AXL inhibition sensitizes mesenchymal cancer cells to
anti-mitotic drugs
Catherine Wilson1, Xiaofen Ye1, Thinh Pham2, Eva Lin1, Sara Chan2, Erin
McNamara3, Richard M. Neve1, Lisa Belmont1, Hartmut Koeppen2, Robert L.
Yauch1, Avi Ashkenazi4, and Jeff Settleman1*
Departments of Discovery Oncology1, Pathology2, Translational Oncology3, and
Research Oncology4, Genentech, 1 DNA Way, South San Francisco, CA 94080
*Correspondence:
Jeff Settleman, Discovery Oncology, Genentech Inc., 1 DNA Way, South San
Francisco, California 94080, USA. Phone: 650-467-7140; Fax: 650-255-5770;
Email: [email protected]
Running title: AXL inhibition sensitizes cancer cells to anti-mitotics
Keywords: AXL, EMT, anti-mitotics, cancer, drug resistance
Precis: Findings challenge a purported role for AXL in drug resistance while
offering a novel rationale to combine AXL targeting drugs with anti-mitotic agents to
eradicate invasive cancers.
COI Disclosure: All authors are employees of Genentech and may be shareholders
of Roche Pharmaceuticals.
2
ABSTRACT
Molecularly-targeted drug therapies have revolutionized cancer treatment, however,
resistance remains a major limitation to their overall efficacy. Epithelial to
mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine
kinase inhibitors (TKIs), independent of mutational resistance mechanisms. AXL is
a receptor tyrosine kinase (RTK) associated with EMT that has been implicated in
drug resistance, and has emerged as a candidate therapeutic target. Across 643
human cancer cell lines that were analyzed, elevated AXL was strongly associated
with a mesenchymal phenotype, particularly in triple negative breast cancer and
non-small cell lung cancer. In an unbiased screen of small molecule inhibitors of
cancer-relevant processes, we discovered that AXL inhibition was specifically
synergistic with anti-mitotic agents in killing cancer cells that had undergone EMT
and demonstrated associated TKI resistance. However, we did not find that AXL
inhibition alone could overcome acquired resistance to EGFR TKIs in the EMT
setting, as previously reported. These findings reveal a novel co-treatment
strategy for tumors displaying mesenchymal features that otherwise render them
treatment-refractory.
3
INTRODUCTION
Epithelial to mesenchymal transition (EMT) is a vital cellular process during normal
development, and contributes to the invasive and metastatic properties of human
tumors (1). EMT has also been implicated in resistance to multiple cancer drug
therapies, including several tyrosine kinase inhibitors (TKIs). Thus, drug-sensitive
cancer cell lines selected in culture for acquired drug resistance can adopt a
mesenchymal phenotype (2-4). Moreover, EMT has been associated with acquired
resistance to Epidermal Growth Factor Receptor (EGFR) TKIs in EGFR mutant
lung cancer patients (2, 5).
The receptor tyrosine kinase AXL is a member of the TAM family kinases, which
also includes TYRO-3 and MER (6). AXL is overexpressed in many solid tumors (4,
7-10), although, activating mutations have not been observed (11). AXL has one
known ligand, Gas6 (12), and ligand binding promotes cell proliferation, survival
and migration through activation of the PI-3K/AKT/S6K and ERK/MAPK pathways
(9, 13-15). AXL expression may be a negative prognostic factor for breast and
pancreatic cancer patients (7, 9), and may be a unique EMT effector in breast
cancer progression (7, 16).
The association of AXL with EMT has prompted interest in AXL as a therapeutic
target. AXL has been associated with a mesenchymal signature in non-small cell
lung cancer (NSCLC) and has been proposed as a therapeutic target in EGFR TKI
resistance (17), and in a model of acquired erlotinib resistance, EMT associated
4
with elevated AXL appeared to underlie erlotinib resistance (4). Moreover, co-
treatment of EGFR wild-type NSCLC xenografts with erlotinib and an anti-AXL
antibody decreased tumor volume and metastasis (18). Similarly, lapatinib-
resistant HER2-positive breast cancer cells demonstrated elevated AXL (19), and
AXL knockdown reportedly re-sensitizes imatinib-resistant CML cell lines to
imatinib (20).
We broadly surveyed AXL expression in a large panel of human cancer cell lines,
and assessed its role in drug resistance associated with a mesenchymal
phenotype. AXL expression was well correlated with a mesenchymal phenotype in
the context of both intrinsic and drug resistance-associated EMT. AXL inhibition did
not detectably increase sensitivity to TKIs as previously reported; however, we
observed a striking synergistic interaction between AXL inhibition and anti-mitotic
agents specifically in mesenchymal tumor cells.
MATERIALS AND METHODS
Cell lines
Cell lines were from American Type Culture Collection or Deutsche Sammlung von
Mikroorganismenund Zelkulturen, and were obtained between 2010 and 2013. All
cell lines were authenticated by STR profiling and SNP fingerprinting, and were
confirmed to be mycoplasma-negative by the Genentech cell line banking facility
5
(gCELL) prior to use (see Supplementary Procedures). Cells were maintained in
culture for a 6 week period and thereafter a fresh vial of cells was obtained from
gCELL. To induce EMT, cells were treated with 2ng/ml of rh-TGF-β1 every three
days over a 2-3 week period.
Inhibitors
Erlotinib and cisplatin were from LC laboratories. Docetaxel and doxorubicin were
from Sigma. PF-03814735, BI-2536, MP-470 and PHA-739358, were from Selleck
Chemicals. Paclitaxel was from Tocris. Gemcitabine was from Toronto Research.
R428 was from Synkinase. Recombinant human AXL-Fc, Gas6 and TGF-β1 were
from R&D Systems.
RNA-seq analysis
RNA from cell lines and was used to prepare libraries using the TruSeq RNA
Sample Preparation kit (Illumina). Libraries were sequenced on the Illumina HiSeq
2000 platform. Expression of AXL and Gas6 RNA was extracted from a database
derived from RNA-seq analysis [Klijn et al 2014, Manuscript in preparation].
