Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer.

Abstract

We describe here the existence of a heregulin-HER3 autocrine loop, and the contribution of heregulin-dependent, HER2-mediated HER3 activation to gefitinib insensitivity in non-small cell lung cancer (NSCLC). ADAM17 protein, a major ErbB ligand sheddase, is upregulated in NSCLC and is required not only for heregulin-dependent HER3 signaling, but also for EGFR ligand-dependent signaling in NSCLC cell lines. A selective ADAM inhibitor, INCB3619, prevents the processing and activation of multiple ErbB ligands, including heregulin. In addition, INCB3619 inhibits gefitinib-resistant HER3 signaling and enhances gefitinib inhibition of EGFR signaling in NSCLC. These results show that ADAM inhibition affects multiple ErbB pathways in NSCLC and thus offers an excellent opportunity for pharmacological intervention, either alone or in combination with other drugs.

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Targeting ADAM-mediated ligand cleavage to inhibit HER3
and EGFR pathways in non-small cell lung cancer
Bin-Bing S. Zhou,1,4,* Michael Peyton,2Biao He,3Changnian Liu,1Luc Girard,2Eian Caudler,1Yvonne Lo,1
Frederic Baribaud,1Iwao Mikami,3Noemi Reguart,3Gengjie Yang,1Yanlong Li,1Wenqing Yao,1Kris Vaddi,1
Adi F. Gazdar,2Steven M. Friedman,1David M. Jablons,3Robert C. Newton,1Jordan S. Fridman,1
John D. Minna,2and Peggy A. Scherle1
1Drug Discovery, Incyte Corporation, Experimental Station, Route 141 and Henry Clay Road, Building 400, Wilmington, Delaware 19880
2Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, 600 Harry Hines
Boulevard, Dallas, Texas 75390
3Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California, San Francisco,
San Francisco, California 94115
4Present address: Tanox, Inc., 10555 Stella Link Road, Houston, Texas 77025.
*Correspondence: binbing_s_zhou@yahoo.com
Summary
We describe here the existence of a heregulin-HER3 autocrine loop, and the contribution of heregulin-dependent, HER2-
mediated HER3 activation to gefitinib insensitivity in non-small cell lung cancer (NSCLC). ADAM17 protein, a major ErbB
ligand sheddase, is upregulated in NSCLC and is required not only for heregulin-dependent HER3 signaling, but also for EGFR
ligand-dependentsignaling in NSCLC cell lines. A selective ADAM inhibitor, INCB3619, prevents the processing and activation
of multiple ErbB ligands, including heregulin. In addition, INCB3619 inhibits gefitinib-resistant HER3 signaling and enhances
gefitinib inhibition of EGFR signaling in NSCLC. These results show that ADAM inhibition affects multiple ErbB pathways in
NSCLCandthusoffersanexcellentopportunityforpharmacologicalintervention,eitheraloneorincombinationwithotherdrugs.
Introduction
The ErbB family of receptor tyrosine kinases and their ligands
are important regulators of tumor cell proliferation, angiogene-
sis, and metastasis (Gschwind et al., 2004; Yarden and Sliw-
kowski, 2001). There are four receptors in the ErbB family:
EGFR (HER1 or ErbB1), HER2 (neu or ErbB2), HER3 (ErbB3),
and HER4 (Erb4). Among them, EGFR, HER2, and HER4 have
tyrosine kinase activities; HER3 has a truncated kinase domain,
which is not functional, and thus can signal only in the context
of receptor heterodimerization (Guy et al., 1994). Eleven ligands
have been reported to bind to the ErbB receptor family, includ-
ing epidermal growth factor (EGF), transforming growth factor
a(TGFa),heparinbindingEGF-likeligand(HB-EGF),amphiregu-
lin (AR), betacellulin (BTC), epiregulin (EPR), epigen (EPG), and
heregulin (HRG)/neuregulin (NRG) family members (Harris
et al., 2003). These ligands bind directly to EGFR, HER3, or
HER4, leading to the formation of hetero- or homodimers that
trigger multiple downstream signaling cascades, including
Ras-ERK and PI3K-Akt pathways (Yarden and Sliwkowski,
2001). Although HER2 lacks a functional ligand binding domain,
it is the preferred partner for heterodimerization upon ligand
binding (Graus-Porta et al., 1997; Mosesson and Yarden, 2004).
