When RON MET TAM in Mesothelioma: All Druggable for One, and One Drug for All?

Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer with a poor survival rate. Treatment options are limited at best and drug resistance is common. Thus, there is an urgent need to identify novel therapeutic targets in this disease in order to improve patient outcomes and survival times. MST1R (RON) is a trans-membrane receptor tyrosine kinase (RTK), which is part of the c-MET proto-oncogene family. The only ligand recognized to bind MST1R (RON) is Macrophage Stimulating 1 (MST1), also known as Macrophage Stimulating Protein (MSP) or Hepatocyte Growth Factor-Like Protein (HGFL). In this study, we demonstrate that the MST1-MST1R (RON) signaling axis is active in MPM. Targeting this pathway with a small molecule inhibitor, LCRF-0004, resulted in decreased proliferation with a concomitant increase in apoptosis. Cell cycle progression was also affected. Recombinant MST1 treatment was unable to overcome the effect of LCRF-0004 in terms of either proliferation or apoptosis. Subsequently, the effect of an additional small molecular inhibitor, BMS-777607 (which targets MST1R (RON), MET, Tyro3, and Axl) also resulted in a decreased proliferative capacity of MPM cells. In a cohort of MPM patient samples, high positivity for total MST1R by IHC was an independent predictor of favorable prognosis. Additionally, elevated expression levels of MST1 also correlated with better survival. This study also determined the efficacy of LCRF-0004 and BMS-777607 in xenograft MPM models. Both LCRF-0004 and BMS-777607 demonstrated significant anti-tumor efficacy in vitro, however BMS-777607 was far superior to LCRF-0004. The in vivo and in vitro data generated by this study indicates that a multi-TKI, targeting the MST1R/MET/TAM signaling pathways, may provide a more effective therapeutic strategy for the treatment of MPM as opposed to targeting MST1R alone.


TMA Patients and histological subtypes
Zurich TMA Tissue specimens of 352 patients with MPM, diagnosed between 1975 and2004, were sent to the Zurich Pneumoconiosis research group for mineralogical assessment of dust exposure, in particular, asbestos, and later included in this study. The total cohort comprised 114 epithelioid, 192 biphasic and 46 sarcomatoid histotypes. A total of 77% of the specimens were derived from autopsy and 23% from biopsy specimens. Tissue was processed according to the guidelines of the Swiss Society of Pathology. All cases were entirely reviewed for histotype classification by two pathologists (33). Clinical data were retrospectively assessed from medical archives of the different hospitals and the local cancer registries. The construction of the TMA was conducted after formal approval from the relevant Hospital Ethics Committee (Stv.29-2009).
Further clinical data could be retrieved from 206 of these 352 patients (59%), who were mainly males (94%). The median age was 62 years. Asbestos exposure was known in 97 patients (47%). Disease was located in 52% of the patients on the right, 36% on the left and 3% on both sides. As tumour stage was not documentable in most cases, a retrospective staging was performed for 102 patients, based on the surgical pathology reports according to the IMIG staging system, stage T4 being predominant accounting for 71%. Both regional and mediastinal lymph nodes as well as distant metastases were present in 90% of the patients. Only 30% of the patients received therapy, 70% were treatment-naıve. Treatment was surgical for 67 patients and comprised 26 extrapleural pneumonectomies, 16 pleurectomies and 25 palliative procedures, such as talc pleurodesis or tumour debulking. A total of eight patients received only chemotherapy in different combinations, mostly platinum-based. Four patients received radiation therapy. The median overall survival of the 128 patients with complete follow-up data was 11.7 months (95% confidence interval (CI): 10.0-13.6).

Sydney TMA
This series consisted of 80 MPM patients who underwent extrapleural pneumonectomy (EPP) at Royal Prince Alfred (RPAH) or Strathfield Private Hospital (SPH) between 1994 and 2009. All diagnoses of MPM and the subtypes were confirmed by a panel of experienced pathologists from whole sections. A biphasic histological subtype was assigned if both epithelioid and sarcomatoid components were present, and exceeded 10% of the cross sectional area in the slides examined.

TMA construction
Zurich: A set of three tissue microarrays (TMA) with quadruple punches per patient (total n = 1408) was accomplished with a custom-made, semi-automatic tissue arrayer (Beecher Instruments, Sun Prairie, WI, USA), as previously described (34). Sections (4.5-µm thick) of TMA blocks were transferred to an adhesive-coated slide system (Instrumedics, Hackensack, NJ, USA) supporting the cohesion of 0.6 mm array elements on glass.
Sydney: A TMA was generated with five to six cores of 1 mm diameter per patient using an Advanced Tissue Arrayer, ATA-100 (Chemicon, USA). Serial 4-µm thick paraffin sections were transferred to slides for immunohistochemical analysis.

