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Modulation of Activation-Loop Phosphorylation by JAK Inhibitors Is Binding Mode Dependent

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Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor–induced activation loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop may or may not be elicited. Significance: This study shows that JAK inhibitors can lead to an increase of activation loop phosphorylation in a manner that is binding mode dependent. Our results highlight the need for detailed understanding of inhibitor mechanism of action, and that it may be possible to devise strategies that avoid target priming using alternative modes of inhibiting JAK kinase activity for the treatment of JAK-dependent diseases. Cancer Discov; 2(6); 512–23. © 2012 AACR. This article is highlighted in the In This Issue feature, p. 473
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Modulation of Activation-Loop
Phosphorylation by JAK Inhibitors
Is Binding Mode Dependent
Rita Andraos1, Zhiyan Qian1, Débora Bonenfant2, Joëlle Rubert1, Eric Vangrevelinghe3,
Clemens Scheufl er4, Fanny Marque1, Catherine H. Régnier1, Alain De Pover1, Hugues Ryckelynck1,
Neha Bhagwat5,6, Priya Koppikar5, Aviva Goel5, Lorenza Wyder1, Gisele Tavares4, Fabienne Baffert1,
Carole Pissot-Soldermann3, Paul W. Manley3, Christoph Gaul3, Hans Voshol2, Ross L. Levine5,
William R. Sellers1, Francesco Hofmann1, and Thomas Radimerski1
RESEARCH ARTICLE
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JUNE 2012CANCER DISCOVERY | 513
INTRODUCTION
The Janus kinase (JAK) family of nonreceptor tyrosine
kinases has crucial roles in cytokine signaling and develop-
ment. In mammals, 4 JAK family members—JAK1, JAK2, JAK3,
and TYK2—have been identifi ed (1–3). The JAKs contain a
carboxyl-terminal tyrosine kinase domain and an adjacent
pseudokinase domain (1), termed JAK homology (JH) 1 and
JH2 domains, respectively. It is thought that the JH2 domains
lack protein kinase activity, although this has been challenged
for the JAK2 JH2 (4), and that they may negatively regulate
the JH1 kinase (5). However, regions of the JH2 domains seem
to be required for signal transduction (6, 7). Through their
amino-terminal domain, the JAKs associate with cytokine
receptors, which upon cytokine binding undergo a confor-
mational change that allows JAK activation via either auto- or
transphosphorylation on tyrosine residues in the activation
loop (8). Subsequently, the JAKs phosphorylate cognate recep-
tors and STAT proteins that then translocate into the nucleus
to initiate transcription of target genes (9).
ABSTRACT Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid
arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these
drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This
type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade
of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the
opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography
to corroborate the binding mode and report for the fi rst time the crystal structure of the JAK2 kinase
domain in an inactive conformation. Importantly, JAK inhibitor–induced activation loop phosphorylation
requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on
the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop
may or may not be elicited.
SIGNIFICANCE:: This study shows that JAK inhibitors can lead to an increase of activation loop phos-
phorylation in a manner that is binding mode dependent. Our results highlight the need for detailed
understanding of inhibitor mechanism of action, and that it may be possible to devise strategies that
avoid target priming using alternative modes of inhibiting JAK kinase activity for the treatment of JAK-
dependent diseases. Cancer Discov; 2(6); 512–23. ©2012 AACR.
JAK3 has emerged as a target for the prevention of transplant
rejection and treatment of autoimmune diseases (10), and a JAK3-
biased inhibitor is currently undergoing evaluation in patients
with rheumatoid arthritis and other immunologic diseases. JAK2
is an important oncology target due to STAT activation in many
cancers, recurrent translocations in leukemias creating consti-
tutively active JAK2 fusion proteins (11), and, most notably,
activation of JAK2 through somatic mutation in polycythemia
vera (12). Interestingly, the latter mutation occurs within the JH2
domain (12), exchanging valine at position 617 to phenylalanine,
and is believed to disrupt the repressive function of the pseudoki-
nase. The JAK2 V617F mutation is also frequently found in the
myeloproliferative diseases essential thrombocythemia (ET) and
primary myelofi brosis (PMF; ref. 13).
Different mechanistic approaches can be taken to inhibit
protein kinases (14). The fi rst JAK inhibitors to be described
were the unselective tyrphostins, such as AG-490 (15), which
are thought to act in a substrate-competitive manner, either
non-competitive or mixed-competitive for ATP (16). More
recently, ATP-competitive compounds binding to the kinase-
active conformation (type I inhibitors) emerged as potent and
selective JAK inhibitors (17, 18). Several type I JAK inhibitors
have been developed and some have shown promising activity
in patients with rheumatoid arthritis, psoriasis, and myelofi -
brosis (19). In contrast, type II inhibitors engage kinases in
their inactive conformation (14). Such type II inhibitors occupy
a hydrophobic pocket adjacent to the ATP-binding site, which
becomes accessible through a conformational shift of the
activation loop to the DFG-out state. Several type II inhibitors
targeting either a spectrum of kinases or directed toward BCR-
ABL have been developed for the treatment of solid tumors or
chronic myeloid leukemia, respectively. To date, however, type
II inhibitors targeting JAK2 have been relatively unexplored.
Here, we analyze the effects of JAK inhibitors having dif-
ferent binding modes on JAK activation loop phosphoryla-
tion. Expanding on previous studies (20–22), we fi nd that
Authors’ Affi liations: 1Disease Area Oncology, 2Developmental and Molecu-
lar Pathways, 3Global Discovery Chemistry, 4Center for Proteomic Chemis-
try, Novartis Institutes for BioMedical Research, Basel, Switzerland; 5Human
Oncology and Pathogenesis Program and Leukemia Service, Department of
Medicine, Memorial Sloan-Kettering Cancer Center; and 6Gerstner Sloan-
Kettering Graduate School of Biomedical Sciences, New York
Note: Supplementary data for this article are available at Cancer Discov-
ery Online (http://cancerdiscovery.aacrjournals.org/).
Current address for L. Wyder: Actelion Pharmaceuticals Ltd., Allschwil,
Switzerland.
R. Andraos and Z. Qian contributed equally to this work.
Corresponding Author: Thomas Radimerski, Disease Area Oncology,
Novartis Institutes for BioMedical Research, Klybeckstrasse 141, 4057
Basel, Switzerland. Phone: 41-61-696-20-64; Fax: 41-61-696-55-11;
E-mail: thomas.radimerski@novartis.com
doi: 10.1158/2159-8290.CD-11-0324
©2012 American Association for Cancer Research.
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Andraos et al.
RESEARCH ARTICLE
514 | CANCER DISCOVERYJUNE 2012 www.aacrjournals.org
compounds that inhibit JAKs with a type I mechanism can
increase JAK activation loop phosphorylation, despite block-
ade of kinase function and inhibition of STAT phosphor-
ylation. Importantly, we show that stabilizing JAKs in the
inactive state by a type II inhibitor leads to the loss of activa-
tion loop phosphorylation. The increase of JAK activation
loop phosphorylation by type I inhibitors is staurosporine-
sensitive, ATP-dependent, and requires receptor interaction,
as well as intact kinase and pseudokinase domains.