While multiple ligands, including EGF, TGFa, HB-EGF, AR,
BTC,EPR,andEPG,canbindEGFR,heregulinistheonlyknown
ligand for HER3. Heterodimerization of HER3 with HER2 results
in the formation of the most oncogenically active ErbB receptor
complex upon heregulin stimulation; coexpression of HER3 and
HER2, butnotHER3 andEGFR,synergizes to transform NIH3T3
cells(Alimandietal.,1995;Holbroetal.,2003).Inaddition, treat-
ment of transformed cell lines with anti-HER3 antibodies has
been shown to reduce their proliferation and migration in vitro,
in part through the alteration of HER2-HER3 dimerization (van
der Horst et al., 2005), suggesting that this pathway is important
for promoting tumorigenesis. However, the properties and clin-
ical significance of a putative autocrine mechanism involving
heregulin, HER3, and HER3’s heterodimerization partners
have not been well characterized.
ErbB ligands are structurally and functionally related mem-
brane proteins that can be proteolytically cleaved and released
from cells for signaling through autocrine and paracrine mecha-
nisms (Normanno et al., 2001; Borrell-Pages et al., 2003). This
S I G N I F I C A N C E
Agents that inhibit EGFR function, such as gefitinib and cetuximab, have shown modest therapeutic efficacy in patients with NSCLC.
One mechanism of drug resistance is the activation of alternative ErbB pathways, such as signaling through HER3, as demonstrated
herein. Recently, ADAMs, particularly ADAM17, have emerged as upstream activators of ErbB ligands. We demonstrate here that a
selective ADAM inhibitor blocks the cleavage of multiple ErbB ligands and thereby inhibits the activation of multiple ErbB pathways,
including HER3 in NSCLC. A strategy targeting ADAMs may be an important complement to existing anti-ErbB approaches and could
be used in combination with various anti-ErbB and chemotherapeutic agents.
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cleavage event is critical for the activation of the ligands under
a variety of circumstances and is thought to be mediated
by ADAMs (a disintegrin and metalloproteases) (Blobel, 2005;
Gee and Knowlden, 2003; Sahin et al., 2004; Zhou et al., 2005),
which are zinc-dependent membrane-associated metallopro-
teases. More specifically, ADAM17-deficient cells have been
shown to be defective in the shedding of TGFa, HB-EGF, EPR,
AR, and heregulin (Merlos-Suarez et al., 2001; Montero et al.,
2000; Peschon et al., 1998; Sahin et al., 2004; Sunnarborg
et al., 2002; Horiuchi et al., 2005). Although ADAM17 has been
suggestedtobethemajorErbBligandsheddase,ADAM10ispo-
tentiallythemainsheddaseofEGFandBTCinmouseembryonic
fibroblasts (Sahin et al., 2004) and possibly other ligands under
specificcircumstances.ItisapparentthattheseADAMsregulate
theavailabilityofErbBligandsbyliberatingthesolublefunctional
forms and, as a result, activate ErbB signaling.
Several anti-EGFR agents, including antibodies and small
molecule kinase inhibitors, have been approved recently to treat
various human cancers (Baselga and Arteaga, 2005; Herbst
et al., 2004). The chimeric monoclonal antibody cetuximab
(Erbitux), which blocks EGFR ligand binding, has been shown
to improve the survival of patients with metastatic colon cancer
and has shown efficacy in patients with non-small cell lung can-
cer (NSCLC) (Baselga and Arteaga, 2005), clinically validating
the approach of targeting EGFR ligands to treat these cancers.
The EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erloti-
nib (Tarceva) have also shown efficacy in patients with NSCLC
(Herbst et al., 2004), particularly in a subgroup of patients that
have mutations in the EGFR kinase domain that are more sus-
ceptible to inhibition by these molecules (Lynch et al., 2004;
Paez et al., 2004; Pao et al., 2004). Unlike other growth factor
receptors, many of these mutant EGFR proteins are not consti-
tutively active and are still sensitive to ligand stimulation (Lynch
et al., 2004; Riemenschneider et al., 2005).
