Preclinical Modeling of KIF5B-RET Fusion Lung Adenocarcinoma

Qingling Huang1, Valentina E. Schneeberger1, Noreen Luetteke2, Chengliu Jin3, Roha Afzal2,

Mikalai M. Budzevich2, Rikesh J. Makanji4, Gary V. Martinez2,5, Tao Shen6, Lichao Zhao7, Kar-

Ming Fung6,7, Eric B. Haura8,9, Domenico Coppola9,10, and Jie Wu1,6,7,9,*

1
Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute,
Tampa, Florida. 2Small Animal Modeling and Imaging Core, H. Lee Moffitt Cancer Center and
Research Institute, Tampa, Florida. 3Transgenic and Gene Targeting Core, Georgia State
University, Atlanta, Georgia. 4Department of Radiology, H. Lee Moffitt Cancer Center and
Research Institute, Tampa, Florida. 5Department of Cancer Imaging and Metabolism, H. Lee
Moffitt Cancer Center and Research Institute, Tampa, Florida. 6Peggy and Charles Stephenson
Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.
7
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City,
Oklahoma. 8Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research
Institute, Tampa, Florida. 9Department of Oncology Sciences, University of South Florida
College of Medicine, Tampa, Florida. 10Department of Anatomic Pathology, H. Lee Moffitt
Cancer Center and Research Institute, Tampa, Florida

*Corresponding Author:

Jie Wu, Ph.D.

Peggy and Charles Stephenson Cancer Center
University of Oklahoma Health Sciences Center
975 N.E. 10th Street, BRC-411B
Oklahoma City, OK 73104
Telephone: (405) 271-8001, ext. 31092
Fax: (405) 271-2141
Email: jie-wu@ouhsc.edu
Running title:

Targeting KIF5B-RET Fusion in Lung Cancer

Key words:

KIF5B-RET, fusion oncogene, lung adenocarcinoma, transgenic mice, protein tyrosine kinase

inhibitor, drug resistance, mutation, ponatinib

Abbreviations list:

AOI, area of interest; B/KR, BaF3/KIF5B-RET; CCSP, Clara cell secretory protein; C/KR,

CCSP-rtTA/tetO-KIF5B-RET; CT, computerized tomography; Dox, doxycycline; GOS, gain-of-

sensitivity; IHC, immunohistochemistry; IL-3, interleukin-3; MRI, magnetic resonance imaging;

NSCLC, non-small cell lung cancer; PTK, protein tyrosine kinase; TCGA, the Cancer Genome

Atlas; TKI, protein tyrosine kinase inhibitor.

Financial information

This study was supported by National Institutes of Health grants R21CA175603 (to J. Wu),

R01CA178456 (to J. Wu), P30CA076292 (to T. Sellers), P50CA119997 (to E. B. Haura), and

P20GM103639 (to K.M. Fung).

2
Abstract

RET fusions have been found in lung adenocarcinoma, of which KIF5B-RET is the most

prevalent. We established inducible KIF5B-RET transgenic mice and KIF5B-RET-dependent

cell lines for preclinical modeling of KIF5B-RET-associated lung adenocarcinoma. Dox-induced

CCSP-rtTA/tetO-KIF5B-RET transgenic mice developed invasive lung adenocarcinoma with

desmoplastic reaction. Tumors regressed upon suppression of KIF5B-RET expression. By

culturing KIF5B-RET-dependent BaF3 (B/KR) cells with increasing concentrations of

cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and

vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and

vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B-RET and its

drug-resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RETG810A mutant displayed

gain-of-sensitivity (GOS) to ponatinib and lenvatinib. Treatment of Dox-induced CCSP-

rtTA/tetO-KIF5B-RET bitransgenic mice with ponatinib effectively induced tumor regression.

These results indicate that KIF5B-RET-associated lung tumors are addicted to the fusion

oncogene and ponatinib is the most effective inhibitor for targeting KIF5B-RET in lung

adenocarcinoma. Moreover, this study finds a novel vandetanib-resistant RETG810A mutation and

identifies lenvatinib and ponatinib as the secondary drugs to overcome this vandetanib resistance

mechanism.

3
Introduction

Lung adenocarcinoma is a major subtype of non-small cell lung cancer (NSCLC) with

increasing incidence in many countries (1). Compared to other histological types of lung cancer,

lung adenocarcinoma frequently harbors KRAS or protein tyrosine kinase (PTK) aberrations (2,

3). While mutant KRAS is difficult to target directly at present, PTKs are targetable, allowing

precision treatment targeting individual patient’s driver oncogene. Targeting PTK driver

oncogenes with small molecule inhibitors in lung adenocarcinoma has shown clinical success in

managing NSCLC. This is exemplified by EGFR and ALK inhibitor therapies in EGFR mutation

and ALK-fusion lung adenocarcinoma (4, 5). However, acquired resistance to EGFR and ALK

kinase inhibitors usually occur within a year after the drug treatment, making it necessary to use

secondary drugs to prolong the therapeutic response (6, 7). Moreover, except for EGFR

mutations and ALK fusions, other PTK alterations in lung adenocarcinoma occur at <3% rates (8,

9), rendering clinical studies of these PTK oncogenes difficult (10).

