MPM Cell Lines Research Articles

BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

AbstractMalignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and the identification of novel druggable targets is urgently needed. In previous work, we identified 15 deregulated genes highly expressed in MPM tissues and correlated with a poor prognosis. Here, we validated these findings on an independent dataset of 211 MPM patients (EGA, EGAD00001001915) and on a panel of MPM cell lines. Furthermore, we carried out in vitro gene silencing followed by proliferation, cytotoxicity, caspase, and migration assays to define whether these targets could be cancer-driver genes. We ended up with three novel candidates (i.e., BAG2, MAD2L1, and MDK), whose encoded proteins could be exploited as druggable targets. Moreover, of novelty, immunohistochemistry analysis on tissues revealed that the overexpression of BAG2 and MAD2L1 could differentiate MPM from RMP patients. Furthermore, when we tested Neratinib (an inhibitor of MAD2L1) and iMDK (an inhibitor of MDK) we found that they are effective on MPM cells, in part phenocopying the effects of MAD2L1 and MDK gene silencing. In summary, in the present work, we report that BAG2, MAD2L1, and MDK are bona fide cancer-driver genes for MPM worth of further studies.

FK228 suppress the growth of human malignant pleural mesothelioma tumor independent to epithelioid or non-epithelioid histology

Human malignant pleural mesothelioma (hMPM) is an aggressive, rare disease with a poor prognosis. Histologically, MPM is categorized into epithelioid, biphasic, and sarcomatoid subtypes, with the epithelioid subtype generally displaying a better response to treatment. Conversely, effective therapies for the non-epithelioid subtypes are limited. This study aimed to investigate the potential role of FK228, a histone deacetylase inhibitor, in the suppression of hMPM tumor growth. We conducted a comprehensive analysis of the histological and molecular characteristics of two MPM cell lines, CRL-5820 (epithelioid) and CRL-5946 (non-epithelioid). CRL-5946 cells and non-epithelioid patient-derived xenografted mice exhibited heightened growth rates compared to those with epithelioid MPM. Both CRL-5946 cells and non-epithelioid mice displayed a poor response to cisplatin. However, FK228 markedly inhibited the growth of both epithelioid and non-epithelioid tumor cells in vitro and in vivo. Cell cycle analysis revealed FK228-induced G1/S and mitotic arrest in MPM cells. Caspase inhibitor experiments demonstrated that FK228-triggered apoptosis occurred via a caspase-dependent pathway in CRL-5946 but not in CRL-5820 cells. Additionally, a cytokine array analysis showed that FK228 reduced the release of growth factors, including platelet-derived and vascular endothelial growth factors, specifically in CRL-5946 cells. These results indicate that FK228 exhibits therapeutic potential in MPM by inducing cytotoxicity and modulating the tumor microenvironment, potentially benefiting both epithelioid and non-epithelioid subtypes.

Evening Primrose Extract Modulates TYMS Expression via SP1 Transcription Factor in Malignant Pleural Mesothelioma.

To determine the mechanism of EPE in downregulating TYMS in MPM cancer. The TYMS mRNA expression with epithelial-to-mesenchymal transition biomarkers and nuclear factor SP1 was assessed using the GEO database in a data set of MPM patients (GSE51024). Invasive MPM cell lines were in vitro models for the investigation of TYMS expression after EPE treatment. The tyms promoter SP1 binding sequences were determined using Genomatix v 3.4 software Electrophoretic mobility shift and dual-luciferase reporter assays revealed specific SP1 motifs in the interaction of EPE and reference compounds. Chromatin immunoprecipitation and Re-ChIP were used for the co-occupancy study. In MPM patients, a positive correlation of overexpressed TYMS with mesenchymal TWIST1, FN1 and N-cadherin was observed. EPE and its major components, gallic and ellagic acid (GA and EA, respectively), downregulated TYMS in invasive MPM cells by interacting with particular SP1 motifs on the tyms promoter. The luciferase constructs confirmed the occupation of two SP1 regulatory regions critical for the promotion of TYMS expression. Both EPE and reference standards influenced SP1 translocation into the nucleus. EPE components reduced TYMS expression by occupation of SP1 motifs on the tyms promoter and reversed the EMT phenotype of invasive MPM cells. Further in-depth analysis of the molecular docking of polyphenol compounds with SP1 regulatory motifs is required.

