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


Abstract and Figures

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
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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 (
Current address for L. Wyder: Actelion Pharmaceuticals Ltd., Allschwil,
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;
doi: 10.1158/2159-8290.CD-11-0324
©2012 American Association for Cancer Research.
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Andraos et al.
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.
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
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
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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Andraos et al.
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
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
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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Andraos et al.
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.
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Binding Mode-Dependent Modulation of JAK Phosphorylation RESEARCH ARTICLE
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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Published OnlineFirst May 3, 2012; DOI: 10.1158/2159-8290.CD-11-0324
Andraos et al.
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
(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.
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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Published OnlineFirst May 3, 2012; DOI: 10.1158/2159-8290.CD-11-0324
Andraos et al.
inhibiting JAK kinase activity might present a novel thera-
peutic strategy for the treatment of JAK-dependent diseases.
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.
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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2012;2:512-523. Published OnlineFirst May 3, 2012.Cancer Discovery
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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Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, arising from immature lymphocytes that show uncontrolled proliferation and arrested differentiation. Genomic alterations affecting Janus kinase 2 (JAK2) correlate with some of the poorest outcomes within the Philadelphia-like subtype of ALL. Given the success of kinase inhibitors in the treatment of chronic myeloid leukemia, the discovery of activating JAK2 point mutations and JAK2 fusion genes in ALL, was a breakthrough for potential targeted therapies. However, the molecular mechanisms by which these alterations activate JAK2 and promote downstream signaling is poorly understood. Furthermore, as clinical data regarding the limitations of approved JAK inhibitors in myeloproliferative disorders matures, there is a growing awareness of the need for alternative precision medicine approaches for specific JAK2 lesions. This review focuses on the molecular mechanisms behind ALL-associated JAK2 mutations and JAK2 fusion genes, known and potential causes of JAK-inhibitor resistance, and how JAK2 alterations could be targeted using alternative and novel rationally designed therapies to guide precision medicine approaches for these high-risk subtypes of ALL.
... 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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Janus kinase (JAK) is a family of cytoplasmic non-receptor tyrosine kinases that includes four members, namely JAK1, JAK2, JAK3, and TYK2. The JAKs transduce cytokine signaling through the JAK-STAT pathway, which regulates the transcription of several genes involved in inflammatory, immune, and cancer conditions. Targeting the JAK family kinases with small-molecule inhibitors has proved to be effective in the treatment of different types of diseases. In the current review, eleven of the JAK inhibitors that received approval for clinical use have been discussed. These drugs are abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, and upadacitinib. The aim of the current review was to provide an integrated overview of the chemical and pharmacological data of the globally approved JAK inhibitors. The synthetic routes of the eleven drugs were described. In addition, their inhibitory activities against different kinases and their pharmacological uses have also been explained. Moreover, their crystal structures with different kinases were summarized, with a primary focus on their binding modes and interactions. The proposed metabolic pathways and metabolites of these drugs were also illustrated. To sum up, the data in the current review could help in the design of new JAK inhibitors with potential therapeutic benefits in inflammatory and autoimmune diseases.
... 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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Aberrant signaling in myeloproliferative neoplasms may arise from alterations in genes coding for signal transduction proteins or epigenetic regulators. Both mutated and normal cells cooperate, altering fragile balances in bone marrow niches and fueling persistent inflammation through paracrine or systemic signals. Despite the hopes placed in targeted therapies, myeloid proliferative neoplasms remain incurable diseases in patients not eligible for stem cell transplantation. Due to the emergence of drug resistance, patient management is often very difficult in the long term. Unexpected connections among signal transduction pathways highlighted in neoplastic cells suggest new strategies to overcome neoplastic cell adaptation.
... 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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Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell’s intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN.
... 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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Somatic mutations in JAK2, calreticulin, and MPL genes drive myeloproliferative neoplasms (MPN), and recent technological advances have revealed a heterogeneous genomic landscape with additional mutations in MPN. These mainly affect genes involved in epigenetic regulation and splicing and are of diagnostic and prognostic value, predicting the risk of progression and informing decisions on therapeutic management. Thus, genetic testing has become an integral part of the current state-of-the-art laboratory work-up for MPN patients and has been implemented in current guidelines for disease classification, tools for prognostic risk assessment, and recommendations for therapy. The finding that JAK2, CALR, and MPL driver mutations activate JAK2 signaling has provided a rational basis for the development of targeted JAK2 inhibitor therapies and has fueled their translation into clinical practice. However, the disease-modifying potential of JAK2 inhibitors remains limited and is further impeded by loss of therapeutic responses in a substantial proportion of patients over time. Therefore, the investigation of additional molecular vulnerabilities involved in MPN pathogenesis is imperative to advance the development of new therapeutic options. Combination of novel compounds with JAK2 inhibitors are of specific interest to enhance therapeutic efficacy of molecularly targeted treatment approaches. Here, we summarize the current insights into the genetic basis of MPN, its use as a diagnostic and prognostic tool in clinical settings, and the most recent advances in targeted therapies for MPN.
