LABORATORY INVESTIGATION

Reduction of sporadic and neurofibromatosis

type 2–associated vestibular schwannoma growth in vitro
and in vivo after treatment with the c-Jun N-terminal
kinase inhibitor AS602801
Mark C. Dougherty, MD,1 Seiji B. Shibata, MD, PhD,2 J. Jason Clark, MS,2 Franklin J. Canady, MD,2
Charles W. Yates, MD,3 and Marlan R. Hansen, MD2
1
Department of Neurosurgery, University of Iowa, Iowa City; 2Department of Otolaryngology–Head and Neck Surgery, University
of Iowa, Iowa City, Iowa; and 3Department of Otolaryngology, Indiana University School of Medicine, Indianapolis, Indiana

OBJECTIVE  Vestibular schwannomas (VSs) are benign nerve sheath tumors that result from mutation in the tumor sup-
pressor gene NF2, with functional loss of the protein merlin. The authors have previously shown that c-Jun N-terminal
kinase (JNK) is constitutively active in human VS cells and plays a central role in their survival by suppressing accumula-
tion of mitochondrial superoxides, implicating JNK inhibitors as a potential systemic treatment for VS. Thus, the authors
hypothesized that the adenosine 5ʹ-triphosphate–competitive JNK inhibitor AS602801 would demonstrate antitumor
activity in multiple VS models.
METHODS  Treatment with AS602801 was tested in primary human VS cultures, human VS xenografts, and a genetic
mouse model of schwannoma (Postn-Cre;Nf2flox/flox). Primary human VS cell cultures were established from freshly
obtained surgical tumor specimens; treatment group media was enriched with AS602801. VS xenograft tumors were
established in male athymic nude mice from freshly collected human tumor. Four weeks postimplantation, a pretreatment
MRI scan was obtained, followed by 65 days of AS602801 (n = 18) or vehicle control (n = 19) treatment. Posttreatment
MRI scans were used to measure final tumor volume. Tumors were then harvested. Finally, Postn-Cre;Nf2flox/flox mice
were treated with AS602801 (n = 10) or a vehicle (n = 13) for 65 days. Posttreatment auditory brainstem responses were
obtained. Dorsal root ganglia from Postn-Cre;Nf2flox/flox mice were then harvested. In all models, schwannoma identity
was confirmed with anti-S100 staining, cell proliferation was measured with the EdU assay, and cell death was mea-
sured with terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining. All protocols were approved
by the local institutional review board and Institutional Animal Care and Use Committees.
RESULTS  Treatment with AS602801 decreased cell proliferation and increased apoptosis in primary human VS
cultures. The systemic administration of AS602801 in mice with human VS xenografts reduced tumor volume and cell
proliferation. Last, the AS602801-treated Postn-Cre;Nf2flox/flox mice demonstrated decreased cell proliferation in glial cells
in the dorsal root ganglia. However, AS602801 did not significantly delay hearing loss in Postn-Cre;Nf2flox/flox mice up to 3
months posttreatment.
CONCLUSIONS  The data suggest that JNK inhibition with AS602801 suppresses growth of sporadic and neurofibroma-
tosis type 2–associated VSs. As such, AS602801 is a potential systemic therapy for VS and warrants further investigation.
https://thejns.org/doi/abs/10.3171/2022.7.JNS22934
KEYWORDS  vestibular schwannoma; acoustic neuroma; JNK inhibitor; bentamapimod

V
schwannomas (VSs) are benign nerve
estibular
sheath tumors that originate from Schwann cells
(SCs) lining cranial nerve VIII. Approximately 95%
of VSs occur sporadically, whereas the remainder occur
secondary to the autosomal dominant syndrome neurofi-
bromatosis type 2 (NF2).1 Currently, the treatment for most
VSs is limited to surgical resection, stereotactic radiation
therapy, or observation with serial imaging.2 There are no
FDA-approved drugs for the treatment of VS. Compounds
with promising preclinical findings, such as lapatinib and

ABBREVIATIONS  ABR = auditory brainstem response; DRG = dorsal root ganglia; JNK = c-Jun N-terminal kinase; NF2 = neurofibromatosis type 2; PBS = phosphate-
buffered saline; SC = Schwann cell; TUNEL = terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling; VS = vestibular schwannoma.
SUBMITTED  April 26, 2022.  ACCEPTED  July 12, 2022.
INCLUDE WHEN CITING  Published online September 9, 2022; DOI: 10.3171/2022.7.JNS22934.

