SUPPLEMENTAL DATA
JAK1, JAK2, and JAK3 in vitro enzyme assay
As part of a broader kinase selectivity profiling effort, the JAK LanthaScreen™ Kinase
Assay (Life Technologies, Grand Island, NY, USA) was used with a time-resolved fluorescence
resonance energy transfer (TR-FRET) format to assess the JAK1, JAK2, and JAK3 inhibitory
activity (IC50) of LY2784544, Compound 1, and other clinical JAK inhibitors. The assay used
20 μM adenosine triphosphate (ATP) with14 nM JAK1, 5 μM ATP with 1nM JAK2, or 2 μM
ATP with 2.5nM JAK3 enzymes, and with serial (1:3) dilutions of inhibitors from 20 000 to 1
nM. LY2784544 showed potent inhibitory activity with all three JAK kinases tested; the IC50
values were 19.8 nM for JAK1, 2.52 nM for JAK2, and 48.0 nM for JAK3, indicating selectivity
for JAK2 over JAK1 and JAK3 (8- and 16-fold, respectively; Supplemental Table 1). These
results are consistent with the cellular selectivity of LY2784544 for JAK2 over JAK1 and JAK3.
LY2784544 displayed modest selectivity for JAK2 over the other JAK family kinases,
JAK1 and JAK3 (8- and 19-fold, respectively; Supplemental Table 1).
Biochemical assessment of LY2784544 inhibitory activity against a panel of
additional kinases
In addition to JAK1, JAK2, and JAK3, 99 kinases were assayed to assess LY2784544
inhibitory activity using standard kinase inhibition profiling as performed by Cerep (Redmond,
WA). The inhibitory effect of LY2784544 on each kinase was determined by measuring
phosphorylation of a substrate by a human recombinant enzyme and the LANCE® detection
method (PerkinElmer, Waltham, MA, USA). The IC50 value was calculated from the 10-point
inhibitor (20 to 0.001 μM) concentration data using a four-parameter logistic equation. Eleven
1
kinases (in addition to JAK1, JAK2, and JAK3) were found to be inhibited by LY2784544 with
an IC50 ≤0.3 µM (Supplemental Table 2).
Cellomics pSTAT5 assay of JAK2V617F-expressing cells
To confirm the potency of LY2784544 in JAKV617F-mediated malignancy models, the
Cellomics pSTAT5 assay was used as it had been with TF-1 and NK-92 cells. (See Methods
section of main paper.) Murine pro-B-cells (Ba/F3) expressing human JAK2V617F were placed
in RPMI-1640 (2x105 cells/96-well) containing vehicle alone (DMSO) or test inhibitors (0.00120 μM). After 60 minutes, cells were stained for phosphorylated STAT5. Ba/F3 cells expressing
human wild-type JAK2 were handled similarly, except inhibitor (2x concentration) was added
for a 10 minutes pre-incubation, followed by addition of IL-3 in a volume that yielded a
concentration of 2 ng/mL and an inhibitor concentration of 1x. LY2784544 showed much more
potent activity against JAK2V617F signaling (IC50=0.055µM) than its inhibition of IL-3-wildtype JAK2-STAT5 signaling (IC50=2.26 µM) (Supplemental Table 3). In contrast, ruxolitinib
was not only potent at inhibiting JAK2V617F signaling (IC50=0.078 µM) but also inhibited IL-3
wild-type JAK2-STAT5 signaling at lower concentrations (IC50=0.529 µM). SAR302503
inhibited IL-3-stimulated wild-type JAK2-dependent signaling at higher concentrations
(IC50=1.999 μM), and was also less potent at inhibiting JAK2V617F compared with LY2784544
(IC50=0.298 μM).