Several laboratories have identified recurrent RET fusion genes in 1-2% of lung

adenocarcinoma cases (8, 11-14). Among the fusion partners, KIF5B is the most prevalent. RET

rearrangements in lung adenocarcinoma are mutually exclusive from other known driver

oncogenic mutations, including KRAS, EGFR, ALK, BRAF, and ERBB2 (8). Moreover, ALK,

RET, and ROS1 oncofusion-associated lung adenocarcinoma tissues harbor significantly fewer

mutations than other lung adenocarcinoma tissues (10). This suggests a strong role of these

fusion oncogenes in driving the tumors, implicating them as excellent therapeutic targets.

A number of PTK inhibitors (TKIs) are known to cross-inhibit RET kinase activity (15).

These include cabozantinib and vandetanib that have been approved by the United States Food

and Drug Administration for the treatment of advanced medullary thyroid cancer, which carries

4
RET point mutations in 30-50% of cases (15). So far, two reports have described the responses

of 6 RET-fusion lung adenocarcinoma patients treated with cabozantinib: 4 patients had partial

responses and 2 patients had stable disease (16, 17). One patient with RET-fusion lung

adenocarcinoma who received vandetanib was reported to have decreased tumor size (18). These

are encouraging initial findings but more studies are needed. Several clinical trials of RET kinase

inhibitors are ongoing (8, 15). However, the rarity of this molecular subtype of the disease

presents a barrier for the current and future clinical studies, as it will take a long time to enroll

adequate number of patients into the clinical trials.

To generate a preclinical model of RET fusion lung adenocarcinoma in an immune

competent environment, we generated tetO-KIF5B-RET transgenic mice and used CCSP-

rtTA/tetO-KIF5B-RET bitransgenic mice to induce KIF5B-RET expression in the lungs by

doxycycline (Dox). We found that KIF5B-RET induced invasive lung adenocarcinoma with

desmoplastic reaction. After tumor development, continuous expression of the KIF5B-RET

fusion gene is required to maintain the lung tumors. Using a parallel in vitro cell-based assay, we

identified ponatinib as the most potent inhibitor against KIF5B-RET, its kinase gatekeeper

mutations, and a novel vandetanib-resistant RETG810A mutant. Treatment of Dox-induced CCSP-

rtTA/tetO-KIF5B-RET transgenic mice with ponatinib resulted in tumor regression.

5
Materials and Methods

Reagents

Antibodies to phospho-RET(Tyr905) and cleaved-PARP were from Cell Signaling

Technology, antibodies to Flag-tag and β-actin were from Sigma, anti-RET antibody was from

Santa Cruz Biotech. Anti-TTF1 (ab137061) antibody was from Abcam. Anti-cytokeratin

antibody was from Dako (cat. No. Z0622). Ponatinib was from LC Laboratories. Cabozantinib,

vandetanib, and lenvatinib were from Selleckchem.

Generation of transgenic mice

The KIF5B-RET (K15;R12, RET51 long form) (12) coding region with a Flag tag coding

sequence at the C-terminus was synthesized by GeneArt Gene Synthesis (Life Technologies) and

cloned into the L3/L2 loxP-tetO plasmid (19). The 6.6-kb BssHII tetO-KIF5B-RET transgene

cassette (Fig. 1A) DNA fragment was used to generate tetO-KIF5B-RET transgenic mice in

FVB/N strain as described in Supplementary Information.

Bitransgenic CCSP-rtTA/tetO-KIF5B-RET (C/KR) mice were produced by crossing tetO-

KIF5B-RET mice with CCSP-rtTA mice (also in FVB/N strain) (19-21) (Fig. 1B). To induce

KIF5B-RET expression, C/KR mice at 4 weeks old were fed with rodent chow containing 200

mg/kg Dox (Dox diet, Bio-Serv). Both male and female mice were used in the experiments.

Genotyping of CCSP-rtTA transgenic mice was as described previously (19, 21). Genotyping

of tetO-KIF5B-RET transgenic mice was performed using GoTaq Hot Start Green Master Mix

(Promega) using primer pairs: K5R-3T, 5’-CACGAGAGCTGATGGCACTAACACT and

TETO-3B, 5’-CACAGCCCAGCCCAGCCCTCTACT. The 25 μl PCR reaction protocol was: 3

min at 94 oC, 30 cycles of 94 oC for 30 sec, 57 oC for 30 sec and 72 oC for 30 sec with a final

6
extension step of 72 oC for 5 min, which yielded a 309-bp product. Animal experiments were

approved by the Institutional Animal Care and Use Committee of University of South Florida.