Triple-negative breast cancer (TNBC) and HER2-positive breast cancer are particularly aggressive and the effectiveness of current therapies for them is limited. TNBC lacks effective therapies and HER2-positive cancer is often resistant to HER2-targeted drugs after an initial response. The recent studies have demonstrated that the combination of JAK2 inhibitors and SMO inhibitors can effectively inhibit the growth and metastasis of TNBC and HER2-positive drug resistant breast cancer cells. In this study, deep reinforcement learning was used to learn the characteristics of existing small molecule inhibitors of JAK2 and SMO, and to generate a novel library of small molecule compounds that may be able to inhibit both JAK2 and SMO. Subsequently, the molecule library was screened by molecular docking and a total of 7 compounds were selected out as dual inhibitors of JAK2 and SMO. Molecular dynamics simulations and binding free energies showed that the top three compounds stably bound to both JAK2 and SMO proteins. The binding free energies and hydrogen bond occupancy of key amino acids indicate that A8976 and A10625 has good properties and could be a potential dual-target inhibitor of JAK2 and SMO.
Hyperactivation of the Janus kinase 2 (JAK2) signaling pathway leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 can be used as an effective strategy for the treatment of MPNs. Here, our study indicated that WWQ-131 was a highly selective JAK2 inhibitor (IC50 =2.36 nM), with 182-fold and 171-fold more selective to JAK1 and JAK3, respectively. In JAK2V617F-dependent cell lines, WWQ-131 efficaciously inhibited cell proliferation, induced cell cycle arrest at the G2/M phase and apoptosis, and blocked the aberrant activation of JAK2 signaling pathway. In a mouse Ba/F3_JAK2V617F driven disease model, WWQ-131 effectively suppressed STAT5 phosphorylation in spleen and liver, and inhibited Ba/F3_JAK2V617F cells spreading and proliferation in vivo. In addition, WWQ-131 suppressed rhEPO-induced extramedullary erythropoiesis and polycythemia in mice, as well as hematocrits and spleen sizes, especially had no effect on white blood cell count. Furthermore, WWQ-131 (75 mg/kg) exhibited stronger therapeutic effects than fedratinib (120 mg/kg) in these two MPN models. Taken together, this study suggests that WWQ-131 will be a promising candidate for the treatment of MPNs.
Cytokines act through their membrane-bound receptors to transmit a variety of signals including cell survival, proliferation, differentiation and functional activity. Cytokine receptors are a conserved family of about 40 members that includes the receptors for hormones, interleukins, interferons and colony stimulating factors. Abnormal cytokine levels or aberrations in their signaling pathways can lead to a variety of diseases including cancers and inflammatory conditions reflecting their importance in normal hematopoiesis and immunity. Determination of the three-dimensional atomic structures of cytokines and their receptors has provided detailed insights into how cytokines transmit biological signals across cell membranes.
JAK2 is a non-receptor tyrosine kinase that regulates hematopoiesis through the JAK-STAT pathway. The pseudokinase domain (JH2) is an important regulator of the activity of the kinase domain (JH1). V617F mutation in JH2 has been associated with the pathogenesis of various myeloproliferative neoplasms, but JAK2 JH2 has been poorly explored as a pharmacological target. In light of this, we aimed to develop JAK2 JH2 binders that could selectively target JH2 over JH1 and test their capacity to modulate JAK2 activity in cells. Toward this goal, we optimized a diaminotriazole lead compound into potent, selective, and cell-permeable JH2 binders leveraging computational design, synthesis, binding affinity measurements for the JH1, JH2 WT, and JH2 V617F domains, permeability measurements, crystallography, and cell assays. Optimized diaminotriazoles are capable of inhibiting STAT5 phosphorylation in both WT and V617F JAK2 in cells.
Philadelphia-negative classical Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hemopathies that emerge in the hematopoietic stem cell (HSC) compartment. MPN driver mutations are restricted to specific exons (14 and 12) of Janus kinase 2 (JAK2), thrombopoietin receptor (MPL/TPOR) and calreticulin (CALR) genes, are involved directly in clonal myeloproliferation and generate the MPN phenotype. As a result, an increased number of fully functional erythrocytes, platelets and leukocytes is observed in the peripheral blood. Nevertheless, the complexity and heterogeneity of MPN clinical phenotypes cannot be solely explained by the type of driver mutation. Other factors, such as additional somatic mutations affecting epigenetic regulators or spliceosomes components, mutant allele burdens and modifiers of signaling by driver mutants, clonal architecture and the order of mutation acquisition, signaling events that occur downstream of a driver mutation, the presence of specific germ-line variants, the interaction of the neoplastic clone with bone marrow microenvironment and chronic inflammation, all can modulate the disease phenotype, influence the MPN clinical course and therefore, might be useful therapeutic targets.