Changes in expression of mesothelial BBS genes in 2D and 3D after lithium chloride and ammonium sulphate induction of primary cilium disturbance: a pilot study

BackgroundMalignant pleural mesothelioma (MPM), a rare and aggressive pleural tumor, has significant histological and molecular heterogeneity. Primary Cilium (PC), an organelle of emerging importance in malignancies, has been scarcely investigated in MPM. A critical molecular complex for the PC function is the BBSome and here we aimed at assessing its expression patterns in ordinary 2D and spheroid 3D cell cultures.MethodsA human benign mesothelial cell line (MeT-5A), MPM cell lines (M14K, epithelioid MPM; MSTO, biphasic MPM), and primary MPM cells (pMPM) were used. Primers specific for the human BBS1, 2, 4, 5, 7, 9, 18 transcripts were designed, and quantitative real-time PCR (qRT-PCR) was done with β-actin as the gene of reference. The relative gene expression across 2D and 3D cultures was analyzed by the expression factor (mean of 1/ΔCt values). With the 2–∆∆Ct method the gene expression fold changes were assessed from qRT-PCR data. Molecular changes using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) were also determined.ResultsPC was present in all cells used in the study at approximately 15% of the observed area. BBSome transcripts were differentially expressed in different dimensions of cell culture (2D vs. 3D) in all cell lines and pMPM. Treatment with AS and LC affected the expression of the ciliary BBS2 and BBS18 genes in the benign as well as in the MPM cells.ConclusionsThese data indicate distinct BBSome molecular profiles in human benign and MPM cells cultured in 2D and 3D dimensions and support the notion that PC genes should be investigated as potential MPM therapeutic targets.

Abstract 1760: ADI-PEG20 and GC7 as a novel anti-metabolite strategy for the treatment of malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with less than 10% of patients surviving 5-years from diagnosis. Arginine deprivation using pegylated arginine deiminase (ADI-PEG20) is a novel treatment for mesothelioma deficient in the arginine biosynthetic enzyme ASS1. However, resistance to ADI-PEG20 in MPM is a significant problem and is characterized in part by reprogramming of the urea and polyamine metabolism pathway (Locke et al., 2016). We sought to identify a targeted metabolism-based therapy to overcome the emerging resistance and increase the efficiency of ADI-PEG20 treatment for patients with MPM. New models of ADI-PEG20 resistance were generated by constant exposure of MPM cell lines to arginine deprivation for 6 months. ADI-PEG20 resistant and sensitive MPM cell lines were treated with a panel of polyamine targeting molecules. Metabolome alterations were studied by targeted metabolomics and glucose flux analyses. Our results confirmed the up-regulation of ASS1 expression from (50 to 3000-fold) alongside upregulation of polyamine catabolism and upon generation of ADI-PEG20 resistance. Treatment of ADI-PEG20 resistant cell lines with a range of different polyamine inhibitors demonstrated that ADI-PEG20 resistant cell lines were significantly more sensitive to the spermidine-analogue GC7 than their respective ASS1-ve parental cell lines. Moreover, the combination of GC7 and ADI-PEG20 prevented the emergence of resistant cells in vitro. We observed a significant synergistic effect of GC7 and ADI-PEG20 in both ASS1-ve and ASS1+ve cell lines in 2D and 3D cell culture models as well as in our mice in vivo model. Metabolomics analysis revealed that GC7 treatment significantly impaired the TCA cycle. We hypothesize that GC7 is repurposing the fate of Acetyl-CoA to polyamine catabolism therefore shutting down the TCA cycle. Our findings provide novel insights into a polyamine targeted therapy that may prevent resistance to ADI-PEG20 treatment in MPM and related arginine-auxotrophic cancers. Citation Format: Joséphine Carpentier, Peter W. Szlosarek, Sarah Anne Martin. ADI-PEG20 and GC7 as a novel anti-metabolite strategy for the treatment of malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1760.