Despitetheefficacyoftheantibodiesandkinaseinhibitorstar-
geting the EGFR pathway, the majority of patients do not expe-
riencelong-termbenefitfromthesetherapies.Datafrompreclin-
ical models suggest that activation ofalternative ErbB pathways
can bypass specific blockade and that combination therapies
are more effective than any single agent against the EGFR path-
way (Huang et al., 2004; Matar et al., 2004; Normanno et al.,
2002). Alternative strategies targeting the ErbB pathway are still
needed.ApotentADAMinhibitorwouldfulfillthisrolebycontrol-
ling the availability of multiple ErbB ligands and could potentially
be used in combination with various anti-ErbB agents. It should
be noted that many of the existing broad-spectrum metallopro-
tease inhibitors, such as marimastat, inhibit not only MMPs (ma-
trix metalloproteinases) but also ADAMs in vitro (Roghani et al.,
1999) and thereby prevent the processing of multiple ErbB li-
gands (Dong et al., 1999; O-Charoenrat et al., 2002). However,
their clinical development has been hampered by side effects,
many of which are likely due to MMP inhibition (Coussens
etal.,2002;Zhouetal.,2005).Inaddition,itremainstobetested
whetheraselectiveADAMinhibitorcanalsoinhibitmultipleErbB
ligand processing and signaling.
Since the importance of the ErbB pathway and the molecular
understanding of its sensitivity to EGFR antagonists are best
documented in NSCLC, and NSCLC is the leading cause of
death from cancer in both men and women in the United States
(Jemal et al., 2005), we focused our current study in this disease
setting and tried to address the following questions. Does the
activation of alternative ErbB signaling pathways, specifically
those involving heregulin and HER3, lead to resistance to anti-
EGFR agents such as gefitinib in NSCLC? Which ADAM should
we target to inhibit the HER3 pathway and overcome heregulin-
dependent gefitinib resistance? Can we find an ADAM inhibitor
that is more selective than marimastat but still capable of inhib-
iting cleavage ofmultipleErbB ligands?CantheselectiveADAM
inhibitor be used in combination with gefitinib and chemothera-
peutic agents in NSCLC?
Results
A HER3 autocrine stimulatory loop in NSCLC
To test our hypothesis that the heregulin-HER3 autocrine loop
may be involved in human lung tumorigenesis, we examined
the expression of heregulin and HER3 in NSCLC tumors using
antibodies against heregulin and HER3. Fourteen fresh NSCLC
tumors, of which seven were squamous cell carcinomas (cases
1, 2, 4, 5, 7, 9, and 10) and seven were adenocarcinomas (cases
3, 6, 8, 11, 12, 13, and 14), and their autologous-matched non-
malignant lung tissues were obtained from patients undergoing
tumor resection as part of their treatment. As shown in Figure 1,
nine out of fourteen freshly resected NSCLC tissues presented
Figure 1. Protein expression of heregulin, HER3, ADAM17, and ADAM10, and activation phosphorylation of HER3 in NSCLC tumor samples
Whole-cell extracts were prepared using tumor tissue (T) and adjacent nonmalignant lung tissue (N) from NSCLC patients (cases 1 to 14) and subjected to
SDS-PAGE and Western blot with various antibodies. b-Actin expression was used as a loading control.
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with increased expression of heregulin compared with nonma-
lignant lung tissue control, while seven out of fourteen had
increased expression of HER3. To show directly that the HER3
autocrine stimulatory loop was active in NSCLC, we used a
phosphospecific antibody against Tyr-1289 of HER3, which is
phosphorylated upon heregulin binding and is in a YXXM motif
that participates in direct signaling to PI3 kinase (Kim et al.,
1994). As shown in Figure 1, at least five out of fourteen freshly
resected NSCLC tissues had activated HER3, and all of them
coexpressed both HER3 and heregulin, suggesting the exis-
tence of a HER3 autocrine stimulatory loop in NSCLC.
Heregulin and HER2-mediated HER3 activation correlate
with insensitivity to gefitinib among NSCLC cell lines
Because the clinical significance of HER3 activation in NSCLC
is currently unknown, we used NSCLC cell lines to ask whether
HER3activationcouldcontributetodrugresistance.Werecently
evaluatedgeneexpressionin44differentNSCLCcelllinesusing
microarrays and determined their in vitro sensitivity to gefitinib
(L.G., M.P., I. Sekine, S. Zachariah, C. Lam, M. Wong, D. Beer,
W. Gerald, A.F.G., and J.D.M., unpublished data; M.P., L.G.,
and J.D.M., unpublished data). To examine the effects of ErbB
ligands and receptors on gefitinib sensitivity, we compared their
mRNA levels against gefitinib sensitivity across the 44 NSCLC
cell lines. Among various genes examined (Figure 2A), only the
expressionofheregulin(HRG)significantlycorrelatedwithinsen-
sitivitytogefitinib(p<0.001).Interestingly,HER3expressionvery
weaklycorrelatedwithgefitinibsensitivity(p=0.135),suggesting
that it is the heregulin-induced HER3 activation rather than the
level of HER3 expression that leads to gefitinib insensitivity.
