Background
Mantle cell lymphoma (MCL), an aggressive B cell-derived malignant tumor of the hematological and lymphatic systems, accounts for 6–8% of non-Hodgkin lymphoma [
1] and is associated with poor prognosis with a median overall survival (OS) of 4–5 years [
2]. Most patients with MCL experience a very short regression duration after standard first-line therapy. In spite of various salvage therapies for MCL, such as temsirolimus, bortezomib, ibrutinib, lenalidomide, and autologous hematopoietic stem cell transplantation (ASCT) [
3‐
9], there is still no curative treatment available for MCL. Therefore, it is important to exploit new therapeutic targets or regimens to further improve the prognosis of MCL.
Mer tyrosine kinase (MerTK), also known as RP38, c-Eyk, c-mer, and Tyro12, was first cloned from a human B lymphoblastoid expression library by Graham et al. [
10] and is one of the TAM (Tyro-3, Axl, and MerTK) receptor tyrosine kinase (RTK) family [
11]. As in other RTK family proteins, aberrant expression of MerTK in various malignant tumors, such as melanoma [
12,
13], gastric cancer [
14], leukemia [
15‐
17], and lung cancer [
18], plays a pivotal role in the process of oncogenesis. By binding to its corresponding ligand (Gas6, protein S, tubby, tubby-like protein 1, and galectin-3), auto-phosphorylation sites (Y749, Y753, and Y754) of MerTK are activated and lead to activation of downstream signaling pathways including extracellular signal-regulated kinase (ERK)1/2, AKT, p38, focal adhesion kinase (FAK) and signal transducer and activator of transcription (STAT)6 [
11]. MerTK is not expressed in normal B or T lymphocytes, but in neoplastic B or T lymphocytes [
19]. Within the hematopoietic malignant tumors, ectopic expression of MerTK has been reported in pre-B cell acute lymphoblastic leukemia (B-ALL), T cell acute lymphoblastic leukemia (T-ALL), and acute myeloid leukemia (AML) [
15‐
17]. shRNA-mediated MerTK knockdown or a series of MerTK-selective small molecular inhibitors, such as UNC1062, UNC569, and UNC2025, reduces activation of downstream signaling, inhibits proliferation and invasion, and promotes apoptosis in tumor cells [
12,
14,
20‐
23]. Therefore, ectopic MerTK may be a pivotal promoter in the development of B or T cell-derived hematopoietic malignant tumors.
Differential gene expression analysis between MCL cells and normal B cell populations identified MerTK as an upregulated oncogene in MCL [
24], but there have been no further studies about the function of MerTK in MCL. Our data revealed that MerTK was ectopically expressed in MCL patients and MCL cell lines. MerTK inhibition by either shRNA or treatment with UNC2250, a MerTK-selective small molecular tyrosine kinase inhibitor, suppressed pro-survival signaling, proliferation, invasion, and migration in MCL cells and promoted chemosensitivity to common chemotherapeutic agents. Additionally, UNC2250 promoted apoptosis and induced G2/M phase arrest in MCL cells and significantly delayed disease progression in MCL-cell-derived xenograft models. These results suggest that ectopic MerTK is a novel therapeutic target in MCL.
Methods
Clinical samples and immunohistochemistry (IHC)
Patients’ samples were collected after obtaining informed consent in accordance with the Declaration of Helsinki. All 132 patients were newly diagnosed or had received treatment between January 1, 2001, and June 1, 2017, in the Peking University Cancer Hospital & Institute. Survival analysis was performed on patients treated with R-CHOP-like regimens (R-CHOP, R-CHO, R-CHOPE, and R-mini-CHOP) as first-line therapy and did not undergo ASCT. Overall survival (OS) was defined as the interval between treatment and date of death, and progression-free survival (PFS) was defined as the interval between treatment and disease progression. The detailed clinical characteristics of patients are listed in Additional file
1: Table S1. Immunohistochemistry stains for MerTK were performed as the standard streptavidin-biotin-peroxidase-immunostaining procedure [
18]. Primary antibody to MerTK was listed in Additional file
1: Table S2. This study was approved by the Review Board of the Peking University Cancer Hospital & Institute.