TMA immunohistochemistry
De-paraffined sections were stained either manually or on a Ventana (Ventana Medical Systems

TMA Data interpretation and statistical analysis
Zurich: The intensity level of immunoreactivity of the MST1R, MST1 proteins and CD68 macrophage was scored semi-quantitatively: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) in the tumor cell cytoplasm, by two observers (A.S. and C.W.). A mesothelioma-cell-associated signal was considered if three or more cells were positive. A global sum score was created from the four cores and was dichotomised closest to the median into low and high. Clinical data were retrospectively assessed as completely as possible from the medical archives of different hospitals and the local cancer registry. The material of the patients came mainly from autopsies and there was no standardised therapy during this time period. Associations and correlations with clinico-pathologic parameters were examined by chi-squared and Kendall's taub tests, respectively, for both nondichotomised as well as dichotomised parameters. OS was calculated by the Kaplan-Meier method and survival time differences were compared using the log-rank test. Significant parameters were further analysed by multivariate Cox proportional hazards regression models to test for independency. All analyses were carried out using the SPSS 16.0.1 software package (SPSS Inc., Chicago, IL, USA).
Sydney: The intensity of staining was assessed for Tyro3 was scored by two observers (SK, KG) and stratified for >10% cells stained. OS was calculated by the Kaplan-Meier method and survival time differences were compared using the log-rank test.

Cellular Viability (High content analysis)
NCI-H226 cells were seeded at 2.5 × 103/well in a 96-well plate and adhered overnight. Cells were treated for 24 -72 h with human recombinant MST1 (250 ng/mL) and LCRF-0004 (200 nM) alone or in combination. Live cells were stained with propidium iodide (PI) (1 μg/mL) and Hoechst (Bisbenzimide H 33342) (5 μg/mL) for 30 min at 37°C. Cells were imaged on a Cytell Cell Imaging System (GE Healthcare Bio-Sciences, PA, USA) and analysed using IN Cell Investigator software (GE Healthcare). Algorithms were set to identify those as PI positive as dead. Various parameters were quantified using these algorithms including cell number and viable cell number.

Cellular Apoptosis (High content analysis)
NCI-H226 cells were seeded at 2.5 × 103/well in a 96-well plate and adhered overnight. Cells were treated for 24 -72 h with human recombinant MST1 (250 ng/mL) and LCRF-0004 (200 nM) alone or in combination. Live cells were stained with PI (1 μg/mL), Hoechst (5 μg/mL) and AnnexinV (1:40) (Enzo Life Sciences, Inc., NY, USA) for 30 min at 37°C, imaged using the Cytell and analysed using the IN Cell Investigator software. Algorithms were set to identify (i) apoptotic but live as those stained with AnnexinV only, (ii) apoptotic (dead) stained with both AnnexinV and PI, and (iii) necrotic stained with PI only.

Cellular Apoptosis (FACS)
NCI-H226 cells were seeded in 6-well plates at a density of 1x10 5 cells per well and were allowed to adhere overnight. Following overnight incubation, cells were treated with appropriate concentrations of drug, diluted in cell culture media, for a further 48 h. Where appropriate, control cells were treated with either vehicle or left untreated with media only. Following treatment, culture media was removed, transferred to labelled FACS tubes and placed on ice. Adhered cells were then trypsinised and transferred to corresponding FACS tubes. Cells were pelleted by centrifugation at 1300 rpm for 3 min and the supernatant removed. The cells were washed in 1 mL 1X binding buffer (BB) diluted in ice cold PBS, pelleted by centrifugation and re-suspended in 100 µL BB. Two µL Annexin V (IQproducts) was added to each tube, with the exception of the negative control and media only samples, and cells were incubated at 4°C for 20 min, protected from light. Cells were again washed in 1 mL 1X binding buffer and supernatant removed. Immediately before analysis by FACS, cells were re-suspended in 400 µL BB containing 0.5 µg/mL PI (Invitrogen), except the negative control and FMO for PI for which BB alone was used, and apoptotic cells were analyzed by Flow cytometry, using a CyAn™ ADP flow cytometer and Summit software (Dako, CO, USA).

Cell cycle analysis (FACS)
Cells were seeded at 1x10 5 in full serum media and allowed to adhere overnight. Subsequently, cells were serum starved (0.5% FBS media) for 24 h and treated with either DMSO (vehicle control) or LCRF-0004 (200 nM) for 24 and 48 h. Cellular supernatants were harvested, as was the PBS wash. Cells were trypsinised and diluted in a further 5 mL PBS and added to appropriate PBS/supernatant tube. Samples were centrifuged for 5 min and pellets re-suspended in 1 mL ice cold 70% EtOH (while slowly vortexing). Samples were stored at -20°C for 20 min and centrifuged. The EtOH supernatant was removed and the pellet washed in 5 mL PBS. Cells were pelleted and re-suspended in a PBS staining solution containing 10 mg/mL RNAse A and 1 mg/mL PI, and incubated at 37°C for 1 h. Samples were run on a BD LSR II flow cytometer (BS Biosciences, San Jose, CA, USA) and analysed using FlowJo vX software (Ashland, OT, USA). Plots were graphed based on PI Area (PI A) versus count as a histogram.

Supplementary Figures and Tables
Supplementary       Figure S6. Analysis of macrophage infiltration and survival using macrophage marker, CD68.
(A) Immunohistochemical staining of an MPM TMA for macrophages using CD68 staining, at 10x and 20x magnification for a positive core. (B) Following scoring by two pathologists and dichotomized closest to the median into low and high using >2, </=2 global scores, the results were analyzed using Cox regression for survival on (n=130) patients for which clinical data was available. No survival benefit was observed. (C) Analysis of the mesothelioma TCGA dataset confirming that CD68 gene expression is not associated with any survival benefit in patients with MPM.