Immunoblotting
Immunodetection of proteins was performed using standard protocols. The AXL,
Mer, Tyro-3, E-Cadherin, vimentin, phospho-CDC2 (Tyr 15), CDC2, phospho-AKT,
total AKT, phospho-S6, total S6, and GAPDH antibodies were from Cell Signaling
Technology, and the PARP antibody was from eBioscience.
6
Gas6 ELISA
Cells were incubated overnight and replenished in fresh RPMI 1640 media.
Medium was collected 24h later and was analyzed using the Human Gas6
Quantikine ELISA Kit from Quantikine, R&D systems. ELISA was performed as per
manufacture’s instructions and normalized to cell number.
Invasion assay
Pre-labeled DiIC12(3) BD Bioscience cell suspensions in serum-free RPMI media
were added to the apical chamber of the BD BioCoat™ Tumor Invasion System,
8µm from BD Biosciences. Invasion assays were performed as per manufacturer’s
instructions.
Kinase Profiling
The R428 kinase profile was performed at KINOMEscan. R428 was screened at
1000nM, and results for primary screen binding interactions are reported as
percentage control, where lower numbers indicate stronger binding.
Cell Viability
Cell viability was assessed as previously described (21).
RNA interference
7
AXL knockdown was achieved by transfection using ON-TARGETplus AXL siRNA
(Dharmacon) and Lipofectamine RNAiMax (Invitrogen). ON-TARGETplus Non-
targeting Pool siRNA (Dharmacon) served as control.
Time-lapse imaging
Cells were seeded in a glass-bottom 24-well plate (Greiner Bio-One). 100 cells per
treatment were tracked and mitotic fate and cell death were scored as described
previously (22).
Xenograft studies
All procedures were approved by and conformed to the guidelines and principles of
the Institutional Animal Care and Use Committee of Genentech and were carried
out in an Association for the Assessment and Accreditation of Laboratory Animal
Care (AALAC)-accredited facility. Five million HeLa or MDA-MB-231 cells were
inoculated in the right flank of Nu/Nu nude mice. When tumors reached 100-200
mm3, mice were treated with vehicle control, R428 (125 mg/kg five times per
week; oral gavage), docetaxel (10 mg/kg three times a week, intravenously for
HeLa and 10 mg/kg once a week, intravenously for MDA-MB-231), or the
combination. Tumors were measured three times weekly using digital calipers and
tumor volumes were calculated as (L × (W × W))/2. Differences between R428
and docetaxel combination groups and individual-treated and control groups were
determined using two-way ANOVA.
8
Statistics
Bliss expectation was calculated as (A + B) – A x B, where A and B are the
fractional growth inhibitions of drug A and B at a given dose. The difference
between Bliss expectation and observed growth inhibition of the combination of
drugs A and B at the same dose is the "Delta Bliss excess." Delta Bliss values
were summed across the dose matrix to generate the Bliss sum. Differences
between two groups were determined using Student’s t-test, p values are
represented as p<0.05 *, p<0.01 ** and p<0.001 ***. Differences between the R428
and docetaxel combination groups and individual-treated and control groups were
determined using two-way ANOVA.
RESULTS
AXL expression correlates with a mesenchymal phenotype
Using RNAseq, we profiled mRNA expression of AXL (Figure 1A and
Supplementary Dataset S1) and its ligand Gas6 (Figure S1A) in 643 human cancer
cell lines. AXL expression in cell lines from breast and lung demonstrated the
greatest dynamic range within a tissue subset, where expression levels could
readily classify most lines as “AXL-high” or “AXL-low”. Gas6 mRNA was more
uniformly expressed. To identify a potential correlation between AXL expression
and the mesenchymal phenotype, we examined expression of the epithelial marker
E-cadherin and the mesenchymal marker vimentin (Figure S1B). AXL-high cells
generally expressed abundant vimentin whereas AXL-low cells demonstrated
9
higher E-cadherin expression. AXL-high cells showed, on average, a 4.6 fold
increase in vimentin relative to AXL-low cells (p value = 2.04e-24) (Figure S1C).
Thus, AXL expression is strongly associated with a mesenchymal phenotype in
human cancer cell lines.
Within breast cancer subtypes, AXL is relatively elevated within the triple negative
breast cancer (TNBC) subset compared to ER-positive/PR-negative and HER2
amplified subsets (Figure 1B). Gas6 is similarly elevated in the TNBC subset
(Figure S1D). Correspondingly, AXL protein was highly elevated in TNBC relative
to the other subtypes (Figure 1C). We expanded the TNBC panel and observed a
particularly strong correlation between AXL expression and the mesenchymal
subset of TNBC lines (Figure 1D, S1E). Expression of the AXL-related TYRO-3
RTK is variable, and is not correlated with any particular subset, and MER
expression is inversely correlated with AXL (Figure 1D).
Immunohistochemistry analysis of TNBC tumors revealed that most expressed E-
Cadherin, with 20/26 cases exhibiting strong membranous staining in all tumor
cells and 4 displaying focal expression in malignant cells (Figure S2). Significantly,
7/26 samples displayed vimentin tumor cell staining in varying proportions of
malignant cells (Figure S2). In all samples, vimentin and AXL were detected in
normal stroma cells. AXL staining of a small proportion of tumor cells was seen in
3/26 tumor samples (Figure S2). The observed percentage of AXL-positive TNBCs
is consistent with recent previous reports (23, 24).
10
In NSCLC, a previous report correlated the mesenchymal phenotype with
sensitivity to the EGFR TKI, erlotinib (25). Indeed, AXL tended to be elevated in
erlotinib-insensitive cells with relatively high vimentin (Figure 1E). Conversely, cell
lines with greater erlotinib sensitivity tended to exhibit lower vimentin and AXL
expression. This relationship was also observed with RNAseq analysis (Figure
S1F). These results indicate a strong correlation between AXL expression and a
mesenchymal/drug-resistant phenotype in breast cancer and NSCLC cells.
TGF--induced EMT is associated with increased AXL and TKI resistance
To explore a functional requirement for AXL in EMT-associated drug resistance,
we experimentally induced EMT in cancer cell lines with previously established
drug sensitivity by treatment with TGF-β (1, 26, 27). Exposure of EGFR- or HER2-
“addicted” cell lines to TGF-β yielded the expected EMT, with transition from a
compact uniform appearance to a spindle-like morphology (Figure 2A), as well as
loss of E-cadherin and gain in vimentin (Figure 2B), and increased invasion
capacity (Figure 2C). EMT is a reversible process, and indeed, upon withdrawal of
TGF- for 2 weeks, the mesenchymal cells reverted to an epithelial phenotype
(Figure S3A).