RESULTS
Structurally Diverse ATP-Competitive JAK Inhibitors
Can Increase JAK Activation Loop Phosphorylation
Upon profi ling JAK inhibitors in JAK2V617F-dependent
SET-2 cells, which have JAK2 amplifi cation and predomi-
nantly express mutant JAK2 (23), we noticed an increase in
JAK2 activation loop phosphorylation with JAK inhibitor
exposure, despite suppression of STAT5 phosphorylation
(Fig. 1A). This phenomenon was seen with different inhibi-
tors, including the pan-JAK inhibitor “JAK inhibitor 1” (17),
the JAK3-biased pyrrolo[2,3-d]pyrimidine CP-690,550 (18),
and the JAK2-biased quinoxaline NVP-BSK805 (24). A simi-
lar disconnect between the changes in phosphorylation of
JAK2 and STAT5 was previously observed for JAK inhibitor
1–treated HEL cells (20–22), which only express JAK2V617F and
have JAK2 amplifi cation (23). Similar results were obtained
in Ba/F3 cells expressing mutant MPLW515L (Supplementary
Fig. S1A), which is found in up to 10% of JAK2V617F-negative
ET and PMF cases (25). In B-cell precursor acute lymphob-
lastic leukemia (ALL) MHH-CALL-4 cells with deregulated
CRLF2 expression and JAK2I682F mutation (26), the different
JAK inhibitors suppressed STAT5 phosphorylation without
appreciably altering JAK2 phosphorylation (Supplementary
Fig. S1B). In Ba/F3 cells expressing TEL-JAK2, a cytoplas-
mic fusion protein of the oligomerization domain of TEL
with the JAK2 kinase domain (27), NVP-BSK805 partially
suppressed activation loop phosphorylation (Supplemen-
tary Fig. S1C). Basal JAK2 phosphorylation was minimal in
SET-2 cells, but incubation with increasing concentrations
of NVP-BSK805 increased activation loop phosphorylation,
reaching a plateau at concentrations of 300 nmol/L or more,
which coincided with suppression of STAT5 phosphorylation
(Fig. 1B). As the JAK2 phospho-Tyr1007/Tyr1008 antibody
can cross-react with the analogous TYK2 phosphoryla-
tion sites, we verifi ed JAK2-specifi city by depleting JAK2 in
HEL92.1.7 cells, which seem to have largely lost dependency
on JAK2V617F for proliferation (28). This approach should
avoid potential confounding effects resulting from apopto-
sis induction after JAK2 depletion in JAK2V617F-dependent
Figure 1. Increase of JAK activation loop phosphorylation by JAK inhibitors. A, SET-2 cells were treated for 30 minutes with JAK inhibitors at 1 μmol/L or
DMSO and then extracted for Western blot analysis of JAK2 Y1007/Y1008 and STAT5 Y694 phosphorylation. JAK2 and STAT5 served as loading controls.
B, SET-2 cells were treated with increasing concentrations of NVP-BSK805 for 30 minutes and then assessed as described above. C, nontargeting (Ctrl) or
JAK2 targeting siRNA oligos were transfected into HEL92.1.7 cells. After 72 hours, cells were treated for 30 minutes with 1 μmol/L NVP-BSK805 or DMSO
and then assessed as described above. D, SET-2 cells were treated with 1 μmol/L NVP-BSK805 or DMSO for 30 minutes. JAK2 was immunoprecipitated (IP)
using an amino- or carboxyl-terminal antibody, followed by Western blot analysis of P-JAK2 and JAK2. E, CMK cells were treated for 30 minutes with JAK
inhibitors at 1 μmol/L or DMSO and then extracted for Western blot analysis of JAK3 Y980 (following JAK3 IP) and STAT5 phosphorylation. F, TF-1 cells were
starved in medium without GM-CSF overnight and then either pretreated with DMSO or JAK inhibitors at 1 μmol/L for 30 minutes. Cells were then stimulated or
not with 10 ng/mL IFN-α for 10 minutes, followed by extraction for Western blot analysis of TYK2 Y1054/Y1055 (after TYK2 IP) and STAT5 phosphorylation.
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Binding Mode-Dependent Modulation of JAK Phosphorylation RESEARCH ARTICLE
JUNE 2012CANCER DISCOVERY | 515
cells. Both baseline and JAK2 inhibitor–induced phospho-
JAK2 levels were blunted in JAK2-depleted HEL92.1.7 cells
(Fig. 1C), supporting specifi c detection of JAK2 activation
loop phosphorylation. TYK2 depletion did not impact induc-
tion of JAK2 phosphorylation upon JAK2 inhibitor treatment
(data not shown).
In SET-2 cells treated with JAK inhibitors, we failed to
immunoprecipitate JAK2 using a carboxyl-terminal–directed
antibody (Fig. 1D), indicating that the inhibitors engage the
kinase either in a conformation or multiprotein complex
that masks the epitope. Accordingly, immunoprecipitation of
JAK2 from inhibitor-treated cells with an antibody recogniz-
ing an amino-terminal epitope was feasible and the kinase
had increased levels of activation loop phosphorylation, as
compared with JAK2 immunoprecipitated from control cell
extracts (Fig. 1D). Similar results were obtained using gran-
ulocyte macrophage colony–stimulating factor (GM-CSF)–
stimulated TF-1 cells with wild-type JAK2 pretreated with
NVP-BSK805 (Supplementary Fig. S1D).
Next, we assessed whether JAK inhibitors could also increase
activation loop phosphorylation on other JAK family mem-
bers. CMK cells express JAK3 bearing an activating A572V
mutation (29), and constitutive STAT5 phosphorylation in
these cells is dependent on both JAK3 and JAK1 (24, 29, 30).
Treatment of CMK cells with JAK inhibitor 1, CP-690,550, or
NVP-BSK805 induced JAK3 activation loop phosphorylation
(Fig. 1E), with the extent being consistent with their rank
order of potency toward JAK3 (18, 24). In TF-1 cells IFN-α
stimulation led to weak TYK2 activation loop phosphoryla-
tion together with robust STAT5 activation. Pretreatment
of TF-1 cells with JAK inhibitor 1, CP-690,550, or NVP-
BSK805 suppressed IFN-α–induced STAT5 phosphorylation
and markedly increased TYK2 phosphorylation (Fig. 1F).
However, pretreatment with JAK inhibitor 1 before IFN-α
stimulation did not discernibly augment JAK1 phosphoryla-
tion (Supplementary Fig. S1E). JAK inhibitor 1 treatment did
increase JAK1 activation loop phosphorylation in HEL92.1.7
cells transiently transfected with JAK1 bearing the V658F
activating pseudokinase mutation (ref. 31; Supplementary
Fig. S1F). These results showed that structurally diverse
ATP-competitive JAK inhibitors (Supplementary Fig. S2) can
induce an increase in JAK activation loop phosphorylation.