Cell culture and knockdown of MerTK via RNAi
Z-138, Mino, JVM-2, Granta519, JeKo-1, and JVM-13 cell lines were generously provided by Dr. Fu, University of Nebraska Medical Center, USA. All cell lines were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies) and penicillin/streptomycin (Gibco) (complete DMEM; cDMEM). Identification of all MCL cell lines was confirmed by short tandem repeat DNA fingerprinting analysis (Applied Biosystems, Foster City, CA, USA). Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from 20 ml blood from healthy volunteers using Lymphoprep (Axis Shield, Oslo, Norway). Normal human B cells were sorted from the PBMCs using a B Cell Isolation Kit II (Miltenyi Biotec, Mecklenburg-Vorpommern, Germany). Lentiviral vectors (GV248) containing green fluorescent protein (GFP) (shControl) or short hairpin RNA (shRNA) sequence targeting MerTK (shMerTK, Oligo ID: TRCN0000000862; shMerTK 4, Oligo ID: TRCN0000000865) were obtained from Genechem (Shanghai, China). Z-138, Mino, and JVM-2 cells were infected with shControl, shMerTK, or shMerTK 4 at MOI 1: 50 and cultured for > 72 h to be used for the downstream experiments.
Western blot and signaling assays
Cells infected with shControl, shMerTK, or shMerTK 4 were harvested after being cultured for ≥ 72 h. Cells treated with UNC2250 (Selleck, Houston, TX, USA) at indicated concentrations were cultured in cDMEM for 1 h for phosphorylation assays or 24 h for proteins associated with apoptosis and cell cycle. Cell lysates were prepared, and signaling proteins were detected by western blot as previous described [
25]. Primary and secondary antibodies were listed in Additional file
1: Table S2. Immunoblot imaging was performed by image processing and analysis software (Fusion SL; Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany).
Cell proliferation assays
For MerTK knockdown, after being infected with shControl, shMerTK, or shMerTK 4 for > 72 h, cells were plated in triplicate at a density of 2000 cells per 100 μl in 96-well black base microplates and cultured for 0, 24, 48, 72, and 96 h. For sensitivity tests, 2000 cells per 100 μl in 96-well black base microplates were cultured in the absence (vehicle) or presence (dosing) of UNC2250 for 72 h. Viable cells were measured by Cell Titer-Glo Luminescent Cell Viability Assay system (Promega, Madison, WI, USA) according to the manufacture’s protocol, and luminescent signals were measured by LMax II (Molecular Devices, Sunnyvale, CA, USA). Inhibition rates were calculated according to the following formula: inhibition rates = (1 − dosing/vehicle) × 100%.
Invasion and migration assays
Cells were infected with shControl or shMerTK for > 72 h or treated with vehicle or indicated concentrations of UNC2250 for 2 h in FBS-free DMEM. For invasion assays, cells were seeded into Corning BioCoat Matrigel invasion chambers with an 8.0-μm polyethylene terephthalate membrane (Costar; Corning Incorporated, Corning, NY, USA) in 24-well plates; for migration assays, cells were seeded into Transwell with 8.0 μm pore polycarbonate membrane insert (Costar; Corning Incorporated, Corning, NY, USA). Cells in the upper chamber were cultured in FBS-free DMEM, while 30% FBS was added to the lower chamber. After 24 h, cells invading or migrating into the lower chamber were harvested and resuspended in 100 μl DMEM. Afterwards, cells were plated in 96-well black base microplates, and viable cells were measured as in the cell proliferation assays. Invasive abilities or migration abilities were determined by the number of viable cells invading or migrating into the lower chamber.
Apoptosis and cell cycle assays
Cells were treated with vehicle or indicated concentrations of UNC2250 for 12, 24, and 48 h for apoptosis assays or for 24 h for cell cycle analysis. For apoptosis assays, cells were stained with annexin-V-FITC and propidium iodide (PI) (Dojindo Laboratories, Kumamoto, Japan) according to the protocol. For cell cycle assays, cells were stained with PI staining buffer (Sigma–Aldrich, Darmstadt, Germany) as previously described [
25]. All samples for apoptosis and cell cycle assays were analyzed by BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). The results of cell cycle assays were reanalyzed by ModFit LT software (Verity Software House, Topsham, ME, USA).
MCL-cell-derived xenograft model
Female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice aged 7–8 weeks were purchased from HFK Bioscience Co. Ltd. (Beijing, China). Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Peking University Cancer Hospital & Institute. Each NOD/SCID mouse was subcutaneously inoculated with 107 Z-138 cells suspended in 0.1 ml PBS on the right side of the back to establish a subcutaneous tumor model. When the tumor volume grew to an average of 100–150 mm3, mice were randomly distributed into groups according to tumor size and weight of mice. UNC2250 (25, 50, or 75 mg/kg) or saline (vehicle) was administered once daily at a dose of 10 ml/kg by oral gavage. For combination treatment, UNC2250 50 mg/kg or saline (vehicle) was administered once daily at a dose of 10 ml/kg by oral gavage, vincristine 0.5 mg/kg, or doxorubicin 3 mg/kg or vehicle was administered once daily at a dose of 10 ml/kg by intraperitoneal injection. The weight and tumor size of the mice were measured twice weekly after initiation of treatment. Tumor volume was calculated according to the following formula: V = ab2/2 (a and b denote respectively long and short diameters of the tumor). Mice were euthanized upon development of advanced tumor (volume > 3000 mm3 or average tumor volume of a group of animals > 2000 mm3, weight loss > 20%, persistent bleeding, and decreased activity). Tumor tissue samples collected from all groups at 4 h after the last dose were embedded in paraffin for IHC. Phosphorylated MerTK in tumor tissues were detected by IHC.