Upon ligand binding, HER3 can form heterodimers with both
EGFR and HER2. To further examine our hypothesis, we added
Figure 2. Heregulin contributes to gefitinib resis-
tance in NSCLC cell lines
A: Heregulin expression correlates with gefitinib
insensitivity in NSCLC cell lines. Microarray ex-
pression data are presented on 42 NSCLC cell
lines, including five gefitinib-sensitive cell lines
(IC50 < 0.4 mM, highlighted in yellow) and 37 ge-
fitinib-insensitive cell lines (IC50 > 4.0 mM, high-
lighted in orange). Calu-3 and H820 cell lines
have IC50s of 1.0 and 3.0 mM, respectively, and
their microarray data are not shown. Log ratios
and p values are shown on the left. Expression
levels are color coded such that darker colors
correspond to higher levels.
B: Effects of soluble heregulin on the gefitinib
sensitivities of five gefitinib-sensitive cell lines.
Shown are gefitinib IC50s with error bars (SD; n =
8) of various cell lines with and without heregulin
(50 ng/ml), and the IC50 shift caused by adding
heregulin. While H3255 and HCC827 have geno-
mic amplification of EGFR, HCC2279, HCC2935,
and HCC4006 do not.
C: Addition of soluble heregulin makes the gefiti-
nib-sensitive cell line HCC2935 more than 60-fold
less sensitive to gefitinib. Cells with or without
heregulin (50 ng/ml) added were treated for
96 hr in the presence of increasing concentra-
tions of gefitinib, and their growth was then
measured using the MTS assay and plotted as
a percentage of the growth of untreated cells
(control) with error bars (SD; n = 3).
D: Heregulin-induced HER3 and Akt phosphory-
lation can bypass the gefitinib blockade in
HCC2935. HCC2935 cells were serum starved
for 6 hr and then grown in the presence of vari-
ous concentration of gefitinib for 12 hr followed
byheregulin(50ng/ml)orEGF(50ng/ml)stimula-
tion for 30 min. Western blots are shown for phos-
pho- and total HER3, ERK1/2, and Akt.
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soluble heregulin to five gefitinib-sensitive cell lines: all of them
have EGFR mutations, but none of them expresses significant
levelsofheregulin(Figure2A).Amongthem,threecelllineswith-
out EGFR amplification (HCC2935, HCC2279, and HCC4006)
showed increased resistance to gefitinib after heregulin was
added (Figure 2B). The change is particularly dramatic for
HCC2935, the only gefitinib-sensitive line in our study that ex-
presses more HER2 than EGFR (Figure 2A; M.P., L.G., and
J.D.M., unpublished data). As shown in Figure 2C, the addition
of soluble heregulin shifts the IC50 of gefitinib from 110 nM
to 6.71 mM. The addition of soluble EGF at similar concentra-
tions did not have much impact on gefitinib sensitivity in the
HCC2935 cell line (data not shown). Furthermore, while the con-
stitutive Akt phosphorylation and EGF-induced Akt phosphory-
lation in HCC2935 are sensitive to gefitinib, heregulin-induced
HER3 phosphorylation and Akt phosphorylation are not inhibit-
able by gefitinib at concentrations up to 5 mM (Figure 2D). On
the contrary, H3255 and HCC827, both of which have genomic
amplification of the EGFR locus (Tracy et al., 2004; Amann et al.,
2005) and presumably predominant EGFR homodimers or het-
erodimers, do not change their sensitivity to gefitinib after the
addition of either heregulin (Figure 2B) or EGF (data not shown).
Since gefitinib is selective toward EGFR, heregulin-dependent
HER3 activation may bypass gefitinib inhibition when HER2-
HER3 is one of the major heterodimers present.
