RESEARCH ARTICLE
Cabozantinib Eradicates Advanced Murine
Prostate Cancer by Activating Antitumor
Innate Immunity
Akash Patnaik
1,2,3,4
, Kenneth D. Swanson
5
, Eva Csizmadia
6
, Aniruddh Solanki
7,8
, Natalie Landon-Brace
7,8
,
Marina P. Gehring
5,9
, Katja Helenius
10
, Brian M. Olson
3,4
, Athalia R. Pyzer
1
, Lily C. Wang
11
, Olivier Elemento
11
,
Jesse Novak
12
, Thomas B. Thornley
13
, John M. Asara
14
, Laleh Montaser
15
, Joshua J. Timmons
5
, Todd M. Morgan
16
,
Yugang Wang
16
, Elena Levantini
1,8,17
, John G. Clohessy
2,18
, Kathleen Kelly
19
, Pier Paolo Pandolfi
2
,
Jacalyn M. Rosenblatt
1,2
, David E. Avigan
1,2
, Huihui Ye
15
, Jeffrey M. Karp
7,8
, Sabina Signoretti
12
,
Steven P. Balk
1,2
, and Lewis C. Cantley
11
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CANCER DISCOVERY | 751
ABSTRACT
Several kinase inhibitors that target aberrant signaling pathways in tumor cells
have been deployed in cancer therapy. However, their impact on the tumor immune
microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed
striking responses in cancer clinical trial patients across several malignancies. Here, we show that
cabozantinib rapidly eradicates invasive, poorly differentiated PTEN/p53-defi cient murine prostate
cancer. This was associated with enhanced release of neutrophil chemotactic factors from tumor cells,
including CXCL12 and HMGB1, resulting in robust infi ltration of neutrophils into the tumor. Critically,
cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte deple-
tion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor plerixafor.
Collectively, these data demonstrate that cabozantinib triggers a neutrophil-mediated anticancer
innate immune response, resulting in tumor clearance.
SIGNIFICANCE: This study is the fi rst to demonstrate that a tyrosine kinase inhibitor can activate
neutrophil-mediated antitumor innate immunity, resulting in invasive cancer clearance. Cancer Discov;
7(7); 750–65.
©2017 AACR.
1
Division of Hematology/Oncology, Department of Medicine, Beth Israel
Deaconess Medical Center, Dana Farber/Harvard Cancer Center, Harvard
Medical School, Boston, Massachusetts.
2
Beth Israel Deaconess Cancer
Center, Department of Medicine, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, Massachusetts.
3
Section of Hematology/
Oncology, Department of Medicine, The University of Chicago, Chicago,
Illinois.
4
The University of Chicago Comprehensive Cancer Center, Chicago,
Illinois.
5
Department of Neurology, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, Massachusetts.
6
Division of Gastroen-
terology, Department of Medicine, Beth Israel Deaconess Medical Center,
Boston, Massachusetts.
7
Department of Medicine, Brigham and Women’s
Hospital, Harvard Medical School, Boston, Massachusetts.
8
Harvard Stem
Cell Institute, Boston, Massachusetts.
9
Laboratório de Farmacologia Apli-
cada, PUCRS, Porto Alegre, Brazil.
10
Koch Institute for Integrative Cancer
Research, Massachusetts Institute of Technology, Cambridge, Massachu-
setts.
11
Meyer Cancer Center, Weill Cornell Medical College, New York, New
York.
12
Department of Pathology, Brigham and Women’s Hospital, Harvard
Medical School, Boston, Massachusetts.
13
Transplant Institute and Immu-
nology Program, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts.
14
Division of Signal Transduction, Beth
Israel Deaconess Medical Center, Harvard Medical School, Boston, Mas-
sachusetts.
15
Department of Pathology, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, Massachusetts.
16
Department of
Urology, University of Michigan, Ann Arbor, Michigan.
17
Institute of Biomedi-
cal Technologies, National Research Council (CNR), Pisa, Italy.
18
Preclinical
Murine Pharmacogenetics Facility, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, Massachusetts.
19
Laboratory of Genitou-
rinary Cancer Pathogenesis , National Cancer Institute, Bethesda, Maryland.
Note: Supplementary data for this article are available at Cancer Discovery
Online (http://cancerdiscov ery.aacrjournals.org/).
M.P. Gehring, K. Helenius, B.M. Olson, and A.R. Pyzer contributed equally
to this article.