TROP2 expression and SN38 antitumor activity in malignant pleural mesothelioma cells provide a rationale for antibody-drug conjugate therapy

ObjectivesMalignant pleural mesothelioma (MPM) is an aggressive cancer which at large is not amenable to curative surgery. Despite the recent approval of immune checkpoint inhibitor therapy, the response rates and survival following systemic therapy is still limited. Sacituzumab govitecan is an antibody-drug conjugate targeting the topoisomerase I inhibitor SN38 to trophoblast cell-surface antigen 2 (TROP-2)-positive cells. Here we have explored the therapeutic potential of sacituzumab govitecan in MPM models. Materials and methodsTROP2 expression was analyzed in a panel of two well established and 15 pleural effusion derived novel lines by RT-QPCR and immunoblotting, TROP2 membrane-localization was studied by flow cytometry and immunohistochemistry. Cultured mesothelial cells and pneumothorax pleura served as controls. The sensitivity of MPM cell lines to irinotecan and SN38 was studied using cell viability, cell cycle, apoptosis and DNA damage assays. Drug sensitivity of cell lines was correlated with RNA expression of DNA repair genes. Drug sensitivity was defined as an IC50 below 5 nM in the cell viability assay. ResultsTROP2 expression was detected at RNA and protein level in 6 of the 17 MPM cell lines, but not in in cultured mesothelial control cells or in the mesothelial layer of the pleura. TROP2 was detectable on the cell membrane in 5 MPM lines and was present in the nucleus in 6 cell models. Ten of 17 MPM cell lines showed sensitivity to SN38 treatment, among those 4 expressed TROP2. High AURKA RNA expression and high proliferation rate correlated with sensitivity to SN38-induced cell death, DNA damage response, cell cycle arrest and cell death. Sacituzumab govitecan treatment effectively induced cell cycle arrest and cell death in TROP2-positive MPM cells. ConclusionTROP2 expression and sensitivity to SN38 in MPM cell lines support biomarker-selected clinical exploration of sacituzumab govitecan in patients with MPM.

Patient-Level Omics Data Analysis Identifies Gene-Specific Survival Associations for a PD-1/PD-L1 Network in Pleural Mesothelioma

Immune checkpoint blockade targeting PDCD1 (PD-1) or CD274 (PD-L1) has demonstrated efficacy and interest across multiple cancers. However, the exact determinants of the response and cancer-specific molecular features remain unclear. A recent pan-cancer study identified a PDCD1/CD274-related immunotherapy network of 40 genes that had differential patient survival associations across multiple cancers. However, the survival relevance of this network in mesothelioma could not be assessed due to a lack of available survival data for the mesothelioma study included. Mesothelioma, a rare cancer that most commonly arises in the pleural membranes around the lung, does have immune checkpoint blockade as an approved treatment strategy, yet questions over its efficacy remain. RNA-seq data from 87 pleural mesothelioma patients were interrogated on cBioPortal to assess the role of the PDCD1/CD274 network identified in a previous study, in addition to identifying repurposed drugs that may have therapeutic efficacy. Extensive literature searches were conducted to identify known information from the literature around the genes shown to impact patient survival (CCR5, GATD3A/GATD3, CXCR6, GZMA, and TBC1D10C). The same literature validation was performed for putative repurposed drugs that were identified as potential immunotherapeutic adjuvants in the context of mesothelioma (disulfiram, terfenadine, maraviroc, clioquinol, chloroxine, and oxyphenbutazone). Only disulfiram returned a specifically focused research article based on the literature search. This article demonstrated cytotoxicity in a panel of five human MPM cell lines of mixed histology (epithelioid, biphasic, and sarcomatoid). There was little information on the remaining five drugs, yet the clear preclinical efficacy of disulfiram validates the methodology used herein and prompts further exploration of the remaining drugs in mesothelioma. This study ultimately sheds light on novel preclinical information of genes related to PDCD1/CD274 in mesothelioma, as well as identifying putative drugs that may have therapeutic efficacy either independently or as an immunotherapeutic adjuvant.

Exploring the mechanism of miR320a in regulating PDL1 upon lung cancer pathogenesis.

