ONO-7475, a Novel AXL Inhibitor, Suppresses the Adaptive Resistance to Initial EGFR-TKI Treatment in EGFR-Mutated Non-Small Lung Cancer

Home , Dacomitinib, Tivantinib

Author Manuscript Published OnlineFirst on January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2321 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Final version2 ONO-7475, a novel AXL inhibitor, suppresses the adaptive resistance to initial EGFR-TKI treatment in EGFR-mutated non-small lung cancer

Naoko Okura1#, Naoya Nishioka1#, Tadaaki Yamada1*, Hirokazu Taniguchi2, Keiko Tanimura1, Yuki Katayama1, Akihiro Yoshimura1, Satoshi Watanabe3, Toshiaki Kikuchi3, Shinsuke Shiotsu4, Takeshi Kitazaki5, Akihiro Nishiyama6, Masahiro Iwasaku1, Yoshiko Kaneko1, Junji Uchino1, Hisanori Uehara7, Mano Horinaka8, Toshiyuki Sakai8, Kohei Tanaka9, Ryohei Kozaki9, Seiji Yano6, & Koichi Takayama1

# N. Okura and N. Nishioka contributed equally to this work and share first authorship.

1. Department of Pulmonary Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine; 465, Kajii-cho, Kamigyo-ku, Kyoto 602–8566, Japan. 2. Department of Respiratory Medicine, Nagasaki University Graduate School of Biomedical Sciences; 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan. 3. Department of Respiratory Medicine and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences; 1–757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. 4. Department of Respiratory Medicine, Japanese Red Cross Kyoto Daiichi Hospital; 15-749 Hon-machi, Higashiyama-ku, Kyoto 605-0981, Japan. 5. Department of Respiratory Medicine, Japanese Red Cross Nagasaki Genbaku Hospital; 3-15 Mori-machi, Nagasaki 852-8511, Japan. 6. Division of Medical Oncology, Cancer Research Institute, Kanazawa University; 13-1, Takara-machi, Kanazawa 920-0934, Japan. 7. Division of Pathology, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503, Japan. 8. Department of Drug Discovery Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine; 465, Kajii-cho, Kamigyo-ku, Kyoto 602–8566, Japan. 9. Research Center of Oncology, Discovery and Research, Ono Pharmaceutical Co., Ltd.

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2321 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running title: ONO-7475 suppresses resistance to initial EGFR-TKI

Key words: AXL, EGFR mutation, initial treatment, lung cancer, drug tolerant cell

Financial support: This study was supported by a research grant for developing innovative cancer chemotherapy from a Grant for Lung Cancer Research, founded by the Japan Lung Cancer

Society (to T. Yamada), a research grant from the Takeda Science Foundation (to T. Yamada), research grants for Joint Research with the Cancer Research Institute of Kanazawa University

(to T. Yamada), and grants from JSPS KAKENHI [Grant Number 19K08608 (to T. Yamada)].

* Corresponding Authors: Tadaaki Yamada, MD, PhD, Department of Pulmonary Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine 465, Kajii-cho, Kamigyo-ku, Kyoto 602–8566, Japan, Phone: +81-75-251-5513, Fax: +81-75-251-5376, E-mail: [email protected]

Conflict of interest: Tadaaki Yamada obtained commercial research grants from Ono

Pharmaceutical Co., Ltd, Chugai Pharmaceutical Co., Ltd, and Takeda Pharmaceutical Co., Ltd.

Satoshi Watanabe received lecture fees from Liliy, Pfizer, Novartis Pharma, AstraZeneca, Chugai

Pharma, Bristol-Myers, Boehringer Ingelheim, Ono Pharmaceutical, Daiichi Sankyo and Taiho

Pharmaceutical. Toshiaki Kikuchi obtained research subsidies from Ono Pharmaceutical Co.,

Ltd, Chugai Pharmaceutical Co., Ltd, Eli Lilly Japan K.K. and Nippon Boehringer Ingelheim Co.,

Ltd., and research funding from AstraZeneca K.K. Kohei Tanaka and Ryohei Kozaki are employees of Ono Pharmaceutical Co., Ltd. No potential conflicts of interest were disclosed by the other authors. Koichi Takayama reports receiving research grants from Chugai-Roche Co.,

and Ono Pharmaceutical Co., and personal fees from AstraZeneca Co., Chugai-Roche Co.,

MSD-Merck Co., Eli Lilly Co., Boehringer-Ingelheim Co., and Daiichi-Sankyo Co.

Word count; 5508

Number of figures; 5, Number of tables; 0

Quantity of supporting information; 10 Figures and 2 Tables

Translational relevance

A recently identified small subpopulation of reversibly “drug-tolerant” cells resulting

from anexelekto (AXL) activation was reported to maintain cell viability in the EGFR

mutant non-small cell lung cancer (NSCLC). We investigated the best therapeutic

strategy to combat AXL-induced tolerance to epidermal growth factor receptor tyrosine

kinase inhibitors (EGFR-TKIs) using the novel AXL inhibitor ONO-7475 in

AXL-expressing EGFR mutated NSCLC cells treated with the new generation

EGFR-TKIs osimertinib and dacomitinib. Our findings demonstrated that the pivotal role

of AXL inhibition in the intrinsic resistance, but not acquired resistance, of EGFR

mutated lung cancer and the emergence of drug tolerant cells. These results showed the

clinical importance of combined initial treatment with osimertinib and ONO-7475 to

suppress the development of intrinsic resistance and the emergence of drug tolerant cells,

and led to the prevention of tumor heterogeneity in EGFR mutated lung cancer.

Abstract Purpose Currently, an optimal therapeutic strategy comprising molecularly targeted agents for treating EGFR-mutated non-small cell lung cancer (NSCLC) patients with acquired resistance to osimertinib is not available. Therefore, the initial therapeutic intervention is crucial for the prolonged survival of these patients. The activation of anexelekto (AXL) signaling is known to be associated with intrinsic and acquired resistance to EGFR-TKIs. In this study, we investigated the best therapeutic strategy to combat AXL-induced tolerance to EGFR-TKIs using the novel AXL inhibitor ONO-7475. Experimental design We examined the efficacy of ONO-7475 in combination with EGFR-TKIs in EGFR mutated NSCLC cells using in vitro and in vivo experiments. We investigated the correlation between AXL expression in tumors and clinical outcomes with osimertinib for EGFR mutated NSCLC patients with acquired resistance to initial EGFR-TKIs. Results ONO-7475 sensitized AXL-overexpressing EGFR-mutant NSCLC cells to the EGFR-TKIs osimertinib and dacomitinib. In addition, ONO-7475 suppressed the emergence and maintenance of EGFR-TKI tolerant cells. In the cell line-derived xenograft models of AXL-overexpressing EGFR mutated lung cancer treated with osimertinib, initial combination therapy of ONO-7475 and osimertinib markedly regressed tumors and delayed tumor re-growth compared to osimertinib alone or the combination after acquired resistance to osimertinib. AXL expression in EGFR-TKI refractory tumors did not correlate with the sensitivity of osimertinib. Conclusion These results demonstrate that ONO-7475 suppresses the emergence and maintenance of tolerant cells to the initial EGFR-TKIs, osimertinib or dacomitinib, in AXL-overexpressing EGFR-mutated NSCLC cells, suggesting that ONO-7475 and osimertinib is a highly potent combination for initial treatment.

Introduction

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are effective for the treatment of

non-small cell lung cancer (NSCLC) harboring EGFR mutations, such as the exon 19

deletion and the L858R mutation in EGFR. Of them, the 1st generation EGFR-TKIs

gefitinib and erlotinib, and the 2nd generation afatinib and dacomitinib have been

approved for untreated EGFR mutated advanced NSCLC patients in countries such as the

U.S. and Japan (1) (2) (3) (4). However, almost all EGFR-positive NSCLC patients

ultimately acquired resistance to EGFR-TKIs after approximately one year. The most

commonly acquired resistance mechanism, known as the secondary resistant mutation,

exon20 T790M mutation in EGFR, is detected in half of the EGFR positive patients (5).

The 3rd generation EGFR-TKI osimertinib showed promising efficacy for overcoming

EGFR T790M mutation positive NSCLC in the AURA2 and AURA3 studies (6,7).

However, an optimal therapeutic strategy using molecularly targeted agents for treating

NSCLC patients without the EGFR T790M mutation after acquired resistance to initial

EGFR-TKIs is not available. Current Phase 3 clinical trials demonstrated that the

treatment with osimertinib or the novel 2nd generation EGFR-TKI dacomitinib showed

better outcomes than that of 1st generation EGFR-TKIs at the first-line setting for

advanced EGFR mutated NSCLC patients (8,9). However, the intervention with

EGFR-TKI treatment for NSCLC cells with EGFR activating mutations has facilitated

tumor evolution and leads to acquired resistance to such EGFR-TKIs, resulting in the lack

of an optimal therapeutic strategy with molecularly targeted therapy after the acquired

resistance to osimertinib. In addition, approximately 20% of EGFR mutated NSCLC

patients show intrinsic resistance to the new generation EGFR-TKIs, dacomitinib and

osimertinib. Therefore, the initial therapeutic intervention plays a crucial role in the

survival of NSCLC patients with EGFR mutations.