Caspase-Glo® 3/7 apoptosis assay
To investigate the anti-proliferative effect of LY2784544, Caspase-3/7 activity was
measured as an early indicator of apoptosis using a Caspase-Glo® 3/7 Assay Kit (Promega,
Madison, WI, USA). Ba/F3 cells expressing JAK2V617F (1x104 cells/96-well) in RPMI-1640
containing 10% FBS were incubated for 18 hours with vehicle alone (DMSO) or a test inhibitor
2
(concentration range, 0.001-20 μM). After addition of Caspase-Glo 3/7 reagent, luminescence
was measured by a Wallac Victor Plate Reader (PerkinElmer, Waltham, MA, USA). The half
maximal effective concentration (EC50) for induction of Caspase-3/7 was calculated using the
GraphPad Prism 4 software (GraphPad Software, Inc., La Jolla, CA, USA). As seen in
Supplemental Table 3 and Supplemental Figure 1, LY2784544 effectively induced apoptosis
(EC50=0.113 µM at 18 hours post treatment). These results suggest that the anti-proliferative
effect of LY2784544 in Ba/F3 cells expressing JAK2V617F is mediated through the
pharmacological inhibition of JAK2V617F-STAT5 pathway and induction of apoptosis. These
results further indicate that LY2784544 inhibits JAK2V617F-mediated hematological
malignancy with little effect on IL-3 wild-type JAK2-mediated STAT5 signaling and cell
growth.
Purification and autophosphorylation of JAK2-Flag proteins
To confirm the constitutive activation of JAK2V617F full-length proteins in vitro, wildtype JAK2-Flag or mutant JAK2V617F-Flag proteins were expressed and purified using the Bacto-Bac Baculovirus Expression Vector System (Invitrogen, Carlsbad, CA, USA) in insect cells
(Sf9 cells, Invitrogen). The purified proteins were separated by electrophoresis (10% SDSPAGE), transferred to nitrocellulose, and incubated with antibodies against either total JAK2
(Cell Signaling #3230; 1:1000) or phosphorylated JAK2 proteins (Upstate #07-606; 1:1000),
then visualized with a fluorescence-labeled secondary antibody. Supplemental Figure 2 shows
comparable quantities of JAK2 protein in both the JAK2 and mutant JAK2V617F (row 1) or
phosphorylated JAK2 (pJAK2, Y1007/1008) (row 2). As shown by the darker band, mutant
JAK2 proteins were autophosphorylated at Y1007/1008, as were purified wild-type JAK2
proteins, although to a lesser extent.
3
KI and KM determinations for JAK2
To understand the mechanism of action of the LY2784544 selectivity against
JAK2V617F over wild-type JAK2 as assessed by STAT5 signaling, the LY2784544 inhibitorbinding constant (KI) values were determined in wild-type JAK2 (full length and the catalytic
domain) and mutant JAK2V617F (full length) purified from insect cells engineered to express
them. The KI values of SAR302503 and ruxolitinib were also determined. The biochemical assay
used a fluorescent peptide substrate Omnia® Y12 (Life Technologies). The initial linear rate of
enzyme activity was assessed by measuring fluorescence (ex/em=360/485 nm) every 2 minutes
for 1-3 hours on an Analyst® GT multimode reader (Molecular Devices Corporation, Sunnyvale,
CA, USA) in a low volume 384-well plate. The KM values of substrate and ATP were
determined using concentrations of substrate or ATP that ranged from 0.05x to 100x KM. The KI
values of the inhibitors were determined by evaluating the initial linear rate of enzyme activity in
a matrix of eight ATP and 10 inhibitor concentrations above and below their respective KM or KI.
The substrate concentration was fixed at its KM. The resulting rates were fit to a noncompetitive
model of enzyme inhibition containing KI and KI' terms.1 The KI' values reported here were
infinite and consistent with ATP competition, with no evidence of tight-binding inhibition that
would have limited the quality of the potency measurement.
As seen in Supplemental Table 4, the JAK2 inhibitors did not show differential effects on
KI values for either wild-type JAK2 (truncated catalytic domain or full length) or JAK2V617F.