The tumour suppressive role of miRNA320a is observed in many cancer types like in colon, lung, breast, and osteosarcoma but it is inversely reported in prostate cancer and in MPM cell lines. miRNA320a targets programmed death ligand 1 (PDL1) negatively in many cancer types and recently in Malignant pleural mesothelioma. In this background it is important to understand the regulatory mechanism of miRNA320a in determining PDL1 expression in different pathological stages of lung cancer. Histology was used to grade the initial and advanced stage of lung cancer following carcinogenic injection. Immunohistochemistry and Western blotting technique were used to analyse PDL1 protein expression. In-situ hybridization was used to determine miRNA320a signals. Initially, using the chemical carcinogen Diethylnitrosamine (25 μg/g), we successfully initiate initial and advanced stage of lung cancer following 6 months and 9 months of carcinogenic injection. The formation of initial and advanced stage of lung cancer is confirmed through histopathological changes which show neoplastic appearance in initial lung cancer and appearance of more mitotic cells along with tissue hardness in the advanced lung cancer stages. In miRNA320a blocked tissue the cancer condition becomes worse with decreased tissue elasticity along with more proliferative cells. Immunohistochemistry and Western blotting studies show that PDL1 is overexpressed in the advanced stages rather than in initial lung cancer because the expression of miRNA320a is overexpressed in initial stages but restricted in advanced stages of lung cancer. miRNA32a blocking studies confirm that miRNA320a expression act as a tumour suppressor that directly controls PDL1 expression that lack of miRNA320a enhances PDL1 expression as well as it triggers lung cancer advances. In summary, miRNA320a possess tumour suppressor function that limits PDL1 expression in initial lung cancer but its control over PDL1 suppression is lost once miRNA320a is downregulated in advanced stage of lung cancer.

P2‐18: Establishment of acquired pemetrexed‐resistant MPM cell lines and evaluation of an IGF‐1R inhibitor picropodophyllin for MPM treatment

P2‐18: Establishment of acquired pemetrexed‐resistant MPM cell lines and evaluation of an IGF‐1R inhibitor picropodophyllin for MPM treatment

Abstract 1918: Coxsackievirus A11 elicits a potent oncolytic activity in human malignant pleural mesothelioma via ICAM-1 receptor

Abstract Malignant pleural mesothelioma (MPM) is an aggressive solid cancer with dismal prognosis. Oncolytic virotherapy is a promising anticancer treatment that directly lyses tumor cells. Coxsackievirus A11 (CVA11) is an emerging oncolytic virus against solid cancers. Herein we tested whether CVA11 infection has an oncolytic activity in human MPM cell lines and identified its cognate receptor. We found that CVA11 infection shows a remarkable oncolytic activity in six of six human MPM cell lines. MPM cells having higher intercellular adhesion molecule-1 (ICAM-1) expression showed higher CVA11-induced cytotoxicity, and that identified ICAM-1 as its surface receptor because coinfection with neutralizing ICAM-1 antibody substantially abrogated CVA11-triggered cytotoxicity in MPM cells. In addition, we unraveled that the activations of both phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) signaling pathways partially but significantly contributed to the CVA11-triggered cytotoxicity of CVA11 as evidenced by treating with respective inhibitors. Taken together, our findings show a novel oncolytic virotherapy using CVA11 could be an alternative therapeutic modality for human MPM. Citation Format: Koji Okamura, Hiroyuki Inoue, Yuki Ikematsu, Rie Furukawa, Keiichi Ota, Yasuto Yoneshima, Eiji Iwama, Kentaro Tanaka, Isamu Okamoto. Coxsackievirus A11 elicits a potent oncolytic activity in human malignant pleural mesothelioma via ICAM-1 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1918.

Abstract 2011: Tumor treating fields induce cellular and morphologic changes including disruption of intercellular communication networks in malignant pleural mesothelioma