Expression of AXL and Gas6 were substantially induced upon EMT, consistent
with AXL’s association with a mesenchymal phenotype (Figure 2B,D). Significantly,
HER2-amplified HCC1954 cells, following EMT, exhibited 10-fold reduced
11
sensitivity to the HER2/EGFR TKI lapatinib (Parental; IC50= 0.456µM, TGF-β; IC50=
4.036µM) (Figure 2E). Similarly, EGFR mutant NSCLC PC9 cells exhibited 4-fold
reduced erlotinib sensitivity following EMT, (Parental; IC50= 0.05µM, TGF-β; IC50=
0.214µM) (Figure 2F). In HCC1954 and PC9 cells, post-EMT, TKI treatment failed
to promote apoptosis (Figure 2G). TKI resistance in the mesenchymal cells did not
reflect drug efflux, as TKIs induced strong suppression of downstream signaling
(Figure S3B). Thus, upon TGF-β-induced EMT in RTK oncogene-addicted cancer
cells lines, the resulting mesenchymal cells exhibit substantially increased AXL,
and become drug-resistant.
EMT-associated drug resistance is independent of AXL function
Recent reports have implicated AXL in acquired drug resistance (4, 19, 20).
Therefore, we employed these models to examine a functional AXL requirement in
EMT-associated drug resistance. Initially, we utilized R428, an AXL kinase inhibitor
(Supplementary Dataset S2) (28). As expected, R428 effectively suppressed
Gas6-induced phospho-AXL (Figure S4A), phospho-AKT and phospho-S6 levels
(Figure S4B&C), and decreased invasion capacity of AXL-expressing cells (Figure
S4D). These effects were not observed in AXL-negative BT-20 cells. We tested the
ability of R428 to restore erlotinib sensitivity in a TGF-β-induced resistance model
and in two models of acquired erlotinib resistance demonstrating EMT and
increased AXL (Figure 3B and S5A). In both parental and drug-resistant cell lines
we observed either very weak R428/erlotinib synergy (positive delta Bliss) or
antagonistic interaction (negative delta bliss) (Figure 3A&C), with no change in cell
12
viability (Figure 3A). Similarly, there was no change in the IC50 value for erlotinib
sensitivity in combination with R428 (1µM) in either model (Figure 3D, E).
Furthermore, AXL knockdown did not restore erlotinib sensitivity in either of the
resistance models (Figure 3F-H, S5B). In addition, inhibition of AXL using an AXL-
Fc fusion protein, an anti-AXL antibody, YW327.6S2 (Figure S5C & D),
doxycycline-inducible AXL knockdown (Figure S5E-G), or overexpression of AXL
(Figure S6A-C), does not detectably impact sensitivity to erlotinib. Finally, co-
treatment of resistant cells with either AXL-Fc fusion protein or the AXL antibody
with erlotinib did not prevent the emergence of drug-resistant clones (Figure S6D).
We next examined R428 activity in an AXL-negative EMT-associated resistance
model. EGFR mutant HCC827 NSCLC cells, upon TGF-β treatment, become
mesenchymal (Figure 1E, lane 48 and 49) and erlotinib-resistant (Figure 3D), but
do not exhibit AXL induction (Figure 1E). R428 did not sensitize these cells to
erlotinib (Figure 3D). Furthermore, MP-470, another AXL inhibitor (4), did not
synergize with erlotinib (Figure S7A). Notably, AXL expression was consistently
suppressed by TKI treatment in the mesenchymal cells (Figure 2G), further
indicating that AXL is not required for the observed resistance. These collective
observations do not support a role for AXL inhibition in overcoming acquired
resistance to TKIs in the EMT context.
AXL inhibition sensitizes erlotinib-resistant mesenchymal cells to docetaxel
13
TGF-β-induced EMT or erlotinib-induced resistance yields mesenchymal cells that
are cross-resistant to many anti-cancer agents (Table S1 and data not shown). We
explored whether AXL inhibition could sensitize resistant mesenchymal cells to
other anti-cancer agents. PC9 cells and mesenchymal derivatives were screened
for sensitivity to 100 anti-cancer agents. Mesenchymal cells were selectively
resistant to many agents—most notably, to docetaxel, an anti-mitotic agent (Table
S1). We extended this observation to other erlotinib resistance models. In both the
erlotinib-induced resistance (ERL-R) and TGF-β-induced mesenchymal resistance
(MES) models the mesenchymal cells were more docetaxel-resistant (Figure 4A).
Co-treatment with docetaxel and R428 was synergistic in both models (Figure 4A,
4B). Similarly, in the drug-resistant HCC827 ERL-R and HCC4006 ERL-R cells,
greater sum Bliss scores were observed (Figure 4B). In contrast, we did not
observe synergy with R428 and gemcitabine (Figure 5A).
Co-treatment of drug-resistant mesenchymal cells with R428 and docetaxel
dramatically shifted docetaxel IC50 values. Thus, for mesenchymal PC9 cells, the
IC50 for docetaxel alone was >300nM while in combination with R428 it was 0.3nM
(Figure 4C). Similarly, in the erlotinib-resistant mesenchymal HCC4006 cells, the
IC50 for docetaxel alone was >300nM whereas in combination with R428, it was
0.191nM (Figure 4D). Conversely, in mesenchymal HCC827 cells, R428 did not
synergistically interact with docetaxel, suggesting that the observed synergy
reflects an AXL-targeted effect of R428 (Figure 4C). We observed similar synergy
between the AXL inhibitor MP-470 and docetaxel (Figure S7A) and no change in
14
IC50 values upon co-treatment with MP-470 and docetaxel in the AXL-negative
mesenchymal HCC827 cells (Figure S7B). Collectively, these findings suggest that
EGFR mutant cancer cells that become resistant to erlotinib and cross-resistant to
docetaxel through innate or drug-induced EMT, can be sensitized to docetaxel by
AXL inhibition.