The lack of biochemical selectivity of the three inhibitors for JAK2V617F prompted an
investigation of the impact of IL-3 receptor alpha (IL3-Rα) on the KI values. For other kinases,
the addition of physiologically relevant binding proteins has produced significant changes to
both substrate-binding (KM) and inhibitor-binding (KI) constants.2 JAK2 has been shown to pre-
4
associate with IL-3Rα prior to the activation of downstream signaling molecules.3 Thus, the
JAK2/IL-3Rα complex was thought to represent a more physiologically relevant enzyme
complex to evaluate in the biochemical setting. The KI determinations were repeated using the
same assay system with the addition of recombinant human IL-3Rα (100 nM) (R&D Systems,
Minneapolis, MN, USA).
The addition of IL-3Rα did not produce change in the KM of substrate for JAK2V617F;
however, there was a concentration-dependent decrease in the KM of substrate for JAK2, with
50-250 nM IL-3Rα sufficient to produce a maximal change in the KM (data not shown). There
was no significant change in the KM of ATP for either JAK2 or JAK2V617F (data not shown).
The KI values of three of the JAK inhibitors were then determined in JAK2 and JAK2V617F
biochemical assays containing 100 nM IL3-Rα (Supplemental Table 4).
Additional Methods
Some results presented in the main paper utilized relatively standard methods, the details
of which are included here.
Cell cultures
TF-1 cells (ATCC, Manassas, VA, USA) were maintained in RPMI-1640 medium with
10% FBS (Life Technologies) and granulocyte-macrophage colony-stimulating factor (GM-CSF)
(2 ng/mL) (Thermo Fisher Scientific, Rockford, IL, USA). NK-92 cells (ATCC) were
maintained in the MEM Alpha medium with 15% FBS, 15% horse serum (Life Technologies),
and interleukin-2 (IL-2) (4 ng/mL) (R&D Systems, Minneapolis, MN).
JAK2-expressing cell lines were created by transducing Ba/F3 cells with a retrovirusexpressing human wild-type or mutant (V617F) JAK2 cDNA controlled by the viral 5'-long
terminal repeat. The expression of green fluorescent protein (GFP) was controlled by the
5
phosphoglycerate kinase (PGK) promoter (MSCV6 wild-type JAK2-PGK/GFP or MSCV6
JAK2V617F-PGK/GFP). Single-cell cloning by serial dilution was performed to select Ba/F3
cells expressing the highest levels of JAK2V617F. Ba/F3 cells expressing JAK2V617F were
maintained in RPMI-1640; wild-type JAK2-expressing Ba/F3 cells were maintained similarly,
but with the addition of murine IL-3 (2 ng/mL) (R&D Systems). Ba/F3 human TEL-JAK1, TELJAK2, and TEL-JAK3 cell lines were kindly provided by Dr. Ross Levine (Brigham and
Women’s Hospital, Boston, MA) and maintained in RPMI-1640. HEK293T cells, grown in
DMEM/F12 (3:1) containing 10% FBS, were transiently transfected with JAK2 constructs using
1µg DNA/5µL FugeneHD (Promega, Madison, WI, USA) for 36 hours.
Western blotting
Cell lystates (10 µg) of proteins were resolved in 4%-20% BioRad Criterion precast gels (Life
Science, Hercules, CA, USA), transferred to nitrocellulose, incubated with antibodies against
phosphorylated STAT5 or total STAT5 proteins (BD 611819, and BD 610192 [BD Bioscience,
San Jose, CA, USA]) followed by then exposed to a secondary antibody, then visualization with
Supersignal West Femto (Pierce Biotechnology, Rockford, IL, USA) and scanning (Fuji camera
system LAS 4000).