Differential Kinetics of JAK2 and STAT5
Phosphorylation Recovery After JAK Inhibitor
Washout in Cells
The fi ndings above raise the question of what happens to
JAK/STAT signaling following inhibitor dissociation from
their activation loop phosphorylated target. To address
this, SET-2 cells were treated with JAK inhibitors possessing
different in vitro binding kinetics (Fig. 2A, Supplementary
Fig. S3A–C). Following a pulse of JAK inhibitor 1, CP-690,550,
or NVP-BSK805, cells were washed, returned to medium
without inhibitor, and extracted at different times. All inhibi-
tors suppressed STAT5 phosphorylation and increased JAK2
phosphorylation during drug treatment (Fig. 2B). After com-
pound washout, STAT5 phosphorylation recovered between
30 minutes to 4 hours. Interestingly, JAK2 hyperphospho-
rylation was only lost in CP-690,550–treated samples (Fig.
2B), whereas in the samples treated with JAK inhibitor 1 or
NVP-BSK805, levels of JAK2 phosphorylation remained ele-
vated. These differences were not readily explained by the
kinetic parameters of the inhibitors (Fig. 2A). Potentially
on-target drug residence time in cells differs from that deter-
mined in biochemical assays with JH1 alone, for example, due
to the conformation adopted by JAK2 in cells. Alternatively,
drugs with a slow off-rate may not have dissociated com-
pletely during the washing step. Incubation of factor-starved
TF-1 cells with CP-690,550 did not lead to detectable induc-
tion of phospho-JAK2, and upon drug washout, there was no
appreciable STAT5 phosphorylation in the absence of GM-
CSF (Supplementary Fig. S4A). These results indicated that
JAK inhibitor–induced activation loop hyperphosphorylation
requires specifi c activating JAK mutations or, in the case of
wild-type JAKs, cytokines engaging receptor complexes.
Increased JAK2 Activation Loop Phosphorylation
Following JAK2 Inhibitor Treatment In Vivo
Induction of JAK2 activation loop phosphorylation fol-
lowing kinase inhibition was also observed in vivo. In a
mouse model of erythropoietin-induced polycythemia (24),
the hormone induced STAT5 phosphorylation in spleen
extracts (Fig. 2C), although signals for phospho-JAK2 were
below detection limits. Treatment with NVP-BSK805 blunted
erythropoietin-induced STAT5 phosphorylation, but increased
phospho-JAK2 levels (Fig. 2C). Similarly, in mice transplanted
with bone marrow expressing MPLW515L, causing signifi cant
thrombocytosis and myelofi brosis (25), NVP-BSK805 treat-
ment increased phospho-JAK2 in spleen extracts, whereas lev-
els of phosphorylated STAT3 and mitogen-activated protein
kinase (MAPK) were reduced (Fig. 2D).
Type II JAK Inhibition Suppresses Activation
Loop Phosphorylation and Downstream STAT
Phosphorylation
The inhibitors assessed stabilize the JAK active confor-
mations through a common binding mode (type I binding
mode), raising the question whether compounds stabilizing
the inactive conformation (type II binding mode) would
differ in terms of effects on activation loop phosphoryla-
tion. Compounds with low activity in enzymatic assays with
activated (phosphorylated) JAK2 kinase, but good potency
in JAK2-dependent cellular assays, as well as structural ele-
ments typical of type II inhibitors were identifi ed by database
mining. The dihydroindole NVP-BBT594, a potent type II
inhibitor of wild-type and T315I mutant BCR-ABL (32),
blocked proliferation of JAK2V617F mutant cells, but displayed
limited activity in the JAK2 enzymatic assay (Supplemen-
tary Table S1). Importantly, incubation of SET-2 cells with
NVP-BBT594 blunted STAT5 and JAK2 activation loop
phosphorylation (Fig. 3A). NVP-BBT594 also inhibited JAK2
activation loop and STAT5 phosphorylation in Ba/F3 TEL-JAK2
cells and in MHH-CALL-4 cells (Supplementary Fig. S4B and C).
Furthermore, NVP-BBT594 suppressed JAK3 and STAT5 phos-
phorylation in JAK3A572V mutant CMK cells (Fig. 3B), indicating
that it inhibits JAK3, JAK1, or both. Accordingly, NVP-BBT594
suppressed growth of CMK cells with a GI50 of 262 nmol/L
in proliferation assays (Supplementary Table S1) and blunted
IFN-α–induced JAK1, TYK2, STAT1, STAT3, and STAT5 phos-
phorylation in TF-1 cells (Supplementary Fig. S4D).
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Immunoprecipitation studies provided additional evidence
for a distinct JAK2 conformation being stabilized by NVP-
BBT594. In contrast to type I compounds (Fig. 1D), the ability
to immunoprecipitate JAK2 from NVP-BBT594–treated cells
was largely indistinguishable using antibodies directed to the
amino- or carboxyl-terminal epitopes (Fig. 3C). In compound
pulse treatment/compound washout experiments with NVP-
BBT594, STAT5 phosphorylation was initially inhibited and
gradually returned to baseline (Fig. 3D). Hence, there is a
marked difference in the changes of JAK activation loop phos-
phorylation imposed by JAK inhibitors, depending on their
binding mode.
NVP-BBT594 Binds to JAK2 in the DFG-Out
Conformation
The type II binding mode hypothesis for NVP-BBT594 (Fig.
4A) was confi rmed by X-ray crystallographic analysis of the
inhibitor in complex with the JAK2 kinase domain at 1.35 Å
resolution (Fig. 4B). In contrast to previous JAK2 crystal struc-
tures obtained with NVP-BSK805 (24) or JAK inhibitor 1 (33),
which are bound to the active conformation of JAK2 (“DFG-
in” conformation), NVP-BBT594 binds to JAK2 in the inactive
conformation (“DFG-out” conformation), where F995 of the
DFG motif is translocated (10 Å) from its position in the
kinase active state. In addition to the usual salt bridge with
catalytic K882, the carboxyl side-chain of conserved helix C
residue E898 is also engaged in an H-bond interaction with
NVP-BBT594. We believe that this is the fi rst report of the
JAK2 JH1 structure in an inactive conformation. The coordi-
nates of residues 1,000 to 1,012 of the activation loop are
missing because of lack of electron density, as frequently
seen in inactive kinase structures. NVP-BBT594 also inter-
acts with the hinge region of JAK2 (Fig. 4B and C), with the
pyrimidine occupying the adenine-binding pocket of the ATP-
binding site and making an H-bond interaction with the
backbone-NH of L932. An H-bond is also observed between
the amide-NH and the backbone-CO of L932. In contrast to
type I inhibitors, NVP-BBT594 binds to the pocket made
accessible through translocation of the DFG-motif with
H-bonds between the urea-CO and the backbone-NH of D994
and side-chain of E898, H-bonds between the protonated
N-methylpiperazine and the backbone-CO of both I973 and
H974, and with the trifl uoromethyl moiety participating in
lipophilic interactions.
Figure 2. JAK inhibitor–induced JAK activation loop phosphorylation can be transient or sustained and is also seen in vivo. A, JAK inhibitor kinetic
parameters determined in biochemical assays with JAK2 JH1. B, SET-2 cells were treated for 2 hours with different JAK inhibitors at 1 μmol/L, followed
by washing, transferring back into medium, and extraction at indicated time points. Control cells were treated with DMSO. JAK2 Y1007/Y1008 and
STAT5 Y694 phosphorylation were detected by Western blotting. JAK2 and STAT5 served as loading controls. C, mice received a subcutaneous injection
of 10 U rhEpo and were orally administered 25, 50, or 100 mg/kg NVP-BSK805. Control animals received either a subcutaneous injection of saline or
10 U rhEpo and were orally administered vehicle. After 3 hours, spleen samples were processed for detection of P-JAK2 and P-STAT5 levels as described
above. D, irradiated mice transplanted with bone marrow transduced with MPLW515L were followed for development of leukocytosis and thrombocytosis.