AXL inhibition sensitizes mesenchymal cancer cells to anti-mitotic agents
To establish that R428/docetaxel synergy is independent of TGF- or drug
exposure, we evaluated this treatment combination in a panel of 100 cancer cell
lines (Supplementary Dataset S3). We observed strong synergy in a subset of
AXL-expressing mesenchymal cell lines (Figure 5A and Supplementary Dataset
S3), and a correlation between AXL expression and the sum Bliss score (Figure
5B). Similarly, R428 synergized with the related taxane paclitaxel to inhibit
proliferation (Figure S8A-D), but not with gemcitabine, doxorubicin, or cisplatin
(Figure 5A,C and S8F&G). We also observed similar synergy between docetaxel
and an AXL-Fc fusion protein, which blocks Gas6 binding to AXL (18), or with MP-
470, in AXL-high cell lines (Figure 5D and S7C), and siRNAs targeting AXL further
confirmed that AXL inhibition mediated the observed synergy (Figure 5E).
Tubulin-binding taxanes cause a sustained mitotic block, resulting in cell death
during mitosis (29). To determine whether R428 synergistically interacts with anti-
mitotic agents with distinct mechanisms of action, we tested two aurora kinase
inhibitors, PHA-739358 and PF-03814735, and the PLK1 (polo kinase) inhibitor BI-
15
2536, which activates the spindle checkpoint, causing mitotic arrest (Figure 5A).
We observed synergy between R428 and the aurora kinase and PLK1 inhibitors in
AXL-expressing mesenchymal cells (Figure 5A,C). We also observed a five-fold
shift in the PHA-739358 IC50 upon combination treatment with R428 for the MDA-
MB-231 cells (PHA-739358 alone, IC50 = 1.63µM and in combination with R428
(1µM), IC50 = 0.34µM (Figure 5C). Thus, AXL inhibition synergistically blocks cell
proliferation in combination with anti-mitotic agents in mesenchymal cancer cells.
AXL kinase inhibition enhances docetaxel activity to suppress tumor growth
We extended the cell line findings to an in vivo context using tumor xenografts.
First, we determined that R428 was pharmacologically active in xenografts (Figure
6A). To assess efficacy, mice bearing 100-200mm3 tumors were treated with R428
and/or docetaxel. R428 or docetaxel slightly inhibited tumor growth; however, co-
treatment caused significant tumor growth suppression (Figure 6B). Similar effects
were seen with MDA-MB-231 xenografts (Figure 6C). A modest decrease in body
weight appeared to be docetaxel-dependent (Figure 6B, 6C). These observations
support the potential in vivo utility of AXL inhibitors combined with anti-mitotic
agents.
AXL inhibition enhances cell death during mitosis when combined with anti-
mitotic agents
To establish whether AXL inhibition alters mitotic cell fate when combined with anti-
mitotic agents, we employed time-lapse microscopy of individual cells to monitor
16
duration of mitosis and cell fate during mitosis (Figure 7A, S9 and Supplemental
Movies 1-4). The duration of completed mitosis in DMSO-treated HeLa cells
ranged from 20-50 minutes (Figure 7B), and R428 did not affect duration (Figure
7B and Supplemental Movie 1&2). By contrast, docetaxel caused mitotic slippage
generating three or four daughter cells (Figure 7A and Supplemental Movie 3) and
a longer duration of mitosis, as expected (Figure 7B). Upon co-treatment with
docetaxel and R428, the percentage of cells that completed mitosis decreased
substantially, and the percentage of cells that died in mitosis was greatly enhanced,
from 46% to 93% (Figure 7A, 7E and Supplemental Movies 3&4).
The duration of the mitotic cell death interval was comparable between docetaxel
treatment alone and co-treatment with R428 (Figure 7C). However, co-treatment
caused most cells to enter mitosis and die without completing mitosis (Figure 7A).
In contrast, with docetaxel alone, the death in mitosis fate occurred during the
second mitosis; thus, cells had completed one mitotic event before experiencing
death in mitosis (explaining why the combined cell fates total >100). To quantify
this event, we recorded the timestamp of the mitotic death event, and observed a
significant effect of combination treatment. Thus, the death in mitosis event
occurred substantially earlier (25.14 ± 0.86 hours for combination versus 41.74 ±
1.98 hours docetaxel alone) (Figure 7D). We similarly observed enhanced mitotic
death in combination-treated MDA-MB-231 cells (Figure S9D-G). Moreover, the
enhanced death in mitosis was dependent on the docetaxel concentration (Figure
S9D&G).
17
As with R428, co-treatment with MP-470, or the AXL-FC protein, and docetaxel
resulted in altered cell fate during mitosis (Figure S9A,B). Similar results were seen
upon co-treatment with PHA-739358 and R428 (Figure 7A). In contrast, co-
treatment with R428 and gemcitabine did not alter mitosis or mitotic fate (Figure
S9C). These findings support the specificity of AXL inhibition in potentiating the
mitotic fate effect of anti-mitotic agents in mesenchymal tumor cells.
AXL’s role in mitotic fate is associated with CDC2 dysregulation
Cell death during mitosis is caspase-dependent (30), and mitotic fate is determined
by both caspase activation and cyclin B1 degradation. Enhanced caspase
activation and slower cyclin B1 decay promotes death in mitosis, whereas, delay in
caspase activation and enhanced cyclin B1 degradation causes mitotic slippage.
Docetaxel induced caspase-3/7 activation, however, upon R428 co-treatment,
caspase-3/7 activity was induced faster and more robustly than with docetaxel
(Figure 7F), paclitaxel (Figure S8D) or PHA-739358 (Figure S8E) alone. In contrast,
no effects on cyclin B1 were observed with docetaxel, even in combination with
R428 (data not shown). Co-treatment with R428 and docetaxel increased G2-M
arrest from 36.2% (docetaxel alone) to 51.2% (Figure 7G). We observed
enhanced PARP cleavage following co-treatment with R428 and either, docetaxel,
paclitaxel, or PHA-739358 (Figure 7H and Figure S8C), confirming that the
observed death in mitosis event was apoptotic.