Immunotoxicology analysis
Flow cytometric analysis was used to characterize the relative percentages and absolute
number of total B-cells (CD45RA+; sIgM+), total T-cells (CD3+; pan-T+), helper T-cells
(CD3+CD4+), cytotoxic/suppressor T-cells (CD3+CD8+), natural killer cells (CD161a+), and
the CD4:CD8 ratio from peripheral blood, spleen, and thymus of male and female SpragueDawley rats either untreated or dosed by gavage with Compound 1 (15 mg/kg) or Compound 2
(LY2784544) (30 mg/kg) for 14 days. All animal studies were performed according to American
6
Association for Laboratory Animal Care institutional guidelines and approved by the Eli Lilly
and Company Animal Care and Use Committee.
Cell proliferation assay.
Ba/F3 cells expressing JAK2V617F were placed in RPMI-1640-containing vehicle
(DMSO) or a test compound (concentration range, 0.001-20 μM) (1x104 cells/96-well). Ba/F3
cells expressing wild-type JAK2 were treated similarly except IL-3 (2 ng/mL) was added. After a
72-hour incubation, cell proliferation was assessed by adding Cell Titer 96 Aqueous One
Solution Reagent (20 μL/well)(Promega) according to manufacturer’s specifications. The IC50
for inhibition of cell proliferation was calculated using the GraphPad Prism 4 software
(GraphPad Software, Inc., La Jolla, CA, USA).
Immunohistochemistry and imaging analysis
Spleen sections (3 µm) from the 14-day response study mice were immunolabeled with a GFP
antibody to detect Ba/F3-JAK2V617F-GFP tumor cells, followed by a rabbit anti-GFP
polyclonal antibody (1 µg/mL) (Abcam, Cambridge, UK). Rabbit immunoglobulin G (IgG; 1
µg/mL) (Invitrogen, Carlsbad, CA, USA) was used as a negative control. All immunolabeled
slides were lightly counterstained with hematoxylin. Hematoxylin-eosin–stained and GFPimmunostained tissue sections were digitally scanned (20x magnification) on Aperio ScanScope
XT (Aperio Technologies, Inc., Vista, CA, USA), and the GFP signal was analyzed by the
Aperio Positive Pixel Count Algorithm (version 9.1). The percentage of the splenic area
occupied by GFP-positive BaF3 cells was determined by adding pixels classified as “positive”
and “strong” and dividing by the total of all positive and negative pixels.
7
References for Supplemental Materials
1. Copeland RA. Enzymes: A Practical Introduction to Structure, Mechanism, and Data
Analysis, 2nd Edition. Hoboken, NJ: Wiley-VCH, Inc.; 2000.
2. Anderson K, Yang J, Koretke K, Nurse K, Calamari A, Kirkpatrick RB, et al. Binding of
TPX2 to Aurora A alters substrate and inhibitor interactions. Biochemistry 2007; 46: 1028710295.
3. Huang HM, Lin YL, Chen CH, Chang TW. Simultaneous activation of JAK1 and JAK2
confers IL-3 independent growth on Ba/F3 pro-B cells. J Cell Biochem 2005; 96: 361-375.
8
Supplemental Table 1. Biochemical data for JAK inhibitors using JAK LanthaScreen™
Kinase Assay with TR-FRET format.
Inhibitors
JAK1
IC50, nM
JAK2
IC50, nM
JAK3
IC50, nM
Compound 1
1.65 ± 0.8
n=3/6a
1.70
n=1/6a
8.0 ± 5.9
n=6
LY2784544
19.8 ± 3.0
n=4
2.52 ± 0.49
n=4
48.0 ± 14.9
n=4
AZD1480
3.38 ± 1.49
n=4
1.26 ± 0.20
n=4
17.5 ± 3.1
n=4
SAR302503
32.7 ± 7.0
n=5
4.22 ± 0.93
n=7
345 ± 114
n=7
Ruxolitinib
<1.00 ± 0
n=5
1.00 ± 0.00
n=6
16.9 ± 12.9
n=6
42.3 ± 39.9
n=4
4.86 ± 2.67
n=6
61.1 ± 39.3
n=6
CYT387
Abbreviations: IC50=half maximal inhibitory concentration; JAK=janus kinase; TR-FRET=time-resolved
fluorescence resonance energy transfer.
a
n=number of experiments (test runs). Results for some runs were less than 0 (compound concentration was too
great); thus, results are reported only from runs for which an IC 50 could be determined.