Anexelekto (AXL) is a tyrosine kinase receptor belonging to the TAM family

of proteins. AXL is usually expressed in epithelial and mesenchymal cells, as well as in

breast, pancreatic, lung, and bone marrow cancers (10) (11) (12) (13) (14). AXL in

malignant tumors plays a pivotal role in contributing to proliferation, migration,

survival, and the epithelial-to-mesenchymal transition (EMT); its overexpression is

consequently correlated with poor prognosis in several cancers (15) (16) (17) (18) (19).

The activation of AXL signaling in tumors is associated with acquired resistance to

several targeted molecular therapy drugs and chemotherapeutic agents (15) (20) (21)

(22). Pre-clinical studies showed that the Gas6-AXL axis induced acquired resistance to

the EGFR-TKI erlotinib or osimertinib in EGFR mutated NSCLC cells, and that the

addition of the AXL inhibitor in combination could overcome this resistance (20) (21)

(22). In melanoma harboring NRAS and BRAF mutations, AXL inhibition has a pivotal

role in overcoming intrinsic or acquired resistance to BRAF/MEK inhibitors (23). We

previously revealed the role of the AXL pathway in the intrinsic resistance to

osimertinib and the emergence of osimertinib-tolerant cells in EGFR mutated NSCLC

cells (24). Hence, the regulation of the AXL pathway is expected to be a promising

therapeutic strategy against malignant progress in several cancers.

Several AXL inhibitors are being developed in clinical trials. Of them,

multi-targeted kinase inhibitors, such as cabozantinib, crizotinib, and sunitinib are

approved for advanced cancers, while bosutinib is applied for refractory tumors that are

resistant to other RTK inhibitors. Recent clinical studies show that AXL inhibitors are

promising combination partners (25). Of them, ONO-7475 is a novel inhibitor of TAM

receptor tyrosine kinase family that has been shown to inhibit the phosphorylation of

AXL and Mer, and to suppress the growth of acute myeloid leukemia with FLT3

mutations(26,27) and solid tumors (28) (29). A phase I clinical trial of ONO-7475 is

currently underway for acute leukemia and advanced or metastatic solid tumors, although

the results remain unpublished (ClinicalTrials.gov identifier NCT03176277 and

NCT03730337).

In this study, we investigated the best therapeutic strategy to combat

AXL-induced tolerance to EGFR-TKIs using the novel AXL inhibitor ONO-7475 in

combination with osimertinib or dacomitinib in AXL-overexpressing EGFR mutated

NSCLC cells.

Materials and Methods

Cell cultures and reagents

Ten human NSCLC cell lines with mutations in EGFR were utilized. As

previously described (24), HCC4011 and H3255 were generously provided by Dr.

David P. Carbone (Ohio State University Comprehensive Cancer Center, Columbus,

OH) and Dr. John D. Minna (University of Texas Southwestern Medical Center, Dallas,

TX), respectively. The H1975 human lung adenocarcinoma cell line with the

EGFR-L858R/T790M double mutation was kindly provided by Dr. Yoshitaka Sekido

(Aichi Cancer Center Research Institute, Japan) and Dr. John D. Minna. The human cell

lines HCC827 and HCC4006 were purchased from the American Type Culture

Collection (Manassas, VA), and the PC-9 cell line was obtained from RIKEN Cell Bank

(Ibaraki, Japan). The PC-9KGR cells, which contain deletions in EGFR exon 19 and the

T790M mutation, were developed from PC-9 cells by stepwise exposure to gefitinib

with limiting dilution analysis (30). The PC-9GXR cells, which contain deletions in

EGFR exon 19 and the T790M mutation, were established at Kanazawa University

(Kanazawa, Japan) from PC-9 cell xenograft tumors in nude mice that had acquired

resistance to gefitinib. The PC-9AR1 cells and HCC827 OR1 cells, which both contain

deletions in EGFR exon 19, were developed from PC-9 cells or HCC827 cells,

respectively, by stepwise exposure to afatinib or osimertinib, respectively, using the

limiting dilution method. All cell lines were maintained in Roswell Park Memorial

Institute (RPMI) 1640 medium (GIBCO, Carlsbad, CA) supplemented with 10% fetal

bovine serum (FBS), penicillin (100 U/mL), and streptomycin (50 g/mL) in a

humidified CO2 incubator at 37 °C. All cells were passaged for less than three months

before being renewed with frozen, early-passage stocks. Cells were regularly screened

for mycoplasma using a MycoAlert Mycoplasma Detection Kit (Lonza). Cell lines were

authenticated by DNA fingerprinting. Patient-derived xenograft (PDX) from a

44-year-old woman with EGFR L858R-positive lung cancer (TM0199) was purchased

from The Jackson Laboratory (Bar Harbor, ME). Female NOD.Cg-Prkdcscid

Il2rgtm1Wjl/SzJ (NSG) mice aged 6–8 weeks were implanted with tumor fragments at

passage 7 at the Jackson Laboratory. The mice were transferred to the animal facility at

Kanazawa University and the tumors were resected. The study protocol was approved

by the Ethics Committee on the Use of Laboratory Animals and the Advanced Science

Research Center, Kanazawa University, Kanazawa, Japan. Osimertinib and dacomitinib

were obtained from Selleckchem (Houston, TX). ONO-7475 was supplied by Ono

Pharmaceutical Co., Ltd.

Kinase inhibitor assays

The IC50 value of ONO-7475 against recombinant human AXLwas

determined using the Off-chip Mobility Shift Assay (MSA) (Carna Biosciences, Inc.,

Kobe, Japan), according to the manufacturer’s instructions. The ATP concentration in

the assay was set at the Km value of AXL for ATP (32 μM). The IC50 value was

calculated from the concentration vs. % inhibition curve by fitting a four-parameter

logistic curve to the data.

For cell-based AXL inhibition, IC50 value of ONO-7475 was determined using

the ACD cell-based tyrosine kinase assay (Advanced Cellular Dynamics, Inc., San

Diego, US). Recombinant human AXL-expressing mouse Ba/F3 cells, which depend on

AXL kinase activity for survival and proliferation, were exposed to ONO-7475. After

48 h, viable cells were analyzed for ATP concentration using CellTiter-Glo®

Luminescent Cell Viability Assay. IC50 value was determined using non-linear

regression analysis.

Kinase selectivity profiling of ONO-7475 in cell-based kinase assay

Inhibitory activity of 100 nmol/L ONO-7475 against 60 tyrosine kinases was

assessed using the ACD cell-based tyrosine kinase assay (Advanced Cellular Dynamics,

Inc.,San Diego, US). For the 52 kinases showing < 50% inhibition, the ratio of IC50 for

each kinase to IC50 for AXL was considered to be >100. For the remaining 8 kinases

(Mer, TYRO3, TrkB, EphB2, PDGFRA, TRKa, TRKc, and FLT3), further studies were

conducted at various concentrations of ONO-7475, and IC50 values were determined.

The ratio of IC50 for each kinase to IC50 for AXL (0.7 nmol/L) was calculated.

Antibodies and western blotting

Protein aliquots of 25 μg each were resolved by SDS polyacrylamide gel

electrophoresis (Bio-Rad, Hercules, CA), as previously described (24) (31).

Electrophoresed protein samples were transferred to polyvinylidene difluoride

membranes (Bio-Rad). After washing three times, the membranes were incubated with

blotting-grade blocker (Bio-Rad) for 1 h at room temperature and overnight at 4 °C with

primary antibodies to p-AXL (Tyr702), t-AXL, p-EGFR (Tyr1068), p-AKT (Ser473),

t-AKT, p-p70S6K (Thr389), t-p70S6K, cleaved PARP, β-actin (13E5) (1:1,000 dilution;

Cell Signaling Technology, Danvers, MA, USA), p-Erk1/2 (Thr202/Tyr204), t-Erk1/2,

and t-EGFR (1:1,000 dilution, R&D systems).

After washing three times, the membranes were incubated for 1 h at room

temperature with HRP-conjugated species-specific secondary antibody. Immunoreactive

bands were visualized using SuperSignal West Dura Extended Duration Substrate

Enhanced Chemiluminescent Substrate (Pierce Biotechnology). Each experiment was

independently performed at least three times.

