Targeting ROR1 in combination with pemetrexed in malignant mesothelioma cells

Journal Pre-proof Targeting ROR1 in combination with pemetrexed in malignant mesothelioma cells Noriko Miyake, Nobuaki Ochi, Hiromichi Yamane, Takuya Fukazawa, Tomoko Ikeda, Etsuko Yokota, Masami Takeyama, Nozomu Nakagawa, Hidekazu Nakanishi, Hiroyuki Kohara, Yasunari Nagasaki, Tatsuyuki Kawahara, Naruhiko Ichiyama, Tomoki Yamatsuji, Yoshio Naomoto, Nagio Takigawa

PII:

S0169-5002(19)30705-6

DOI:

https://doi.org/10.1016/j.lungcan.2019.10.024

Reference:

LUNG 6182

To appear in:

Lung Cancer

Received Date:

24 August 2019

Revised Date:

24 September 2019

Accepted Date:

24 October 2019

Please cite this article as: Miyake N, Ochi N, Yamane H, Fukazawa T, Ikeda T, Yokota E, Takeyama M, Nakagawa N, Nakanishi H, Kohara H, Nagasaki Y, Kawahara T, Ichiyama N, Yamatsuji T, Naomoto Y, Takigawa N, Targeting ROR1 in combination with pemetrexed in malignant mesothelioma cells, Lung Cancer (2019), doi: https://doi.org/10.1016/j.lungcan.2019.10.024

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Targeting ROR1 in combination with pemetrexed in malignant mesothelioma cells

Noriko Miyakea, Nobuaki Ochib, Hiromichi Yamaneb, Takuya Fukazawaa,c, Tomoko Ikedaa, Etsuko Yokotaa, Masami Takeyamab, Nozomu Nakagawab, Hidekazu Nakanishib, Hiroyuki Koharab, Yasunari Nagasakib, Tatsuyuki Kawaharab,

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Naruhiko Ichiyamab, Tomoki Yamatsujic, Yoshio Naomotoc, Nagio Takigawaa,b*

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General Medical Center Research Unit, Kawasaki Medical School, 2-6-1 Nakasange,

Kita-ku, Okayama 700-8505, Japan

Department of General Internal Medicine 4, Kawasaki Medical School, 2-6-1

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Nakasange, Kita-ku, Okayama 700-8505, Japan

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b

Department of General Surgery, Kawasaki Medical School, 2-6-1 Nakasange, Kita-ku,

Correspondence should be addressed to:

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*

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Okayama 700-8505, Japan

Nagio Takigawa, M.D., Ph.D.

Department of General Internal Medicine 4, Kawasaki Medical School, 2-6-1 Nakasange, Kita-ku, Okayama 700-8505, Japan

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Tel: +81-86-225-2111

Fax: +81-86-232-8343 E-mail: [email protected]

Highlights 1

ROR1 appears to be a targetable molecule in malignant mesothelioma. ROR1 inhibition in malignant mesothelioma cells suppressed growth and colony formation.

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Thymidylate synthase was downregulated in mesothelioma cells transfected with siROR1.

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A combination of pemetrexed with siROR1 was effective in malignant mesothelioma cell lines.

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Abstract

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Objective: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in a subset of malignant cells. However, it remains unknown whether ROR1 is targetable

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in malignant mesothelioma (MM). Therefore, in this study, we investigated the effects of ROR1 inhibition in mesothelioma cells. Materials and Methods: Growth inhibition,

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colony formation, apoptosis, and mRNA/protein levels using siRNA-transfected MM cells were evaluated. Cluster analysis using Gene Expression Omnibus repository of

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transcriptomic information was also performed. Results: Our results indicated that in three (H2052, H2452, and MESO-1) among four MM cell lines, ROR1 inhibition had anti-proliferative and apoptotic effects and suppressed the activation of AKT and STAT3. Although growth inhibition by siROR1 was minimal in another mesothelioma

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cell line (H28), colony formation was significantly suppressed. Microarray, quantitative polymerase chain reaction, and Western blot analyses showed that there were differences in the suppression of mRNA and proteins between H2452 and H28 cells transfected with siROR1 compared with those transfected with control siRNA. Cluster analysis further showed that MM tumors had relatively high ROR1 expression, although the cluster in them was different from that in MM cell lines. Thymidylate synthase, a 2

target of pemetrexed, was downregulated in H2452 cells transfected with siROR1. Accordingly, a combination of pemetrexed with siROR1 was found to be effective in the four MM cell lines we studied. Conclusion: Our findings may provide novel therapeutic insight into the treatment of advanced MM.