©AANS 2022, except where prohibited by US copyright law J Neurosurg  September 9, 2022 1
Dougherty et al.
everolimus, have shown limited efficacy in clinical trials.3,4
Bevacizumab—an anti–vascular endothelial growth factor
(VEGF) antibody used for other neoplasms—has demon-
strated clinical efficacy in patients with NF2 who have an
advanced tumor burden; however, its effect is temporary
and long-term use is typically precluded due to systemic
side effects.5,6 Thus, there is an urgent need, especially in
patients with NF2, for the development of novel therapies
that limit VS growth and/or reduce tumor volume.
Inactivation of the NF2 gene is essential for tumori-
genesis in both sporadic and NF2-associated VSs.7–9 NF2
resides on chromosome 22q12 and encodes the protein
merlin. Merlin shares homology with the ezrin–radixin–
moesin (ERM) family of membrane–cytoskeleton-linking
proteins. Merlin regulates interactions of transmembrane
and signaling molecules with cytoskeletal actin, thereby
affecting cell motility, intercellular attachments, and sub-
cellular localization in response to cellular contact inhibi-
tion.10–12 Therefore, merlin plays a central role in contact
inhibition. Depletion of merlin disrupts contact inhibition,
thus promoting cell proliferation and tumorigenesis. Sev-
eral lines of evidence suggest that merlin interacts with
several progrowth kinase signaling cascades, leading
to the suppression of mitogenic activity at both the cell
membrane and nucleus.13,14 Merlin blocks signaling at the
cell membrane by integrins and receptor tyrosine kinases
(RTKs) such as ErB2 and platelet-derived growth factor
(PDGF) receptor; this in turn regulates multiple down-
stream pathways including the Rac/PAK/JNK, mTORC1,
PI3K/AKT, Wnt/β-catenin, Ras/Raf/MEK/ERK, and
FAK/Src pathways.15 Furthermore, upstream suppression
of the Hippo pathway by merlin attenuates Yes-associated
protein 1 (YAP1) function.16 Within the nucleus, merlin
inhibits proliferation via downregulation of the E3 ubiqui-
tin ligase CRL4 (DCAF1).17
The c-Jun N-terminal kinase (JNK) is an evolutionarily
conserved member of the mitogen-activated protein kinase
(MAPK) family. The canonical JNK signaling cascade
regulates numerous cellular processes, particularly in-
flammatory responses and cell death.18,19 Previous reports
indicate that merlin suppresses JNK activity. In the case of
nonneoplastic SCs with intact merlin, nerve injury results
in JNK activation via the p75NTR death domain, ultimately
leading to SC death.20 In contrast, reduced merlin expres-
sion in human VS cells leads to persistent phosphorylation
and activation of JNK. This persistent JNK activity para-
doxically enhances VS cell survival in part by suppress-
ing oxidative stress in the mitochondria, which implicates
JNK inhibitors as potential therapeutic agents.21–23 Indeed,
it has been demonstrated that JNK inhibitors SP6000125
and I-JIP lead to VS cell mitochondrial superoxide accu-
mulation and subsequent cell death in vitro, and increase
the sensitivity of human VS to radiation treatment.24
The adenosine 5ʹ-triphosphate–competitive JNK inhib-
itor AS602801 (also called bentamapimod or PGL5001)
has demonstrated cytotoxicity against cancer stem cells
derived from pancreatic cancer, lung cancer, ovarian can-
cer, prostate cancer, and glioblastoma.25 Importantly, the
safety of systemic AS602801 administration has already
been demonstrated in phase 2 clinical trials for endome-
triosis (NCT01630252, www.ClinicalTrials.gov).26
2 J Neurosurg  September 9, 2022
Given previous findings, we hypothesized that
AS602801 would increase VS apoptosis, decrease prolif-
eration, decrease tumor size, and improve auditory out-
comes. We therefore explored the impact of AS602801
on VS tumor growth, cell proliferation, and cell death in
primary human VS cultures, patient-derived VS mouse
xenografts, and a genetic schwannoma mouse model with
conditional Nf2 deletion using Postn-Cre;Nf2flox/flox mice.
Methods
All animal procedures followed NIH guidelines and
were approved by the University of Iowa’s and the Uni-
versity of Indiana’s Institutional Animal Care and Use
Committees. All protocols involving human tissues were
reviewed and approved by the local institutional review
board.
Primary VS Cultures
Primary VS cultures were prepared as previously de-
scribed.27–29 In brief, following tumor harvest, the tissue
was minced into 1-mm3 fragments, digested in 0.25% tryp-
sin and 0.1% collagenase at 37°C for 30–40 minutes, and
then dissociated by trituration. Cells were resuspended in
DMEM supplemented with N2 supplement (Sigma), bo-
vine insulin (Sigma; 10 µg/mL), and 10% fetal calf serum.
The suspensions were then plated on cell culture slides
that had been pretreated with poly-l-ornithine followed by
laminin (20 µg/mL). Cultures were grown in a humidi-
fied incubator at 37°C with 6.0% CO2. After 1–2 days the
medium was exchanged for serum-free medium. The cells
were subsequently kept in serum-free conditions with reg-
ular media exchanges for 7–10 days without passage. The
JNK inhibitor AS602801 (Selleck Chemicals) was added
to the treatment group cultures and kept at a constant con-
centration for the duration of the experiment. Doses of 2,
20, and 50 µg/mL AS602801 were tested to establish a
dose-response profile. Control cultures were treated in an
identical manner but were not given AS602801.
VS Xenograft Mice
VS xenograft models were developed from tumor spec-
imens harvested from 4 separate patients without NF2 as
previously described.2 In short, acutely resected VS tissue
was placed in Hanks balanced salt solution (Invitrogen)
on ice and transported to the laboratory. The specimens
were then separated into 10-mm3 fragments and implant-
ed in the interscapular fat pad of male athymic Ncr Nu/
Nu mice (National Cancer Institute, NIH; n = 37). Mice
were anesthetized with a cocktail of ketamine (100 mg/
kg; Hospira) and xylazine (10 mg/kg; Phoenix Scientific)
during the procedure. Grafts were developed for 4 weeks
prior to initiating treatment. Animals had unlimited food
and water access and were housed in constant temperature
and humidity conditions with a 12-hour light/dark cycle.
VS xenograft mice were randomly assigned to receive
60 mg/kg/day of AS602801 (n = 18; treatment group) or
vehicle (H2O, n = 19; control group) twice daily for 65
days. Treatments were delivered by oral gavage. Pretreat-
ment MRI scans of the VS xenograft mice were obtained
4 weeks after human VS implantation to determine initial