Cell cultures and analyses

The mouse BaF3 cells were obtained from Dr. H.G. Wang (H. Lee Moffitt Cancer) in 2000

and have been stored in liquid nitrogen. BaF3 cells were cultured in RPMI-1640/10% fetal

bovine serum (FBS) supplemented with 2 ng/ml interleukin-3 (IL-3) as described (22). The

identity of BaF3 cells were evaluated based on their dependency on mouse IL-3. The authors

have not authenticated BaF3 cells using DNA-based method. To generate KIF5B-RET-

transformed BaF3 cells (B/KR), BaF3 cells were infected with lentiviruses containing a flag-

tagged KIF5B-RET gene. Individual puromycin-resistant cell lines were isolated, screened for

the presence of KIF5B-RET by immunoblotting, and evaluated for IL-3-independence.

To identify drug-resistant mutations, established B/KR cells were incubated with increasing

concentrations of cabozantinib or vandetanib in RPMI-1640/10%FBS as described in the Results.

Individual drug-resistant cell lines were isolated from semi-solid methylcellulose cultures, which

contained 4:6 ratio of MethoCult H4100 (Stemcell Technologies) and 20%FBS in RPMI-1640.

Genomic DNA of drug-resistant cell lines was isolated and the RET kinase domain coding

region was sequenced in both strands of DNA following PCR amplification as described (23).

To determine drug sensitivity, B/KR cells (1,500 cells/well) were incubated in RPMI-

1640/10% FBS in the presence of indicated concentrations of drugs in 96-well plates in

triplicates. On day 5, viable cells were measured using the CellTiter-Glo reagent (Promega). IC50

measurements were obtained from at least two independent experiments.

7
 Analyses of cell lysates by immunoprecipitation and immunoblotting were as described (19,

21, 23). Immune complex kinase assay using GST-Gab1 as the substrate was performed as

described previously (24).

Histological examination

Mice were euthanized by CO2 asphyxiation. Lungs were flushed with 10 ml phosphate-

buffered saline and insufflated with 10% buffered formalin. After fixing overnight in 10%

buffered formalin, paraffin blocks were prepared. Tissue sections were stained with hematoxylin

and eosin (H&E). H&E stain tissue slides were examined by three pathologists and scanned

using a ScanScope XT (Aperio). The Genie V1 histology pattern recognition software (Aperio)

was used to segment hyperproliferative lesions/tumors from other lung tissue areas and

background using the same parameters as described previously (21). Trichrome staining was

performed by the Tissue Pathology Core at Stephenson Cancer Center.

Immunohistochemistry (IHC) was performed with a Leica Bond III automated IHC protocol

using a Leica Bond-III Polymer Refine Detection reagents (Cat. No.: DS 9800). Epitope retrieval

was citrate buffer (pH 6.0) at 100 oC for 20 min. Anti-TTF1 and anti-cytokeratin antibodies were

used at 1:400 dilution for 30 min.

Magnetic resonance imaging (MRI) and computerized tomography (CT) protocols

For MRI, mice were anesthetized with 2% isoflurane and transferred to mouse cradle

mounted on an insertion device and positioned within the RF coil of the magnet and kept under

anesthesia for the duration of the experiment. The mice were physiologically monitored and

maintained with a Model 1030 Monitoring and Gating System (SA Instruments, Inc). A

8
respirator sensor pad was placed under each animal to manually control anesthesia mixture

whereas a fiber optic rectal thermometer was used for temperature feedback control, which was

set to maintain a body temperature of 37 ± 1 °C. Respiration rate was maintained at 50 – 60

breaths per minute and respiratory-gated MR images were acquired during the resting phase after

exhalation. MRI was performed using a 7-T horizontal magnet (ASR 310, Agilent Technologies)

equipped with nested 205/120/HDS gradient insert in a bore size of 310 mm. Two RF coils, a 35-

mm Litzcage coil (Doty Scientific, Inc.) and a 24-mm Litzcage coil (Doty Scientific, Inc), were

used depending on the sizes and weights of the mice. Temperature control of the imaging

gradients was achieved by means of a water chiller (Neslab Waters) and maintained at 12 °C for

all experiments.

Coronal multislice T2-weighted fast spin-echo (FSEMS) respiratory-gated sequences were

acquired with TE/TR = 30.05/1497.60 ms, data matrix = 256x128, 20 slices, field of view (FOV)

= 90x40 mm2, 16 averages and slice thickness of 1.20 mm over about 10-12 minutes. In T2–

weighted images, tumor volumes and dimensions were quantified using manually drawn regions

of interest (ROI) within lung on a slice-by-slice basis, where pixels above a given threshold were

counted as part of diffuse lesions. Analysis was done with MATLABTM using a script that

employed multi-thresholding segmentation method from the image processing toolbox of

MATLABTM. Care was taken to exclude the heart, vessels, and mediastinum.