Chemosensitivity assays
Cells were plated in triplicate at a density of 2000 cells per 100 μl in 96-well black base microplates. For MerTK knockdown, cells infected with shControl or shMerTK were cultured in the absence (vehicle) or presence (dosing) of vincristine or doxorubicin for 72 h. For UNC2250 inhibition, cells were cultured in cDMEM containing vehicle, or vincristine (doxorubicin), or UNC2250, or combination of vincristine (doxorubicin) and UNC2250 at indicated concentrations for 72 h. Inhibition rates were calculated as in the cell proliferation assays. The combination index values were calculated using CalcuSyn software and were based on that described by Chou and Talalay [
26].
Statistical analysis
All experiments in vitro were repeated at least three times. SPSS Statistics version 20 was used to analyze correlation between clinical parameters and MerTK expression in MCL patients. Otherwise, statistical analyses were performed using GraphPad Prism version 6.01. Data were presented as the mean ± SEM. Data were analyzed using an unpaired t test for comparisons of two cohorts. One-way ANOVA was used to analyze the remaining data. P < 0.05 was considered to be significant.
Discussion
Most patients with MCL, a clinically aggressive B cell lymphoma, respond to standard first-line therapies but then relapse within a short duration. Despite a wide variety of agents (temsirolimus, bortezomib, ibrutinib, and lenalidomide) being applied in salvage therapies [
3‐
9], outcomes for relapsed and refractory MCL patients are still far from optimistic. Therefore, the search for new therapeutic targets is ongoing to improve prognosis of MCL patients. Here, for the first time, our data showed that ectopic MerTK expression was seen in a large proportion of MCL tumor samples and four of six MCL cell lines. To establish the function of MerTK in MCL, we conducted MerTK inhibition in vitro and in vivo, by either shRNA or treatment with UNC2250. MerTK inhibition suppressed activation of downstream pro-survival pathways, proliferation, invasion, and migration in MCL cells. In addition, UNC2250 promoted apoptosis and induced G2/M phase arrest in MCL cells and delayed disease progression in MCL-cell-derived xenograft models. Importantly, MerTK inhibition promoted chemosensitivity to common therapeutic agents both in vitro and in vivo.
Previous studies proved that MerTK is not expressed in normal B and T lymphocytes, but in neoplastic B or T lymphocytes (such as B-ALL and T-ALL) [
15,
17], so MerTK may be a therapeutic target in B or T cell-derived malignant tumors. Here, western blot proved that four of six MCL cell lines expressed MerTK ectopically, whereas normal B cells did not express MerTK. For the first time, we analyzed MerTK expression retrospectively in a large number of MCL patients by IHC assays, which revealed that approximately half of patients showed positive expression of MerTK. Loss of miR-335 and miR-126 was revealed as a possible reason for MerTK overexpression in metastatic breast cancer [
27], but our data demonstrated that Z-138, Mino, and JVM-2 cells did not express microRNA-126 and microRNA-335. Further studies are needed to establish the exact mechanism of aberrant MerTK expression in MCL and other malignant tumors. The high expression rate of MerTK in MCL patients provides a specific and applicable population for MerTK targeting therapy. Because first-line therapies affect the prognosis of MCL patients, we only analyzed the relationship between survival and MerTK expression in patients receiving R-CHOP-like regimens. Even though survival analysis indicated that MerTK expression had little effect on OS and PFS of MCL patients who received R-CHOP-like regimens, median OS of the MerTK-positive group (36.5 months) was shorter than the 53.2 months of the MerTK-negative group, which indicated that MCL patients with MerTK expression may experience shorter OS. Studies with larger samples are needed to analyze further the relationship between MerTK expression and prognosis of MCL patients.