tumor volume. Posttreatment MRI scans were obtained
to determine final tumor volume. Xenograft tumors were
subsequently harvested and fixed in 4% paraformaldehyde.
Periostin Mouse Strain (Postn-Cre;Nf2flox/flox)
Transgenic mice carrying Cre expression upstream of
Periostin gene (Postn-Cre) were crossed with mice car-
rying loxp sequences flanking exon 2 of the Nf2  gene​
(Nf2flox/flox), generating Postn-Cre;Nf2flox/flox mice and their
Cre-negative control littermates. This Postn-Cre;Nf2flox/flox
mouse model has a 100% penetrance of developing schwan-
nomas in their sensory ganglia, including both cranial
and dorsal root ganglia (DRG), by the age of 10 months.
Nf2flox2 and Nf2∆2 bands were detected by polymerase
chain reaction analysis as described in Giovannini et al.30
The following primers were used to detect the Postn-Cre
transgene via polymerase chain reaction: P1 (CAT TTG
GGC CAG CTA AAC AT) and P2 (CCC GGC AAA
ACA GGT AGT TA), as previously described.31
Postn-Cre;Nf2flox/flox mice were randomized to treat-
ment (n = 10) or control (n = 13) groups. Mice then re-
ceived oral treatment of 60 mg/kg/day of AS602801 (n =
10) or H2O vehicle (n = 13) divided into 2 daily doses for
65 days. Serum levels of AS602801 were measured at 24
hours. At the end of the experiment, auditory brainstem
responses (ABRs) were obtained as described below. The
animals were then euthanized and DRG were harvested
and fixed in 4% paraformaldehyde. Because all sensory
ganglia in this model demonstrate similar SC character-
istics and tumorlet formation, we evaluated DRG rather
than vestibular ganglia, because DRG are more numerous
and allow for more consistent evaluation of cell activity.
This practice is consistent with previous literature.31,32
Tumor Volume
Tumor volume was assessed in VS xenografts as de-
scribed previously.2 In brief, T2-weighted images were
acquired in the axial and sagittal planes using a small-
animal MRI unit (Varian Unity/INOVA 4.7-T scanner;
Varian, Inc.). Tumor volume was measured on ImageJ
software (NIH) using the ABC/2 method by two indepen-
dent reviewers who were blinded to the experimental con-
ditions.33 Final dimensions were the average of each in-
dependent measurement. Tumor growth was calculated as
final tumor volume (Vf ) minus initial tumor volume (Vi)
divided by initial tumor volume (i.e., [Vf − Vi]/Vi), thus
accounting for variability in the initial xenograft volumes.
Immunohistochemistry
Following fixation, the VS xenografts or Postn-
Cre;Nf2flox/flox lumbar DRG were harvested, washed in
phosphate-buffered saline (PBS), cryoprotected in 30%
sucrose, embedded in optimum cutting temperature com-
pound (OCT; Thermo Fischer Scientific), and finally cryo-
sectioned at 10–15 µm. Frozen sections were treated with
2 N HCl for 30 minutes, permeabilized with 0.8% Triton
X-100 in PBS for 20 minutes, and then blocked with block-
ing buffer (5% goat serum, 2% bovine serum albumin, and
0.8% Triton X-100) for 30 minutes. Next, the samples were
incubated in polyclonal rabbit anti-S100 antibody (1:400;
Dougherty et al.
Sigma-Aldrich) and diluted in blocking buffer for 1 hour at
37°C. Following several washes in PBS, a secondary anti-
body (goat anti–rabbit Alexa Fluor 488, 1:800; Invitrogen)
was applied for 1 hour at room temperature. Sections were
mounted with Prolong Gold + DAPI (Life Technologies).
Images were obtained using Metamorph software (Mo-
lecular Devices, LLC) on a Leica DMIRE 2 microscope
equipped with epifluorescent filters (Leica Microsystems).
Nuclei were counted using ImageJ. Analogous methods
were used to stain for S100 and DAPI in VS cultures.
Cell Proliferation
VS cultures were labeled with EdU (10 µM; Life Tech-
nologies) for 48 hours prior to fixation as previously de-
scribed.27 Briefly, following fixation, VS cultures were
treated with 2 N HCl for 15 minutes, immunostained
for DAPI, S100, and EdU. EdU was detected using the
Click-iT EdU detection kit (Life Technologies). Cell pro-
liferation was estimated by dividing the total number
of EdU-positive/S100-positive nuclei by the number of
DAPI-positive/S100-positive nuclei. Nuclei were counted
from 4 randomly selected fields at ×20 magnification per
condition.
Starting 24 hours prior to euthanasia, mice bearing
VS xenografts or Postn-Cre;Nf2flox/flox mice were injected
with 50 mg/kg EdU intraperitoneally 4 times at regular
intervals (10 µM, Life Technologies) in order to label ac-
tively replicating cells. Once the mice were euthanized,
the tumor and 2 pieces of small intestine (EdU-positive
control) were collected. The xenograft tumors and DRG
were dissected and prepared as described above. EdU in-
corporation into tumor cell nuclei was detected using the
Click-iT EdU kit per the manufacturer’s instructions (Life