18
PI-3 kinase inhibition can enhance docetaxel efficacy in breast cancer models (31),
and consistent with this observation, suppression of phospho-AKT and phospho-
S6 by R428 were further enhanced by docetaxel (Figure S10A). A critical regulator
of mitotic entry is the cyclin-dependent kinase-1 (CDC2) (32), via a regulatory
phosphorylation at Tyr15 (33), and co-treatment with R428 and docetaxel
enhanced and sustained CDC2 dephosphorylation (Figure 7I and S10B).
Furthermore, suppression of phospho-CDC2 was observed in R428-treated
xenografts (Figure S10C); however, CDC2 protein levels are not affected by AXL
inhibition (Figure S11). These observations suggest that CDC2 is regulated by AXL,
and upon exposure to anti-mitotic agents, cancer cells rapidly enter mitosis and
remain in mitosis until the death in mitosis event, implicating CDC2 regulation in
sensitization to anti-mitotic agents in mesenchymal cancer cells by AXL inhibitors.
DISCUSSION
EMT is an increasingly recognized driver of tumor progression, and accumulating
evidence implicates EMT in both innate and acquired resistance to various anti-
cancer agents. Our RNA-seq analysis confirmed that AXL expression is indeed
widely associated with a mesenchymal phenotype, particularly within TNBCs and
erlotinib-resistant NSCLCs. However, the low percentage of AXL-positive tumors in
TNBC does not correlate with the in vitro findings and remains to be fully
understood. Targeting AXL might therefore be an attractive therapeutic approach
to overcoming resistance associated with EMT.
19
Our observations do not support a role for AXL inhibition in overcoming acquired
drug resistance in the EMT context. This may reflect in part the fact that many of
the tool compounds used in recent reports are multi-kinase inhibitors, lacking AXL
selectivity (4, 17). Furthermore, there may be off-target cytotoxic effects of AXL-
targeted RNAi reagents. Notably, a recently reported analysis of NSCLC cells with
acquired erlotinib resistance and increased AXL levels similarly excluded a
functional role for AXL (34).
Although our findings do not support a rationale for AXL inhibition in the context of
acquired TKI resistance, we found that AXL inhibition may be effective in
mesenchymal tumors, specifically in combination with anti-mitotic agents. In TNBC,
patients typically present a significant clinical challenge, as they do not respond to
the various targeted cancer therapies due to an apparent lack of RTK activation.
However, TNBC patients do show some response to taxane-based chemotherapy
(35), and our studies suggest that combining anti-mitotics with AXL inhibition may
be an appropriate combination therapy in this disease setting.
Taxane-based therapies exert their cytotoxic effects by binding and stabilizing
microtubules, resulting in cell cycle arrest in mitosis. The response to anti-mitotic
agents can vary significantly among cancer cells. Thus, some cells undergo death
during prolonged mitosis, while others undergo slippage and die in interphase (29).
The degree of caspase activation and the level of cyclin B1 degradation can
determine cell fate during mitosis (30). Several treatments reportedly enhance the
20
efficacy of taxane-based therapy, such as Navitoclax (Bcl-2/Bcl-xl inhibitor), and
the PI-3 kinase inhibitor GDC-0941 (22, 31, 36). In these studies the enhanced
efficacy of combination treatment reflected a decrease in the duration of mitosis
until mitotic cell death, an enhanced rate of apoptosis, the down-regulation of cyclin
D1 and pAkt, and altered kinetics of Bcl-2 family protein stabilization. AXL engages
some of these pathways, particularly the PI-3K/Akt pathway (13, 14), which we
found was suppressed by R428, particularly upon co-treatment with docetaxel,
which may contribute to the observed synergy. Our findings also indicate that the
observed synergy with AXL inhibition reflects altered kinetics of dephosphorylation
of CDC2, a cell cycle-dependent kinase that governs mitotic entry. However, it
remains unclear as to how AXL, relative to other RTKs that transduce signals to
largely overlapping downstream effectors, specifically regulates this signaling event
in the context of mitosis.
ACKNOWLEGEMENTS
The authors thank members of the Settleman laboratory and Amitabha
Chadhuri for helpful discussions, members of the Genentech cell line screening
facility and cell line bank. We thank Nancy Yu and Tom Januario for providing cell
line lysates, and the in vivo pharmacology group for guidance on xenograft studies.
21
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FIGURE LEGENDS
Figure 1: AXL expression correlates with a mesenchymal signature. (A)
Scatter plot representing AXL RNA expression for 643 human cancer cell lines.
Red line represents mean expression within each tissue type. (B) Scatter plot
illustrating AXL expression in 46 breast cancer cell lines based on Estrogen-
positive (ER)/Progesterone-positive (PR), and HER2 status. Differences between
TNBC subtype compared to the HER2 amp (p= 0.0058), and ER+/PR- (p= 0.0081)
were calculated using a Student t-test. No differences were observed between
HER2 amp and ER+/PR- (NS). Black line represents the median expression within
each subtype. (C) Immunoblots indicating AXL protein in triple-negative, Estrogen-
positive (ER)/Progesterone-positive (PR), and HER2-amplified breast cancer cells.
(D) Immunoblots indicating AXL, MER, and TYRO-3 protein in TNBC cells. We
note reported controversy regarding the tissue origin of MDA-MB-435 cells, which
clusters with cell lines of melanoma origin by expression profiling. Asterisks
indicate cell lines with high Vimentin expression. (E) Immunoblots showing AXL
and vimentin expression in NSCLC cell lines. Cell lines were arranged according to
erlotinib sensitivity as IC50 (25). #1 indicates the most erlotinib-sensitive and #46 is
the least sensitive. Lanes 47 and 49 correspond to HCC827 and PC9 cell lines,
respectively, treated with TGF-β for 14 days. GAPDH levels demonstrate protein
normalization. Asterisks indicate cell lines with EGFR mutation.