9
Supplemental Table 2. Biochemical kinase profiling for LY2784544.
Kinasesa
IC50, µM
FLT3
0.004
FLT4
0.025
FGFR2
0.032
TYK2
0.044
TRKB
0.095
FGFR3
0.106
KDR
0.109
ALK
0.138
MUSK
0.147
AURKA
0.168
MAP3K9
0.299
Abbreviation: IC50=half maximal inhibitory concentration.
a
Only kinases with IC50≤0.3 µM are shown.
10
Supplemental Table 3. Inhibition of JAK2-mediated signaling and induction of apoptosis in
Ba/F3 cells expressing JAK2V617F.
pSTAT5 Cellomics Assays
Clinical JAK2
inhibitorsa
Caspase3/7 Assay
V617F
IC50 ± SD, µM
WT + IL-3
IC50 ± SD, µM
V617F
EC50 ± SD, µM
LY2784544
0.055 ± 0.0207
n=9
2.26 ± 0.704
n=5
0.113 ± 0.023
n=2
SAR302503
0.298 ± 0.301
n=5
1.999 ± 0.30
n=2
Ruxolitinib
0.078 ± 0.074
n=3
0.529 ± 0.030
n=2
Abbreviations: EC50= maximal effective concentration; IC50=half maximal inhibitory concentration; IL=interleukin;
JAK=Janus Kinase; n=number of experiments; SD=standard deviation; STAT5=signal transducer and activator of
transcription 5; WT=wild-type.
a
LY2784544 and SAR302503 (JAK2-selective inhibitors), as well as ruxolitinib (JAK1/2 dual inhibitor), were tested
in pSTAT5 Cellomics assays. LY2784544 was tested in Caspase3/7 cell-based assay for understanding the
mechanism of action of anti-proliferative effect.
11
Supplemental Table 4. Biochemical assays do not reveal selectivity of LY2784544 and other
clinical JAK2 inhibitors for mutant JAK2V617F.
KI (nM) as determined in a kinetic ATP-based mechanism of action assay
Without IL-3Rα
With 100 nM IL-3Rα
JAK2
inhibitorsa
JAK2 (catalytic
domain)
JAK2
(full length)
JAK2V617F
JAK2
(full length)
JAK2V617F
LY2784544
1.090
0.203
0.246
0.288
0.245
SAR302503
1.030
1.089
1.062
0.787
0.537
Ruxolitinib
0.082
0.272
0.234
0.268
0.119
Abbreviations: ATP=adenosine triphosphate; IL3-Rα=interleukin-3 receptor alpha; JAK=Janus Kinase;
KI,=inhibitor-binding constant.
a
The KI values were determined only for the JAK2-selective molecules LY2784544 and SAR302503, as well as
JAK1/2 dual inhibitor ruxolitinib. CYT387 and AZD1480 were not tested because their cellular JAK-selectivity is
similar to that of SAR302503 and ruxolitinib, respectively.
12
Supplemental Figure 1. Induction of apoptosis in Ba/F3 cells expressing JAK2V617F by
LY2784544.
Caspase3/7 (Luminescence/RLU)
Abbreviations: Ba/F3=Murine pro-B-cells; EC50= half maximal effective concentration.
150000
100000
50000
EC50=113 ±23nM
0
-4
-3
-2
-1
0
LY2784544 uM
13
1
Supplemental Figure 2. Autophosphorylation of purified mutant JAK2 proteins.
Purified proteins were analyzed by western blot with antibodies against total JAK2 (row 1) or
phosphorylated JAK2 (pJAK2, Y1007/1008) (row 2).
Total JAK2
pJAK2 (Y1007/1008)
JAK2-Flag
Mutant JAK2-Flag
14