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Keywords: ROR1, thymidylate synthase, pemetrexed, mesothelioma

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Introduction

Although malignant mesothelioma (MM) is rare among thoracic malignancies,

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approximately 1500 and 3000 patients are newly diagnosed each year in Japan and the

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United States1, respectively. Treatment of MM, including surgery, radiotherapy, and chemotherapy, is recommended according to stage, performance status, pulmonary

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function, complications, and patient preference. First-line chemotherapy for advanced MM involves a combination of cisplatin and pemetrexed as the standard treatment.

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Although this combination has been proven to prolong survival over cisplatin alone in a phase III trial (12.1 months vs. 9.3 months), the prognosis remains poor2. To improve the poor prognosis in these patients, clinical trials involving the integration of molecular targeting agents in the treatment of advanced MM have begun.

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Accordingly, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor, vascular endothelial growth factor (VEGF), VEGF receptor, insulin-like growth factor 1 receptor, mammalian target of rapamycin, histone deacetylases, tumor necrosis factor, mesothelin, and proteasome have been all investigated as molecular targets. However, prolonged overall survival using these agents has not been demonstrated in randomized studies3,4. 3

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) was originally reported to be involved in the development of the nervous, circulatory, and respiratory systems5,6. In addition, ROR1 has been shown to regulate the epithelial–mesenchymal transition (EMT) and cancer metastasis, with antibodies targeting ROR1 shown to inhibit cancer progression and metastasis in preclinical research7. In malignant cells, ROR1 is

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occasionally overexpressed or mutated in various cancer types, including chronic lymphocytic leukemia (CLL), melanoma, ovarian cancer, breast cancer, lung cancer,

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colorectal cancer, and pancreatic cancer8,9,10,11,12. Among thoracic malignancies, the role of ROR1 during the disease progression of non-small cell lung cancer was extensively

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investigated, and its importance as a therapeutic target was suggested13,14.

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In this study, we investigated the feasibility of targeting ROR1 for MM in vitro. In

Material and Methods

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Reagents

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addition, the combination of targeting ROR1 with pemetrexed was evaluated.

Pemetrexed and cisplatin were purchased from Selleck Chemicals.

Cell lines and cell culture

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The human MM cell lines, H2452, H2052, and H28 were purchased from the American Type Culture Collection (Manassas, VA). ACC-MESO-1 (MESO-1)15 was obtained from RIKEN. Cells were cultured in RPMI1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FBS (Gibco 10487-028, Thermo Fisher) at 37°C in a 5% CO2 incubator.

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siRNA transfection Predesigned siRNAs targeting ROR1 were purchased from Sigma-Aldrich: Mission siRNA Hs_siROR1_#1: 5’-CAGCAAUGGAUGGAAUUUCAAdTdT-3’, #2: 5’CCCAGUGAGUAAUCUCAGUdTdT-3’, #3: 5’CCCAGAAGCUGCGAACUGUdTdT-3’). AllStars Negative Control siRNA (1027280,

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QIAGEN) was used as a control. Cells were seeded and incubated for 24 h prior to the transfection. Ten to 40 nM siRNA was then transfected into the cells using

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lipofectamine RNAiMAX (13778075, Thermo Fisher), according to the manufacturer's

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instruction.

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Trypan blue exclusion assay

Cells were seeded in 12-well plates at a density of 2 to 5 × 104 cells/well, depending on

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the cellular proliferation rate. siRNA-transfected cells were incubated for 5 days prior to cell counting. Cell viability was measured by the trypan blue dye exclusion assay (0.4%

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trypan blue solution, 15250-061, Thermo Fisher) using a Countess™ II FL Automated Cell Counter and Countess Cell Counting Chamber Slides (Thermo Fisher).