Technologies).32 Sections were mounted on coverslips
with Prolong Gold + DAPI (Life Technologies). Stained
sections were imaged using a Leica confocal microscope.
Cell proliferation was estimated by dividing the total num-
ber of EdU-positive/S100-positive nuclei by the number of
DAPI-positive/S100-positive nuclei. A total of 4 randomly
selected microscopic fields per section and 2 sections per
slide were counted. During cell counting, the investigator
was blinded to treatment conditions.
Cell Apoptosis
Apoptotic cells were detected in VS cultures and in
VS xenografts by terminal deoxynucleotidyl transferase–
mediated dUTP nick-end labeling (TUNEL) using the In
Situ Cell Death Detection Kit (Roche Diagnostics).2,20,27,34
Tumor cell apoptosis was estimated as the percentage of
TUNEL-positive/S100-positive nuclei with typical con-
densed morphology identified in 4 randomly selected
microscopic fields at ×20 magnification, as previously
described.23,27 The investigator was blinded to treatment
conditions.
ABR Measurement
ABR measurements were recorded as described previ-
ously in Gehlhausen et al.31 ABRs were first performed on
Postn-Cre;Nf2flox/flox mice at 4 months of age as a baseline.
Mice were then randomized to receive either AS602801
J Neurosurg  September 9, 2022 3
Dougherty et al.
FIG. 1. Treatment with AS602801 reduces proliferation of primary hu-
man VS cultures. Primary VS cultures were grown in the presence or
absence of JNK inhibitor AS602801 for 24 hours. A and B: Representa-
tive cultures immunolabeled with anti-EdU (red) and anti-S100 (green)
antibodies. Nuclei were identified with DAPI (blue). Arrows indicate EdU-
positive nuclei determined by overlap of red and blue channels. C: The
percentages of EdU-positive, S100-positive nuclei to total S100-positive
nuclei were counted by ×20 microscopic fields and are expressed in the
graph as numbers of EdU-positive nuclei per DAPI-stained nuclei. Treat-
ment with AS602801 significantly reduced proliferation of primary hu-
man VS cells in a dose-dependent fashion (p < 0.0001, 1-way ANOVA).
Error bars represent SEMs. Bar = 50 µm.
or vehicle control for 65 days, after which posttreatment
ABR measurements were obtained.
Statistical Analysis
Differences in tumor growth, cell proliferation, and
cell death were analyzed by a Student 2-tailed t-test using
GraphPad Prism software (GraphPad Software). Differ-
ences in relative tumor growth were analyzed by 1-way
ANOVA followed by a post hoc Kruskal-Wallis test using
SigmaStat (Systat Inc.).
Results
JNK Inhibitor Reduces Cell Proliferation and Increases
Apoptosis in Human VS Culture
S100 staining confirmed successful culture of primary
VS. Schwannoma cells were immunolabeled with S100
and counterstained for EdU (Fig. 1) or TUNEL (Fig. 2).
There were significantly fewer EdU-positive VS cells in
AS602801-treated cultures (1.01 ± 0.15 [mean ± SEM],
4 J Neurosurg  September 9, 2022
FIG. 2. Treatment with AS602801 increases apoptosis in primary human
VS cultures. VS cultures were treated with JNK inhibitor AS602801 for
24 hours. A and B: Representative images of control culture staining
with S100 antibody (green). Apoptotic cells were determined by TUNEL
(red) and nuclei were labeled with DAPI (blue). Arrows indicate TUNEL-
positive nuclei. C: The percentages of TUNEL-positive cells, S100-posi-
tive nuclei to total S100-positive nuclei were counted by ×20 microscopic
fields and are expressed here as numbers of TUNEL-positive nuclei per
DAPI-stained nuclei. Treatment with AS602801 significantly increased
the percentage of TUNEL-positive cells in dose-dependent fashion (p <
0.0001, 1-way ANOVA). Error bars represent SEMs. Bar = 50 µm.
50-µM group) compared to vehicle control cultures (3.29
± 0.43; p < 0.0001, 1-way ANOVA), indicating decreased
cellular proliferation following AS602801 treatment; this
reduction occurred in a dose-dependent manner (Fig. 1).
Likewise, there was a significant increase (p < 0.0001,
1-way ANOVA) in TUNEL-positive VS cells in cultures
treated with 20 or 50 µM AS602801 (2.97 ± 0.39, 50-µM
group) compared to nontreated cells (1.03 ± 0.33). Thus,
there is a dose-dependent increase in apoptotic schwan-
noma cells following AS602801 treatment (Fig. 2).
Systemic Administration of AS602801 Reduces Tumor
Cell Proliferation In Vivo but Does Not Slow Progressive
Hearing Loss in Postn-Cre;Nf2flox/flox Mice
Building on these in vitro results, we sought to inves-
tigate whether AS602801 reduces glial cell prolifera-
tion in the Postn-Cre;Nf2flox/flox genetic mouse model of
schwannoma.31,32,35 In order to establish standard experi-
mental dosing protocols, we measured the serum levels
of AS602801 in Postn-Cre;Nf2flox/flox mice at multiple