Mice were then administered either vehicle or NVP-BSK805 at indicated dose levels. After 24 hours, levels of JAK2, STAT3, and MAPK phosphorylation in
spleen extracts were assessed by Western blotting. Actin served as loading control.
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Binding Mode-Dependent Modulation of JAK Phosphorylation RESEARCH ARTICLE
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JAK Type I Versus Type II Inhibition Differentially
Affects JAK Activation Loop Phosphorylation as
Assessed by Mass Spectrometry
Tyrosine phosphorylated peptide enrichment followed by
mass spectrometry was used to assess the effects of JAK
type I versus type II inhibitors at the phosphotyrosine pro-
teome level. SET-2 cells were treated with dimethyl sulfox-
ide (DMSO), NVP-BSK805, CP-690,550, or NVP-BBT594.
Around 100 to 200 different phosphotyrosine peptides were
identifi ed (Supplementary Table S2), with a subset display-
ing marked changes upon JAK inhibition. Consistent with
Western blotting, JAK2 activation loop phosphorylation was
elevated in NVP-BSK805 and CP-690,550–treated samples
and below baseline in NVP-BBT594–treated samples (Table 1).
Similar fi ndings were made for a peptide encompassing
the TYK2 activation loop phosphorylation site Tyr1054.
As expected, treatment with the different JAK inhibitors
suppressed STAT tyrosine phosphorylation (Table 1). Fur-
thermore, JAK inhibition decreased JAK2 Tyr570 phosphor-
ylation, which is a negative regulatory site (34). Loss of Tyr570
phosphorylation upon JAK2 inhibition, irrespective of inhibi-
tor binding mode, was confi rmed in JAK2V617F mutant cells by
Western blotting (Supplementary Fig. S5A–F).
Type I Inhibitor–Induced Increase of
JAK Activation Loop Phosphorylation Is
Staurosporine-Sensitive and ATP-Dependent
In principle, very different mechanisms could cause increased
JAK activation loop phosphorylation upon kinase inhibition by
type I inhibitors: In the frame of feedback regulation, JAKs
might phosphorylate and regulate their own phosphatase in the
removal of activation loop Tyr-phosphorylation (35). However,
this hypothesis was inconsistent with RNA interference (RNAi)-
mediated depletion of tyrosine phosphatases SHP-1, SHP-2, and
PTP1B (data not shown). A second model favoring a JAK kinase
conformation no longer recognized by negative regulatory pro-
teins of the SOCS family seems unlikely because SOCS proteins
interact with the phosphorylated activation loop (36). Thirdly,
JAK activation loops exposed by type I inhibitor binding (33)
might be hyperphosphorylated by another tyrosine kinase. To
investigate this possibility, SET-2 cells were fi rst treated with the
pan-kinase inhibitor staurosporine and then with NVP-BSK805.
Staurosporine alone had little effect on basal JAK2 phosphoryla-
tion, but suppressed the NVP-BSK805–induced increase (Fig. 5A).
Because staurosporine can bind JAK2 in a type I fashion (37),
consistent with phospho-STAT5 suppression (Fig. 5A), SET-2
cells were pretreated (5 or 10 minutes) with NVP-BSK805,
Figure 3. Type II mode of JAK inhibition suppresses both JAK activation loop and substrate phosphorylation. A, SET-2 cells were treated for 30
minutes with 1 μmol/L NVP-BBT594 or DMSO and then extracted for Western blot analysis of JAK2 Y1007/Y1008 phosphorylation and STAT5 Y694
phosphorylation. JAK2 and STAT5 were probed for as loading controls. B, SET-2 or CMK cells were treated with increasing concentrations of NVP-
BBT594 for 1 hour and then extracted for detection of P-JAK2, P-JAK3 (Y980), and P-STAT5 by Western blotting. C, SET-2 cells were treated with 1
μmol/L NVP-BBT594 or DMSO for 30 minutes. JAK2 was immunoprecipitated using an amino- or carboxyl-terminal antibody, followed by Western blot
analysis of levels of P-JAK2 and JAK2 protein that was immunoprecipitated. D, SET-2 cells were treated for 2 hours with 1 μmol/L NVP-BBT594, followed
by washing, transferring back into medium, and extraction at the indicated time points. Control cells were treated with DMSO. P-JAK2 and P-STAT5 levels
were assessed by Western blotting. IP, immunoprecipitation.
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RESEARCH ARTICLE
Figure 4. JAK2 JH1 in complex with
NVP-BBT594 at 1.34 Å resolution.
A, chemical structure of NVP-BBT594.
B, overall ribbon representation of the
JAK2 kinase domain with the bound
inhibitor NVP-BBT594 illustrated as
stick model. Inhibitor binding occurs to
the DFG-out conformation of the kinase
domain. Residues 1,000 to 1,012 from
the activation loop did not show electron
density and are omitted from the fi nal
model. C, stereo view of NVP-BBT594
bound to JAK2. Polar contacts between
the protein, the inhibitor molecule, and
solvent are indicated with dotted green
lines. More distant structural elements
are depicted in a lighter tone.
Table 1.Analysis of changes in tyrosine phosphorylation by mass spectrometry following treatment of SET-2 cells with
different JAK inhibitors
P-Tyr site BSK805 Exp. 1 BSK805 Exp. 2 CP-690,550 Exp. 1 CP-690,550 Exp. 2 BBT594
JAK2 Y570 1.59 2.80 3.62 2.10 2.68
JAK2 Y1007  3.94  6.95  5.34  3.58 0.88
JAK2 Y1007, Y1008 ND ND ND ND 2.16
LYN Y266  6.71 0.76  0.25 0.31 2.85
PTPN11 Y63  2.50 0.88  0.07  0.30 1.09
PTPN18 Y389 1.64 3.40 ND 2.13 2.78
PTPN6 Y564 2.50 2.65 0.74 0.53 2.37
PTPRA Y798 8.64 6.01 0.49  0.11 3.28
SKAP2 Y11  3.89 0.27  2.36  1.69 1.83
SKAP2 Y197 1.09  0.28  0.26  0.63 0.75
STAT1 Y701 ND ND 3.84 ND 3.96
STAT3 Y705 2.32 3.67 7.73 2.80  1.37
STAT5A Y90 0.98  0.85 0.40  0.49 0.54
STAT5A Y694 2.47  0.32 2.60 3.25 2.24
TYK2 Y292  4.99 1.32 5.35 0.89  1.94
TYK2 Y1054  1.51  1.02  5.18  3.22 4.56
Note: SET-2 cells were treated for 30 minutes with the indicated JAK inhibitors and extracted. Changes in phosphopeptide abundance
following JAK inhibitor versus DMSO treatment in the different experiments are displayed as log2 ratios calculated from duplicate runs.
The table shows a subset of the detected peptides, and the respective affected phosphotyrosine residues are indicated. ND, Not detected.