Corresponding Author: Akash Patnaik , The University of Chicago , Knapp
Center for Biomedical Discovery, Room 7152, 900 East 57th Street, Chicago,
IL 60637. Phone: 773-834-3519; Fax: 773-834-0778; E-mail: apatnaik1@
uchicago.edu
doi: 10.1158/2159-8290.CD-16-0778
©2017 American Association for Cancer Research.
INTRODUCTION
Malignant cells and stromal cells cooperate to create an
immunosuppressive microenvironment that protects devel-
oping tumors from immune eradication ( 1 ). Paul Ehrlich fi rst
postulated a pathologically important relationship between
the immune system and cancer, suggesting that a break-
down in normal antitumor immune surveillance occurs as
a part of tumor evolution ( 2 ). The growing tumor subverts
the immune system’s normal wound-healing mechanisms,
exploiting them for tumor protection, maintenance, and
progression ( 3 ). Attempts to perturb this collaborative rela-
tionship between the tumor and its immune stroma date
back approximately 125 years to William Coley, who injected
bacterial lysates into the tumor bed in an attempt to stimu-
late tumor rejection ( 4 ). There has been a resurgent interest
in cancer immunotherapy, partly based on the profound and
durable clinical responses to T lymphocyte checkpoint con-
trol antibodies against CTLA-4 and PD-1/PD-L1. However,
there are still subsets of patients across all malignancies that
fail to respond to these therapies. Although there have been
several approaches developed to activate the adaptive arm
of the immune system for cancer elimination, strategies to
activate antitumor innate immunity have remained elusive.
Cabozantinib (also known as XL-184) is a promiscuous
receptor tyrosine kinase (RTK) inhibitor with potent activ-
ity against c-MET, VEGFR2, RET, KIT, AXL, and FLT3, all
of which have been implicated in tumor growth and sur-
vival ( 5 ). Cabozantinib has been approved by the FDA for
the treatment of medullary thyroid cancer (MTC), a RET-
driven malignancy ( 6 ). In a phase II randomized discontinu-
ation trial in patients with castrate-resistant prostate cancer
(CRPC), 72% of patients exhibited regression in soft-tissue
lesions, whereas 68% of patients had improvement in technec-
tium-99m bone scan response, including complete resolution
in 12%. This dramatic bone scan response is unprecedented in
CRPC with bone metastases treated with current standard-of-
care therapies. The trial exhibited a median progression-free
survival (PFS) of 23.9 versus 5.9 weeks for the cabozantinib-
and placebo-treated cohorts, respectively, with signifi cant
reductions in bone turnover markers and bone pain with
cabozantinib treatment ( 7 ).
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Despite promising phase II clinical trial results, a recent
phase III trial of cabozantinib in patients with heavily pre-
treated metastatic CRPC (COMET-1) failed to demonstrate
a statistically significant increase in overall survival (OS)
with cabozantinib versus prednisone alone. Consistent with
the previous phase II results, median radiographic PFS (HR,
0.48; 95% CI, 0.40–0.57; stratified log-rank P < 0.001) was 5.6
versus 2.8 months for the cabozantinib versus prednisone
arms, respectively (8). Moreover, a randomized phase III trial
(METEOR) of cabozantinib versus everolimus in VEGFR
inhibitor–resistant renal cell cancer (RCC) showed a 42%
decrease in disease progression (9, 10). Therefore, a deeper
understanding of cabozantinib’s antitumor mechanism is
critical for biomarker-based stratification of patients most
likely to respond to the drug.
Loss of PTEN and p53 function are frequent genetic events
in human CRPC (11, 12). Mice with probasin Cre-driven
conditional prostate-specific knockout of Pten and Trp53
(Pb-Cre; Pten
fl/fl
Trp53
fl/fl
) develop invasive CRPC (13) as early
as 9 weeks and locally aggressive tumors by 3 months of age,
that are invariably lethal to the host by 7 months of age (14).
Here, we show that cabozantinib eradicated poorly differenti-
ated invasive adenocarcinoma in these mice within 48 hours,
with concomitant infiltration of neutrophils into the tumor
bed. Strikingly, neutrophil depletion or chemotaxis blockade
with either an HMGB1-neutralizing monoclonal antibody or
the CXCR4 inhibitor plerixafor reversed the tumor clearance
elicited by cabozantinib. These data provide strong evidence
that cabozantinib elicits a neutrophil-mediated anticancer
innate immune response that results in tumor eradication.