Colony formation assay

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Cells were seeded in 6-well plates at a density of 2 × 105 cells/well. After siRNA transfection and overnight incubation, cells were trypsinized and seeded at a density of 250, 500, and 1000 cells/well. After incubation for 2 weeks for the H2052, H2452, and MESO-1 cells lines, or 3 weeks for the H28 cell line, colonies were fixed and stained with Diff-Quik (#16920, Sysmex). The stained wells of each plate were then photographed and printed in order to count colonies. 5

MTT assay Cells were seeded in 96-well plates (2500 cells/well) and incubated overnight in a CO2 incubator. siRNA was transfected and incubated for 24 h. Serially diluted pemetrexed or cisplatin were then added and incubated for 72 h. Viable cells were measured using the

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MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay according to the method described previously16,17. The inhibitory concentration of 50% (IC50) was

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defined as the concentration resulting in a 50% reduction in growth compared with the

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control cell growth.

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Anoikis assay

siRNA-transfected cells were incubated for 24 h, then trypsinized and seeded in

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Anchorage Resistant Plates or Control Plates (CytoSelect 96-Well Anoikis Assay Kit, CBA-081, Cell Biolabs) at a density of 5 × 104 cells/well. After a 48-h incubation

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period, MTT colorimetric detection was performed.

Apoptosis detection by fluorescence microscope siRNA-transfected cells were incubated for 48 h and then trypsinized and stained with

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Hoechst 33342 (H3570, Life Technologies) and propidium iodide (PI) solution (P4864, Sigma-Aldrich). The nucleic morphological changes in apoptotic cells were observed using an Eclipse E600 fluorescence microscope (Nikon).

Flow cytometry Cells were seeded in 6-well plates and treated with siRNA (control) or siROR1 for 72 h. 6

After removing the culture medium, cells were harvested and washed with PBS. To detect apoptosis, cells were stained using the FITC-Annexin V Apoptosis Detection kit I (#556547), PE Active Caspase-3 Apoptosis kit (#550914), or FITC Mouse anti-cleaved PARP kit (#55876) (BD Biosciences). Stained cells were analyzed using a BD

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FACSVerse system (Becton Dickinson).

Western blotting analysis

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Cells treated with siRNA and/or pemetrexed were lysed with lysis buffer (10%

Glycerol, 2.3% SDS, 62.5 mM Tris-HCl, pH 6.8) to obtain protein extracts. Protein

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concentrations were determined with a Pierce BCA Protein Assay Kit (23225, Thermo

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Fisher). Protein samples were mixed with 4× Laemmli Sample Buffer (161-0747, BioRad) and heat-denatured, then applied to SDS-PAGE gels (TGX FastCast Acrylamide

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kit [10%], 161-0173, Bio-Rad), followed by transfer to a membrane (Immobilon-P Transfer Membranes, IPVH00010, Millipore). The antibodies used for immunoblotting

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were as follows: anti-ROR1 (#4102, Cell Signaling), AKT (#9272, Cell Signaling), phospho-AKT (#13038, Cell Signaling), p44/42 MAPK(Erk1/2) (#9102, Cell Signaling), phospho-p44/42 MAPK (Erk1/2) (#9101, Cell Signaling), STAT3 (#12640, Cell Signaling), phospho-STAT3 (#9145, Cell Signaling), TYMS (14586-1-AP,

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Proteintech), and β-actin (sc-69879, Santa Cruz). Goat anti-Rabbit IgG and Goat antiMouse IgG (111-035-003 and 115-035-003, respectively; Jackson ImmunoResearch Laboratories) were used as secondary antibodies. The chemiluminescent signal was detected with the SuperSignal West Pico PLUS Chemiluminescent Substrate (34577, Thermo Fisher) using an Amersham Imager 600 (GE Healthcare).

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Quantitative real-time polymerase chain reaction (PCR) RNA was extracted 72 h after siRNA transfection using the RNeasy Mini Kit (74104, Qiagen). The synthesis of cDNA from prepared total RNA was then performed using the Prime Script RT Reagent Kit with the gDNA Eraser (RR047A, Takara). mRNA expression was analyzed by quantitative real-time PCR using primer sets obtained from

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Sigma-Aldrich and the Power SYBR Green PCR Master Mix (4367659, Applied Biosystems) according to the manufacturer's protocol. PCR amplification and

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measurements were performed using a StepOne Plus Real-Time PCR instrument

(Applied Biosystems). For each target, the relative mRNA expression to GAPDH was

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determined.

