAK 99 91 14EIF2AK4 GCN2(Kin.Dom.2,S808G) 91 100 100GRK1 GRK1 100 100 93GRK4 GRK4 100 100 100GRK7 GRK7 77 76 88GSK3A GSK3A 78 100 100GSK3B GSK3B 100 100 100GSG2 HASPIN 94 92 93HCK HCK 87 85 17HIPK1 HIPK1 83 100 97HIPK2 HIPK2 78 81 74HIPK3 HIPK3 76 68 67HIPK4 HIPK4 92 100 70MAP4K1 HPK1 94 53 65HUNK HUNK 100 74 87ICK ICK 100 100 100IGF1R IGF1R 100 96 54CHUK IKK-­‐alpha 100 100 100IKBKB IKK-­‐beta 100 100 100IKBKE IKK-­‐epsilon 72 65 51INSR INSR 91 83 33INSRR INSRR 86 66 84

IRAK1 IRAK1 68 71 37IRAK3 IRAK3 92 90 58IRAK4 IRAK4 82 87 75ITK ITK 100 100 100JAK1 JAK1(JH1domain-­‐catalytic) 95 100 100JAK1 JAK1(JH2domain-­‐pseudokinase) 100 100 100JAK2 JAK2(JH1domain-­‐catalytic) 83 74 58JAK3 JAK3(JH1domain-­‐catalytic) 100 90 76MAPK8 JNK1 82 91 100MAPK9 JNK2 84 91 100MAPK10 JNK3 90 97 96KIT KIT 93 85 32KIT KIT(A829P) 100 100 100KIT KIT(D816H) 100 89 69KIT KIT(D816V) 100 82 23KIT KIT(L576P) 77 88 19KIT KIT(V559D) 90 81 24KIT KIT(V559D,T670I) 95 88 77KIT KIT(V559D,V654A) 100 94 100KIT KIT-­‐autoinhibited 100 100 93LATS1 LATS1 100 100 100LATS2 LATS2 100 93 83LCK LCK 100 65 7LIMK1 LIMK1 100 100 100LIMK2 LIMK2 100 78 55STK11 LKB1 100 100 100STK10 LOK 100 99 27LRRK2 LRRK2 100 100 65LRRK2 LRRK2(G2019S) 100 100 55LTK LTK 83 13 0.55LYN LYN 100 100 65MAP3K13 LZK 100 55 53MAK MAK 70 74 89MAP3K1 MAP3K1 100 100 100MAP3K15 MAP3K15 100 100 69MAP3K2 MAP3K2 100 100 94MAP3K3 MAP3K3 93 100 100MAP3K4 MAP3K4 96 99 100MAP4K2 MAP4K2 100 100 94MAP4K3 MAP4K3 100 100 79MAP4K4 MAP4K4 100 100 100MAP4K5 MAP4K5 100 100 100MAPKAPK2 MAPKAPK2 100 100 100MAPKAPK5 MAPKAPK5 100 100 100MARK1 MARK1 100 80 80MARK2 MARK2 46 42 53MARK3 MARK3 100 100 88MARK4 MARK4 100 78 69MAST1 MAST1 97 89 49

MAP2K1 MEK1 86 89 89MAP2K2 MEK2 100 92 99MAP2K3 MEK3 74 78 75MAP2K4 MEK4 100 100 86MAP2K5 MEK5 100 100 100MAP2K6 MEK6 96 86 92MELK MELK 100 100 100MERTK MERTK 83 100 44MET MET 100 100 76MET MET(M1250T) 100 100 60MET MET(Y1235D) 98 91 76MINK1 MINK 100 100 100MAP2K7 MKK7 100 94 96MKNK1 MKNK1 100 86 10MKNK2 MKNK2 20 0.25 0MYLK3 MLCK 100 100 100MAP3K9 MLK1 100 100 100MAP3K10 MLK2 85 86 82MAP3K11 MLK3 100 100 100CDC42BPA MRCKA 100 100 100CDC42BPB MRCKB 100 100 100STK4 MST1 100 100 100MST1R MST1R 94 100 100STK3 MST2 98 99 100STK24 MST3 99 100 100MST4 MST4 100 72 50MTOR MTOR 100 87 95MUSK MUSK 100 85 100MYLK MYLK 100 100 98MYLK2 MYLK2 100 96 100MYLK4 MYLK4 99 100 100MYO3A MYO3A 91 100 100MYO3B MYO3B 100 100 78STK38 NDR1 100 100 100STK38L NDR2 100 97 100NEK1 NEK1 100 93 100NEK11 NEK11 100 100 98NEK2 NEK2 100 100 100NEK3 NEK3 100 90 100NEK4 NEK4 100 93 100NEK5 NEK5 89 86 89NEK6 NEK6 100 100 100NEK7 NEK7 100 43 100NEK9 NEK9 100 86 100MGC42105 NIM1 100 100 100NLK NLK 99 99 96OXSR1 OSR1 100 100 74MAPK14 p38-­‐alpha 93 83 100MAPK11 p38-­‐beta 100 86 100