RESULTS
Cabozantinib Eradicates Murine Prostate Cancer
via a c-MET–Independent Immune Mechanism
Cabozantinib was developed as a c-MET/VEGFR2 inhibi-
tor (5). Previous studies have shown Met amplification in
67% of prostate tumors from Pb-Cre; Pten
fl/fl
Trp53
fl/fl
mice
and in approximately 30% of metastatic PTEN and p53-defi-
cient human prostate cancer specimens (15). As a first step
toward evaluating whether the antitumor mechanism of
cabozantinib is c-MET dependent, we tested its effect on
aggressive prostate cancers that develop in 5- to 6-month-old
Pb-Cre; Pten
fl/fl
Trp53
fl/fl
mice. The mice were treated with vehi-
cle, cabozantinib, or PF-04217903 (c-MET–specific inhibitor)
after the solid tumor had reached a long-axis diameter of at
least 5 mm by ultrasound and MRI analysis. Cabozantinib-
treated mice showed an approximately 70% reduction in
tumor volume (Fig. 1A and B), which was accompanied by
decreased FDG-PET signal (Supplementary Fig. S1). Strik-
ingly, histopathology revealed a near-complete clearance of
the poorly differentiated, invasive prostate carcinoma in 4
days, which was sustained over 3 weeks of cabozantinib
treatment (Fig.1C). In contrast, PF-04217903 failed to inhibit
tumor growth (Fig. 1A and B) over 3 weeks of treatment,
despite similar inhibition of intratumoral phospho-MET
with both cabozantinib and PF-04217903 (Supplementary
Fig. S2A). Prostate tumors from PF-04217903–treated mice
exhibited a persistence of poorly differentiated, invasive pros-
tate carcinoma after 3 weeks of treatment (Fig. 1C, top
middle). These data demonstrated that c-MET inhibition
alone was insufficient to explain cabozantinib’s antitumor
mechanism of action.
Cabozantinib Treatment Results in Rapid
Neutrophil Infiltration into the Tumor Bed
In light of this profound antitumor response to cabozan-
tinib in 4 days, we performed detailed histopathologic evalu-
ation of prostate tumors from treated mice over the initial
72-hour time period. This analysis revealed a near-complete
eradication of poorly differentiated invasive adenocarcinoma
within 48 to 72 hours of treatment (Fig.2A, hematoxylin and
eosin panel). This was accompanied by increased perivascular
ICAM1 staining and hypersegmented (see the inset in Fig.2A),
Ly6G
+
, myeloperoxidase
+
(MPO
+
) neutrophil infiltration into
the tumor within 24 to 48 hours of treatment (Fig.2A). Flow
cytometry also showed an increase in CD11b
+
GR1
+
tumor-
infiltrating immune cells following 72 hours of cabozantinib
treatment (Fig.2B). Further immune surface marker analysis
demonstrated that these cells were Ly6G
hi
Ly6C
lo
, consist-
ent with a granulocytic (and not monocytic) predominance
of tumor-infiltrating immune cells following cabozantinib
treatment (Supplementary Fig.S2B).
Cabozantinib Induces In Vivo Tumor Cell Death
via CXCL12–HMGB1–CXCR4–Dependent
Neutrophil Recruitment
The near-complete tumor clearance elicited by cabozantinib
was preceded by a significant increase in caspase-3 stain-
ing in vivo (Fig. 3A). If cabozantinib exerts its antitumor
effects via a cell-autonomous mechanism, then treatment
of murine PTEN/p53-deficient tumor-derived prostate can-
cer cells in vitro would be expected to result in a similar
dramatic induction of apoptosis. To determine the physi-
ologically relevant cabozantinib dose for in vitro apoptosis
experiments, we performed mass spectrometry analysis of
prostate tumor–extracted metabolites from cabozantinib-
treated mice. This revealed a steady-state intratumoral con-
centration of approximately 10
µmol/L within 72 hours
after treatment (Supplementary Fig.S3A), which is in a simi-
lar range to the steady-state serum concentration observed
in cabozantinib-treated patients (16). RTK profiling of the
human androgen-independent prostate cancer cell line PC3
revealed that cabozantinib at 10
µmol/L concentration inhib-
its multiple RTKs (Supplementary Fig. S3B). We therefore
tested three murine PTEN/p53 deficient prostate tumor cell
lines, SC1, AC1, and AC3, for apoptosis induction in vitro at
physiologic concentrations. In contrast to the rapid induc-
tion of caspase-3 staining by cabozantinib in vivo (Fig.3A), we
detected only modest apoptosis in vitro at similar time points,
even at a high concentration of 30
µmol/L (Fig. 3B). These
results suggest that the direct induction of apoptosis is not
the dominant cell death mechanism observed in vivo follow-
ing cabozantinib treatment.
To further explore the mechanism of cabozantinib-mediated
acute tumor clearance in vivo, we performed transcriptional pro-
filing and gene set enrichment analysis of prostate tumors
recovered from Pb-Cre; Pten
fl/fl
Trp53
fl/fl
mice following 48 hours
of cabozantinib treatment versus control untreated tumors.