Cell viability assay

Cell viability was determined using the MTT dye reduction method, as

previously described (24) (31). Briefly, tumor cells (2–3 × 103 cells/100 μL/well) in

RPMI 1640 medium supplemented with 10% FBS were plated in 96-well plates and

cultured with the indicated compound for 72 h. After culturing, 50 μg of MTT solution

(2 mg/mL, Sigma, St. Louis, MO) was added to each well. Plates were incubated for 2

h, the medium was removed, and the dark blue crystals in each well were dissolved in

100 μL of dimethyl sulfoxide (DMSO). Absorbance was measured with a microplate

reader at a test wavelength of 550 nm and a reference wavelength of 630 nm. The

percentage of growth was determined relative to untreated controls. Experiments were

repeated at least three times with triplicate samples. As another cell viability assay, cells

were treated with DMSO, EGFR-TKI osimertinib or dacomitinib, ONO-7475, or a

combination for 7 and 15 days where the drugs were replenished every 72 h. The plates

were stained with crystal violet and visually examined. A plate representative of three

independent experiments is shown.

Wound healing assay

For the wound healing assay, the PC-9 and HCC4011 cells (1×105 cells) were

seeded and incubated for 24 hours at 37 °C. After achieving confluence, the cellular

layer in each plate was scratched using a plastic pipette tip. The migration of the cells at

the edge of the scratch was analyzed at 0 and 24 hours after treatment with the

EGFR-TKIs osimertinib or dacomitinib, ONO-7475, or a combination, when

microscopic images of the cells were captured.

Transfection of siRNAs

Duplexed Silencer® Select siRNAs against AXL (s1845 and s1846; Invitrogen,

Carlsbad, CA) were transfected into cells using Lipofectamine RNAiMAX (Invitrogen),

according to the manufacturer’s instructions. In all experiments, Silencer® Select

Negative Control no.1 siRNA (Invitrogen) was used as the scrambled control, as

previously described (24). Knockdown of AXL was confirmed by western blotting. Each

sample was tested in triplicate in three independent assays.

Plasmid construction

pWPXL plasmids expressing empty vector (Vector) and human AXL

(AXL-WT) were purchased from Addgene, Inc. (32). X-tremeGene HP DNA

Transfection Reagent (Roche) was used to transfect HCC827 cells with Vector or

AXL-WT plasmid, following the manufacturer's instructions. Whole-cell lysates (25

µg) were analyzed by western blot.

Cytokine production

Cells (2 × 105) were cultured in RPMI‐1640 medium with 10% FBS for 24 h,

washed with PBS, and incubated for 48 h in 2 mL of the same medium. The culture

medium was harvested and centrifuged, and the supernatant stored at −70°C until

analysis, as previously described (24) (31). Level of Gas6 was determined with Human

Gas6 DuoSet ELISA kit (R&D Systems), according to the manufacturer's protocols. All

culture supernatants were tested twice. Color intensity was measured at 450 nm using a

spectrophotometric plate reader. Concentrations of growth factors were determined

from standard curves.

Cell-line-derived xenograft models

Suspensions of 5×106 cells were injected subcutaneously into the flanks of

five-week-old male mice with severe combined immunodeficiency (SCID) obtained from

Clea Japan (Tokyo, Japan), as previously described (24). Once the mean tumor volume

reached approximately 100–200 mm3, five or six mice each were injected with the PC-9

and PC-9KGR cell-line-derived xenografts (CDX) at the time of initial treatment or at the

time of sequential therapy. Drugs were administered seven days a week by oral gavage

and the body weight and general condition of the mice were monitored daily. Tumors

were measured twice weekly using calipers and their volumes were calculated as width2 ×

length/2. Approval was obtained from the institutional review board at University

Hospital, Kyoto Prefectural University of Medicine for a study using mice (approval no.

M29-529). According to institutional guidelines, surgery was performed after the animals

were anesthetized with sodium pentobarbital and efforts were made to minimize animal

suffering.

DNA extraction

Genomic DNA was extracted from frozen samples using a nucleospin tissue kit

(Macherey-Nagel, D€uren, Germany) according to the manufacturer’s instructions.

Concentrations of the extracted DNAs were measured using a Qubit 2.0 fluorometer

(Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNAs were stored at -20

°C until use.

PNA-LNA PCR clamp

The PNA-LNA PCR clamp reaction was carried out using the LightCycler 480 II

(Roche, Pleasanton, CA, USA) as previously reported (33). Quantitative PCR was then

carried out with a 30 second hold at 95 °C followed by 45 cycles at 95 °C for 3 seconds,

and 62 °C for 30 seconds.

Patients

Specimens of EGFR-positive tumors were obtained from 7 lung adenocarcinoma patients

hospitalized at University Hospital, Kyoto Prefectural University of Medicine (Kyoto,

Japan) prior to osimertinib treatment. Re-biopsy specimens of tumors containing

EGFR-activating mutations with the T790M mutation, after acquired resistance to initial

treatments with the EGFR-TKIs gefitinib, erlotinib, afatinib and prior to treatment with

osimertinib, were obtained from 28 lung adenocarcinoma patients hospitalized at the

Kanazawa University Hospital (Kanazawa, Japan), Japanese Red Cross Kyoto Daiichi

Hospital (Kyoto, Japan), Niigata University Hospital (Niigata, Japan), Nagasaki

University Hospital (Nagasaki, Japan), or Japanese Red Cross Nagasaki Genbaku

Hospital (Nagasaki, Japan). The study was conducted according to the Declaration of

Helsinki. All patient participated in the Institutional Review Board of University

Hospital, Kyoto Prefectural University of Medicine (approval no. ERB-C-812 and

ERB-C-1349-2) and each hospital-approved study, and provided written informed

consent.

Histological analyses of tumors

Formalin-fixed, paraffin-embedded tissue sections (4 μm thick) were

deparaffinized. Antigen was retrieved by microwaving the tissue sections in 10 mM

citrate buffer (pH 6.0). Proliferating cells were detected by incubating the tissue sections

with Ki-67 antibody (Clone MIB-1; DAKO Corp, Glostrup, Denmark), as previously

described (24). Cell apoptosis was quantitated using the terminal deoxynucleotidyl

transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method, according to the

manufacturer’s instructions. Based on expression patterns, tumor cells in tissue

specimens were separately evaluated for expression of AXL using an anti-AXL antibody

(1:200; goat polyclonal, R&D SYSTEMS). Since immunohistochemical studies have

shown that AXL is present primarily in the cytoplasm of cells and that its staining varies

in intensity, we quantified its expression as negative (0), weak (1+), moderate (2+), and

strong (3+) compared with vascular endothelial cells as an internal control (34). After

incubation of the specimens with the secondary antibody and treatment using the

Vectastain ABC Kit (Vector Laboratories, Burlingame, CA), peroxidase activity was

visualized using 3,3'-diaminobenzidine (DAB) as a chromogen. The sections were

counterstained with hematoxylin.

Quantification of immunohistochemistry results

The five areas containing the highest numbers of positively stained cells within

each section were selected for histological quantitation using light microscopy at a

400-fold magnification, as previously described (24) (31).

Statistical analysis

Data from the MTT assays and tumor progression of xenografts were expressed

as means ± standard deviation (SD) and as means ± standard error (SE), respectively. The

statistical significance of differences was analyzed using one-way ANOVA and

Spearman rank correlations. Progression-free survival (PFS) and 95% confidence

intervals (CIs) were determined using the Kaplan-Meier method and compared using the

log-rank test. Hazard ratios (HRs) of clinical variables for PFS were determined using a

univariate Cox proportional hazards model. All statistical analyses were performed using

GraphPad Prism Ver. 7.0 (GraphPad Software, Inc., San Diego, CA, USA), with a

two-sided P-value less than 0.05 being considered statistically significant.

Results

The AXL inhibitor ONO-7475 sensitized AXL-overexpressing EGFR-mutant

NSCLC cells to EGFR-TKIs

We first tested ten EGFR-mutated NSCLC cell lines and one PDX tumor, which

were divided into those with high levels of AXL expression and those with low levels of

AXL expression. Phosphorylated EGFR tended to be higher in cells with lower relative

AXL expression levels, as we previously described (24) (Figure 1A). The chemical

structure of ONO-7475 is shown in Figure 1B. IC50 values of ONO-7475 against

recombinant human AXL were 0.414 and 0.7 nmol/L using Off-chip MSA and ACD

cell-based tyrosine kinase assay, respectively (supplementary Figure 1A). Ratio of

IC50 for each kinase to that for AXL (0.7 nmol/L) was calculated (supplementary

Figure 1B).