A549 is an example of a HER3 autocrine line that is
insensitivetogefitinibanddependentonEGFRandHER2
for HER3 signaling
Among the few NSCLC cell lines expressing high levels of here-
gulin, HER3, and HER2 (Figure 2A), A549 has been previously
suggested to be a heregulin-HER3 autocrine line (Gollamudi
et al., 2004). We confirmed that A549 has constitutively active
HER3 under serum-free conditions (Figure 3A). To further char-
acterize A549 as a heregulin-HER3 autocrine line, we exoge-
nously added anti-HER3 antibody that can block heregulin
binding to HER3. As shown in Figure 3A, blocking of heregulin
binding inhibits the constitutive phosphorylation of HER3 as
well as Akt downstream in the pathway, suggesting that heregu-
lin functions as an autocrine growth factor in A549. On the other
hand, the constitutive ERK activity in A549 was not affected
by blocking heregulin binding and may be mediated by mutant
K-ras (Valenzuela and Groffen, 1986). In contrast to the anti-
HER3 antibody, gefitinib does not have a significant impact on
this autocrine pathway at the concentrations tested (Figure 3A),
implicating the role of HER2 in this autocrine loop as well. This
hypothesis is supported by RNA interference experiments:
knockdown of either EGFR or HER2 is not sufficient to signifi-
cantly inhibit the HER3 autocrine loop in A549, while knocking
down both leads to a dramatic reduction in both HER3 and
Akt phosphorylation (Figure 3B).
ADAM17 protein correlates with HER3 activation in
NSCLC and is required for HER3 signaling in A549 cells
ADAM17 was suggested to be a major sheddase for heregulin
(Horiuchi et al., 2005) and, therefore, could contribute to the
autocrine activation of HER3 in NSCLC. To study the role of
ADAM17 in NSCLC, we analyzed the expression of ADAM17
in NSCLC tumors using antibodies against ADAM17. As shown
in Figure 1, ten out of fourteen freshly resected NSCLC tissues
had increased expression of ADAM17 compared with autolo-
gous-matched nonmalignant lung tissue control. In addition,
there was a high rate of activation of HER3 in tumors with in-
creased ADAM17 levels compared with those tumors with low
ADAM17 expression (p % 0.05). Although many patients have
similar ADAM10 protein expression in tumors versus control,
several of them (including cases 2, 4, and 11) have a higher ratio
of the processed versus the proform of ADAM10 in the tumor
samples, indicative of ADAM10 activation in these patients.
However, ADAM10 activation does not correlate with HER3 ac-
tivation (Figure 1), and neither do the other ADAMs previously
Figure 3. Characterization of A549 as a HER3 autocrine line and its dependence on ADAM17
A: Effects of anti-HER3 antibody and gefitinib on phosphorylation of HER3 and downstream Akt and ERK1/2 in A549. A549 cells were serum starved for 6 hr and
then grown with various treatments for 48 hr. Western blots are shown for phospho- and total HER3, Akt, and ERK1/2. Phospho- and total Akt and HER3 signals
were quantitated, and the ratios are presented relative to control.
B:BothEGFRandHER2arerequired forheregulin-dependentHER3signalinginA549. A549cellsweretransfected withappropriatesiRNAs twice, serumstarved
for 48 hr, and then harvested for Western blot.
C: ADAM17 is important for heregulin-dependent HER3 signaling in A549. siRNA experiments were performed as in B.
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implicated in ErbB ligand processing (Figure S1 in the Supple-
mental Data available with this article online).
To test if either ADAM17 or ADAM10 is involved in HER3
autocrine signaling in A549, we used RNA interference to selec-
tively knock down the expression level of either protein. As
shown in Figure 3C, ADAM10 knockdown had no significant
effect on HER3 or Akt activity in A549, whereas knockdown of
ADAM17 mirrored the effect of the anti-HER3 antibody and
affected both HER3 and Akt phosphorylation. On the other
hand, knocking down ADAM9 or ADAM15,two ADAMs with sig-
nificant expression in A549, had no apparent effect on HER3 au-
tocrine signaling in A549 cells (Figure S2). Together, these re-
sults support ADAM17 as a potential target for modulating the
HER3 autocrine loop in NSCLC.