FIG. 3. Pharmacokinetics of AS602801 in Postn-Cre;Nf2flox/flox mice.
Serum levels of AS602801 were measured for 24 hours after a single
60-mg/kg treatment. The AS602801 serum levels peak within the first
2 hours, followed by a sharp decline within the next 8 hours. Error bars
represent SDs.
time points after oral administration. This demonstrated
a peak drug level of 1500 ng/mL within the first 3 hours,
followed by a decrease to 100 ng/mL at 8 hours (Fig. 3).
Animals tolerated the diet without difficulty, body weight
was tracked daily, and no systemic side effects were noted.
We next assessed the functional impact of AS602801
on hearing in Postn-Cre;Nf2flox/flox mice. Mice were ran-
domized into 2 groups and treated twice a day for 65
days with 60 mg/kg/day of AS602801 (n = 10) or vehicle
control (H2O; n = 13) by gavage. After the completion of
treatment, mice were followed for an additional 65 days,
at which time ABRs were measured to determine hearing
thresholds (Fig. 4). Control mice had an average hearing
threshold of 51.0 ± 9.7 dB (mean ± SEM), and AS602801-
treated mice had an average threshold of 49.2 ± 8.1 dB.
Although an overall trend toward delayed elevation of
ABR thresholds was noted, no significant difference was
reached (p = 0.61, Student 2-tailed t-test). Thus, AS602801
did not affect hearing thresholds in Postn-Cre;Nf2flox/flox
mice at the time point assessed. No systemic toxicities
were noted throughout the treatment, including weight
loss, decreased oral intake, loss of fur, or signs of distress
such as hunched posture or decreased activity.
Finally, we used the EdU assay to measure glial cell
proliferation in Postn-Cre;Nf2flox/flox mice DRG. We found
that AS602801 significantly decreased EdU uptake in
Postn-Cre;Nf2flox/flox mice DRG, suggesting that AS602801
decreases cellular proliferation (Fig. 5).
AS602801 Reduces Human VS Xenograft Tumor Size and
Cell Proliferation but Does Not Induce Apoptosis
Pretreatment and posttreatment MRI scans were used
to calculate relative tumor growth in patient-derived mouse
xenograft VSs. Relative tumor growth was determined by
comparing the percent change in tumor volume from the
initial to final MRI scan (Fig. 6A and B). Control tumors (n
= 19) demonstrated a mean increase of 19.0 ± 10.3 mm3 in
tumor volume during the 65-day treatment period, where-
as AS602801-treated tumors had a mean decrease in tumor
Dougherty et al.
FIG. 4. Auditory function in Postn-Cre;Nf2flox/flox mice treated with
AS602801 (n = 14) or vehicle control (n = 15). ABR thresholds were
measured at 6 months of age. The mice were administered AS602801
30 mg/kg twice daily (60 mg/kg/day) throughout the treatment period.
ABR threshold levels were widely variable in both groups; although there
is tendency of reduced average threshold shift in AS602801, this did not
reach statistical significance (p = 0.61, Student 2-tailed t-test). Error bars
represent SEMs. SPL = sound pressure level.
volume of 29.2 ± 7.7 mm3 (mean ± SEM) (Fig. 6C).36,37
This difference was statistically significant (p = 0.025,
1-way ANOVA followed by Kruskal-Wallis). Eleven of 19
VSs in control mice demonstrated tumor growth, whereas
tumor shrinkage was seen in 14 of 18 AS602801-treated
mice (Fig. 6D). Thus, although on average the tumors de-
creased in size with AS602801 treatment, there was some
variability in the treatment response. No systemic toxici-
ties were noted throughout the treatment, including weight
loss, decreased oral intake, or signs of distress such as
hunched posture or decreased activity.
To determine whether these differences in tumor size
were related to altered cell proliferation and/or apoptosis,
VS xenografts were also stained with EdU (DNA repli-
cation marker) and TUNEL (apoptotic nucleus marker).
There was a significant reduction in EdU uptake in VS
xenografts that were treated with AS602801 (0.60% ±
1.04%, mean ± SEM) relative to those treated with H2O
vehicle control (1.24% ± 1.71%, mean ± SEM; p = 0.02,
Student 2-tailed t-test) (Fig. 7A–E). However, there was
no significant difference in the percentage of TUNEL-
positive cells between treatment (0.85% ± 1.07%, mean
± SEM) and control (0.94% ± 1.15%, mean ± SEM; p =
0.686) xenograft groups (Fig. 7F). Hence, AS602801 does
not appear to significantly increase apoptosis in VS cells.
Discussion
JNK Inhibitors as Potential Therapies for VSs
Three genes (jnk 1–3) encode the JNK proteins, and
J Neurosurg  September 9, 2022 5
Dougherty et al.