518 | CANCER DISCOVERYJUNE 2012 www.aacrjournals.org
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Binding Mode-Dependent Modulation of JAK Phosphorylation RESEARCH ARTICLE
JUNE 2012CANCER DISCOVERY | 519
followed by treatment with staurosporine, which also sup-
pressed the induction of phospho-JAK2 (data not shown). To
investigate which tyrosine kinase activity might account for JAK
activation loop phosphorylation following JAK type I inhibition,
cells were pretreated with inhibitors of ABL/KIT/PDGFR/DDR
(imatinib), SRC-family kinases (dasatinib), FAK (NVP-TAE226),
or IGF-1R (NVP-AEW541), but these drugs failed to suppress
NVP-BSK805–induced JAK2 phosphorylation (Fig. 5A and data
not shown). siRNA-mediated depletion of BTK, LYN, or PYK2,
which have been implicated in JAK2 signaling (9, 38), did not
suppress NVP-BSK805–induced JAK2 phosphorylation (Fig. 5B
and C and data not shown). JAK2V617F transphosphorylation by
another JAK family member seemed unlikely, as increased activa-
tion loop phosphorylation was also seen with pan-JAK type I
inhibitors (Fig. 1A) and was not suppressed by simultaneous
depletion of JAK1, JAK3, and TYK2 by RNAi in SET-2 cells (data
not shown). Acute ATP depletion in SET-2 cells, by blocking
both glycolysis and oxidative phosphorylation, suppressed the
NVP-BSK805–induced JAK2 activation loop hyperphosphoryla-
tion (Fig. 5D and E), further implicating a kinase activity in the
effect. These experimental conditions led to an approximately
5-fold reduction of cellular ATP levels, triggering AMPK activa-
tion (Fig. 5E).
Intact FERM, JH2, and JH1 Domains Are Required
for Type I Inhibitor–Induced Increase of JAK
Activation Loop Phosphorylation
These results led us to assess whether JAK2 itself might be
involved in increasing activation loop phosphorylation when a
type I inhibitor engages the JH1 domain in the active conforma-
tion. Accordingly, we probed the impact of introducing defi ned
point mutations in the FERM, JH2, and JH1 domains, as previ-
ous studies indicated they interact in vivo (5, 39). First, mutation
of the JAK2 pseudokinase was investigated. Modeling of the
JH2 domain revealed a structure in accordance with a previous
model (40) and possibly compatible with nucleotide binding.
Indeed, it was recently reported that the JAK2 pseudokinase
can bind ATP and autophosphorylate both Ser523 and Tyr570
(4). Thus, if the JH2 domain assumes an active-like conforma-
tion and maintains catalytic activity under particular condi-
tions, mutation of Lys581 in β-strand 3 would be predicted
to abrogate catalysis (4). This lysine and the surrounding
amino acids are conserved in the pseudokinase domains of
JAK1, JAK3, and TYK2. JAK2V617F or JAK2K581R/V617F were tran-
siently transfected into JAK2V617F mutant HEL92.1.7 cells, fol-
lowed by NVP-BSK805 treatment. Compared with controls,
Figure 5. JAK2 type I inhibitor–induced increase of JAK2 activation loop phosphorylation is staurosporine-sensitive and suppressed by reduction of
ATP levels. A, SET-2 cells were pretreated for 30 minutes with DMSO control (Ctrl), 10 μmol/L staurosporine or imatinib, followed by treatment for 1
hour with 1 μmol/L NVP-BSK805 or DMSO. JAK2 Y1007/Y1008 and STAT5 Y694 phosphorylation were assessed by Western blotting. B, nontargeting
or BTK targeting siRNA oligos were transfected into SET-2 cells. After 72 hours, cells were treated for 1 hour with 1 μmol/L NVP-BSK805 or DMSO and
P-JAK2 and P-STAT5 levels were assessed as above. JAK2, STAT5, and BTK served as loading controls and to verify siRNA-mediated target knockdown.
C, nontargeting or LYN targeting siRNA oligos were transfected into SET-2 cells, followed by treatment and analysis as described above. D, SET-2 cells
were treated for 30 minutes with 10 mmol/L 2-deoxy--glucose and 20 μmol/L oligomycin A or drug vehicle. Cells were then treated for 20 minutes with
1 μmol/L NVP-BSK805 or DMSO. P-JAK2 and P-STAT5 levels were assessed as above. E, extracts from the experiment shown in D were also probed (left)
for phosphorylated AMPKα. AMPKα and β-tubulin served as loading controls. The histogram depicts relative ATP levels (means of 4 independent experi-
ments ± SD) in SET-2 cells treated as described in D.
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Andraos et al.
RESEARCH ARTICLE
520 | CANCER DISCOVERYJUNE 2012 www.aacrjournals.org
JAK2V617F-transfected cells had higher levels of both basal
and JAK2 inhibitor–induced phospho-JAK2. Importantly, in
JAK2K581R/V617F–transfected cells phospho-JAK2 levels were com-
parable with control cells and induction of phospho-JAK2 levels
by NVP-BSK805 treatment was suppressed (Fig. 6A). As a nega-
tive control, we mutated K607, situated adjacent to the predicted
helix-αC in the JH2 domain. In cells transfected with JAK2K607R/
V617F NVP-BSK805–induced activation loop phosphorylation
was not suppressed (Supplementary Fig. S6A). Transfection of
JH1 domain catalytically dead JAK2V617F/K882E also suppressed
NVP-BSK805–induced activation loop phosphorylation (Sup-
plementary Fig. S6B). These results were corroborated in Ba/
F3 cells stably expressing the human erythropoietin receptor
by transient transfection with JAK2V617F, JAK2K581R/V617F, and
JAK2V617F/K882E (Fig. 6B). To assess the potential involvement
of the FERM domain, we transiently transfected cells with
JAK2Y114A/V617F, a FERM domain mutation that precludes recep-
tor binding (41), which also suppressed NVP-BSK805–induced
activation loop phosphorylation (Fig. 6B).
These results suggested that defi ned conformations at recep-
tor complexes are required for increased JAK2 activation loop
phosphorylation after type I inhibitor treatment. In JAK2V617F
mutant cells, to enable this phenomenon, we believe that
the enzyme has to be bound to homodimeric type I cytokine
receptor complexes. To assess JAK signaling from oligomeric
cytokine receptor complexes, we used JAK3A572V mutant CMK
cells (29). As JAK3 signals in conjunction with JAK1, which
plays a dominant role in signaling via common γ-chain con-
taining cytokine receptor complexes (30), we tested the effect
of JAK1 depletion on JAK inhibitor–induced JAK3 activation
loop phosphorylation. JAK1 depletion reduced basal levels of
phospho-JAK3 and phospho-STAT5 and also strongly sup-
pressed CP-690,550–mediated induction of phospho-JAK3
(Supplementary Fig. S6C). We then investigated IFN signal-
ing via the heterodimeric IFN-α receptor complex. Depletion
of JAK1 in TF-1 cells suppressed STAT5 phosphorylation
and JAK inhibitor 1–mediated induction of phospho-TYK2
upon IFN-α stimulation (Supplementary Fig. S6D). As we
could not preclude that JAK1 depletion might impact recep-
tor complex conformations and/or receptor interaction of the
respective cooperating JAK family members, we assessed the
impact of exogenous JAK1 expression. TF-1 cells were either
transiently transfected with empty vector, wild-type JAK1, or
a variant with a mutation in the conserved lysine in β-strand 3
(K622R). Enforced expression of JAK1 resulted in constitutive
JAK1 phosphorylation but modest inducibility of TYK2 and
STAT5 phosphorylation by IFN-α. Nevertheless, JAK inhibitor
1 pretreatment resulted in elevated levels of phospho-TYK2,
although less than that of vector control (Supplementary Fig.