Microarray

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Total RNA was extracted at 48 h post siRNA transfection. Prepared RNA samples were submitted for microarray analysis, which was performed at Filgen, Inc. (Nagoya, Japan)

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using the CodeLink Human Whole Genome Bioarray for H28 and GeneChip Gene ST Array for H2452.

Cluster analysis

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For microarray analysis, gene expression data (Gene Expression Omnibus platform accession nos. GSE2549) was downloaded from the Gene Expression Omnibus repository of transcriptomic information (http://www.ncbi.nlm.mih.gov/geo/), which was obtained using the microarray platform Affymetrix Human Genome U133 Plus 2.0 Array. This dataset includes surgically resected human MM tumor specimens (n = 40), normal lung specimens (n = 4), normal pleura specimens (n = 5), and MM and SV408

immortalized mesothelial cell lines (n = 5). The values were log2 transformed and normalized, heatmaps were generated, and hierarchical clustering performed using the Institute for Genomic Research MeV software package version 4.9 (http://mev.tm4.org/)18.

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Statistical analysis All data are expressed as the mean ± standard deviation (SD), with statistical analyses

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performed by two-tailed paired Student t-test (Stata for Windows, ver. 14.0, Stata,

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College Station, TX, USA). A value of p < 0.05 was regarded as statistically significant.

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Results

Cell growth and colony suppression by siROR1

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The four MM cell lines15,19 we studied were first confirmed for their response to siROR1. Any cells were partially agglutinated 72 h after exposure to siROR1#1,

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although there were morphological differences depending on the cell line (Fig. 1A). Cell numbers were significantly reduced by exposure to siROR1 (Fig. 1B). However, only the H28 cell line poorly responded to siROR1 compared with the other cell lines. Subsequently, colony formation assays after exposure to siROR1 were performed (Fig.

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2A). Although colony formation was significantly suppressed in all the cell lines studied, the MESO-1 cell line had, at most, a 20% regression (Fig. 2B). H28 colonies were reduced by approximately half by siROR1#1.

Apoptosis by siROR1 From the results of MM cell growth inhibition by siROR1, the effect on apoptosis 72 h 9

after siROR1 transfection was investigated using flow cytometry. Three cell lines (H2052, H2452, and MESO-1) showed an increase in apoptosis (Fig. 3). The proportions of early and late apoptosis are inserted in the lower right and upper right panels of Figure 3, respectively. Apoptosis in H28 cells was not confirmed using either fluorescence microscopy (Supplementary Fig. 1A) or Anoikis assays (Supplementary

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Fig. 1B).

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Differences in the growth inhibitory mechanism between H2452 and H28 cells

H2052, H2452, and MESO-1 cells transfected with siROR1 appeared to be led to cell

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death as part of the apoptotic process. However, the mechanism of suppressed colony

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formation by siROR1 in H28 cells remains unclear. Thus, microarray analysis was performed to determine if any differences existed between H28 and H2452 cells

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transfected with siROR1#1 or control siRNA. Decreased mRNA levels (>50%) and the expression value of samples > mean of the negative control are shown in Supplementary

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Fig. 2, and 52 and 166 genes were identified, respectively. However, common downregulated genes in both cell lines included coenzyme Q3 homolog methyltransferase (COQ3) and metaxin 3. Next, we examined the downstream signals after 96-h of exposure to siROR1. Activated AKT and STAT3 were reduced in H2052,

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H2452, and MESO-1 cells treated with siROR1, but these proteins in H28 cells remained unchanged in response to siROR1 (Fig. 4A). TYMS is a molecule known to regulate the sensitivity to pemetrexed20. Microarray

analysis showed TYMS was reduced more than twice in H2452 cells treated with siROR1 (Supplementary Fig. 2). ROR1 has been shown to sustain caveolae and survival signaling as a scaffold of cavin-1 and caveolin-114, which are downstream targets of 10

PTBP3. PTBP3 induces EMT in breast tumor cells and promotes their invasive growth and metastasis21. Caveolin-1 and cyclin-dependent kinase 1 (CDK1) genes have been shown to express big changes in the transcriptome of porcine granulosa cells during the short-term in in vitro cultures22. MNAT1, a CDK-activating kinase complex, is highly expressed in various cancers, with MNAT1-knockdown dramatically decreasing cell

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motility and invasion in colorectal cancer cells23. TOP2A and RAD51AP1, DNA-repair genes, are overexpressed in mesothelioma24. Thus, we examined the mRNA levels of

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TYMS, PTBP3, MNAT1, TOP2A, RAD51AP1, and CDK1 using quantitative RT-PCR. Cells were all treated with siROR1#1 and siROR1#2 (Supplementary Fig. 3). Only

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TYMS was significantly reduced in both H2452 and H28 cells treated with siROR1#1

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and siROR1#2. Next, the protein expression of TYMS in both H2452 and H28 cells was examined using Western blotting (Fig. 4B) and was found to be significantly reduced in

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both cell lines (Fig. 4C).

