AXL inhibition sensitizes mesenchymal cancer cells to antimitotic drugs

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.

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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.

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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

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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

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(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.

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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

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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.

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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

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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).

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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

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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

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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

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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

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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-

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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

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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).

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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.

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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.

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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

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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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27

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.

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DT

X (

nM

) E

rlo

tinib

(µ

M)

R428 (µM) R428 (µM) R428 (µM) R428 (µM)

HCC827 ERL-R


HCC827 Parental


DT

X (

nM

) E

rlo

tinib

(µ

M)

R428 (µM) R428 (µM) R428 (µM) R428 (µM)

HCC827 ERL-R


HCC827 Parental


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


HCC4006 Parental


DT

X (

nM

) E

rlotin

ib

(µM

)

R428 (µM) R428 (µM) R428 (µM) R428 (µM)

HCC4006 ERL-R


HCC4006 Parental


DT

X (

nM

) E

rlotin

ib

(µM

)

R428 (µM) R428 (µM) R428 (µM) R428 (µM)

PC9 Mesenchymal


DT

X (

nM

)

PC9 Parental


Erl

otin

ib

(µM

)

R428 (µM) R428 (µM) R428 (µM) R428 (µM)

HCC4006 ERL-R


HCC4006 Parental


DT

X (

nM

) E

rlotin

ib (µ

M)

R428 (µM) R428 (µM) R428 (µM) R428 (µM)

HCC827 ERL-R


HCC827 Parental


DT

X (

nM

) E

rlo

tin

ib

(µM

)

R428 (µM) R428 (µM) R428 (µM) R428 (µM)

HCC4006 ERL-R


HCC4006 Parental


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


HeLa


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)


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


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


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

AXL inhibition sensitizes mesenchymal cancer cells to antimitotic drugs

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