S6E). In contrast, phospho-JAK1 was undetectable in cells
expressing JAK1K622R, either at baseline following factor starva-
tion, or after IFN-α stimulation, and levels of phospho-TYK2
and phospho-STAT5 were lowest. Finally, in transient transfec-
tion experiments using HEL92.1.7 cells, the JAK1K622R protein
Figure 6. JAK type I inhibitor–
induced increase of JAK activation
loop phosphorylation requires
intact JH1 and JH2 domains. A,
HEL92.1.7 cells were transiently
transfected with the indicated
JAK2 constructs. Control cells
were transfected with empty
vector (Ctrl). After 24 hours, cells
were treated with 1 μmol/L NVP-
BSK805 or DMSO for 30 minutes,
followed by extraction for Western
blot detection of P-JAK2 (Y1007/
Y1008) and P-STAT5 (Y694) levels.
B, Ba/F3 EpoR cells were transiently
transfected with the indicated JAK2
constructs or empty vector (Ctrl).
After 24 hours cells were treated
as described above. C, HEL92.1.7
cells were transiently transfected
with the indicated JAK1 constructs
or empty vectors (Ctrl). After 24
hours, cells were either treated with
1 μmol/L JAK inhibitor 1 or DMSO
for 1 hour, followed by extraction
for Western blot detection of JAK1
Y1022/Y1023 phosphorylation, and
of P-JAK2 and P-STAT5 as above.
Total levels of JAK1, JAK2, and
STAT5 served as loading controls.
D, HEL92.1.7 cells were transiently
transfected and treated as in C to
assess the impact of the JAK1K648R
control mutant on JAK inhibitor 1–
induced activation loop phosphor-
ylation of cotransfected JAK1V658F.
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Binding Mode-Dependent Modulation of JAK Phosphorylation RESEARCH ARTICLE
JUNE 2012CANCER DISCOVERY | 521
(Fig. 6C), but not wild-type JAK1 (Supplementary Fig. S6F) or
a negative control mutant JAK1K648R (Fig. 6D), suppressed JAK
inhibitor 1–mediated increases in JAK1V658F activation loop
phosphorylation. Thus, the increase of JAK activation loop
phosphorylation by type I inhibitors requires JAK receptor
interaction, as well as intact kinase and pseudokinase domains.
DISCUSSION
Several examples of inadvertent kinase activation by small-
molecule inhibitors have been reported (42, 43), one of the
most striking being the cross-activation of RAF family mem-
bers by B-RAF kinase inhibitors in B-RAF wild-type cells (44).
Often the involved kinases exhibit elevated phosphorylation
on regulatory sites, which may enable rapid catalytic reactiva-
tion upon dissociation of the inhibitors. Thus, it is important
to recognize and understand feedback and priming mecha-
nisms in the process of drug discovery, as it may be possible
to devise strategies that avoid target priming.
We fi nd that inhibitors targeting the active conforma-
tion of JAK2 can increase activation loop phosphorylation,
corroborating and expanding on previous studies (20, 21). In
cells with either deregulated CRLF2 expression together with
JAK2I682F mutation or expressing TEL-JAK2, type I kinase
inhibitors either did not enhance or partially suppressed
activation loop phosphorylation. We speculate that JAK2 may
already be maximally phosphorylated in MHH-CALL-4 cells,
whereas chimeric TEL-JAK2 lacks the regulatory elements of
native JAK2. Importantly, complete loss of JAK activation loop
phosphorylation occurred with a compound that stabilized
the inactive state. We also report the fi rst cocrystal structure of
JAK2 in complex with a type II inhibitor, which might enable
the design of more potent inhibitors. It will be of particular
interest to assess if type II inhibitors differ in terms of myelo-
proliferative neoplasm disease modifi cation as compared with
type I inhibitors, which could be explored in animal models of
myeloproliferative neoplasm–like disease (12, 25).
Currently, it is unclear how engagement of JAKs in their
active conformation by type I inhibitors results in increased
activation loop phosphorylation. The process seems to be
ATP-dependent and staurosporine-sensitive. It is thought that
JAK activation involves JAK trans- and/or autophosphoryla-
tion. Furthermore, there seems to be an interplay between the
JH1 and JH2 domains, and the latter may have both negative
and positive impacts on JH1 kinase activation. On one hand,
disruption of the JAK2 JH2 domain dramatically increases
JH1 activity in vitro (5), but on the other hand, an intact JH2
domain is crucial for JAK2, JAK3, and TYK2 activation in cells
by cytokines and IFNs (6, 7, 45). In severe combined immuno-
defi ciency, JAK3 JH2 mutations (e.g., C759R) were described
that paradoxically result in a hyperphosphorylated, but cata-
lytically inactive kinase (46). Interestingly, the phosphoryla-
tion is lost when K855 is mutated in the kinase domain of
C759R mutant JAK3 (45). In JAK3, either deletion of the
JH2 domain or its inclusion in a construct otherwise only
containing JH1, abrogated kinase activity (45). These fi ndings
were reconciled in a model in which the FERM, JH2, and JH1
domains interact to form a signaling competent kinase.
From this perspective, our fi ndings that specifi c mutations
either in the FERM, JH1, or JH2 domain suppress JAK type I
inhibitor–induced increase in JAK2V617F activation loop phos-
phorylation are intriguing. This suggests that JAK receptor
complex interactions, ligand binding or activating mutations
are required to permit increased activation loop phosphoryla-
tion after type I inhibitor treatment. Furthermore, the kinase
domain itself needs to be intact, which could be interpreted as
transphosphorylation of the inhibitor bound JAK by the other
JAK partner at the receptor complex, if its conformation pre-
cludes inhibitor binding, but not ATP binding and phosphor-
ylation of activation loop tyrosines. However, we cannot exclude
the possibility that JAK2V617F/K882E predominates in the inactive
conformation, providing a potential explanation for reduced
type I inhibitor–induced activation loop phosphorylation.
JH2 β-strand 3 mutation also suppressed JAK inhibitor–
induced JAK activation loop phosphorylation, indicating that
an intact pseudokinase domain is required for the phenom-
enon to occur. Functional impact of specifi c JH2 domains
is underscored by the recurrence of the V617F mutation in
myeloproliferative neoplasms (12, 13) and of R683 mutations
in ALL (47), which suggests these hotspots could constitute a
gain of function, rather than a loss of function. Interestingly,
modeling and mutagenesis approaches have identifi ed F595 in
the JH2 helix-αC to be required for constitutive JAK2V617F acti-
vation (40). The model postulates that F595 and F617 interact,
which may impact orientation of the JH2 helix-αC and thereby
may lead to modulation of JH1 activity. Notwithstanding this
stacking model, F595 is also required for constitutive activity
of JAK2 exon 12 mutants and R683G mutant JAK2 (40).