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Treatment with pemetrexed after siROR1 transfection Although siROR1 inhibited the growth of MM cells, this effect was not considered satisfactory. Since pemetrexed is more effective at lower TYMS levels25, we supposed that the MM cells might be more sensitive to a combination of pemetrexed with siROR1

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than with pemetrexed alone. Several concentrations of pemetrexed with siROR1#1 were shown to significantly reduce cell viability compared with pemetrexed with control siRNA (Fig. 5). MTT assays further showed that the IC50 value of pemetrexed was substantially reduced from 16.8M to 0.15M (Supplementary Fig. 5). Next, we investigated whether sensitivity to cisplatin, one of the standard chemotherapeutic agents for MM, was affected by siROR1. However, after transfection 11

with siROR1#1, cisplatin induced the same degree of growth suppression as the control did in H2452 cells (Supplementary Fig. 6).

Cluster analysis For microarray analysis, ABCG1, ASNS, CALB2, CXCL5, IL1A, RNF4, TOP2B, and

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WNT5A demonstrated decreased mRNA levels (>50%) in H28 cells, with EGR1, TOP2A, and TYMS demonstrated decreased levels in H2452 cells. MAGEA3 and

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NRN1 were shown to have increased mRNA levels (>200%) in H28 cells. ROR2

belongs to the same transmembrane glycoprotein family of ROR1. Because ROR1 and

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ROR2 are reported to be reciprocally regulated and play opposing roles in melanoma

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invasion10, ROR2 was incorporated in cluster analysis. To this end, 40 human MM tumors and four MM cell lines were integrated into the cluster analysis (Supplementary

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Fig. 4). In addition, five normal pleurae, four normal lungs, and one non-malignant mesothelial cell line (Met-5A) were incorporated into the analysis. Samples were

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divided clearly into three large clusters. The color bar above represents the relative expression values, with the green color representing the lowest expression levels, white representing medium expression levels, and red signifying the highest expression levels. Cluster A contained all MM tumor specimens, in which the ROR1, ROR2, ABCG1,

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TOP2B, and CALB2 genes were largely upregulated. Cluster C contained all MM cell lines, which were different cell lines we actually used. Seven genes, including WNT5A, TYMS, TOP2A, IL1A, MAGEA3, ASNS, and RNF4 were found to be significantly expressed in Cluster C, but not in Cluster A (MM tumor specimens) or Cluster B (normal pleura and lung tissues). Thus, MM tumor specimens had relatively high ROR1 expression with variable TYMS expression. Meanwhile, the four cell lines in Cluster C 12

had high TYMS expression with comparatively low ROR1 expression.

Discussion Our results suggested that ROR1 was a possible targetable molecule in MM. Indeed, MM cells treated with siROR1 were more sensitive to pemetrexed than those treated

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with control siRNA. Suppression of TYMS by the ablation of ROR1 may be one of the mechanisms of pemetrexed treatment.

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Our results indicated that normal tissues, including lung and pleura, have low levels of

ROR1 expression (Supplementary Fig. 4), which is consistent with the findings of other

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studies6. Therefore, we believed that ROR1 might represent a targetable molecule in

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MM. The ROR1 signature of the mesothelioma samples that showed ROR1 amplification was clustered with TOP2B, ABCG1, ROR2, and CALB2. Saji et al.