Abstract Tumor Treating Fields (TTFields) is a novel therapeutic strategy that uses alternating electric fields to disrupt mitosis in actively dividing cells through exertion of dielectrophoretic force and dipole alignment on microtubule subunits. However, the additional effects of TTFields on cellular morphology and communication remain unclear. Tunneling nanotubes (TNTs) are ultrafine F-actin-based protrusions that facilitate intercellular communication through cell-cell contact, including efficient transport of molecular cargo that accelerate invasive potential and chemoresistance. We hypothesized that by creating dielectrophoretic force on polar actin subunits, treatment with TTFields would lead to sustained disruption or prevention of formation of MPM TNTs. TTFields (200 kHz) were applied at 0.5 or 1.0 V/cm to VAMT and MSTO MPM cell lines using the Inovitro system (Novocure). TNT index (average # of TNTs/cell) was determined at 0, 24, 48, and 72 hours of TTFields application. At the 72 hour period, TTFields were discontinued and assessment for recovery of TNT formation was performed after an additional 24 hours. Cell viability was determined by staining with NucGreen 488 dye. We also used time-lapse microscopy with concurrent application of TTFields and the chemotherapeutic agents cisplatin and pemetrexed to analyze effects on TNTs and functional cargo transfer. Application of continuous TTFields at 1.0 V/cm, but not at 0.5 V/cm, suppressed TNT formation by 48.9% in MSTO (p=0.005). This suppression was achieved at the 48-hour time point and was independent of cell proliferation. No significant differences in TNT index were noted for VAMT. Cell viability was consistently above 95% at all time points for both cell lines at the stated frequency and intensities. Cargo transfer rates were lower in experimental groups treated with TTFields and either cisplatin, pemetrexed, or both. Here, we show that treatment with TTFields suppresses formation of TNTs between MSTO cells, but not VAMT, suggesting additional factors that may determine susceptibility to TTFields treatment. Additionally, TTFields treatment of MSTO decreased the function of TNTs in these cells, as demonstrated by lower cargo transfer rates. In sum, these data identify effects of TTFields on TNTs as a novel mechanism for this therapeutic modality. Citation Format: Akshat Sarkari, Sophie Korenfeld, Katherine Ladner, Phillip Wong, Antonia Martinez, Eyal Dor-On, Moshe Giladi, Amrinder Nain, Emil Lou. Tumor treating fields induce cellular and morphologic changes including disruption of intercellular communication networks in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2011.

Impact of metallothionein-knockdown on cisplatin resistance in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rare, but aggressive tumor with dismal prognosis. Platinum-based chemotherapy is regularly used as part of multimodality therapy. The expression of metallothioneins (MT) has been identified as a reason for cisplatin resistance, which often leads to early therapy failure or relapse. Thus, knockdown of MT expression may improve response to cisplatin treatment. The MT gene- and protein expression of the MPM-cell lines MSTO-211H, NCI-H2052 and NCI-H2452 and the human fibroblast cell line MRC-5, as well as their sensitivity to cisplatin treatment have been evaluated. Knockdown of MT1A, 1B and 2A expression was induced by RNA interference. MT expression was measured using quantitative real-time PCR. An in vitro Assay based on enzyme activity was used to detect cell viability, necrosis and apoptosis before and after incubation with cisplatin. MT2A gene expression could be detected in all MPM cell lines, showing the highest expression in NCI-H2452 and NCI-H2052, whereas gene expression levels of MT1A and MT1B were low or absent. The immunohistochemically protein expression of MT-I/II reflect MT2A gene expression levels. Especially for MSTO-211H cell presenting low initial MT2A levels, a strong induction of MT2A expression could be observed during cisplatin treatment, indicating a cell line-specific and platin-dependent adaption mechanism. Additionally, a MT2A-dependent cellular evasion of apoptosis during cisplatin could be observed, leading to three different MT based phenotypes. MSTO-211H cells showed lower apoptosis rates at an increased expression level of MT2A after cisplatin treatment (from sixfold to fourfold). NCI-H2052 cells showed no changes in MT2A expression, while apoptosis rate is the highest (8–12-fold). NCI-H2452 cells showed neither changes in alteration rate of MT2A expression nor changes in apoptosis rates, indicating an MT2A-independent resistance mechanism. Knockdown of MT2A expression levels resulted in significantly induced apoptotic rates during cisplatin treatment with strongest induction of apoptosis in each of the MPM cell lines, but in different markedness. A therapeutic meaningful effect of MT2A knockdown and subsequent cisplatin treatment could be observed in MSTO-211H cells. The present study showed MT2A to be part of the underlying mechanism of cisplatin resistance in MPM. Especially in MSTO-211H cells we could demonstrate major effects by knockdown of MT2A expression, verifying our hypothesis of an MT driven resistance mechanism. We could prove the inhibition of MT2A as a powerful tool to boost response rates to cisplatin-based therapy in vitro. These data carry the potential to enhance the clinical outcome and management of MPM in the future.