The conserved lysine of JH2 β-strand 3 might interact with a
conserved glutamate in the helix-αC to impose the correct JH2
architecture. Our fi ndings with JAK2K581R and JAK1K622R are
consistent with a model in which the JH2 assumes an active-
like conformation and perhaps even catalytic activity under
particular circumstances. Recent studies suggested that pro-
teins previously assumed to be pseudokinases, such as CASK
(48) or ErbB3 (49), might have protein kinase activity in vivo,
despite lacking the canonical catalytic residues. The JAK JH2
domains lack the Asp in the HRD motif of the catalytic loop.
Whether key residues required for catalysis might be provided
under specifi c conditions by a substrate, structurally compen-
sated for by appropriate residues in adjacent subdomains or
by interacting domains, can only be speculated.
Ungureanu and colleagues (4) reported that the JH2
domain of JAK2 has dual-specifi city protein kinase activity
in vitro, autophosphorylating the negative regulatory sites
Ser523 and Tyr570. This suggests that JH2 catalytic activity
maintains low basal JAK2 JH1 activity, supported by the fi nd-
ing that K581A mutation led to elevated phosphorylation of
the JAK2 activation loop, STAT1 and STAT5, along with loss
of JAK2 Ser523 and Tyr570 phosphorylation (4). However,
in our studies with JAK2V617F/K581R and JAK1K622R proteins,
we did not detect elevated baseline JAK activation loop (as
compared with JAK2V617F and JAK1, respectively) or STAT
phosphorylation. Our results suggest that when JH1 is inhib-
ited by a type I inhibitor exposing the activation loop, JH2 has
a positive role in regulating its phosphorylation. A potential
shift in the phosphorylation pattern on regulatory sites upon
JAK2 inhibition is also supported by the fi nding of reduced
Tyr570 phosphorylation in our phosphoproteomics analysis.
Taken together, our data suggest that alternate modes of
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RESEARCH ARTICLE
522 | CANCER DISCOVERYJUNE 2012 www.aacrjournals.org
inhibiting JAK kinase activity might present a novel thera-
peutic strategy for the treatment of JAK-dependent diseases.
METHODS
Compounds
NVP-BSK805 (50), NVP-BBT594, staurosporine, and imatinib were
synthesized internally. JAK inhibitor 1 was from Calbiochem and
CP-690,550 from Cardiff Chemicals. Compounds were prepared as
10 mmol/L stock solutions in DMSO.
Cell Culture
SET-2, CMK, and TF-1 cells were cultured as described (24).
HEL92.1.7 cells (American Type Culture Collection) were cultured in
RPMI medium supplemented with 10% of fetal calf serum, 2 mmol/L
L-glutamine, 1% sodium pyruvate, and 1% (v/v) penicillin/streptomy-
cin. Cell lines were verifi ed in 2006 by isolating genomic DNA and
sequencing JAK2 exon 14 (and JAK3 exon 13 in CMK cells), and later
by genotyping using Affymetrix Genome Wide Human SNP Array
6.0. MHH-CALL-4 cells (DSMZ, Braunschweig, Germany: Carries out
characterization by short-tandem repeat DNA typing) were cultured
in 24-well plates in medium as described above, but supplemented
with 1% HEPES and 1% sodium pyruvate.
Western Blotting
Cells were treated with inhibitors and extracted essentially as described
(24). Typically, 20 μg of protein lysates were resolved by NuPAGE Novex
4% to 12% Bis-Tris Midi Gels (Invitrogen) and transferred to polyvinyli-
dene difl uoride membranes by semi-dry blotting. The following antibod-
ies were used: Phospho-STAT5 (Y695; #9359), phospho-JAK1 (Y1022/
Y1023; #3331), JAK1 (#3332), phospho-JAK2 (Y1007/Y1008; #3776),
JAK2 (#3230), phospho-TYK2 (Y1054/Y1055; #9321), TYK2 (#9312),
phospho-STAT1 (Y701; #9171), phospho-STAT3 (Y705; #9131),
phospho-AMPKα (Τ172; #2535), AMPKα (#2532), BTK (#3533), and
LYN (#2796) were from Cell Signaling Technology. JAK2 (#sc-34480),
phospho-JAK3 (Y980; #sc-16567), TEL (#sc-11382), and STAT5 anti-
bodies (#sc-835) were from Santa Cruz Biotechnology. Phospho-JAK2
(Y570; #09–241) was from Millipore and 4G10 was made in-house. The
β-tubulin (#T4026) antibody was from Sigma. Antibodies were typically
incubated overnight at 4°C followed by washes and incubation with
horseradish peroxidase–conjugated secondary antibodies. Immunoreac-
tive bands were revealed with ECL reagents.
JAK Immunoprecipitation
Cells were extracted as described above. Typically, lysates were
adjusted to 0.5 mg total protein input in 200 μL of lysis buffer. Anti-
bodies for immunoprecipitation of JAK2 (#sc-34480, amino-terminal
epitope) or JAK3 (#sc-513) were from Santa Cruz Biotechnology, and
of JAK2 (#3230, carboxyl-terminal epitope) or TYK2 (#9312) from Cell
Signaling Technology. Then, 25 μL of UltraLink Immobilized Protein
A/G beads (Pierce) were added and samples were incubated for 1 hour
with rotation at 4°C. After washing, bound fractions were released by
heating at 70°C for 10 minutes in 20 μL NuPAGE LDS sample buffer
for Western blot analysis as described above.
X-ray Crystallography
Crystals were grown at 20°C using the hanging drop vapor diffusion
method. Purifi ed JAK2 kinase domain [JAK2 (840–1,132)::Y1007F,
Y1008F] with mutations Y1007F and Y1008F in complex with NVP-
BBT594 (molar ratio 1:3) at 9 mg/mL in 20 mmol/L Tris, 250 mmol/L
NaCl, 1 mmol/L DTT, pH 8.5 was mixed with an equal volume of a
reservoir solution containing 1.8 mol/L sodium malonate pH 6,
0.1 mol/L glycyl–glycine pH 8.2. Plate-like crystals grew within a few
days. Before fl ash cooling the sample in liquid nitrogen, it was trans-
ferred to a cryoprotectant solution consisting of 2.7 mol/L sodium
malonate pH 6, 0.1 mol/L glycyl–glycine pH 8.2. During data collec-
tion, the crystal was cooled at 100 K. Diffraction data were collected
at the Swiss Light Source (beamline X10SA) using a Marresearch CCD
detector and an incident monochromatic X-ray beam with 0.9999 Å
wavelength. Raw diffraction data were processed and scaled with
XDS/XSCALE software. The structure was determined by molecular
replacement with PHASER as implemented in CCP4 using as search
model the coordinates of JAK2 in complex with a pan-JAK inhibitor
(PDB code 2B7A; ref. 33). The program BUSTER was used for struc-
ture refi nement (Supplementary Table S3) using all diffraction data
between 20 and 1.34 Å resolution, excluding 5% of the data for cross-
validation. The fi nal model converged at Rwork = 17.6% (Rfree = 18.9%).