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recently reported that ROR1 is expressed in both MM clinical specimens and cell lines26. Yamaguchi et al. reported that ROR1 is a target of the TTF-1/NKX2-1 lineage-

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survival oncogene and sustains the pro-survival signaling mediated by EGFR in lung adenocarcinoma cells13. The authors also described the interaction of ROR1-CAVIN3 with PI3K-AKT pro-survival signaling in lung cancer cells27. In this study, we showed that AKT and STAT3 were inactivated in three among four MM cell lines treated with

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siROR1. However, phospho-AKT and phospho-STAT3 in H28 cells were similar irrespective of siROR1 exposure. Recently, Obradović et al. reported that ROR1 ablation reduced breast cancer metastatic outgrowth and prolonged survival in preclinical models28. ROR1 signaling was mediated at least partly by Rac1 and Cdc42 to promote the invasion and proliferation of H2452 and H28 cells26. Although the mechanisms of ROR1 inhibition remain unclear, solid tumors, including lung, breast, 13

and pleura, seem to be candidates for ROR1-targeting therapy. A phase I study of the anti-ROR1 monoclonal antibody cirmtuzumab in patients with relapse/refractory CLL was reported29. Cirmtuzumab was found to be safe and effective at inhibiting tumor cell ROR1 signaling in patients with CLL. In addition, treatment of patient-derived xenograft mice with cirmtuzumab and paclitaxel was more effective

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than treatment with either alone30. Currently, a phase I study to investigate the safety of combining cirmtuzumab with paclitaxel for patients with HER2-negative, metastatic

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breast cancer is ongoing (NCT02776917).

A small molecule inhibitor (KAN0439834) targeting ROR1 has also been developed31.

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KAN0439834, in combination with gemcitabine, induced significantly greater

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cytotoxicity in pancreatic cancer cells than KAN0439834 alone32. Thus, the combination of targeting ROR1, using a monoclonal antibody or small molecule, in

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conjunction with a chemotherapeutic drug, is an attractive treatment regimen in preclinical studies. A further study using cirmtuzumab or KAN0439834 instead of

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siROR1 is warranted.

T cells that express the ROR1-specific chimeric antigen receptor (CAR) had improved survival in xenograft models of ROR1-positive human tumors33. An anti-ROR1 CAR-T therapeutic strategy is already in a phase I clinical trial for CLL, mantle cell lymphoma,

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acute lymphoblastic leukemia, non-small cell lung cancer, and triple-negative breast cancer (NCT02706392). Although ROR1 is believed to be only minimally expressed in normal cells34, it has actually been found to be expressed in several normal tissues,

including parathyroid, pancreatic islets, and regions of the esophagus, stomach, and duodenum35. Thus, further ROR1-specific targeted therapy is currently being developed. ROR1-targeted CAR-T cells expressing synthetic Notch receptors for EpCAM or B714

H3, which are expressed on tumor cells but not stromal cells, induce tumor regression without toxicity when ROR1-positive tumor cells and ROR1-positive normal cells are segregated, but not when they are co-localized36. There were some limitations to the present study. First, COQ3 and metaxin 3 mRNA in H2452 and H28 cells were significantly downregulated by siROR1. As COQ3 belongs

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to a class of membrane-binding proteins37 and metaxin is a mammalian mitochondrial outer membrane protein38, they may be conjugated to ROR1. Thus, the association

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between ROR1 and COQ3 or metaxin 3 should be further examined. Second,

microarray analysis showed that PTBP3, MNAT1, TOP2A, RAD51AP1, and CDK1

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were all reduced in H2452 cells treated with siROR1#1. siROR1#2 also suppressed the

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expressions of their mRNA in H2452 cells, although the degree of suppression was slightly different. However, RT-PCR showed that there were some differences in terms

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of the degree of regression in H28 cells treated with siROR1#1 or siROR1#2. Thus, the significance of reducing ROR1 may differ between H2452 and H28 cells. A further

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study elucidating the association between ROR1 and PTBP3, MNAT1, TOP2A, RAD51AP1, or CDK1 is warranted. Third, H2452 cells were shown to have decreased TYMS by microarray analysis (GeneChip Gene ST Array), but this did not show up for H28 using CodeLink Human Whole Genome Bioarray. Two microarray analyses were

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performed at different times (2013 and 2017) and CodeLink Human Whole Genome Bioarray was closed at the time of 2017. The inconsistent results between microarray (Supplementary Fig. 4) and RT-PCR (Supplementary Fig. 3) or Western blot (Fig. 4B, C) might be due to cut-offs/sensitivity used in the normalization etc of the array analysis. Although we cannot validate the result from CodeLink Human Whole Genome Bioarray, RT-PCR and Western blot analyses showed that the decrease of TYMS mRNA 15

and protein levels by the inhibition of ROR1 seemed to be parallel. Fourth, we did not perform in vivo experiments as part of this study. The combination treatment of pemetrexed with a pharmaceutical ROR1 inhibitor in a xenograft model using MM cells should be performed. In conclusion, TYMS seems to be downregulated in response to the suppression of

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ROR1 at least in the MM cell lines, which served to critically increase the sensitivity of MM cells to pemetrexed. Our findings should provide therapeutic insight into treatment

Disclosure of Potential Conflicts of Interest

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options for intractable MM.