28
Figure 2: AXL expression is increased upon TGF-β-induced EMT. (A)
Microscopically observed morphological changes in breast cancer cell lines
HCC1954 and BT474, and lung cancer cell line PC9 treated with TGF-β for 14
days. (B) Immunoblot demonstrating loss of E-Cadherin and MER and increased
vimentin and AXL upon TGF-β treatment of BT474, HCC1954, and SUM149PT,
and PC9 cells. (C) Bar graph illustrating enhanced invasion capacity of TGF-β-
treated (day 14) HCC1954 cells. Differences between Parental and TGF-β-treated
(p= 0.033) cells were calculated by Student t-test. (D) Bar graph demonstrating
increased Gas6 secretion (by ELISA) in HCC1954 and PC9 cells following TGF-β-
induced EMT (day 14). Differences between Parental compared to TGF-β-treated
(HCC1954, p<0.0001 and PC9, p=0.0003) were calculated by Student t-test. Error
bars represent the mean ± SEM. (E) Cell viability assay demonstrating the effect of
lapatinib in TGF-β-treated (day 14) HCC1954 cells. IC50 values for lapatinib were
calculated in Prism, Parental; IC50= 0.456µM, TGF-β; IC50= 4.036µM. (F) Cell
viability assay demonstrating the effect of erlotinib in TGF-β-treated (day 14) PC9
cells. IC50 values for erlotinib were calculated in Prism, Parental; IC50= 0.05µM,
TGF-β; IC50= 0.214µM (G) Immunoblot showing the effect of erlotinib (ERL; 50nM)
and lapatinib (LAP; 1µM) on PARP cleavage and AXL expression after 72h. All
Error bars represent the mean ± SEM.
Figure 3: AXL inhibition does not re-sensitize erlotinib-resistant NSCLC cells.
(A) Drug matrix heat-map grid illustrating percentage inhibition and delta Bliss for
R428 in combination with erlotinib in PC9 parental and mesenchymal (MES) cell
29
lines (Top), HCC4006 parental and erlotinib-resistant (ERL-R) cells (Middle), and
HCC827 parental and erlotinib-resistant (ERL-R) cells (Bottom). Drug matrix heat-
map grids correspond to one representative experiment out of three independent
experiments. (B) Immunoblot demonstrating increased AXL following prolonged
erlotinib treatment (ERL-R) in HCC4006 and HCC827 cells. (C) Table illustrating
the sum Bliss score for erlotinib in combination with R428. (D) Cell viability assay
demonstrating the effect of R428 (1µM) in combination with erlotinib in PC9
parental and mesenchymal (MES) cells (Left) and HCC827 parental and
mesenchymal (MES) cells (Right). (E) Cell viability assay illustrating the effect of
R428 (1µM) in combination with erlotinib in HCC4006 parental and erlotinib-
resistant (ERL-R) cells (Left), and HCC827 parental and erlotinib-resistant (ERL-R)
cells (Right). All error bars represent the mean ± SEM. (F) Immunoblot
demonstrating decreased AXL expression following knockdown (10nM) for 72h
with four different AXL siRNAs in PC9 mesenchymal cells. NTC siRNA serves as a
control. (G) Immunoblot showing the effect of AXL knockdown (10nM) for 72h and
in combination with erlotinib (50nM) for a further 72h on PARP cleavage and
vimentin expression in PC9 parental (PAR) and mesenchymal (MES) cells. (H) Cell
viability assay demonstrating the effect of AXL knockdown (20nM) for 24h and in
combination with erlotinib for a further 72h in HCC4006 erlotinib-resistant (ERL-R)
cells. Error bars represent the mean ± SEM.
30
Figure 4: AXL inhibition synergizes with docetaxel in erlotinib-resistant
mesenchymal NSCLC. (A) Drug matrix heat-map grid illustrating percentage
inhibition and delta Bliss for R428 in combination with docetaxel (DTX) for PC9
parental and mesenchymal (MES) cells (Top), HCC4006 parental and erlotinib-
resistant (ERL-R) cell lines (Middle), and HCC827 parental and erlotinib-resistant
(ERL-R) cells (Bottom). Drug matrix heat-map grids correspond to one
representative experiment out of three independent experiments. (B) Table
illustrating the sum Bliss score for docetaxel in combination with R428. (C) Cell
viability assay demonstrating the effect of R428 (1µM) in combination with
docetaxel. IC50 values for docetaxel single agent or in combination R428 (1µM)
were calculated in Prism, PC9: PAR, 2.265nM; PAR + R428, 0.535nM; MES,
>300nM; MES + R428, 0.3nM. Parental (PAR), Mesenchymal (MES). (D) Cell
viability assay illustrating the effect of R428 (1µM) in combination with docetaxel.
IC50 values for docetaxel single agent or in combination with R428 (1µM) were
calculated in Prism, HCC4006 (Left): PAR, 0.429nm; PAR + R428, 0.223nM; ERL-
R, >300nM; ERL-R + R428, 0.191nM, HCC827 (Right): PAR, 2.759nm; PAR +
R428, 1.012nM; ERL-R, >300nM; ERL-R + R428, 100nM. All error bars represent
the mean ± SEM.
Figure 5: AXL inhibition enhances the efficacy of anti-mitotic agents. (A)
Table summarizing the sum Bliss score upon co-treatment with R428 and
docetaxel, mesenchymal cell lines indicated in blue (left panel). Right-hand panel,
31
drug matrix heat-map grid (right) illustrating percentage inhibition and delta Bliss
for HeLa (Left) and MDA-MB-231 (Right) cell lines upon co-treatment with R428
and docetaxel (DTX) (Top), PHA-739358 (Middle), and gemcitabine (Bottom). Drug
matrix heat-map grids correspond to one representative experiment out of three
independent experiments. (B) Plot demonstrating the correlation between AXL
expression (RPKM) and sum Bliss score of the R428 and docetaxel combination.
AXL-negative cell lines were removed from analysis. R2 value equals 0.5044 and
was calculated using Pearson correlation coefficient. (C) Cell viability assay
demonstrating the effect of R428 (1µM) in combination with docetaxel (Left), PHA-
739358 (Middle) and gemcitabine (Right). Error bars represent the mean ± SEM.
MDA-MB-231 IC50 values for docetaxel single agent treatment or in combination
with R428 were calculated in Prism, DMSO, 0.275nM; R428 (1µM), 0.147nM;
R428 (3µM), 0.053nM. IC50 values for PHA-739358 single agent treatment or in
combination R428 were calculated in Prism, DMSO, 1.626µM; R428 (1µM),
0.336µM; R428 (3µM), 0.17µM. (D) Syto 60 cell staining of HeLa cells treated for
72h with docetaxel (DTX; 3nM), R428 (1µM), AXL-FC (10µg/ml), or MP-470 (1µM).