FIG. 5. Treatment with AS602801 reduced proliferation of merlin-deficient SCs in Postn-Cre;Nf2flox/flox mice DRG. A–D: EdU uptake
in adult (4-month-old) mice was analyzed with confocal microscopy. Mice were injected with EdU (50 mg/kg intraperitoneally 4
times in a 24-hour period: at 0, 4, 8, and 24 hours), and DRG were dissected within 72 hours. Arrows depict EdU-positive cells.
E: DRG glial cells of Postn-Cre;Nf2flox/flox mice treated with vehicle control (n = 13) demonstrated significantly more EdU-positive
cells than AS602801-treated mice (n = 10; p < 0.001, Student t-test). Bar = 50 µm.

alternative splicing leads to at least 10 isoforms of JNK.
Whereas proteins derived from the JNK1 and JNK2 genes
are ubiquitously expressed in all cells and tissue types,
JNK3 protein is thought to be neuron-specific.38 The JNK
proteins are activated by multiple stimuli in a wide range
of tissues and regulate diverse extracellular activities, play-
ing a critical role in cellular proliferation, apoptosis, and
differentiation.39 As such, the JNK proteins are often im-
plicated in contradictory cellular responses.40 In human VS
cells, we previously identified that JNK1 and JNK2 are ex-
pressed in higher levels in normal nerve tissue than in VS,
although the significance of this finding remains unclear.23
6 J Neurosurg  September 9, 2022
We further identified that JNK is persistently activated
(phosphorylated) in primary VS cultures and promotes VS
survival, at least in part, by suppressing mitochondrial su-
peroxides. Furthermore, JNK inhibition led to increased
oxidative stress and apoptosis in VS cells following irra-
diation.23,24 Together, such studies suggested that further
investigation of JNK inhibition in VS was warranted.
In this study we used in vitro and in vivo models to
demonstrate that the JNK inhibitor AS602801 reduces
VS cell proliferation. In primary human VS culture,
AS602801 significantly reduced cell proliferation and in-
creased apoptosis (Figs. 1 and 2). We also explored the
 Dougherty et al.