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This research was partially supported by Japanese pharmaceutical companies, including Takeda Pharmaceutical Company, Pfizer Inc, Boehringer-Ingelheim, Eli Lilly,

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Ono Pharmaceutical Co. Ltd, Chugai Pharmaceutical, and Nippon Kayaku Co. Ltd. Dr. Takigawa reports grants and personal fees from Eli Lilly Japan, grants and

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personal fees from AstraZeneca, grants and personal fees from Daiichi-Sankyo Pharmaceutical, grants and personal fees from Chugai Pharmaceutical, grants and personal fees from Taiho Pharmaceutical, grants and personal fees from Pfizer Inc. Japan, grants and personal fees from Boehringer-Ingelheim Japan, grants and personal

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fees from Ono Pharmaceutical, grants from Kyowa Hakko Kirin, grants from Nippon Kayaku Co. Ltd, grants from Takeda Pharmaceutical Co. Ltd, personal fees from MSD, personal fees from Bristol-Myers Squibb Company Japan, personal fees from Astellas Japan, personal fees from Eisai Co., Ltd. , outside the submitted work. Other authors have no interests to declare.

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Grant Support This research is supported by Research Project Grant (28-18, 29-114) from Kawasaki Medical School and by a Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology,

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Japan (S1291010).

Acknowledgments

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We thank Prof. Takashi Takahashi and Dr. Tomoya Yamaguchi, Division of Molecular

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Carcinogenesis, Nagoya University Graduate School of Medicine, for their technical

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assistance.

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Figure Legends Fig. 1 A) Morphology of cells treated with siROR1. Cells 72 h after exposure to siROR1#1 were partially agglutinated, although there were other morphological differences depending on the cell line (100× magnification). B) Growth inhibition by siROR1.

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Bars represent relative cell numbers compared with control (mean +/− SD). Cell numbers were significantly reduced by exposure with siROR1. However, the H28 cell

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line alone poorly responded to siROR1 compared with other cell lines.

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Fig. 2 A) Colony formation after exposure to siROR1. Representative images of stained colonies are shown. Although colony formation was

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significantly suppressed in all the cell lines used, MESO-1 cells had, at most, a 20%

B) Relative colony numbers after exposure to siROR1.

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regression.

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Mean colony numbers derived from triplicate wells were calculated. Bars represent mean +/− SD of relative colony numbers compared with control. H28 cell colonies were

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reduced by approximately half by siROR1#1.

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Fig. 3

A) Flowcytometric analysis showing apoptosis after exposure to siROR1.

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The scatter plots show representative flow cytometry analysis data from FITC-Annexin V/PI staining. The proportions of early and late apoptosis are inserted in the lower right and upper right quadrant of each graph. Increased apoptosis was shown in three cell lines, but not for H28 cells.

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Fig. 4

A) Western blotting analysis showing AKT, ERK, and STAT3 signals.

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Cells were treated with siROR1 (#1, #2) or siControl for 96 h, and protein extracts were analyzed by Western blotting. Phospho-AKT (pAKT) and phospho-STAT3 (pSTAT3) were suppressed in H2052, H2452, and MESO-1 cells treated with siROR1. B) Western blotting analysis showing TYMS expression. TYMS was suppressed in H2452 and H28 cells treated with siROR1. C) Relative expression levels of TYMS. 24

The histogram shows the densitometric analysis of the WB bands with a significant

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decrease in levels of TYMS in the cells treated with siROR1.

Fig. 5

Sensitivity of pemetrexed combined with siROR1. Cells were treated with siRNA and pemetrexed (PEM), and cell viability was measured by the trypan blue exclusion assay. Bars show the mean +/− SD of the relative cell number to the treatment without PEM. Some concentrations of PEM with siROR1 significantly reduced cell viability compared with PEM and control siRNA.

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