MAPK13 p38-­‐delta 81 100 100MAPK12 p38-­‐gamma 89 100 93PAK1 PAK1 99 100 100PAK2 PAK2 100 95 100PAK3 PAK3 100 89 65PAK4 PAK4 95 93 100PAK6 PAK6 100 100 100PAK7 PAK7 89 100 93CDK16 PCTK1 100 90 100CDK17 PCTK2 95 91 100CDK18 PCTK3 97 91 100PDGFRA PDGFRA 100 100 93PDGFRB PDGFRB 92 84 39PDPK1 PDPK1 99 96 83CDPK1 PFCDPK1(P.falciparum) 100 100 100MAL13P1.279 PFPK5(P.falciparum) 100 100 73CDK15 PFTAIRE2 100 93 100CDK14 PFTK1 96 96 100PHKG1 PHKG1 91 94 44PHKG2 PHKG2 100 100 75PIK3C2B PIK3C2B 100 72 84PIK3C2G PIK3C2G 100 100 100PIK3CA PIK3CA 100 100 100PIK3CA PIK3CA(C420R) 100 100 100PIK3CA PIK3CA(E542K) 98 100 100PIK3CA PIK3CA(E545A) 100 92 100PIK3CA PIK3CA(E545K) 94 81 97PIK3CA PIK3CA(H1047L) 99 100 100PIK3CA PIK3CA(H1047Y) 90 94 93PIK3CA PIK3CA(I800L) 92 91 90PIK3CA PIK3CA(M1043I) 100 100 100PIK3CA PIK3CA(Q546K) 100 95 100PIK3CB PIK3CB 97 100 100PIK3CD PIK3CD 83 86 100PIK3CG PIK3CG 88 87 94PI4KB PIK4CB 100 100 100PIM1 PIM1 100 100 100PIM2 PIM2 79 84 64PIM3 PIM3 89 77 100PIP5K1A PIP5K1A 92 100 100PIP5K1C PIP5K1C 100 100 100PIP4K2B PIP5K2B 97 90 100PIP4K2C PIP5K2C 100 100 67PRKACA PKAC-­‐alpha 100 93 93PRKACB PKAC-­‐beta 89 94 100PKMYT1 PKMYT1 100 96 82PKN1 PKN1 100 100 100PKN2 PKN2 72 48 63pknB PKNB(M.tuberculosis) 100 100 100