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BACKGROUND: Children with Down's syndrome have a greatly increased risk of acute megakaryoblastic and acute lymphoblastic leukaemias. Acute megakaryoblastic leukaemia in Down's syndrome is characterised by a somatic mutation in GATA1. Constitutive activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) pathway occurs in several haematopoietic malignant diseases. We tested the hypothesis that mutations in JAK2 might be a common molecular event in acute lymphoblastic leukaemia associated with Down's syndrome. METHODS: JAK2 DNA mutational analysis was done on diagnostic bone marrow samples obtained from 88 patients with Down's syndrome-associated acute lymphoblastic leukaemia; and 216 patients with sporadic acute lymphoblastic leukaemia, Down's syndrome-associated acute megakaryoblastic leukaemia, and essential thrombocythaemia. Functional consequences of identified mutations were studied in mouse haematopoietic progenitor cells. FINDINGS: Somatically acquired JAK2 mutations were identified in 16 (18%) patients with Down's syndrome-associated acute lymphoblastic leukaemia. The only patient with non-Down's syndrome-associated leukaemia but with a JAK2 mutation had an isochromosome 21q. Children with a JAK2 mutation were younger (mean [SE] age 4.5 years [0.86] vs 8.6 years [0.59], p<0.0001) at diagnosis. Five mutant alleles were identified, each affecting a highly conserved arginine residue (R683). These mutations immortalised primary mouse haematopoietic progenitor cells in vitro, and caused constitutive Jak/Stat activation and cytokine-independent growth of BaF3 cells, which was sensitive to pharmacological inhibition with JAK inhibitor I. In modelling studies of the JAK2 pseudokinase domain, R683 was situated in an exposed conserved region separated from the one implicated in myeloproliferative disorders. INTERPRETATION: A specific genotype-phenotype association exists between the type of somatic mutation within the JAK2 pseudokinase domain and the development of B-lymphoid or myeloid neoplasms. Somatically acquired R683 JAK2 mutations define a distinct acute lymphoblastic leukaemia subgroup that is uniquely associated with trisomy 21. JAK2 inhibitors could be useful for treatment of this leukaemia. FUNDING: Israel Trade Ministry, Israel Science Ministry, Jewish National Fund UK, Sam Waxman Cancer Research Foundation, Israel Science Foundation, Israel Cancer Association, Curtis Katz, Constantiner Institute for Molecular Genetics, German-Israel Foundation, and European Commission FP6 Integrated Project EUROHEAR.
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tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-α/β binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-α8 and partial signaling. While no contribution of this protein toward interferon-β binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-α and -β binding and signaling.
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Theprotein-tyrosine kinases (PTKs) area burgeoning family ofproteins, eachofwhichbears a conserved domainof250to300aminoacids capable ofphosphorylating substrate proteins on tyrosine residues. We recently exploited theexistence oftwohighly conserved sequenceelements within thecatalytic domainto generate PTK-specific degenerate oligonucleotide primers (A.F.Wilks, Proc. Natl. Acad.Sci. USA86:1603- 1607, 1989). Byapplication ofthepolymerase chain reaction, portions ofthecatalytic domains ofseveral novel PTKswere amplified. We describe heretheprimary sequenceofoneofthese new PTKs,JAK1(fromJanus kinase), amemberofa new class ofPTKcharacterized bythepresenceofasecond phosphotransferase-related domainimmediately N terminal tothePTK domain. Thesecond phosphotransferase domainbearsallthe hallmarks ofa protein kinase, although its structure differs significantly fromthatofthePTKandthreonine/ serine kinase family members. A second memberofthis family (JAK2)hasbeenpartially characterized and exhibits a similar arrayofkinase-related domains. JAK1isa large, widely expressed membrane-associated phosphoprotein ofapproximately 130,000 Da.ThePTKactivity ofJAK1hasbeenlocated intheC-terminal PTK-like domain. Therole ofthesecond kinaselike domainisunknown. Protein-tyrosine kinases (PTKs)are structurally well suited toa roleinintracellular signal transduction. Many growthfactor receptors, forexample, transduce theextra- cellular stimulus theyreceive through interaction withtheir cognateligand viaan intracellular tyrosine kinase domain(5, 33,52;reviewed inreference 60).MembersofthePTK family eachbeara highly related "catalytic" domain. The phylogenetic relationships established by an aminoacid sequencecomparison ofthecatalytic domains (10) areborne outintheoverall structure ofthePTKs.Forexample, families ofPTKs,suchasthose based on thestructure ofthe