Our data showed that Z-138, Mino, and JVM-2 cells expressed Gas6 at both mRNA and protein level. Therefore, MerTK may be activated in MCL cells by its corresponding ligand (Gas6) in an autocrine action [
28,
29]. AKT and p38 are commonly activated in MCL tumors [
30,
31], and elevated levels of phosphorylated AKT and p38 are associated with shorter survival in MCL [
30,
32]. We demonstrated that MerTK inhibition by shRNA or increasing doses of UNC2250 suppressed activation of AKT and p38, thus blocking key nodes of proliferation and pro-survival-related signaling in MCL. Two lentiviral shRNA targeting MerTK (shMerTK and shMerTK 4) were constructed and applied in MerTK knockdown assays, and results of the downstream signaling assays and cell proliferation assays were consistent between the two lentiviral shRNA, which demonstrated that the effects of lentiviral shRNA are indeed due to MerTK knockdown but not due to off-target effects of shRNA. UNC2250 significantly suppressed proliferation of MerTK-positive Z-138, Mino, JVM-2, and JVM-13 cells, but had little effect on MerTK-negative JeKo-1 and Granta519 cells; thus, the effects of UNC2250 on MCL cells are MerTK specific and not due to off-target inhibition. UNC2250 induced different response in suppression of proliferation in JVM-2 cells compared with the other MerTK-positive cell lines; the possible reason is that JVM-2 cell line is a bimodal female cell line immortalized in vitro with Epstein-Barr virus (EBV) with approximately half the cells being pseudodiploid and the other half pseudotetraploid [
33].
MerTK is closely related to tumor cell migration and invasion. In melanoma and glioblastoma cells, MerTK inhibition mediated abrogation of migration and invasion by altering signaling through total and phosphorylated FAK, RhoA, and total and phosphorylated myosin light chain 2 (MLC2) [
12,
34,
35]. In this study, all MCL cell lines are suspension cells; thus, invasive abilities of MCL cells were determined by the ability of secreting secreted extracellular hydrolytic enzymes to digest Matrigel and pass through the membrane to enter the lower chamber. Consistent with previous reports about MerTK in other malignant tumors, MerTK inhibition by either shRNA or treatment with UNC2250 attenuated invasion and migration in MCL cells, accompanied by decrease in phosphorylated FAK and total RhoA.
MerTK not only modulates activation of downstream signaling, but also regulates gene transcription [
15]. In B-ALL, knockdown of MerTK attenuates expression of pro-survival protein kinase C and increases expression of pro-apoptotic proteins (BAX and PUMA) [
15]. Here, we demonstrated that MerTK inhibition by treatment with UNC2250 led to increased expression of pro-apoptotic proteins, including Bax, cleaved caspase 3, and PARP, and decreased expression of pro-survival proteins, including Bcl-2, Mcl-1, and Bcl-xL in MCL cells. For JVM-2 cells, we did not detect cleaved PARP when apoptosis was obvious, so apoptosis in JVM-2 cells may not depend on the classical caspase pathway. Furthermore, cell-cycle-associated proteins including cyclin B1 and phosphorylated Cdc-2 also decreased due to MerTK inhibition by treatment with UNC2250, accompanied by G2/M phase arrest in MCL cells.
Although MerTK inhibition by shRNA or treatment with UNC2250 suppressed oncogenic potential of MCL cells in vitro, UNC2250 mediated potent but limited effects on MCL-cell-derived xenograft mouse models. UNC2250 at a dose of 75 mg/kg produced ~ 50% inhibition of tumor growth relative to the vehicle-treated group in MCL-cell-derived xenograft mouse models, indicating that effects of UNC2250 monotherapy were limited. However, we demonstrated here that MerTK inhibition sensitized MCL cells to treatment with vincristine in vitro and doxorubicin both in vitro and in vivo. Thus, combination of MerTK inhibition and common chemotherapeutic agents produced greater effects on MCL cells, suggesting that co-treatment with MerTK inhibition and common chemotherapeutic agents could be a new strategy to treat MCL.
Conclusions
In summary, our data revealed that MerTK was ectopically expressed in MCL. MerTK inhibition by either shRNA or treatment with UNC2250 apparently suppressed oncogenic potential in MCL cells, delayed disease progression in MCL-cell-derived xenograft models, and prompted chemosensitivity to vincristine and doxorubicin in vitro or in vivo. Therefore, ectopic MerTK may be a novel therapeutic target in MCL, and it is essential to perform pre-clinical or even clinical studies on UNC2250 or new MerTK inhibitors.
Acknowledgements
The authors thank Dr. Fu from the University of Nebraska Medical Center in USA for the kind gifts of the MCL cell lines, Dr. Bin Dong from the Peking University Cancer Hospital & Institute for the analysis of immunohistochemistry stains, and Dr. Xijuan Liu from the Peking University Cancer Hospital & Institute for the analysis of flow cytometry.