This analysis revealed a statistically significant upregulation
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Activation of Antitumor Innate Immunity in Prostate Cancer
RESEARCH ARTICLE
JULY 2017
CANCER DISCOVERY | 753
Figure 1. Cabozantinib causes tumor regression and near-complete clearance of invasive poorly differentiated murine prostate cancer. A, Repre-
sentative MRI images showing the relative impact of vehicle (top), cabozantinib (middle), and the c-MET inhibitor PF-04217903 (bottom), on regression
of established murine PTEN/p53-deficient prostate tumors. Mice were treated with the indicated drugs at the following concentrations: vehicle control,
cabozantinib (100 mg/kg), and PF-04217903 (50 mg/kg). The yellow borders mark solid tumor boundaries during the 3-week course of treatment.
B, Volumetric analysis showed significant tumor regression in mice treated with cabozantinib, but not vehicle or PF-04217903 treatment. C, Hematoxylin
and eosin (H&E) staining of tumors from cabozantinib-treated mice revealed near-complete eradication of poorly differentiated prostate tumors at 4,
10, 18, and 21 days of treatment, not observed with vehicle- or PF-04217903–treated mice (n = 4 mice per treatment arm/time point).
CabozantinibPF-04217903
A
1 WeekBaseline 2 Week
s3
Weeks
Vehicle
0714 21
0
100
200
300
400
500
800
1,200
Vehicle
PF-04217903
Cabozantinib
Treatment days
Percent tumor volume relative to baseline
B
Vehicle
Cabozantinib 10 days
Cabozantinib 4 days
Cabozantinib 21 days
Cabozantinib 18 days
PF-04217903 21 days
50 µm
C
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Figure 2. Cabozantinib induces extensive infiltration of neutrophils into the tumor bed and near-complete clearance of invasive poorly differentiated
murine prostate cancer within 48 to 72 hours of drug treatment. A, H&E, Ly6G, MPO, and ICAM1 staining of established prostate tumors harvested from
Pb-Cre; Pten
fl/fl
Trp53
fl/fl
mice treated with vehicle, 24, 48, and 72 hours of cabozantinib (100 mg/kg) treatment, respectively. Mice were treated with vehi-
cle or cabozantinib for the indicated times, and tumor tissues were stained with either H&E or the indicated antibodies by IHC. These data show an increase
in Ly6G-positive and MPO-positive neutrophils, and an increase in ICAM1-postive endothelium within 48 hours of cabozantinib treatment. B, Flow cytometry
analysis of dissociated tumor showed a similar increase in CD11b
+
GR1
+
tumor-infiltrating neutrophils with cabozantinib treatment.
Vehicle Cabozantinib 24 hCabozantinib 48 hCabozantinib 72 h
H&E
A
B
MPO Ly6G
ICAM1
50 µm
Vehicle
Cabozantini
b
80
60
40
20
0
P = 0.002
% CD11b
+
GR1
+
34 64.8
GR1
CD11b
10
0
10
1
10
1
10
2
10
3
10
4
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
72 hoursVehicle
of immune response transcripts following cabozantinib treat-
ment in vivo (Fig. 4A). Subsequent qPCR-based RNA profil-
ing of cabozantinib-treated tumors revealed a spike in gene
expression of the chemokine CXCL12 and its receptor CXCR4
within the tumor microenvironment following 24 hours of
cabozantinib treatment (Fig. 4B). CXCR4 is implicated in
lymphocyte (17) and neutrophil chemotaxis (18) from the
periphery. CXCR4 can engage CXCL12 as a homodimer or a
2:1 heterocomplex of CXCL12 and HMGB1, the latter serving
as a danger signal during immunogenic cell death (19, 20).
The tumor microenvironment is a complex admixture of
transformed tumor cells and nontransformed stromal cells,
including a diverse population of immune cells (21). Tumor
cells can secrete a number of chemokines that cross-talk with
different stromal cells within the microenvironment and alter
innate and adaptive immune function (21, 22). To determine
the predominant cell type within the tumor responsible
for cabozantinib-induced production of CXCL12, we per-
formed RNA in situ hybridization (RISH) for CXCL12 followed
by PTEN IHC on prostate tumors from mice treated with
vehicle or 24 hours of cabozantinib. Consistent with our qPCR
results, we observed an increase in intratumoral CXCL12 stain-
ing by RISH following cabozantinib treatment specifically in
PTEN-deficient cells within the microenvironment (Fig.4C;
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