PLK1 PLK1 91 94 69PLK2 PLK2 98 100 93PLK3 PLK3 98 100 61PLK4 PLK4 95 43 4PRKCD PRKCD 97 81 95PRKCE PRKCE 100 83 100PRKCH PRKCH 100 92 100PRKCI PRKCI 99 100 100PRKCQ PRKCQ 100 100 100PRKD1 PRKD1 100 92 72PRKD2 PRKD2 94 100 91PRKD3 PRKD3 93 89 98PRKG1 PRKG1 97 100 100PRKG2 PRKG2 100 100 100EIF2AK2 PRKR 100 100 97PRKX PRKX 100 87 100PRPF4B PRP4 100 100 100PTK2B PYK2 94 53 10KIAA0999 QSK 100 100 85RAF1 RAF1 100 100 100RET RET 100 100 100RET RET(M918T) 89 100 100RET RET(V804L) 93 91 99RET RET(V804M) 100 100 100RIOK1 RIOK1 97 92 100RIOK2 RIOK2 100 100 100RIOK3 RIOK3 70 56 100RIPK1 RIPK1 93 88 94RIPK2 RIPK2 58 45 87RIPK4 RIPK4 100 90 96DSTYK RIPK5 85 82 60ROCK1 ROCK1 93 62 82ROCK2 ROCK2 87 66 80ROS1 ROS1 51 19 6RPS6KA4 RPS6KA4(Kin.Dom.1-­‐N-­‐terminal) 96 91 89RPS6KA4 RPS6KA4(Kin.Dom.2-­‐C-­‐terminal) 100 100 100RPS6KA5 RPS6KA5(Kin.Dom.1-­‐N-­‐terminal) 95 100 100RPS6KA5 RPS6KA5(Kin.Dom.2-­‐C-­‐terminal) 100 100 100RPS6KA1 RSK1(Kin.Dom.1-­‐N-­‐terminal) 95 99 100RPS6KA1 RSK1(Kin.Dom.2-­‐C-­‐terminal) 99 98 76RPS6KA3 RSK2(Kin.Dom.1-­‐N-­‐terminal) 100 92 65RPS6KA3 RSK2(Kin.Dom.2-­‐C-­‐terminal) 99 100 90RPS6KA2 RSK3(Kin.Dom.1-­‐N-­‐terminal) 100 100 100RPS6KA2 RSK3(Kin.Dom.2-­‐C-­‐terminal) 93 100 98RPS6KA6 RSK4(Kin.Dom.1-­‐N-­‐terminal) 99 86 88RPS6KA6 RSK4(Kin.Dom.2-­‐C-­‐terminal) 98 98 54RPS6KB1 S6K1 85 63 14SBK1 SBK1 79 33 9.1SGK1 SGK 100 96 100

SgK110 SgK110 100 100 55SGK3 SGK3 100 100 90SIK1 SIK 100 85 22SIK2 SIK2 74 27 5.2SLK SLK 95 77 9.8NUAK2 SNARK 100 98 24SNRK SNRK 88 92 100SRC SRC 82 51 2.2SRMS SRMS 96 91 40SRPK1 SRPK1 76 75 100SRPK2 SRPK2 100 100 100SRPK3 SRPK3 100 100 100STK16 STK16 100 100 100STK33 STK33 93 91 32STK35 STK35 100 100 100STK36 STK36 100 100 96STK39 STK39 100 100 82SYK SYK 100 100 100MAP3K7 TAK1 96 91 80TAOK1 TAOK1 100 100 88TAOK2 TAOK2 100 100 100TAOK3 TAOK3 100 100 100TBK1 TBK1 63 56 52TEC TEC 62 48 30TESK1 TESK1 95 92 100TGFBR1 TGFBR1 100 100 100TGFBR2 TGFBR2 97 77 100TIE1 TIE1 97 72 30TEK TIE2 100 87 57TLK1 TLK1 96 93 97TLK2 TLK2 98 100 100TNIK TNIK 76 100 100TNK1 TNK1 85 18 2.1TNK2 TNK2 35 4 1.7TNNI3K TNNI3K 100 100 95NTRK1 TRKA 95 97 100NTRK2 TRKB 96 100 100NTRK3 TRKC 71 79 96TRPM6 TRPM6 100 100 100TSSK1B TSSK1B 77 100 83TTK TTK 100 100 53TXK TXK 99 69 60TYK2 TYK2(JH1domain-­‐catalytic) 100 100 100TYK2 TYK2(JH2domain-­‐pseudokinase) 100 77 12TYRO3 TYRO3 96 80 86ULK1 ULK1 100 76 9.8ULK2 ULK2 100 90 20ULK3 ULK3 93 100 94KDR VEGFR2 100 100 100

VRK2 VRK2 100 98 85WEE1 WEE1 100 100 100WEE2 WEE2 100 100 100WNK1 WNK1 94 94 100WNK3 WNK3 95 100 100STK32A YANK1 100 100 43STK32B YANK2 100 75 27STK32C YANK3 94 100 79YES1 YES 100 67 18STK25 YSK1 100 91 88YSK4 YSK4 100 100 100ZAK ZAK 92 89 100ZAP70 ZAP70 97 95 99