For µCT, mice were scanned on an Inveon µCT scanner (Inveon micro CT/PET/SPECT,

Siemens, Knoxville, TN, USA) using a free-breathing protocol. Animals were anesthetized with

a mixture of 2% isoflurane and oxygen. Breathing rate was controlled by a BioVet physiological

monitoring system (m2m Imaging Corporation, USA) and kept at average 60 breaths per minute.

The 440 projections were taken over 220 degree arc trough 12 minutes over a FOV of 2.7 x 1.5

9
cm. The X-ray tube scanning parameters were 80 kVp voltage, 480 µA current, 1000ms

exposure, we also applied 1.5 mm Al filter.

The acquired data were reconstructed 3D volume with dimensions 1024x576x441 voxels

with 27.2669 um effective isotropic voxel size. CCD detector binning was 2x2. Reconstructed

data of absorption coefficients were converted to Hounsfield units (HU) in range of -1000 Hu to

1000 HU, where the value of -1000 HU corresponds to the air and 0 HU corresponds to the water

(we used 50 ml water phantom for calibration). Image data were evaluated by board certified

radiologist using preclinical Inveon Research Workstation 3D Visualization module (Siemens,

Knoxville, TN). The lung µCT images were viewed in coronal, sagittal, and axial planes as well

as in 3D. The 3D model of lung’s air pathways were rendered from original HU data after

applying intensity threshold procedure.

Statistical analysis

Statistical analyses were performed using unpaired t test with Welch’s correction, without

assuming equal SDs. The statistical significance was set to 0.05.

10
Results

Inducible KIF5B-RET transgenic mice

We generated a tetO-KIF5B-RET transgenic mouse line (A1) as described in the

supplementary information. These monotransgenic mice had no detectable phenotype and no

KIF5B-RET transcript in the tissues that we have examined (Fig. 1C, top panel). CCSP-

rtTA/tetO-KIF5B-RET bitransgenic mice (C/KR) were generated by crossing tetO-KIF5B-RET

mice with CCSP-rtTA mice (19, 20) and evaluated for Dox-induced expression of KIF5B-RET.

As illustrated in Fig. 1C (bottom panel), Dox specifically induced KIF5B-RET mRNA in the

lungs but not in the heart, intestine, liver, kidney, or spleen of C/KR mice. Immunoprecipitation

of lung tissue lysates with an anti-Flag antibody followed by immunoblotting detected KIF5B-

RET protein and phosphorylated KIF5B-RET in the lungs of Dox-induced C/KR mice (Fig. 1D).

In another microinjection experiment performed later, we obtained two other tetO-KIF5B-

RET transgenic mouse lines (A2, G6). CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice

generated from A2 and G6 lines displayed the same phenotype as that observed in the A1 line

(Supplementary Fig. 1). Since the A1 line was generated much earlier, Most of our experiments

were conducted using this line.

KIF5B-RET transgenic mice develop lung adenocarcinoma with desmoplastic reaction.

As early as one month after Dox induction, the lungs of C/KR bitransgenic mice developed

areas of hyperplastic and early tumor lesions that were often accompanied by thickening of

pleural stroma (Fig. 2A). Pleural thickening was observed in human lung adenocarcinoma

harboring RET fusion (Supplementary Fig. 2A). By 4-5 months after Dox induction of KIF5B-

RET expression, extensive lung tumors were observed in these C/KR mice (Fig. 2B, C). These

11
tumors displayed predominantly lepidic growth pattern with focal solid pattern, visceral pleural

involvement, and desmoplastic reaction (Fig. 2B, C). Lymphocytes were often observed near the

desmoplastic reaction areas and macrophages were observed in the tumor lesion areas. In total,

we examined 12 lungs from C/KR mice induced with Dox for 4-7 months (4 four months, 6 five

months, 1 six months, and 1 seven months). Invasive tumors were observed in all 12 lungs,

which were confirmed by cytokeratin IHC in every case. We have not observed metastasis of

tumors to distal organs. However, we have not ruled out the possibility that distal metastasis may

occur when the Dox-induced C/KR mice are kept for a longer period of time.

Desmoplastic reaction is associated with invasive lung adenocarcinoma in human lung

cancer patients (Supplementary Fig. 2B) and was reported in the first case of KIF5B-RET lung

adenocarcinoma when KIF5B-RET fusion was discovered (14). In the Cancer Genome Atlas

(TCGA) Research Network lung adenocarcinoma study (2), desmoplastic stroma are visible in

the tissue sections of RET-fusion lung adenocarcinoma cases (Supplementary Fig. 2A).

Desmoplastic reaction also occurred in the lung tumors in Dox-induced C/KR mice derived

from Line A2 and Line G6 of tetO-KIF5B-RET transgenic mice (Supplementary Fig. 1A, B). In

comparison, we have not observed desmoplastic reaction in lung adenomas and adenocarcinomas

in KrasLA1 mice that express KrasG12D (25) (~ 6 months of age, Supplementary Fig. 1C), Dox-

induced CCSP-rtTA/tetO-EGFRL858R transgenic mice (21, 26), or Dox-induced CCSP-rtTA/tetO-

SHP2E76K transgenic mice (19) (n >20 lungs examined in each case).