We examined the potency of the AXL inhibitor ONO-7475 on the sensitivity of

EGFR-mutated NSCLC cells to the EGFR-TKIs osimertinib or dacomitinib. Two

different doses with ONO-7475 for 72 h increased the sensitivity to osimertinib and

dacomitinib and reduced viability of high AXL-expressing PC-9 and HCC4011 cells, but

not of low-AXL-expressing HCC827 cells (Figure 1C, supplementary Figure 2). In

addition, ONO-7475 enhanced osimertinib efficacy on the viability of cell lines PC-9,

PC-9KGR, and HCC4011, and H1975, all of which express high levels of AXL, but had

only a marginal effect on the viability of cell lines HCC827, HCC4006, and H3255,

which express low levels of AXL. Similar results were observed in the treatment with

dacomitinib (Figure 1D). In addition, the continuous co-treatment of EGFR-TKI

osimertinib or dacomitinib and ONO-7475 in PC-9 and HCC4011 cells reduced the cell

viability for 15 days of incubation, but did not affect viability of low-AXL-expressing

HCC827 cells (Figure 1E). To prove further evidence that AXL is an important mediator

of the therapeutic action of osimertinib on EGFR-mutated NSCLC cells, we surmised that

enforced expression of AXL in low-AXL-expressing cells might suppress their

sensitivity to osimertinib. To test this hypothesis, we transfected HCC827 cells with

Vector or AXL-WT. Western blot showed an increase in total AXL protein in the

AXL-WT-expressing cells (supplementary Figure 3A), which reduced their sensitivity

to osimertinib, as expected. Moreover, ONO-7475 in combination with osimertinib

restored viability of AXL-WT-transfected cells (supplementary Figure 3B).

To elucidate the underlying mechanisms of the combined therapy of ONO-7475

and the EGFR-TKIs osimertinib or dacomitinib, we investigated protein expression by

western blot. The combination of osimertinib and ONO-7475 for 4 h markedly inhibited

the phosphorylation of AXL, AKT, and p70S6K compared with treatment of the

high-AXL-expressing cell lines treated with osimertinib alone (Figure 1F). The

combined use of ONO-7475 with osimertinib for 48 h increased cleaved PARP in PC-9

and HCC4011 cells compared with the treatment with osimertinib alone. These findings

were also observed in the dacomitinib treatment. Moreover, the co-treatment of

ONO-7475 with osimertinib on PC-9 and HCC4011 cells inhibited cell migration for 24 h

incubation, compared to osimertinib alone (Supplementary Figure 4).

These results showed that cell sensitivity to the EGFR-TKIs osimertinib or

dacomitinib is enhanced by the combined treatment with an AXL inhibitor ONO-7475,

resulting in reduced viability and migration, and induced apoptosis of

high-AXL-expressing EGFR- mutated NSCLC cell lines, but not with cells expressing

low levels of AXL.

The AXL inhibitor ONO-7475 suppressed the emergence and maintenance of

EGFR-TKI tolerant cells

Drug-tolerant (DT) cells are reported as a small subpopulation of cells with

remarkably reduced sensitivity to targeted drugs. They are generated within several days

to several weeks of exposure to target drugs (35). Thus, we isolated DT cells from PC-9,

HCC4011, PC-9KGR, and H1975 cells after nine days of exposure with high doses of

osimertinib (3 µM) or dacomitinib (1 µM). As expected, these DT cells were resistant to

the EGFR-TKIs osimertinib or dacomitinib, compared with their parental cells (Figure

2A, Supplementary Figure 5A). We next examined the expression level of AXL, and its

ligand Gas6, in both parental cells and DT cells. The DT cells from osimertinib and

dacomitinib expressed high level of total AXL compared with parental cells in

high-AXL-expressing cells, but not low-AXL-expressing cells (Figure 2B,

Supplementary Figure 6). Production of Gas6 could not be detected in culture medium

of either parental cells or osimertinib-DT cells isolated from PC-9 and HCC4011 cells,

indicating that AXL activation in DT cells is independent of ligand stimulation

(Supplementary Table 1). ONO-7475 treatment decreased the viability of DT cells, but

not that of the parental PC-9, HCC4011, PC-9KGR, or H1975 cells (Figure 2C,

Supplementary Figure 5B, 7A). In addition, knockdown of AXL decreased the viability

of DT cells compared to scrambled control (Supplementary Figure 7B, 7C).

Western blot analysis showed that while osimertinib and dacomitinib slightly

inhibited the phosphorylation of EGFR in the DT cells, ONO-7475 suppressed the

phosphorylation of AKT, p70S6K, ERK, as well as EGFR (Figure 2D). Moreover, the

continuous treatment of PC-9DT and HCC4011DT cells with ONO-7475 inhibited the

viability of these DT cells for 7- and 15-days of incubation (Figure 2E, 2F).

These results indicated that activated AXL played a critical role in the

emergence and the maintenance of DT cells by treatment with the EGFR-TKIs

osimertinib or dacomitinib, and that ONO-7475 could suppress the formation of these DT

cells though AKT and ERK signals.

Initial combination therapy of ONO-7475 and osimertinib delayed re-growth of

cell-line derived xenograft tumors

We evaluated the effect of ONO-7475 and osimertinib in a CDX model using

high-AXL-expressing PC-9KGR cells, which has an exon 19 deletion and the

exon21-T790M mutation in EGFR. Mice were continuously administered osimertinib

alone, ONO-7475 alone, or a combination of the two drugs seven days a week by oral

gavage until day 29. Treatment with osimertinib alone caused tumor regression within

one week, but the tumors reappeared within three weeks, indicating that recurrence was

induced by acquired resistance. In contrast, treatment with ONO-7475 alone had little

effect on the tumor growth. The combined initial treatment with osimertinib and

ONO-7475 caused tumor regression compared to osimertinib alone, and the size of

tumors was maintained for 4 weeks (Figure 3A). No apparent adverse events, including

weight loss, were observed during these treatments (Supplementary Figure 8A).

To further elucidate the best therapeutic strategy of ONO-7475 plus osimertinib,

we next evaluated the effect of the combination in a CDX model of PC-9 cells, using two

different treatment schedules, 1) during the initial phase, and 2) during the acquired

resistance phase with osimertinib. For the initial phase studies, mice were treated as

above with PC-9KGR cells (Figure 3B). The results were similar, except that the

combined initial treatment with osimertinib and ONO-7475 caused tumor regression

within one week and the size of the regressed tumors was maintained for 7 weeks. The

number of Ki-67 positive proliferating tumor cells was significantly lower in the

combination-treated tumors than in osimertinib-treated tumors derived from PC-9 cells,

whereas the number of TUNEL-positive tumor cells was not significantly different

between the two (Figure 3C, 3D, supplementary Figure 9A, 9B). In the PC-9

cell-derived tumors, expressions of phosphorylated AKT and p70S6K were inhibited by a

combination of ONO-7475 with osimertinib, compared to osimertinib alone (Figure 3E).

These results indicated that combined treatment with osimertinib and

ONO-7475 during the initial phase prevented the growth of high-AXL-expressing

EGFR-mutated NSCLC cells with or without the EGFR-T790M mutation in vivo. For the

acquired resistance phase studies, the PC-9 tumor-bearing mice were initially treated with

osimertinib alone. The size of tumors gradually increased, despite the continued

osimertinib treatment, indicating that the tumor cells had acquired resistance to

osimertinib. On day 29, the mice were randomly divided into two groups. One group was

continuously treated with osimertinib alone and the other was additionally treated by a

combination with ONO-7475. The tumors treated with osimertinib alone grew

persistently until day 57. Tumor growth when treated with the combination of osimertinib

with ONO-7475 from the acquired resistance phase was slightly slower than those treated

with osimertinib alone until day 47. From day 47 to day 57, tumor growth was similar to

osimertinib alone (Figure 3B). No apparent adverse events, including weight loss, were

observed during these treatments (Supplementary Figure 8B). Overall, the results from

the rapid recurrence model using PC-9 cells indicated that the therapeutic intervention of

combined treatment with osimertinib and ONO-7475 prevents more tumor re-growth at

the initial phase than at the osimertinib-resistant phase, indicating the crucial role of

initial therapeutic intervention for high-AXL-expressing EGFR-mutated NSCLC cells.

PC-9OR1 cells established from acquired in vivo resistance to osimertinib are

insensitive to combined therapy with osimertinib and ONO-7475

We harvested subcutaneous tumors from mice on day 29 after osimertinib

treatment and cultured them in vitro. The expanded tumor cells were named PC-9OR1

(Figure 4A). We first examined the EGFR-mutation status of both PC-9 and PC-9OR1

cells. Both the PC-9 and PC-9OR1 cells possessed deletions in the EGFR exon 19 gene,

however the C797S EGFR-mutation reported as the osimertinib resistance-inducing

mutation, was not detected in both cell lines (Supplemental Table 2). While the growth

of PC-9OR1 cells was slightly faster than that of PC-9 cells, both cell lines grew at a

constant rate in vitro (Figure 4B). To evaluate the role of AXL activation between the

initial and acquired resistance phases of osimertinib treatment, we assessed PC-9OR1

cells compared to PC-9 cells in subsequent experiments. The expression of AXL and

phosphorylation of AKT, but not phosphorylation of EGFR and ERK, increased in

PC-9OR1 cells compared to PC-9 cells by Western blot (Figure 4C). Next, we examined

the drug sensitivity of PC-9OR1 cells in vitro. As expected, PC-9OR1 cells were resistant

to osimertinib compared with PC-9 cells (Figure 4D; IC50 PC-9OR1, 1424 nmol/L;

IC50 PC-9, 2 nmol/L). Importantly, consistent with in vivo experiments, PC-9OR1 cells

were also less sensitive to osimertinib in combination with ONO-7475 than PC-9 cells,

even though PC-9OR1 cells have higher AXL expression than PC-9 cells (Figure 4E).