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SB1518 is an innovative pyrimidine-based macrocycle that shows a unique kinase profile with selective inhibition of Janus Kinase-2 (JAK2; IC50=23 and 19 nM for JAK2WT and JAK2V617F, respectively) within the JAK family (IC50=1280, 520 and 50 nM for JAK1, JK3 and TYK2, respectively) and fms-like tyrosine kinase-3 (FLT3; IC50=22 nM). SB1518 shows potent effects on cellular JAK/STAT pathways, inhibiting tyrosine phosphorylation on JAK2 (Y221) and downstream STATs. As a consequence SB1518 has potent anti-proliferative effects on myeloid and lymphoid cell lines driven by mutant or wild-type JAK2 or FLT3, resulting from cell cycle arrest and induction of apoptosis. SB1518 has favorable pharmacokinetic properties after oral dosing in mice, is well tolerated and significantly reduces splenomegaly and hepatomegaly in a JAK2V617F-driven disease model. SB1518 dose-dependently inhibits intra-tumor JAK2/STAT5 signaling, leading to tumor growth inhibition in a subcutaneous model generated with SET-2 cells derived from a JAK2V617F patient with megakaryoblastic leukemia. Moreover, SB1518 is active against primary erythroid progenitor cells sampled from patients with myeloproliferative disease. In summary, SB1518 has a unique profile and is efficacious and well tolerated in JAK2-dependent models. These favorable properties are now being confirmed in clinical studies in patients with myelofibrosis and lymphoma.Keywords: SB1518; JAK2; myelofibrosis; lymphoma; kinase inhibitor
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Two distinct types of interferon, IFN-/ and IFN-, commonly exhibit antiviral activities by transmitting signals to the interior of the cell via their homologous receptors. Receptor stimulation results in the activation of distinct combinations of Janus family protein tyrosine kinases (Jak PTKs); Jak1/Tyk2 and Jak1/Jak2 for IFN-/ and IFN-, respectively. Jak PTK activation by these IFNs is commonly followed by tyrosine phosphorylation of the transcription factor Stat1 at Y701, which is essential for dimerization, translocation to the nucleus and DNA-binding activity. To gain full transcriptional activity, Stat1 also requires serine phosphorylation at S727. In this paper we demonstrate that Pyk2, which belongs to another PTK family, is critical for the Jak-mediated MAPK and Stat1 activation by IFN-, but not IFN-. Pyk2 is selectively associated with Jak2 and activated by IFN-. Overexpression of PKM, a dominant interfering form of Pyk2, in NIH 3T3 cells results in a strong inhibition of the IFN--induced activation of Erk2, serine phosphorylation of Stat1 and Stat1-dependent gene transcription. Finally, the antiviral action of IFN-, but not IFN-, is severely impaired by PKM overexpression. Thus, the two types of IFN may utilize distinct Jak-mediated Erk2, and possibly other MAPK activation pathways for their antiviral action.
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Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2.
ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues-including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region--although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K(d) of approximately 1.1 microM. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened alphaC-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the "inactive-like" configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.
Genetic deficiency of Jak3 leads to abrogation of signal transduction through the common gamma chain (γc) and thus to immunodeficiency suggesting that specific inhibition of Jak3 kinase may result in immunosuppression. Jak1 cooperates with Jak3 in signaling through γc-containing receptors. Unexpectedly, a Jak3-selective inhibitor was less efficient in abolishing STAT5 phosphorylation than pan-Jak inhibitors. We therefore explored the roles of Jak1 and Jak3 kinase functionality in signaling using a reconstituted system. The presence of kinase-inactive Jak1 but not kinase-inactive Jak3 resulted in complete abolishment of STAT5 phosphorylation. Specific inhibition of the "analog-sensitive" mutant AS-Jak1 but not AS-Jak3 by the ATP-competitive analog 1NM-PP1 abrogated IL-2 signaling, corroborating the data with the selective Jak3 inhibitor. Jak1 thus plays a dominant role over Jak3 and these data challenge the notion that selective ATP-competitive Jak3 kinase inhibitors will be effective.
Recent advances in our understanding of the pathogenesis of the Philadelphia chromosome-negative myeloproliferative neoplasms, polycythaemia vera, essential thrombocythaemia and myelofibrosis have led to the identification of the mutation V617F in Janus kinase (JAK) as a potential therapeutic target. This information has prompted the development of ATP-competitive JAK2 inhibitors. Therapy with JAK2 inhibitors may induce rapid and marked reductions in spleen size and can lead to remarkable improvements in constitutional symptoms and overall quality of life. Because JAKs are involved in the pathogenesis of inflammatory and immune-mediated disorders, JAK inhibitors are also being tested in clinical trials in patients with rheumatoid arthritis and psoriasis, as well as for the treatment of other autoimmune diseases and for the prevention of allograft rejection. Preliminary results indicate that these agents hold great promise for the treatment of JAK-driven disorders.