The KIF5B-RET oncogene is required to maintain lung tumors

To determine if lung tumors in Dox-induced C/KR mice were addicted to the KIF5B-RET

oncogene, we identified 7 Dox-induced C/KR mice (4-5 months) with MRI-detectable lung

12
tumors. These mice were then fed regular chow without Dox. One month after Dox withdrawal,

these mice were examined by MRI again. Lung tissue sections were examined by histology and

tissue lysates analyzed by immunoprecipitation and immunoblotting for the expression of RET.

As shown in Fig. 3A, Dox withdrawal resulted in regression of MRI-detected tumor lesions

in all 7 mice. Lungs from 5 of these mice were examined by histology as illustrated in Fig. 3B.

The remaining lesions in the lungs from Dox withdrawn mice were mostly of incompletely

resolved fibrotic tissues. Using a histology pattern recognition program (Aperio) as described

previously (21), the hyperproliferation/tumor lesion areas were semi-quantified. The area of

interest (AOI), which included some of bronchiolar epithelia in addition to

hyperproliferation/tumor lesions, in these 5 lungs was 4.33+1.76% (mean + std). In comparison,

the AOI from 5 Dox-induced C/KR mice was 38.80+25.34%, whereas the AOI from the 5

wildtype mice was 2.47%+1.17%. Thus, Dox withdrawal from C/KR mice resulted in a

significant reduction (p = 0.008) in the AOI to a level similar to that of normal wildtype mice (p

= 0.98) (Fig. 3B).

Both KIF5B-RET protein and phosphorylated (pY905) KIF5B-RET were detected in Dox-

induced C/KR mice (Fig. 3C). They were no longer detected in the lungs one month post-Dox

withdrawal. Taken together, these data show that after tumor formation, continuous expression

of KIF5B-RET is required to maintain the lung tumors.

Ponatinib is the most potent KIF5B-RET kinase inhibitor

BaF3 cells depend on IL-3 for survival. We established stable BaF3 cells expressing KIF5B-

RET (B/KR). When cells were incubated in medium without IL-3, the parental cells and vector-

control cells underwent apoptosis in 6 hours as measured by PARP cleavage, whereas B/KR

13
cells continued to grow (Fig. 4A, B). Thus, KIF5B-RET transformed BaF3 cells into cytokine-

independence. The KIF5B-RET transformation was inhibited by cabozantinib that suppressed

the KIF5B-RET kinase activity (Fig. 4C, D).

Cabozantinib and vandetanib inhibited B/KR cells with IC50s of 0.175 µM and 0.90 µM,

respectively. To isolate cabozantinib- or vandetanib-resistant KIF5B-RET mutations, we

incubated B/KR cells (2x107 cells) with sequentially increasing concentrations of cabozantinib

(from 0.175 µM to 0.65 or 0.85 µM in two independent experiments) or vandetanib (from 0.90

µM to 4.5 or 5.5 µM in two independent experiments). Individual drug resistant cells were

cloned by methylcellulose culture. After verification of constitutive KIF5B-RET tyrosine kinase

activity in these cells in the presence of cabozantinib or vandetanib in the culture medium

(Supplementary Fig. 3A), the protein tyrosine kinase domain coding regions of KIF5B-RET

from these drug-resistant cell clones were sequenced. The RETV804L gatekeeper mutation was

identified in all 10 cabozantinib-resistant cell lines. A novel RETG810A mutation was found in 8

of 10 vandetanib-resistant cell lines. We next immunoprecipitated KIF5B-RET, KIF5B-

RETV804L, and KIF5B-RETG810A from these cells and assayed their PTK activity in vitro in the

presence of increasing concentrations of cabozantinib or vandetanib. The results confirmed that

KIF5B-RETV804L was resistant to cabozantinib and that KIF5B-RETG810A was resistant to

vandetanib (Supplementary Fig. 3B).

To confirm that the novel RETG810A mutation renders vandetanib resistance and to evaluate if

drug resistance may be attributed to overexpression of KIF5B-RET protein, we recreated a

BaF3/KIF5B-RETG810A cell line (B/KRG810A-REC) using lentivirus encoding the KIF5B-RETG810A.

We then compared expression of KIF5B-RET and KIF5B-RETG810A protein in these cells and

vandetanib resistance between B/KRG810A and B/KRG810A-REC. As shown in Supplementary Fig.