Western blot analysis showed that osimertinib inhibited phosphorylation of EGFR and

ERK in both cell lines, whereas the EGFR downstream molecule AKT was activated in

PC-9OR1 cells, but not in PC-9 cells. These findings suggest that osimertinib failed to

inhibit AKT phosphorylation in PC-9OR1 cells, resulting in acquired osimertinib

resistance. Moreover, the combination of ONO-7475 and osimertinib did not completely

inhibit AKT phosphorylation in PC-9OR1 cells compared with PC-9 cells (Figure 4F).

To characterize DT cells from the acquired resistance phase, we tested the

growth of PC-9OR1 DT cells selected by osimertinib treatment. PC-9OR1 DT cells were

less sensitive to ONO-7475 compared with the PC-9 DT cells, suggesting that DT cells

from the osimertinib-resistant phase reduced AXL-dependent cell viability (Figure 4G).

These results showed that the survival of osimertinib-acquired resistant cells was

independent of the AXL pathway. Cells could not recover sensitivity to osimertinib by

treatment with a combination of an AXL inhibitor, suggesting the progression of drug

tolerance with clonal evolution during osimertinib treatment.

AXL expression in tumors correlated with sensitivity to osimertinib in the

pre-treatment EGFR-mutant NSCLC patients but not those with the T790M

mutation diagnosed by re-biopsy

To assess the role of AXL expression in osimertinib-treated tumors, we

performed a retrospective study in patients with TKI-naive EGFR-mutant NSCLC (N=7).

Pre-treatment expression of AXL in the cytoplasm of tumor cells was evaluated using

immunohistochemistry (IHC) staining, and scored as high (3+), intermediate (2+), low

(1+), or negative (0). High AXL protein expression was detected in 1 (14.3%),

intermediate in 0 (0%), low in 3 (42.9%), and negative in 3 (42.9%) of the 7 tumors. We

next assessed whether AXL expression in tumors could serve as a negative predictor for

treatment with osimertinib. The response to osimertinib was high (83.3%) and low (0%)

in patients with AXL expression scores of 0 to 1+ and 3+, respectively. Additionally, the

average tumor shrinkage rate relative to baseline was high (53.3%) and low (-19%) in

osimertinib-treated patients with AXL expression scores of 0 to 1+ and 3+, respectively

(Figure 5A, 5B).

To assess the role of AXL expression in tumors after acquired resistance to

EGFR-TKIs with osimertinib treatment, we next performed a retrospective study in

patients with EGFR mutant tumors containing the T790M mutation (N=28). Time to

progression with osimertinib ranged from 0.3 to 28 months. Among 28 tumors obtained

from 28 patients with the EGFR-T790M mutation, high AXL protein expression was

detected in 2 (7.1%), intermediate in 7 (25.0%), low in 19 (67.9%), and negative

expression in 0 (0%). The response rate to osimertinib for the patients with AXL

expression scores of 0 to 1+ was low (62.3%), while for those patients with AXL

expression scores of 2+ to 3+, the response rate to osimertinib was relatively higher

(77.8%) (p = 0.67). Progression-free survival with osimertinib treatment was not

significantly altered in patients with low and high AXL expression (p = 0.111)

(Supplemental Figure 10A, 10B).

These findings show that AXL expression in tumors may be a good predictor of

osimertinib sensitivity, although the sample size was small. In contrast, after acquired

resistance to initial EGFR-TKIs, AXL expression in tumors may not be a good predictor

for the acquired resistance to osimertinib. It appears to be difficult to select promising

populations for combined therapy with osimertinib and an AXL inhibitor in EGFR

mutated tumors obtained from the acquired resistance phase.

Discussion

Acquired resistance to 1st and 2nd generation EGFR-TKIs is caused by various

mechanisms, such as gatekeeper mutations like the EGFR-T790M second site mutation,

activation of an alternate pathway, activation of EGFR downstream signals,

transformation to small cell lung cancer, and the EMT (36). Several clinical trials

targeting the bypass signals related to drug resistance have been conducted in EGFR

mutated NSCLC patients with acquired resistance to initial EGFR-TKIs. Of these, several

clinical trials with an AXL inhibitor have been ongoing for EGFR-mutated NSCLC

patients at the acquired resistance setting (37) (38). However, previous clinical studies

demonstrated that the outcomes are limited for NSCLC patients with acquired resistance

to EGFR-TKIs (39) (40) (41). These observations suggest that maintenance of residual

tumor cells during EGFR-TKI treatment may be accelerated by various molecular

mechanisms, such as minor subpopulations with a resistance mechanism, the tumor

microenvironment, and the reversible drug-tolerant state (42), which ultimately leads to

tumor heterogeneity and drug resistance.

Currently, DT cells are thought to play a pivotal role in the progression of

tumor heterogeneity because these cells are considered to eventually enhance tumor

recurrence (43). However, it is not completely understood how to prevent the

development of drug tolerant cells and tumor heterogeneity, which is related to

epigenetic changes in treated cells (44). Previous studies demonstrated that a small

subpopulation of reversibly DT cells maintain viability through the activation of the

IGF-1 receptor, aurora kinase A, and GPX4 (35) (45) (46). However, a clinical study

using the combination of an IGF1-receptor inhibitor plus the EGFR-TKI erlotinib in

unselected patients with lung cancer failed to demonstrate clinical benefits (47), although

pre-clinical studies showed that the combination reduced these DT cells in EGFR

mutated NSCLC (35). These pre-clinical and clinical observations suggest that a

predictive biomarker for the emergence of DT cells needs to be developed for the clinical

setting.

We previously reported that addition of an AXL inhibitor NPS1034 during the

initial phase of osimertinib treatment reduced tumor growth in AXL-expressing

EGFR-mutated NSCLC cells. In addition, in analysis of clinical specimens, high AXL

expression in the pre-treatment of EGFR tumors correlates with a poor response to initial

EGFR-TKIs, including osimertinib, suggesting the high AXL expression in tumors may

be a promising negative biomarker to detect the response to EGFR-TKIs (24). However,

in cases of refractory EGFR-mutated tumors to initial EGFR-TKIs, a significant

correlation was not observed between those with AXL expression and clinical outcomes

with osimertinib. Additionally, CDX mice models and cell line-based analysis showed

that the combination of ONO-7475 and osimertinib is more effective at intervention of

the initial phase than at the osimertinib-acquired resistance phase in

high-AXL-expressing EGFR mutated NSCLC cells. These results indicate that in high

AXL-expressing tumors, intervention with initial EGFR-TKIs may not be sufficient to

combat the resistance to initial EGFR-TKIs by inhibition of the AXL-dependent

pathway. Interestingly, PC-9OR1 expressed more AXL proteins compared to the

pre-treatment PC-9 and their DT cells, whereas the DT cells from PC-9OR1 showed less

sensitivity to ONO-7475 than that of PC-9 DT cells, indicating that the intervention of

EGFR-TKIs expanded the diversity of drug tolerant mechanisms in addition to AXL

activation and led to insensitivity to an AXL inhibitor. In contrast, our results showed that

EGFR mutated NSCLC cells harboring the T790M mutation are also sensitive to

combined therapy of ONO-7475 and osimertinib at the initial therapeutic intervention,

suggesting that the combination of ONO-7475 and osimertinib is effective for

EGFR-T790M mutated NSCLC cells, without the progression of tumor heterogeneity.

Collectively, our findings support the idea that therapeutic intervention with initial

EGFR-TKI leads to clonal evolution and facilitates tumor heterogeneity in EGFR

mutated NSCLC cells. Therefore, an AXL-targeted therapy to conquer drug tolerant

mechanisms at the initial phase might be promising to reduce the emergence of tumor

heterogeneity compared with those at the resistance phase of initial treatment in

AXL-overexpressing EGFR mutated NSCLC cells.

In summary, a novel AXL inhibitor ONO-7475 suppresses the emergence and

maintenance of cells tolerant to the initial EGFR-TKI osimertinib or dacomitinib in

AXL-overexpressing EGFR-mutated NSCLC cells, suggesting that the combination of

ONO-7475 and osimertinib is highly potent for the initial treatment phase. Further

clinical investigations are warranted for the development of novel strategies using an

AXL inhibitor at the initial therapeutic setting to overcome the tolerance to the

EGFR-TKIs osimertinib and dacomitinib in AXL-overexpressing EGFR mutated

NSCLC patients.

Acknowledgments

We appreciate the gifts of the cell lines HCC4011 and H3255, provided by Dr. David P.

Carbone (The Ohio State University Comprehensive Cancer Center, Columbus, OH)

and Dr. John D. Minna (University of Texas Southwestern Medical Center), and the

H1975 cells kindly provided by Dr. Yoshitaka Sekido (Aichi Cancer Center Research

Institute, Japan) and Dr. John D. Minna (University of Texas Southwestern Medical

Center).