FIG. 6. Treatment with AS602801 reduces growth of patient-derived VS xenografts. A and B: Representative MRI scans of
xenografts in axial planes from the beginning and end of experiment, with increased tumor size in a control xenograft (A) and a de-
creased size in an AS602801-treated tumor (B). C: Treatment with AS602801 reduced the growth of the VS xenografts compared
with controls (p = 0.025; error bars represent mean ± SEM). D: Most AS602801-treated tumors (14/18) shrank over the treatment
period, whereas most control tumors (11/19) grew. Each bar on the x-axis represents 1 mouse.

effects of AS602801 in Postn-Cre;Nf2flox/flox mice. These
mice develop proliferation of SCs—predominantly in
the sensory ganglia including the DRG, geniculate gan-
glia, and vestibular ganglia—mirroring the propensity of
schwannomas to occur in sensory ganglia in humans.31,32,41
In these mice, SC proliferation forms tumorlets centered
on the cell bodies of the sensory neurons. Systemic admin-
istration of AS602801 reduced proliferation of schwanno-
ma cells in Postn-Cre;Nf2flox/flox mice. However, AS602801
did not preserve auditory function (Figs. 4 and 5). In the
J Neurosurg  September 9, 2022 7
Dougherty et al.

FIG. 7. Treatment with AS602801 reduces proliferation of patient-derived VS xenograft cells. EdU and TUNEL uptake in adult
(3-month-old) xenograft mice was analyzed (6 AS602801-treated mice, 9 control). A–D: EdU was detected in tumor cells (red)
and counterstained with DAPI (blue); arrows indicate EdU-positive nuclei. E: Quantification of EdU demonstrates clear reduction
of proliferation in AS602801-treated mice (p = 0.02, Student 2-tailed t-test). F: In contrast, TUNEL staining did not show increased
apoptotic cells in AS602801 compared to control (p = 0.686). Bar = 50 µm.

DRG of Postn-Cre;Nf2flox/flox mice, AS602801 treatment
reduced cell proliferation.
VS tumor–bearing xenograft mice treated with
AS602801 demonstrated radiographic reduction in tumor
size (Fig. 6). Likewise, in VS xenograft mice, AS602801
reduced cell proliferation but did not increase apoptosis
(Figs. 6 and 7). Taken together, these data indicate that JNK
inhibition may be effective in slowing the growth of VSs.
When combined with the fact that systemic administration
of AS602801 has already been demonstrated in phase 2
clinical trials, these findings implicate JNK inhibition via
AS602801 as a potential systemic therapy for VS. These
8 J Neurosurg  September 9, 2022
data are particularly relevant to patients with NF2, who are
often afflicted with too many tumors to treat with radiation
and/or surgery alone.
Limitations
Although these data are promising, discrepancies in find-
ings between our different model systems and variability in
tumor growth responses underscore the difficulty of con-
trolling schwannoma growth by targeting a single aberrant
pathway. This is consistent with previous reports from our
group and others.32,42,43 Optimal VS treatment will be likely