14
3C, KIF5B-RET and KIF5B-RETG810A protein levels were similar between B/KR and the B/KR-

derived B/KRG810A cells. The independently generated B/KRG810A-REC cells had a 20% higher

level of KIF5B-RETG180A protein. The vandetanib IC50s were 5.310 µM and 5.675 µM for

B/KRG810A and B/KRG810A-REC cells (sFig. 3D). These data confirm that the RETG810A mutant is

resistant to vandetanib. While we do not rule out the possibility that increased KIF5B-RET

expression may confer some extent of drug tolerance, our results indicate that vandetanib

resistance of B/KRG810A cells is mainly due to the G810A mutation.

Cabozantinib, vandetanib, ponatinib and lenvatinib are multi-kinase TKIs that have been

reported in on-going clinical trials of RET-fusion NSCLC (15). We compared these four drugs in

the KIF5B-RET-dependent growth of B/KR cells. As shown in Fig. 4D, ponatinib was the most

potent inhibitor with an IC50 of 0.07 µM for B/KR cells. B/KRV804L gatekeeper mutation cells

were pan-resistant to all four TKIs, resulting in 6.2, 8.7, 15.7, and 76-fold increase in IC50s for

ponatinib, vandetanib, cabozantinib, and lenvatinib, respectively (Fig. 4D). However, ponatinib

had the least fold increase in IC50 and remained the most potent inhibitor for B/KRV804L cells

with a sub-micromolar IC50.

Vandetanib IC50 for B/KRG810A cells was increased 5.7-fold to 5.1 µM compared to B/KR

cells. Interestingly, while cabozantinib had similar IC50s in B/KRG810A cells and B/KR cells,

ponatinib and lenvatinib had lower IC50s in B/KRG810A cells by 8.7- and 3.6-folds, respectively.

Thus, while the RETG810A mutation causes vandetanib resistance, it becomes hypersensitive to

ponatinib and lenvatinib. We hereby term this type of mutation as a gain-of-sensitivity (GOS)

mutation.

To further compare the potencies of these four drugs for RET and RETV804L and also to

determine the potencies of these drugs for another known RET gatekeeper mutant RETV804M,

15
IC50s for inhibition of recombinant RET, RETV804L, and RET V804M in an in vitro kinase assay

were measured in parallel via a commercial service (Reaction Biology, Malvern, PA). As shown

in Supplementary Fig. 4, ponatinib again displayed the most potent inhibitory activities against

RET, RETV804L, and RETV804M among these four compounds.

Ponatinib induces lung tumor regression in Dox-induced C/KR bitransgenic mice

Since ponatinib was the most potent TKI for KIF5B-RET and its gatekeeper mutations, we

chose it for the in vivo study in our KIF5B-RET transgenic animal model of NSCLC. Dox-

induced C/KR mice (4-5 months) were examined by MRI and micro-CT. Eight mice with MRI-

detected lung tumor lesions were divided into two groups and treated with ponatinib or vehicle

(4 mice/group) by oral gavage (30 mg/kg/day, 5 days/week) (see Supplementary Fig. 5 for

treatment schedule and body weight monitoring data). After 4 weeks of drug treatment, the lungs

of these mice were examined again by MRI and µCT, and lung tissue sections were analyzed by

histology.

Lung tumor lesions in the vehicle-treated mice were similar or worse based on MRI and µCT

examination (Fig 5A, B, Supplementary Fig. 6, 7, Supplementary Information 2, sMovie). In

contrast, the lung lesions were greatly reduced in ponatinib-treated mice. Semi-quantitative

measurement of tumor lesions by MRI showed that the mean VOI was significantly (p = 0.0286)

decreased from 494+83 mm3 (prior to treatment) to 68+51 mm3 (post treatment) in the ponatinib-

treated cohort (Fig. 5B). In comparison, the mean MRI VOI was not significantly changed (p =

0.6571) in the vehicle-control cohort before (580+227 mm3) and after (480+176 mm3) treatment.

Analysis of H&E stained lung tissue sections (Supplementary Fig. 6) by the histology pattern

recognition program show that vehicle-treated cohort had a mean 23.25+3.20% of AOI with

16
hyperplasia or neoplasia, whereas ponatinib-treated mice had significantly (p = 0.0286) less AOI

(mean = 2.79+0.63%) similar to that of the wildtype mice (2.47%+1.17%, Fig. 3B, 5C). In

another experiment, lung tissue lysates from Dox-induced C/KR mice treated with vehicle or

ponatinib for 1 month were analyzed for tyrosine phosphorylation of KIF5B-RET. As shown in

Fig. 5D, KIF5B-RET protein was decreased and its tyrosine phosphorylation was suppressed in

the lungs of ponatinib-treated animals. Thus, ponatinib is effective in inhibiting KIF5B-RET

kinase activity and reducing the lung tumors of C/KR mice.

17
Discussion

RET oncogene fusions occur in 1-2% of lung adenocarcinoma. The rarity of the molecular

lesion presents a barrier for clinical studies of RET fusion-associated lung adenocarcinoma (10).

To facilitate in vivo studies of RET-associated lung adenocarcinoma, we have established an

inducible transgenic mouse model of KIF5B-RET-associated lung adenocarcinoma.