Reference

1. Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. The New England journal of medicine 2010;362(25):2380-8 doi 10.1056/NEJMoa0909530. 2. Zhou C, Wu YL, Chen G, Feng J, Liu XQ, Wang C, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. The Lancet Oncology 2011;12(8):735-42 doi 10.1016/s1470-2045(11)70184-x. 3. Sequist LV, Yang JC, Yamamoto N, O'Byrne K, Hirsh V, Mok T, et al. Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. Journal of clinical oncology : official

journal of the American Society of Clinical Oncology 2013;31(27):3327-34 doi 10.1200/jco.2012.44.2806. 4. Wu YL, Cheng Y, Zhou X, Lee KH, Nakagawa K, Niho S, et al. Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial. The Lancet Oncology 2017;18(11):1454-66 doi 10.1016/s1470-2045(17)30608-3. 5. Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB, Fidias P, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Science translational medicine 2011;3(75):75ra26 doi 10.1126/scitranslmed.3002003. 6. Goss G, Tsai CM, Shepherd FA, Bazhenova L, Lee JS, Chang GC, et al. Osimertinib for pretreated EGFR Thr790Met-positive advanced non-small-cell lung cancer (AURA2): a multicentre, open-label, single-arm, phase 2 study. The Lancet Oncology 2016;17(12):1643-52 doi 10.1016/s1470-2045(16)30508-3. 7. Mok TS, Wu YL, Ahn MJ, Garassino MC, Kim HR, Ramalingam SS, et al. Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer. The New England journal of medicine 2017;376(7):629-40 doi 10.1056/NEJMoa1612674. 8. Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee KH, et al. Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer. The New England journal of medicine 2018;378(2):113-25 doi 10.1056/NEJMoa1713137. 9. Mok TS, Cheng Y, Zhou X, Lee KH, Nakagawa K, Niho S, et al. Improvement in Overall Survival in a Randomized Study That Compared Dacomitinib With Gefitinib in Patients With Advanced Non–Small-Cell Lung Cancer and EGFR-Activating Mutations. Journal of Clinical Oncology 2018;36(22):2244-50 doi 10.1200/jco.2018.78.7994. 10. Stitt TN, Conn G, Gore M, Lai C, Bruno J, Radziejewski C, et al. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995;80(4):661-70. 11. Koorstra JB, Karikari CA, Feldmann G, Bisht S, Rojas PL, Offerhaus GJ, et al. The Axl receptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target. Cancer biology & therapy 2009;8(7):618-26. 12. Neubauer A, Fiebeler A, Graham DK, O'Bryan JP, Schmidt CA, Barckow P, et al. Expression of axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis. Blood 1994;84(6):1931-41.

13. Varnum BC, Young C, Elliott G, Garcia A, Bartley TD, Fridell YW, et al. Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6. Nature 1995;373(6515):623-6 doi 10.1038/373623a0. 14. Okimoto RA, Bivona TG. AXL receptor tyrosine kinase as a therapeutic target in NSCLC. Lung Cancer (Auckland, NZ) 2015;6:27-34 doi 10.2147/lctt.S60438. 15. Graham DK, DeRyckere D, Davies KD, Earp HS. The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer. Nature reviews Cancer 2014;14(12):769-85 doi 10.1038/nrc3847. 16. Gjerdrum C, Tiron C, Hoiby T, Stefansson I, Haugen H, Sandal T, et al. Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. Proceedings of the National Academy of Sciences of the United States of America 2010;107(3):1124-9 doi 10.1073/pnas.0909333107. 17. Wu CW, Li AF, Chi CW, Lai CH, Huang CL, Lo SS, et al. Clinical significance of AXL kinase family in gastric cancer. Anticancer research 2002;22(2b):1071-8. 18. Rankin EB, Fuh KC, Taylor TE, Krieg AJ, Musser M, Yuan J, et al. AXL is an essential factor and therapeutic target for metastatic ovarian cancer. Cancer research 2010;70(19):7570-9 doi 10.1158/0008-5472.Can-10-1267. 19. Holland SJ, Powell MJ, Franci C, Chan EW, Friera AM, Atchison RE, et al. Multiple roles for the receptor tyrosine kinase axl in tumor formation. Cancer research 2005;65(20):9294-303 doi 10.1158/0008-5472.Can-05-0993. 20. Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, et al. Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nature genetics 2012;44(8):852-60 doi 10.1038/ng.2330. 21. Namba K, Shien K, Takahashi Y, Torigoe H, Sato H, Yoshioka T, et al. Activation of AXL as a Preclinical Acquired Resistance Mechanism Against Osimertinib Treatment in EGFR-Mutant Non-Small Cell Lung Cancer Cells. Molecular cancer research : MCR 2019;17(2):499-507 doi 10.1158/1541-7786.Mcr-18-0628. 22. Kim D, Bach DH, Fan YH, Luu TT, Hong JY, Park HJ, et al. AXL degradation in combination with EGFR-TKI can delay and overcome acquired resistance in human non-small cell lung cancer cells. Cell Death Dis 2019;10(5):361 doi 10.1038/s41419-019-1601-6. 23. Boshuizen J, Koopman LA, Krijgsman O, Shahrabi A, van den Heuvel EG, Ligtenberg MA, et al. Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nature medicine 2018;24(2):203-12 doi 10.1038/nm.4472.

24. Taniguchi H, Yamada T, Wang R, Tanimura K, Adachi Y, Nishiyama A, et al. AXL confers intrinsic resistance to osimertinib and advances the emergence of tolerant cells. Nature communications 2019;10(1):259 doi 10.1038/s41467-018-08074-0. 25. Shen Y, Chen X, He J, Liao D, Zu X. Axl inhibitors as novel cancer therapeutic agents. Life sciences 2018;198:99-111 doi 10.1016/j.lfs.2018.02.033. 26. Yasuhiro T, Yoshizawa T, Fujikawa R, Tanaka K, Koike T, Kawabata K. Development of an Axl/Mer Dual Inhibitor, ONO-9330547: Promising Single Agent Activity in an Acute Myeloid Leukemia (AML) Model. Blood 2014;124(21):999-. 27. Ruvolo PP, Ma H, Ruvolo VR, Zhang X, Mu H, Schober W, et al. Anexelekto/MER tyrosine kinase inhibitor ONO-7475 arrests growth and kills FMS-like tyrosine kinase 3-internal tandem duplication mutant acute myeloid leukemia cells by diverse mechanisms. Haematologica 2017;102(12):2048-57 doi 10.3324/haematol.2017.168856. 28. Tanaka K, Yoshizawa T, Yasuhiro T, Fujikawa R, Koike T, Kawabata K. Abstract LB-259: The potent and selective Axl/Mer dual inhibitor ONO-9330547, shows promising single agent activity in non-small cell lung cancer (NSCLC). Cancer research 2015;75(15 Supplement):LB-259-LB- doi 10.1158/1538-7445.Am2015-lb-259. 29. Yoshizawa T, Tanaka K, Yasuhiro T, Fujikawa R, Ri S, Kawabata K. Abstract LB-218: Development of Axl/Mer inhibitor, ONO-9330547: preclinical evidence supporting the combination with immunotherapeutics. Cancer research 2016;76(14 Supplement):LB-218-LB- doi 10.1158/1538-7445.Am2016-lb-218. 30. Nanjo S, Yamada T, Nishihara H, Takeuchi S, Sano T, Nakagawa T, et al. Ability of the Met kinase inhibitor crizotinib and new generation EGFR inhibitors to overcome resistance to EGFR inhibitors. PloS one 2013;8(12):e84700 doi 10.1371/journal.pone.0084700. 31. Taniguchi H, Takeuchi S, Fukuda K, Nakagawa T, Arai S, Nanjo S, et al. Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors. Cancer science 2017;108(1):53-60 doi 10.1111/cas.13111. 32. Meyer AS, Zweemer AJ, Lauffenburger DA. The AXL Receptor is a Sensor of Ligand Spatial Heterogeneity. Cell systems 2015;1(1):25-36 doi 10.1016/j.cels.2015.06.002. 33. Tanzima Nuhat S, Sakata-Yanagimoto M, Komori D, Hattori K, Suehara Y, Fukumoto K, et al. Droplet digital polymerase chain reaction assay and peptide nucleic acid-locked nucleic acid clamp method for RHOA mutation detection in