to require simultaneously targeting multiple pathways; thus,
AS602801 may be most effective as one of several drugs
used together. For example, VS cells can survive in the
presence of the high-affinity p75NTR ligand proNGF, and
proNGF rescues VS cells from JNK inhibitor–mediated
cell death via activation of NF-κB. This suggests a paradox-
ical antiapoptotic role of p75NTR leading to VS growth.20,44
Therefore, targeting the p75NTR and JNK pathways together
may simultaneously impair VS growth and spare normal
SCs. Thus, although our results are encouraging, they also
highlight the limitation of a monotherapy for VSs due to the
complexity and multifactorial nature of VS tumor growth.
Further research is necessary to investigate multiple molec-
ular targets to optimize therapy against VS growth.
The development of animal models that accurately
recapitulate human NF2-related schwannomas has also
been challenging. Previous reports have shown that deaf-
ness in Postn-Cre;Nf2flox/flox mice first becomes apparent
at 6 months and progresses further at 8 and 10 months.31
Mice in our study were 6–7 months of age at the time of
ABR testing; hence, it is possible that hearing loss at this
age is too mild to detect significant differences between
treatment groups, even when differences are present. The
mechanism of hearing loss in this model is incompletely
understood, further complicating the interpretation of our
finding. Further research is necessary to study the un-
derlying mechanism of hearing loss phenotype in Postn-
Cre;Nf2flox/flox mice.
In addition, the proliferative and apoptotic biomarkers
we use to study tumor response provide only indirect evi-
dence of JNK pathway inhibition. Due to the small number
of animals and the size of tissue samples, we were unable
to directly quantify JNK pathway inhibition in this study.
Despite this, we believe that our approach is supported
by previous publications from our laboratory and others,
which have shown that JNK is persistently phosphorylated
(active) in VS primary human tumors and cell cultures;
this activation is driven by lack of merlin and can be sup-
pressed by reexpression of wild-type NF2/merlin.21–24
Another limitation of our study is our use of a single
dose and concentration of AS602801 in the mouse models.
We did not perform an assay testing different dosages of
oral AS602801 in our in vivo models; rather, the 60 mg/
kg/day dosage of AS602801 was determined based on our
in vitro data (Fig. 1). Twice daily dosing (30 mg/kg/dose)
was selected based on the observed pharmacokinetics of
AS602801 in Postn-Cre;Nf2flox/flox mice; however, it is pos-
sible that 3 doses per day would be more effective than 2.
Thus, further quantitative screening assays for dose titra-
tion will be necessary. Nonetheless, the administered dose
of AS602801 was well tolerated in our mice, and the safety
profile of AS602801 in humans has been validated in a
phase 2 clinical trial for endometriosis (NCT01630252).26
This suggests that AS602801 may be suitable for long-
term therapy, which is especially important for patients
with NF2.
Conclusions
Current treatment for VS consists of resection, stereo-
tactic radiation therapy, or observation with serial imag-
Dougherty et al.
ing. So far, bevacizumab is the only pharmacotherapy
to show efficacy in phase 3 clinical trials, but this use
is off-label, the effect is temporary, and long-term use
can have severe side effects.5,6 Here we have shown us-
ing in vitro and in vivo models of VS that the adenosine
5ʹ-triphosphate–competitive JNK inhibitor AS602801 re-
duces cell proliferation and tumor growth. This suggests
that AS602801 may be developed as a systemic therapy
to limit schwannoma growth in patients in whom surgery
and radiation treatment fail. Such pharmacotherapies are
especially relevant for patients with NF2.
Acknowledgments
We acknowledge the assistance of Linjing Xu and Brian
Mostaert. Financial support was provided to Dr. Hansen by DOD
CDMRP/NFRP NF130072, NIDCD-R01 DC009801, and NIDCD
5T32DC000040.
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Disclosures
Dr. Hansen has ownership in IotaMotion, Inc.
Author Contributions
Conception and design: Hansen, Clark, Canady, Yates. Acquisition
of data: Clark, Canady, Yates. Analysis and interpretation of data:
all authors. Drafting the article: Dougherty, Shibata, Canady.
Critically revising the article: Dougherty. Reviewed submit-
ted version of manuscript: Hansen, Dougherty, Shibata, Yates.
Approved the final version of the manuscript on behalf of all
authors: Hansen. Administrative/technical/material support:
Clark, Yates. Study supervision: Hansen, Yates.
Supplemental Information
Previous Presentations
A portion of this work has been previously presented at the
Association for Research in Otolaryngology’s annual meeting,
held in Baltimore, MD, January 29, 2017. Presented as “JNK
Inhibitor AS602801 (Bentamapimod) Decreases Schwannoma
Tumor Volume and Cell Proliferation.”
Current Affiliations
Dr. Shibata: Rick and Tina Caruso Department of Otolaryn­
gology–Head and Neck Surgery, Keck School of Medicine of the
University of Southern California, Los Angeles, CA.
Mr. Clark: CI-CG LLC, Louisville, KY.
Dr. Canady: Department of Anesthesiology, Stanford University
School of Medicine, Palo Alto, CA.
Correspondence
Marlan R. Hansen: University of Iowa, Iowa City, IA.
marlan-hansen@uiowa.edu.