KIF5B-RET displayed a constitutively active PTK activity as measured by pRET when it

was expressed in the lungs of Dox-induced C/KR transgenic mice. Hyper-proliferative lesions

and early tumors in the lungs were observed as early as one month after Dox induction of the

C/KR mice, and progressed to invasive lung tumors in 4-5 months after Dox induction of

KIF5B-RET expression in the C/KR mice. Lung tumors formed in this Dox-induced C/KR

model resemble human invasive lung adenocarcinoma and are associated with desmoplastic

reaction and visceral pleural involvement. Desmoplastic stroma was also evident in the RET

fusion-positive lung adenocarcinoma cases in TCGA. Various histological patterns of RET

fusion-positive lung adenocarcinoma have been observed, including the prevalence of poorly

differentiated, solid-predominant histologic features in RET fusion cases of lung

adenocarcinoma (17, 27-29). Lung tumors in our C/KR mouse model recapitulate the invasive

desmoplastic feature of RET fusion-associated tumors.

Similar to the absence of desmoplastic stroma in the lung tumors of KrasLA1 mice that we

observed in this study, a previous study using the adenovirus-Cre recombinase/LSL-RasG12D

mouse model also found that lung adenoma and adenocarcinoma developed in that mouse model

lack stromal reaction (30). Knockout of tgfbr2 in the LSL-KrasG12D model allows the KrasG12D-

driven lung tumors to progress to invasive lung adenocarcinoma with desmoplastic reaction (30).

Human NSCLC often can cause desmoplasia when they invade into stroma. Among 230 cases of

18
human lung adenocarcinoma characterized in TCGA (2), there are 1 case with TGFBR2 deletion

and 1 case with TGFBR2 point mutation (E257D), both of them occur in KRASG12C tumors

(www.cbioportal.org). While many other lung adenocarcinoma cases in the TCGA cohort show

the desmoplastic histology, such as RET-fusion cases, they are not associated with TGFBR2

deficiency. Thus, although TGFBR2 deficiency could promote desmoplastic stroma deposition,

other factors are also likely to contribute to the stromal response to the invasive tumors.

Genomic analysis has suggested the exclusive dependency of lung adenocarcinoma on ALK,

RET, or ROS fusion oncogenes (10). Consistently, data from our Dox withdrawal experiment

indicate that lung tumors developed in Dox-induced C/KR mice require the continuing

expression of the KIF5B-RET fusion oncogene to maintain the malignancy. This property

suggests that KIF5B-RET is an effective target for drug treatment to eliminate KIF5B-RET-

associated lung adenocarcinoma.

By culturing KIF5B-RET-dependent B/KR cells with RET kinase inhibitors, we identified

cabozantinib-resistant RETV804L gatekeeper mutation and a novel vandetanib-resistant RETG810A

mutation. Interestingly, while the RETG810A mutant remained sensitive to cabozantinib, it

acquired GOS to ponatinib and lenvatinib. Recent clinical experience in EGFR and ALK

inhibitor therapy of lung adenocarcinoma has shown that a major mechanism of developing drug

resistance is secondary mutations in the targeted PTK kinase domain. Mutation-sensitive

secondary TKIs can be developed and used to prolong the therapeutic response (31-33). Our

finding here suggests that ponatinib or lenvatinib are the secondary drugs to use when RETG810A

mutation occurs in vandetanib-treated RET-associated cancer, including lung adenocarcinoma

and metastatic thyroid cancer.

19
 Among cabozantinib, lenvatinib, ponatinib, and vandetanib, our data show that ponatinib is

the most potent inhibitor of RET PTK. Treatment of lung tumors induced by KIF5B-RET in the

transgenic animals resulted in tumor regression. Thus, ponatinib is an effective drug for treating

KIF5B-RET-associated lung adenocarcinoma in this genetically engineered mouse model and is

predicted to show anti-tumor efficacy in RET fusion lung adenocarcinoma patients. While the

gatekeeper RETV804L and RETV804M mutations are less sensitive to ponatinib than the wildtype

RET, ponatinib remains the most potent inhibitor (Fig. 4 and Supplementary Fig. 4). Thus,

ponatinib appears to be the drug of choice for treating RET fusion-associated lung

adenocarcinoma.

It is envisioned that more drugs will be developed, re-purposed, and evaluated for therapy of

RET fusion-associated lung adenocarcinoma, either as novel single agent or in combination

treatment. The immune competent C/KR mouse model that we developed here provides a

resource allowing the direct side-by-side comparison of two drugs in preclinical studies or co-

clinical trials (34, 35) of RET fusion-associated lung adenocarcinoma.

20
Disclosure of Potential Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

We thank H. Lee Moffitt Cancer Center Core facility staff, including Animal, DNA

sequencing, Histology, Microscopy Core facilities, and Stephenson Cancer Center/University of

Oklahoma Health Sciences Center Histology Core facility staff for assistance. We also thank J.A.