angioimmunoblastic T-cell lymphoma. Cancer science 2018;109(5):1682-9 doi 10.1111/cas.13557. 34. Sato K, Suda K, Shimizu S, Sakai K, Mizuuchi H, Tomizawa K, et al. Clinical, Pathological, and Molecular Features of Lung Adenocarcinomas with AXL Expression. PloS one 2016;11(4):e0154186 doi 10.1371/journal.pone.0154186. 35. Sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, Maheswaran S, et al. A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 2010;141(1):69-80 doi 10.1016/j.cell.2010.02.027. 36. Takeuchi S, Yano S. Clinical significance of epidermal growth factor receptor tyrosine kinase inhibitors: sensitivity and resistance. Respiratory investigation 2014;52(6):348-56 doi 10.1016/j.resinv.2014.10.002. 37. Reckamp KL, Frankel PH, Ruel N, Mack PC, Gitlitz BJ, Li T, et al. Phase II Trial of Cabozantinib Plus Erlotinib in Patients With Advanced Epidermal Growth Factor Receptor (EGFR)-Mutant Non-small Cell Lung Cancer With Progressive Disease on Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Therapy: A California Cancer Consortium Phase II Trial (NCI 9303). Frontiers in oncology 2019;9:132 doi 10.3389/fonc.2019.00132. 38. Levin PA, Brekken RA, Byers LA, Heymach JV, Gerber DE. Axl Receptor Axis: A New Therapeutic Target in Lung Cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2016;11(8):1357-62 doi 10.1016/j.jtho.2016.04.015. 39. Johnson ML, Riely GJ, Rizvi NA, Azzoli CG, Kris MG, Sima CS, et al. Phase II trial of dasatinib for patients with acquired resistance to treatment with the epidermal growth factor receptor tyrosine kinase inhibitors erlotinib or gefitinib. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2011;6(6):1128-31 doi 10.1097/JTO.0b013e3182161508. 40. Azuma K, Hirashima T, Yamamoto N, Okamoto I, Takahashi T, Nishio M, et al. Phase II study of erlotinib plus tivantinib (ARQ 197) in patients with locally advanced or metastatic EGFR mutation-positive non-small-cell lung cancer just after progression on EGFR-TKI, gefitinib or erlotinib. ESMO open 2016;1(4):e000063 doi 10.1136/esmoopen-2016-000063. 41. Wu YL, Zhang L, Kim DW, Liu X, Lee DH, Yang JC, et al. Phase Ib/II Study of Capmatinib (INC280) Plus Gefitinib After Failure of Epidermal Growth Factor Receptor (EGFR) Inhibitor Therapy in Patients With EGFR-Mutated, MET Factor-Dysregulated Non-Small-Cell Lung Cancer. Journal of clinical oncology : official journal of

the American Society of Clinical Oncology 2018;36(31):3101-9 doi 10.1200/jco.2018.77.7326. 42. Suda K, Bunn PA, Jr., Rivard CJ, Mitsudomi T, Hirsch FR. Primary Double-Strike Therapy for Cancers to Overcome EGFR Kinase Inhibitor Resistance: Proposal from the Bench. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2017;12(1):27-35 doi 10.1016/j.jtho.2016.09.003. 43. Hammerlindl H, Schaider H. Tumor cell-intrinsic phenotypic plasticity facilitates adaptive cellular reprogramming driving acquired drug resistance. Journal of cell communication and signaling 2018;12(1):133-41 doi 10.1007/s12079-017-0435-1. 44. Vallette FM, Olivier C, Lezot F, Oliver L, Cochonneau D, Lalier L, et al. Dormant, quiescent, tolerant and persister cells: Four synonyms for the same target in cancer. Biochemical pharmacology 2019;162:169-76 doi 10.1016/j.bcp.2018.11.004. 45. Shah KN, Bhatt R, Rotow J, Rohrberg J, Olivas V, Wang VE, et al. Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer. Nature medicine 2019;25(1):111-8 doi 10.1038/s41591-018-0264-7. 46. Hangauer MJ, Viswanathan VS, Ryan MJ, Bole D, Eaton JK, Matov A, et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 2017;551(7679):247-50 doi 10.1038/nature24297. 47. Moran T, Felip E, Keedy V, Borghaei H, Shepherd FA, Insa A, et al. Activity of dalotuzumab, a selective anti-IGF1R antibody, in combination with erlotinib in unselected patients with Non-small-cell lung cancer: a phase I/II randomized trial. Experimental hematology & oncology 2014;3(1):26 doi 10.1186/2162-3619-3-26.

Figure legends

Figure 1. The AXL inhibitor ONO-7475 sensitized AXL-expressing EGFR-mutant

NSCLC cells to the EGFR-TKIs osimertinib and dacomitinib.

(A) The EGFR-mutated NSCLC cell lines PC-9, PC-9KGR, PC-9GXR, PC-9AR1,

HCC4011, H1975, HCC827, HCC827OR1, HCC4006, H3255, and PDX tumor cells

(TM0199), were lysed and the indicated proteins were detected by western blot. (B)

Structure of ONO-7475. (C) PC-9 and HCC4011 cells were incubated with osimertinib

or dacomitinib in the presence or absence of the AXL inhibitor ONO-7475 (0.1, 1

μmol/L) for 72 h and then cell viability was determined using MTT assays. Data are

representative of three independent experiments that produced similar results. (D)

Quantitative determination of the inhibition of cell viability of high-AXL-expressing and

low-AXL-expressing EGFR-mutant cells treated with the EGFR-TKIs osimertinib or

dacomitinib in the presence or absence of ONO-7475. Paired Student’s t tests were used

for comparisons. (E) Cells were treated with DMSO, 300 nmol/L osimertinib or 10

nmol/L dacomitinib, 1 μmol/L ONO-7475, or a combination of 300 nmol/L osimertinib

or 10 nmol/L dacomitinib, and 1 μmol/L ONO7475 for 15 days where the drugs were

replenished every 72 h. The plates were stained with crystal violet and visually examined.

A plate representative of three independent experiments is shown. (F) The indicated cells

were incubated with osimertinib (100 nmol/L) or dacomitinib (100 nmol/L) with or

without ONO-7475 (1 μmol/L) for 4 h or 48 h. The cells were lysed and the indicated

proteins were detected by western blot.

Figure 2. The AXL inhibitor ONO-7475 suppressed the emergence and

maintenance of tolerant cells resulting from treatment of EGFR-TKIs.

(A) Drug-tolerant (DT) cells previously treated with 3 μmol/L osimertinib or 1 μmol/L

dacomitinib for 9 days were treated with the indicated concentrations of osimertinib or

dacomitinib for 72 h and their viability was assessed using MTT assays. (B) PC-9

parental cells, and DT cells (generated by treatment of parental cells with 3 μmol/L

osimertinib or 1 μmol/L dacomitinib for 9 days) were lysed and the indicated proteins

were detected by western blotting. (C) PC-9, HCC4011 parental cells, and each DT cells

generated by treatment with 3 μmol/L osimertinib (top panel) or 1 μmol/L dacomitinib

(bottom panel) were treated with the EGFR-TKI osimertinib or dacomitinib, 1 μmol/L of

ONO-7475, or a combination of these agents for 72 h and the cell viability was assessed

using MTT assays. (D) The indicated DT cells from PC-9 were incubated with

osimertinib (100 nmol/L) or dacomitinib (100 nmol/L) with or without ONO-7475 (1

μmol/L) for 4 h. The cells were lysed and the indicated proteins were detected by western

blot. (E) PC-9 DT cells were generated for 9 days of treatment with 3 μmol/L of

osimertinib (top panel) or 1 μmol/L of dacomitinib (bottom panel) exposed to either

EGFR TKI alone (osimertinib 300 nmol/L or dacomitinib 10 nmol/L) or with the addition

of ONO-7475 (1 μmol/L) for the indicated times. Paired Student’s t tests were used for

comparisons. (F) PC-9 DT and HCC4011 DT cells were treated with DMSO, 300 nmol/L

osimertinib or 10 nmol/L dacomitinib, 1 μmol/L ONO-7475, or a combination of 300

nmol/L osimertinib or 10 nmol/L dacomitinib and 1 μmol/L ONO-7475 for 15 days

where the drugs were replenished every 72 h. The plates were stained with crystal violet

and visually examined. A plate representative of three independent experiments is shown.

Figure 3. Initial combination therapy of ONO-7475 and osimertinib delayed

re-growth of cell-line derived xenograft tumors.

(A) PC-9KGR cell-line-derived xenograft (CDX) tumors were treated with vehicle

(control), ONO-7475 10 mg/kg, osimertinib 5mg/kg, or ONO-7475 10 mg/kg plus

osimertinib 5mg/kg (n = 6 per group). Tumor volumes were measured over time from the

start of treatment and the results are shown (mean ± SEM). *, p < 0.05. (B) PC-9 CDX

tumors were treated with vehicle (control), ONO-7475 10 mg/kg, osimertinib 5mg/kg, or

ONO-7475 10 mg/kg plus osimertinib 5mg/kg (n = 6 per group). In the osimertinib

treatment group, tumors on day 29 were randomized, and treated with continuous

administration osimertinib 5mg/kg or ONO-7475 10 mg/kg plus osimertinib 5mg/kg until

day 57 (n = 5 per group). The results of tumor volume are plotted (mean ± SEM). *, p <

0.001. (C) Quantification of proliferating cells, as determined by their Ki-67-positive

proliferation index (percentage of Ki-67-positive cells) as described in the Methods.