(E) Syto 60 cell staining of HeLa cells upon siRNA knockdown of AXL (10nM) or
non-targeting control (NTC) for 72 hours and in combination with docetaxel (DTX,
1nM) for a further 72 hours, left panel. Immunoblot demonstrating AXL protein
expression following knockdown in combination with docetaxel (1nM), right panel.
Figure 6: AXL inhibition in combination with docetaxel retards the growth of
mesenchymal tumor cells in vivo. (A) Immunoblots showing phospho-AKT
32
(pAKT), total AKT, phospho-S6 (pS6) and total S6, in individual MDA-MB-231
xenograft tumors following R428 treatment. (B) Tumor growth assay showing the
anti-tumor effect of R428 in combination with docetaxel in Hela xenografts.
Differences between the R428 and the docetaxel combination group compared to
the Vehicle group (p<0.0001), and individual treatment with R428 (p=0.0001) and
docetaxel (p<0.0001) were calculated using two-way ANOVA. Error bars represent
the mean ± SEM. Percentage body weight change is shown at the bottom. (C)
Tumor growth assay showing the anti-tumor effect of R428 in combination with
docetaxel in MDA-MB-231 xenografts. Differences between the R428 and the
docetaxel combination group compared to the Vehicle group (p=0.0033), and
individual treatment with R428 (<0.0001) and docetaxel (p<0.0001) compared to
the combination group were calculated using two-way ANOVA. Error bars
represent the mean ± SEM. Percentage body weight change is shown at the
bottom.
Figure 7: Axl inhibition in combination with anti-mitotic agents promotes
mitotic death. (A) Table representing the fate of 100 individual cells following
exposure to R428 (1µM), docetaxel (DTX; 3nM), PHA-739358 (PHA; 100nM), or
the indicated combinations based on a 72h microscopy assay. (B) Scatter plot
demonstrating the duration of completed mitosis (h) for 100 individual cells upon
each drug treatment: R428 (1µM), docetaxel (DTX), PHA-739358 (PHA) and
combinations in HeLa cells in a 72h assay. Red line indicates Mean. (C) Scatter
plot demonstrating the duration of the mitotic death interval in hours (h) of 100
33
individual cells upon each drug treatment: R428 (1µM), docetaxel (DTX) and the
combination in HeLa cells in a 72h assay. Red line indicates Mean. (D) Scatter plot
demonstrating a significant difference as assessed using Student’s t-test (HeLa p=
<0.0001) of the time of mitotic death in hours (h) of 100 individual cells upon each
drug treatment: R428 (1µM), docetaxel (DTX; 3nM) and the combination in HeLa
cells in a 72h assay. Red line indicates Mean. (E) Microscopy images
demonstrating mitosis following treatment with R428 (1µM), docetaxel (DTX; 3nM)
or the combination in HeLa cells at 18h (left panel) and 36h (right panel). (F) Bar
graph representing caspase-3/7 activation following treatment with R428 (1µM),
docetaxel (DTX; 10nM) or the combination in HeLa cells during a 18h, 24h and 36h
time course. Error bars represent the mean ± SEM. (G) Histogram plots
demonstrating DNA content upon exposure of HeLa cells to R428 (1µM) in
combination with docetaxel (DTX; 10nM) after 8h. (H) Immunoblot demonstrating
apoptosis (cleaved PARP) in HeLa cells after PHA-739358 (PHA) or docetaxel
(DTX) treatment in combination with R428 (1µM) for 72 hours. (I) Immunoblots
showing phospho-CDC2 (pCDC2) and total CDC2 in HeLa cells following
treatment with R428 (1µM), docetaxel (DTX; 3nM) or the combination (COMBO)
during an 8 hour assay period.
TNBC HER2 amp ER+/PR-2-10
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Figure 3
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R428 (µM) R428 (µM) R428 (µM) R428 (µM)
HCC4006 ERL-R
Percentage Inhibition Delta. Bliss
HCC4006 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlo
tinib
(µ
M)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
HCC827 ERL-R
Percentage Inhibition Delta. Bliss
HCC827 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlo
tinib
(µ
M)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
HCC827 ERL-R
Percentage Inhibition Delta. Bliss
HCC827 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlotin
ib (µ
M)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
Erlo
tinib
(µ
M)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
B
Total AXL
GAPDH
NT
C S
iRN
A
AX
L S
iRN
A #
1
AX
L S
iRN
A #
2
AX
L S
iRN
A #
3
AX
L S
iRN
A #
4
Vimentin
Cleaved PARP
Vimentin
GAPDH
AXL#2 NTC AXL#2
MES
NTC
- + - + - + - + ERL 50nM 72h
PAR
Figure 4
A
HCC4006 ERL-R
Percentage Inhibition Delta. Bliss
HCC4006 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlotin
ib
(µM
)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
HCC4006 ERL-R