Whitsett for the CCSP-rtTA transgenic mice, D.C. Radisky and A.P. Fields for advice and

assistance, and K. Politi and G. Felsenfeld for reagents.

21
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Figure legends

Figure 1. Derivation and analysis of tetO-KIF5B-RET transgenic mice. A, schematic

representation of tetO-KIF5B-RET transgene. B, scheme for production of C/KR bitransgenic

mice and induction of the KIF5B-RET transgene expression in the mouse lung type II epithelial

cells. C, RT-PCR analysis of KIF5B-RET mRNA expression in various tissues in

monotransgenic tetO-KIF5B-RET mouse and in C/KR bitransgenic mouse fed with Dox diet for

1 month. D, after induction with Dox diet for 1 month, cell lysates were prepared from lung

tissues of CCSP-rtTA/tetO-KIF5B-RET (C/KR) bitransgenic or wildtype (W) mice.

Immunoprecipitation-immunoblotting analysis of KR expression and phosphorylation was

performed.

Figure 2. Dox-induced bitransgenic C/KR mice develop lung tumors. A, H&E stained sections

showing C/KR bitransgenic mice developed areas of hyperplastic lesions and early tumors after

one month Dox induction. B, lung sections of a C/KR mouse induced with Dox for 4 months.

Left, an H&E stained lung section showing lung with desmoplastic tumors. Right, tumor cells

were stained positive of nuclear TTF1. C, lung sections of a C/KR mouse induced with Dox for 5

months. Right, H&E stained lung section with extensive tumors and desmoplasia. Right,

cytokeratin stain confirmed invasive lung tumor.

Figure 3. KIF5B-RET expression is required to maintain lung tumors. A, C/KR mice developed

MRI-detectable lung adenocarcinoma after Dox induction for 5 months. MRI images of

representative tumor bearing mouse prior to and one month after Dox withdrawal. The graph

27
shows comparison of MRI-detectable lung tumor burden prior to and post Dox withdrawal. Each

line represents an individual mouse (n = 7). VOI, Volume of interest. B, H&E stained tissue

section from lung of a mouse after Dox withdrawal (left). Lung tissue image from Genie® v1

histology pattern recognition software (Aperio) analysis (middle). Lung tumor burden was

analyzed from H&E stained lung tissues (right) (n = 5 in each group). AOI, areas of interest.

White, not lung tissue, area excluded; pink, background; blue, normal; purple:

hyperplasia/neoplasia including some areas of normal bronchial epithelia (21). C, comparison of

pRET and RET protein in the lungs of C/KR mice induced with Dox (prior to Dox withdrawal)

and 1 month post Dox withdrawal. W, wildtype mouse.

Figure 4. Ponatinib is the most potent KIF5B-RET inhibitors and the vandetanib resistant

RETG810A mutation is hypersensitive to ponatinib. A, growth curve of parental, vector control

(B/V) and stable BaF3 cells expressing KIF5B-RET (B/KR) without IL-3. B, cells were grown in

medium without IL-3 for 6, 16, 24 hours. The cell lysates were immunoblotted with anti-cleaved

PARP or anti-β actin. P, parental; V, vector control; KR, KIF5B-RET. C, vector control and

KIF5B-RET cells were treated with or without 1 µM cabozantinib (CBT) for 3 hours. The cell

lysates were immunoblotted with anti-RET-pY905, anti-Flag, anti-cleaved PARP or anti-beta

actin. D, Ba/F3 expressing KIF5B-RET, KIF5B-RETV804L, or KIF5B-RETG810A were seeded on

96-well plates and treated with indicated concentration of ponatinib (PNT), cabozantinib (CBT),

vandetanib (VDT) or lenvatinib (LVT) for 5 days. Cell viability was measured using the

CellTiter-Glo assay and IC50s were determined. The data were from two triplicate experiments (n

= 6).

28
Figure 5. Ponatinib treatment results in tumor regression. A, C/KR mice with MRI-detectable

lung tumors were treated with vehicle (upper panel) or ponatinib (30 mg/kg/day, 5 days/week,

p.o., lower panel) for 1 month and then analyzed by MRI again (n = 4 in each group). Lung

tissue sections at the end point were stained with H&E. Representative images of MRI, H&E

sections, and image from Genie® v1 histology pattern recognition software analysis (right) are

shown. B, comparison of MRI-detectable lung tumor burden prior to and post vehicle or

ponatinib treatment. Each line represents an individual mouse. Blue, vehicle-treated; Black,

ponatinib-treated. C, lung tumor burden analyzed from samples of H&E stained lung tissue

sections. V, vehicle-treated; P, ponatinib-treated. D, comparison of pRET, RET and β-actin

proteins in the lungs of CCSP and C/KR mice treated with vehicle or ponatinib. CCSP, negative

control mouse.

29