Inhibition of ADAM protease activity prevents the
cleavage of heregulin and EGFR ligands
To studythe impactofADAMinhibition onshedding ofheregulin
as well as EGFR ligands and its potential to inhibit multiple
ErbB pathways in the clinic, we identified a selective, orally bio-
available small molecule inhibitor, INCB3619 (Figure 4A; Liu
et al., 2006), which has potent inhibitory activity against both
ADAM10 and ADAM17 and a better selectivity profile than
marimastat across a panel of metalloproteases (Figure 4B).
The selectivity and inhibitory activity of the structurally related
molecules INCB8765, INCB4298, and INCB3420 are also pre-
sented in Figure 4B. Since INCB3619 has some MMP2 and
MMP12 inhibitory activities, INCB3420, which lacks ADAM10
and ADAM17 activity, was identified as a negative control. As
shown in Figure 4C, the dual ADAM inhibitor INCB3619 inhibits
heregulin cleavage with an IC50 value of 0.24 mM. In addition
to heregulin, INCB3619 can prevent the cleavage of TGFa,
HB-EGF, AR,EGF (Figure 4D), andHER2(Liu et al.,2006)similar
tomarimastat. Inagreement withdataobtained fromstudiesus-
ing knockout cells (Sahin et al., 2004; Horiuchi et al., 2005), we
found that the ADAM17-selective inhibitor INCB4298 inhibits
the shedding of heregulin, TGFa, HB-EGF, and AR, whereas
the ADAM10-selective inhibitor INCB8765 blocks EGF ligand
processing. These results suggest that pharmacological inter-
vention of ADAM activity has a similar effect to knocking out
the protein. In contrast, the MMP2/12-active, ADAM10/17-inac-
tive INCB3420 did not block the shedding of any tested ErbB
ligands (Figure 4D), further strengthening the argument that it
is ADAMs, particularly ADAM17 and ADAM10, not MMPs, that
are involved in ErbB ligand shedding. To maximize the effect
on ErbB ligand processing and signaling, the dual inhibitor
INCB3619 was chosen for further characterization.
INCB3619 abrogates heregulin activation and heregulin-
dependent HER3 signaling and increases gefitinib
sensitivity in A549 cells
To examine the effect of ADAMs and their inhibitors on the func-
tional release and activation of heregulin, conditioned medium
from A549 cells treated with inhibitors or transfected with
siRNAs was added to the medium of a reporter cell, MCF-7,
which expresses HER3 but insignificant amount of heregulin.
As shown in Figure 5A, INCB3619 inhibits the heregulin release
and activation, measured by HER3 activation in MCF-7, similar
to the effect of ADAM17 siRNA. The ADAM17-selective inhibitor
INCB4298 showedasimilar effect,whereas the ADAM10-selec-
tive inhibitor INCB8765 or ADAM10 siRNA had no effect. As a
control, the heregulin neutralizing antibody and the anti-HER3
antibody can block the activity in A549 conditioned medium
(FigureS3),confirmingthatitistheheregulinactivitythatismea-
sured in the MCF-7 reporter cell assay.
Similar to antibody blockage of heregulin binding to HER3,
INCB3619 can inhibit the heregulin-dependent HER3-Akt path-
way but not ERK activity in A549 cells; the ADAM17-selective
inhibitor INCB4298 has a similar effect (Figure 5B). Addition of
soluble heregulin can bypass the inhibition, consistent with the
hypothesis that heregulin cleavage is the target of inhibition.
As controls, the ADAM10-selective inhibitor INCB8765 (Fig-
ure 5B) and the ADAM10/17-inactive inhibitor INCB3420 (data
not shown) did not have a significant impact on this pathway.
Since a HER3 autocrine mechanism is believed to contribute
to the gefitinib insensitivity in A549 cells, we tested whether
INCB3619 can sensitize A549 cells to gefitinib. As shown in Fig-
ure 5C, INCB3619 shifts the IC50 of gefitinib in A549 cells quite
significantly, further supporting the role of a heregulin-HER3
Figure 4. Selective ADAM inhibitor INCB3619 inhibits the cleavage of here-
gulin and EGFR ligands
A: Chemical structure of INCB3619.
B: Selectivity and inhibition profile of INCB3619 and related ADAM inhibitors
against a panel of metalloproteases.
C: INCB3619 inhibits the heregulin cleavage measured by ELISA assay. Data
are presented as mean percentage inhibition values with error bars (SD;
n = 4).
D:Effects ofseveralADAMinhibitorsonthecleavageofvarious ErbBligands.
IC50s are presented here.
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