Columns, mean of five evaluated areas; bars, SD. Comparisons by paired Student’s t

tests. *, p < 0.05. (D) Representative images of PC-9 xenografts containing the following

immunohistochemical staining with antibodies specific for human Ki-67. Bar, 100 μm.

Figure 4. Osimertinib acquired resistant cells are insensitive to combined therapy

with osimertinib and ONO-7475.

(A) PC-9 parental and the osimertinib acquired resistant PC-9OR1 cells were evaluated

using a light microscope. Bar, 200 μm. (B) Cells were incubated for the indicated times

and their viability assessed using MTT assays. (C) Protein expression was analyzed by

Western blot with the indicated antibodies. (D) Growth inhibition was assessed by MTT

assay of PC-9 and PC-9OR1 cells treated with the indicated concentrations of osimertinib

for 72h. (E) Growth inhibition assessed by MTT assay of PC-9 and PC-9OR1 cells

treated with 300 nmol/L osimertinib or 1 μmol/L of ONO-7475, or a combination of these

agents for 72 h. *, p < 0.01. (F) The indicated cells were incubated with osimertinib (100

nmol/L) or dacomitinib (100 nmol/L) with or without ONO-7475 (1 μmol/L) for 4 h. The

cells were lysed and the indicated proteins were detected by western blot. (G) The

indicated cells were treated with DMSO, 300 nmol/L osimertinib, 1 μmol/L ONO-7475,

or a combination of 300 nmol/L osimertinib and 1 μmol/L ONO-7475 for 15 days where

the drugs were replenished every 72 h. The plates were stained with crystal violet and

visually examined. A plate representative of three independent experiments is shown.

Figure 5. The correlation between osimertinib sensitivity and AXL expression in

pre-treatment tumors derived from EGFR-mutated NSCLC patients.

(A) Waterfall plot of EGFR-mutated NSCLC patients treated with osimertinib. (B)

Correlation between cytoplasmic AXL protein expression levels (determined

immunohistochemically) and the response to osimertinib treatment, including the average

tumor shrinkage rate relative to baseline in seven patients with EGFR-mutated NSCLC

treated with osimertinib.

A High AXL Low AXL E Medium Osimertinib Dacomitinib

Osimertinib Dacomitinib

ONO7475 + +

AR1

ONO7475 ONO7475 9 9 KGR 9 GXR 9 - - - - HCC827 HCC827OR1 H3255 HCC4006 H1975 TM0199 AuthorPC ManuscriptPC PC PublishedPC HCC4011 OnlineFirst on January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2321 Author manuscripts have been peer reviewed and accepted for publication but have not yet beenPC edited.-9 HCC4011 AXL pEGFR EGFR βactin HCC827

F C PC-9 HCC4011 ONO7475 - - + + - - + + PC-9 Medium

PC-9 120 120 ONO 0.1 μM Osimertinib - + - + - + - + 100 100 ONO 1 μM 80 80 pAXL

60 viability 60

40 40 AXL 20 20 pEGFR %cell %cell viability 0 %cell 0 EGFR

pERK h 4 Osimertinib(μM) Dacomitinib(nM) ERK HCC4011

HCC4011 120 120 pAKT 100 100 AKT 80 80 60 60 pp70S6K 40 40 20 p70S6K 20 h 48 %cell %cell viability %cell %cell viability 0 0 cPARP βactin Osimertinib(μM) Dacomitinib(nM) PC-9 HCC4011 D high AXL low AXL ONO7475 - - + + - - + +

100 p<0.005 100 p=0.127 Dacomitinib - + - + - + - +

PC-9 HCC827 80 PC-9KGR 80 H3255 HCC4011 pAXL 60 60 HCC4006 H1975 AXL 40 40 pEGFR

%cell viability 20

%cell viability 20 EGFR 0 0 Medium ONO 1μM Medium pERK h 4 +Osimertinib 100nM +Osimertinib 100nM ERK p<0.005 p=0.200 100 100 pAKT

80 80 AKT 60 60 pp70S6K 40 40 p70S6K 48 h 48 %cell viability %cell viability 20 20 cPARP 0 0 βactin Medium ONO 1μM Medium ONO 1μM +DacomitinibDownloaded 100nM from clincancerres.aacrjournals.org+Dacomitinib on 100nMSeptember 24, 2021. © 2020 American Association for Cancer Research. Fig.2 A Osimertinib or Dacomitinib B

Parent 9days Drug-tolerant(DT) cells

PC-9 PC-9 AXL Author Manuscript Published OnlineFirst on January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2321 Author manuscripts have been peer reviewed andPC-9 accepted DT for publication but have not yet been edited.

120 120 βactin 100 100 80 80 60 60 40 40 C PC-9 HCC4011 20 20 %cell %cell viability %cell %cell viability 0 0 Control Osimertinib ONO COMB

Osimertinib(μM) Dacomitinib(nM) 120 120

100 100 HCC4011 HCC4011 80 80 HCC4011 DT 60 60

120 120 40 40 100 100

20 %cell viability 20 80 80 %cell viability

viability 60 60 0 0

40 40 PC-9 PC-9 DT HCC4011 HCC4011 DT 20 20 %cell %cell viability %cell 0 0 Control Dacomitinib ONO COMB Osimertinib(μM) Dacomitinib(nM) 120 120

D 100 100 DT by Osimertinib DT by Dacomitinib 80 80 60 60 - - + + - - + + ONO7475 40 40

%cell %cell viability 20 EGFR-TKI - + - + - + - + %cell viability 20 0 0 pEGFR HCC4011 HCC4011 DT PC-9 PC-9 DT EGFR pERK E ERK pAKT Osimertinib Osimertinib/ONO AKT 700 pp70S6K 600 p70S6K 500 400 βactin p<0.001 300 200 %cell %cell viability F 100 PC-9 HCC4011 0 1 3 5 7 Days Medium ONO7475 Dacomitinib Dacomitinib/ONO Osimertinib Osimertinib + 700 ONO7475 600 500 400 Medium ONO7475 300 p<0.05 200 %cell %cell viability Dacomitinib Dacomitinib 100 + 0 ONO7475 1 3 5 7 Days Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Fig.3

A Control ONO Osimertinib Combined 1200

)

3 1000

Author Manuscript Published OnlineFirst on January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2321 Author manuscripts800 have been peer reviewed and accepted for publication but have not yet been edited. 600

400 * * * *

Tumor volume (mm volume Tumor 200

0 1 4 8 11 15 18 22 25 29 Days

B Control ONO Osimertinib Combined Osimertonib→OsimertinibOsimertinib Osimertinib→Combined Combined Osimertinib→Osimertinib Treatment Osimertinib→Combined 1200

) 3 1000

800 N.S. Randomization 600 * 400

Tumor volume (mm volume Tumor 200

0 1 5 8 12 15 19 22 26 29 33 36 40 43 47 50 54 57 Days

C D 60 HE Ki67 50 40 * 30 Control 20 100μm 100μm 10 0

Osimertinib Ki67 index (% of positive cells)

100μm 100μm

E ONO7475 - - + + ONO7475 Osimertinib - + - +

A B PC-9 PC-9OR1 PC-9 PC-9OR1 1200 Author Manuscript Published OnlineFirst on January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2321 1000 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 800 N.S. 600 400

%cell %cell growth 200 50μm 50μm 0 1 2 3 4 5 Days C D E

PC-9 PC-9OR1 PC-9 PC-9OR1 AXL 140 pEGFR 140 120 120 * EGFR 100 100 pERK 80 80 * ERK 60 60 pAKT 40 40 %cell viability 20

%cell viability 20 AKT 0 0 βactin

Osimertinib(μM)

F PC-9 PC-9OR1 G Medium ONO7475 ONO7475 - - + + - - + +

Osimertinib - + - + - + - + 9DT pEGFR - EGFR PC

pERK ERK pAKT

AKT 9OR1DT - βactin PC

Author Manuscript2 Published0 OnlineFirst on January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2321 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

) 0

(

t a

r -30

e s

n -50

p s

e AXL 0

R AXL 1+ AXL 3+ -100

Cases

The average tumor AXL expression No. of case No. of responder shrinkage rate relative to baseline (Range) 0 3 2 (66.7%) 52.3 (15.5-80.6) 1+ 3 3 (100%) 54.2 (41.5-64.9) 2+ 0 0 (0%) NE 3+ 1 0 (0%) -19 (-19) Total 7 5 (71.4%) 43.0 (-19-80.6)

ONO-7475, a novel AXL inhibitor, suppresses the adaptive resistance to initial EGFR-TKI treatment in EGFR-mutated non-small lung cancer

Naoko Okura, Naoya Nishioka, Tadaaki Yamada, et al.

Clin Cancer Res Published OnlineFirst January 17, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-2321

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/01/17/1078-0432.CCR-19-2321.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2020/01/17/1078-0432.CCR-19-2321. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.