The refi ned coordinates of the complex structure have been depos-
ited in the RCSB Protein Data Bank under accession code 3UGC.
Mouse Models of Myeloproliferative Neoplasm–like Disease
The mouse models of erythropoietin-induced polycythemia (24) and
MPLW515L-driven myelofi brosis-like disease (25) have been described
previously. Animal experiments were conducted in strict adherence to
the Swiss law for animal welfare and approved by the Swiss Cantonal
Veterinary Offi ce of Basel-Stadt, or carried out according to an animal
protocol that has been approved by the Memorial Sloan-Kettering
Cancer Center Instructional Animal Care and Utilization Committee.
Disclosure of Potential Confl icts of Interest
N. Bhagwat, P. Koppikar, A. Goel, and R.L. Levine declare no com-
peting fi nancial interests. Note that all other authors are or have been
full-time employees of Novartis Pharma AG.
Acknowledgments
The authors thank Hans Drexler for the generous gift of SET-2
cells, Sébastien Rieffel, Bernard Mathis, Markus Kroemer, Céline Be,
Nina Baur, Francesca Santacroce, and Violetta Powajbo for excellent
technical assistance, Michael Eck for fruitful discussions, and Ralph
Tiedt, Patrick Chène, and David Weinstock for critical reading and
comments on the manuscript.
Received December 6, 2011; revised March 28, 2012; accepted
April 10, 2012; published OnlineFirst May 3, 2012.
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Rita Andraos, Zhiyan Qian, Débora Bonenfant, et al.
Is Binding Mode Dependent
Modulation of Activation-Loop Phosphorylation by JAK Inhibitors
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... The absence of any JAK2 point mutations in MF patients who acquired resistance to ruxolitinib suggests a role for a mutation-independent mechanism that enables persistent JAK/ STAT signaling in the setting of long-term JAK2 inhibition (Koppikar et al., 2012;Harrison et al., 2020a;Ross et al., 2021). Ruxolitinib resistance in MF has been modelled in vitro by culturing cell lines expressing JAK2 p. V617F long-term with ruxolitinib and demonstrated that ruxolitinib resistance occurs due to heterodimeric activation of JAK2 p. V617F pJAK2 by other JAK family members, a mechanism now known as ruxolitinib persistence (Andraos et al., 2012;Koppikar et al., 2012;Tvorogov et al., 2018). Interestingly, ruxolitinib persistent cells could be resensitized following ruxolitinib withdrawal (Koppikar et al., 2012), consistent with a number of clinical reports following ruxolitinib rechallenging (Gisslinger et al., 2014;Gerds et al., 2018). ...
... Imatinib also binds within the highly conserved ATP-binding site of ABL1, however, ruxolitinib inhibits a significantly higher number of kinases compared to imatinib (Davis et al., 2011), which may contribute to ruxolitinib's increased treatment-related toxicity. Furthermore, clinical resistance to ruxolitinib occurs primarily through heterodimeric activation (Andraos et al., 2012;Koppikar et al., 2012;Tvorogov et al., 2018), rather than the emergence of point mutations, enabling therapeutic resistance despite ruxolitinib binding to WT and/or JAK2 p. V617F-mutant JAK2. The adverse events associated with ruxolitinib therapy in MPNs suggests that similar clinical challenges will be observed when incorporating ruxolitinib into treatment approaches for JAK2-altered ALL. ...
... This type-II binding mode is similar to the inhibition of BCR:ABL1 with imatinib (Druker and Lydon, 2000;Schindler et al., 2000). The first type-II JAK inhibitor identified was BBT594 ( Figure 3C), which was originally designed to inhibit BCR:ABL1 harboring the TKI-resistant ABL1 p. T315I-mutation (Andraos et al., 2012). BBT594 inhibited STAT5 phosphorylation in cell models expressing either TEL::JAK2 or JAK2 p. V617F-mutant JAK2, albeit with low specificity and limited potency (Andraos et al., 2012). ...
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... Type II JAK inhibitors also bind to the ATP-binding site of the kinase domain in the inactive conformation of JAKs [27,34]. NVP-BBT594 and NVP-CHZ868 ( Figure 4) are representative examples of type II inhibitor, which target JAK2 [37,38]. ...
... Type II JAK inhibitors also bind to the ATP-binding site of the kinase domain in the inactive conformation of JAKs [27,34]. NVP-BBT594 and NVP-CHZ868 ( Figure 4) are representative examples of type II inhibitor, which target JAK2 [37,38]. • Allosteric JAK Inhibitors ...
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... The main JAK2 downstream targets involved in disease persistence are listed in Table 3. NLRP3 inflammasome Cleavage of the precursors form of IL-1β and IL-18 Cytoplasm Maturation and secretion of pro-inflammatory IL-1β and IL-18 [69] Paradoxically, mutant JAK2 has been shown to be overexpressed and hyperphosphorylated in cells treated with ruxolitinib and other JAK2 type I inhibitors. These types of inhibitors are ATP-competitor compounds, able to stabilize the kinase in its active conformation and block its downstream signaling [90,91]. The increased phosphorylation of the JAK2 activation loop seems to be dependent on the binding mode of the type I inhibitor; it is not mediated by type II inhibitors, which stabilize JAK2 in its inactive state [91]. ...
... These types of inhibitors are ATP-competitor compounds, able to stabilize the kinase in its active conformation and block its downstream signaling [90,91]. The increased phosphorylation of the JAK2 activation loop seems to be dependent on the binding mode of the type I inhibitor; it is not mediated by type II inhibitors, which stabilize JAK2 in its inactive state [91]. It has been demonstrated that the reactivation of the JAK/STAT pathway after prolonged ruxolitinib treatment depends on the formation of heterodimers between JAK2 and TYK2 or JAK1 and their subsequent transphosphorylation [35,90] ( Figure 3A). ...
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... Currently, two FDA-approved JAK2 inhibitory drugs [12][13][14] provide some clinical benefit, however, both inhibit mutated JAK2 and wt JAK2, which leads to limited clinical effectiveness. Drug resistance is another challenge, due to insufficient inhibition of phosphorylation of the activation loop on JAK2-Y1007 [15][16][17]. Thus, an alternative therapeutic approach is needed that is able to selectively target mutated JAK2 without disrupting the activity of wt JAK2 and that is able to override the resistance that is observed with available JAK2 inhibitors. ...
... In addition to the limitations resulting from inhibition of wildtype JAK2, drug resistance is also a problem due to insufficient inhibition of phosphorylation of the activation loop on JAK2-Y1007 [15][16][17]. Resistance may also result from compensatory activities by other JAK family members that form complexes with drug-inhibited JAK2 [16,35,36]. Thus, there is an urgent need for more efficacious, targeted approaches that inhibit aberrant JAK2 activity without affecting JAK2 signaling in normal cells. ...
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... Refined JAK2 inhibitors truly reducing the MPN clone and halting clonal progression are highly desirable. Investigative efforts are ongoing, as, e.g., for JAK inhibitors with a type II mode of binding similar to BCR-ABL inhibitors [86,87]. In addition, mutant calreticulin, which is exposed at the cell surface in association with MPL, could be addressed as an therapeutic target, directly relating to JAK2-STAT signaling in CALR mutant patients [38,88,89]. ...
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Chapter
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