Percentage Inhibition Delta. Bliss
HCC4006 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlotin
ib
(µM
)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
PC9 Mesenchymal
Percentage Inhibition Delta. Bliss
DT
X (
nM
)
PC9 Parental
Percentage Inhibition Delta. Bliss
Erl
otin
ib
(µM
)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
HCC4006 ERL-R
Percentage Inhibition Delta. Bliss
HCC4006 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlotin
ib (µ
M)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
HCC827 ERL-R
Percentage Inhibition Delta. Bliss
HCC827 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlo
tin
ib
(µM
)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
HCC4006 ERL-R
Percentage Inhibition Delta. Bliss
HCC4006 Parental
Percentage Inhibition Delta. Bliss
DT
X (
nM
) E
rlotin
ib (µ
M)
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
C
D
B
Combination with R428 and docetaxel
Cell line Sum bliss score
PC9 Mesenchymal 2536
PC9 Parental 108
HCC827 ERL-R 575
HCC827 Parental 11
HCC4006 ERL-R 470
HCC4006 110
Percentage Inhibition Delta. Bliss
HeLa
Percentage Inhibition Delta. Bliss
MDA-MB-231
R428 (µM) R428 (µM) R428 (µM) R428 (µM)
PH
A-7
39
35
8 (µ
M)
DT
X (
nM
) G
em
cita
bin
e
(µM
)
A
DMSO Axl-FC R428
-DT
X
+D
TX
D DMSO MP-470
-DT
X
+D
TX
C
E NTC SiRNA Axl SiRNA
-DT
X
+D
TX
AXL
GAPDH
- + - + DTX
NTC AXL SiRNA
Figure 5:
0.1 1 10 100 1000-400
-200
0
200
400
600
Su
m b
liss s
co
re
AXL RPKM (Log10)
R2 = 0.5044B
Docetaxel PC9 Mesenchymal 2536
anti-mitotic HCC827 ERL-R 575
HCC4006 ERL-R 470
HeLa 371
CAL-120 292
HCC1937 233
HCC1143 133
MDA-MB-231 121
HCC4006 110
PC9 Parental 108
HCC827 Parental 11
PHA-739358 MDA-MB-231 528
Aurora Kinase Inhibitor PC9 Mesenchymal 524
anti-mitotic HeLa 250
PC9 Parental -568
HCC1143 -305
PF03814735 PC9 Mesenchymal 491
Aurora Kinase Inhibitor MDA-MB-231 333
anti-mitotic PC9 Parental 68
HCC1143 -45
HeLa -124
BI-2536 PC9 Mesenchymal 411
PLK-1 inhibitor MDA-MB-231 368
HeLa 156
PC9 Parental 114
HCC1143 -127
Gemcitabine MDA-MB-231 30
Nucleoside analog HCC1143 -304
HeLa -169
PC9 Parental -509
PC9 Mesenchymal -592
Cisplatin HeLa 2
DNA synthesis inhibitor MDA-MB-231 -10
Doxorubicin HeLa -114
DNA intercalator MDA-MB-231 -27
Figure 6:
MDA-MB-231 Xenograft
0 2 4 6 8 10 12 14 160
200
400
600
800Vehicle (n=8)
R428 (n=9)
DTX (n=8)
DTX + R428 (n=9)
Time (Days)
Tu
mo
r v
olu
me
(m
m3)
MDA-MB-231 Xenograft
0 2 4 6 8 10 12 14 160
200
400
600
800Vehicle (n=8)
R428 (n=9)
DTX (n=8)
DTX + R428 (n=9)
p<0.0001
Day
Tu
mo
r v
olu
me
(m
m3)
HeLa Xenograft
0 5 10 15 200
200
400
600
800Vehicle (n=6)
R428 (n=6)
DTX (n=6)
DTX + R428 (n=8)
Time (Days)
Tu
mo
r v
olu
me
(m
m3)
HeLa xenograft
0 5 10 15 200
200
400
600
800Vehicle (n=6)
R428 (n=6)
DTX (n=6)
DTX + R428 (n=8)
Time (Days)
Tu
mo
r v
olu
me
(m
m3)
p<0.0001
B C
MDA-MB-231 Xenograft
0 2 4 6 8 10 12 14 16-20
-10
0
10
20Vehicle (n=8)
R428 (n=9)
DTX (n=8)
DTX + R428 (n=9)
Time (Days)
Pe
rce
nta
ge
bo
dy
we
igh
t c
ha
ng
e
MDA-MB-231 Xenograft
0 2 4 6 8 10 12 14 160
200
400
600
800Vehicle (n=8)
R428 (n=9)
DTX (n=8)
DTX + R428 (n=9)
p<0.0001
Day
Tu
mo
r v
olu
me
(m
m3)
HeLa Xenograft
0 5 10 15 20-20
-10
0
10
20Vehicle (n=6)
R428 (n=6)
DTX (n=6)
DTX + R428 (n=8)
Time (Days)
Pe
rce
nta
ge
b
od
y w
eig
ht c
ha
ng
e
HeLa xenograft
0 5 10 15 200
200
400
600
800Vehicle (n=6)
R428 (n=6)
DTX (n=6)
DTX + R428 (n=8)
Time (Days)
Tu
mo
r v
olu
me
(m
m3)
p<0.0001
A
pS6 (Ser235/236)
Total S6
- - - + + + + + + + + R428
8hr 2hr
pAKT (ser473)
Total AKT
GAPDH
Figure 7:
A HeLa DMSO R428 (1µM) DTX (3nM) DTX + R428 PHA (100nM) PHA + R428
Total Cells 100 100 100 100 100 100
Enter Mitosis 100% 100% 100% 100% 98% 98%
Complete Mitosis 99% 99% 46% 6% 74% 6%
Mitotic Slippage 0% 0% 8% 1% 15% 73%
Die in Mitosis 1% 1% 46% 93% 9% 19%
Die in Interphase 0% 0% 39% 3% 3% 30%
20
30
40
50 HeLa
ratio
n o
f mito
tic
ea
th in
terv
al (h
)
4
6
8
10
12 HeLa
tio
n o
f co
mp
lete
d
mito
sis
(h
)
R428
DTX
DTX + R428
p<
0.0
00
1
B C D
p % % % % % %
R428 - + - +0
10
DTX (3nM)DMSO
Du
de
- + - + - +0
2
R428
DTX (3nM) PHA (100nM)DMSO
Du
ra
0 10 20 30 40 50 60 70 80
DMSO
Time of mitotic death event (h)
E
SO
-R428 +R428 -R428 +R428 FDMSO
R4285
6
ng
e
U)
DM
SD
TX
0
1
2
3
4
5
Ca
sp
ase
-3/7
fo
ld c
ha
n
lum
ine
sce
nce
(R
FU
t
H
IDT
X
PHA DTX
pCDC2 (Tyr15)
pCDC2 (Tyr15)
DMSO
R428
0.5 1 2 3 4 5 6 7 8 R428 (h) (h)
- + - + - + DTX
18h 24h 36h
G
ou
nt
Co
un
t
PARP
Cleaved PARP
GAPDH
- + - + - + - + - + R428
50
nM
0.5
µM
1n
M
3n
M pCDC2 (Tyr15)
pCDC2 (Tyr15)
Total CDC2
Total CDC2
DTX
COMBO
DMSO
R428
C
FL2-A FL2-A
Total CDC2
Total CDC2
DTX
COMBO
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