Abstract 3954: Validation of the extracellular matrix protein fibulin-3 as a molecular target in malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor of the pleura with poor prognosis and limited therapeutic options. Fibulin-3 (gene EFEMP1) is an extracellular matrix protein found in the parenchyma and pleural effusions of MPM, which has been proposed as prognostic biomarker complementing diagnostic biomarkers -such as mesothelin- for this cancer type. However, the functions and potential mechanisms of fibulin-3 in MPM remain completely unknown. To further define the relevance of fibulin-3 in MPM we performed gain- and loss-of-function experiments by respectively overexpressing fibulin-3 in normal mesothelial cells or knocking down its expression in MPM cells, followed by evaluation of cell viability, colony formation, invasion and chemoresistance. We also evaluated changes in gene expression and signaling mechanisms after fibulin-3 downregulation. Furthermore, a novel anti-fibulin-3 antibody was developed and tested for its ability to recognize fibulin-3 in mesothelioma, inhibit pro-tumoral signaling, and disrupt tumor growth in orthotopic MPM models. Fibulin-3 downregulation decreased viability, clonogenic capacity, and invasiveness of MPM cell lines, whereas overexpression of this protein increased the same phenotypic traits in normal mesothelial cells. At the molecular level, fibulin-3 regulated the activation of PI3K/Akt and NFkB signaling and correlated with a gene expression signature required for cell adhesion and motility, matching its cellular effects. Loco-regional delivery of anti-fibulin-3 had a marked cytostatic effect and significantly increased median survival and the number of long-term surviving animals with stable disease. Our work reveals that fibulin-3 is a relevant therapeutic target in mesothelioma, adding to its relevance as prognostic biomarker and encouraging further development of anti-fibulin-3 targeted therapies for this cancer type. Citation Format: Arivazhagan Roshini, Chandra Goparaju, Mohan S. Nandhu, Sharon L. Longo, John A. Longo, Joan Chou, Frank A. Middleton, Harvey I. Pass, Mariano S. Viapiano. Validation of the extracellular matrix protein fibulin-3 as a molecular target in malignant pleural mesothelioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3954.

Involvement of the M-CSF/IL-34/CSF-1R pathway in malignant pleural mesothelioma.

BackgroundMalignant pleural mesothelioma (MPM) is a rare and aggressive cancer related to asbestos exposure. The tumor microenvironment content, particularly the presence of macrophages, was described as crucial for the development of the disease. This work aimed at studying the involvement of the M-CSF (CSF-1)/IL-34/CSF-1R pathway in the formation of macrophages in MPM, using samples from patients.MethodsPleural effusions (PEs), frozen tumors, primary MPM cells and MPM cell lines used in this study belong to biocollections associated with clinical databases. Cytokine expressions were studied using real-time PCR and ELISA. The Cancer Genome Atlas database was used to confirm our results on an independent cohort. An original three-dimensional (3D) coculture model including MPM cells, monocytes from healthy donors and a tumor antigen-specific cytotoxic CD8 T cell clone was used.ResultsWe observed that high interleukin (IL)-34 levels in PE were significantly associated with a shorter survival of patients. In tumors, expression of CSF1 was correlated with ‘M2-like macrophages’ markers, whereas this was not the case with IL34 expression, suggesting two distinct modes of action of these cytokines. Expression of IL34 was higher in MPM cells compared with primary mesothelial cells. Particularly, high expression of IL34 was observed in MPM cells with an alteration of CDKN2A. Finally, using 3D coculture model, we demonstrated the direct involvement of MPM cells in the formation of immunosuppressive macrophages, through activation of the colony stimulating factor-1 receptor (CSF1-R) pathway, causing the inhibition of cytotoxicity of tumor antigen-specific CD8+ T cells.ConclusionsThe M-CSF/IL-34/CSF-1R pathway seems strongly implicated in MPM and could constitute a therapeutic target to act on immunosuppression and to support immunotherapeutic strategies.

MA23.03 BAP1 Loss Induces Genome Instability Through BRCA1-Dependent and Independent Mechanisms in Mesothelioma

BRCA1 associated protein 1 (BAP1) is a tumor suppressor that is the most frequently mutated in the majority of mesotheliomas. We have previously reported that loss of BRCA1 expression in mesothelioma is a common event, and mediates resistance to spindle checkpoint activator vinorelbine, a drug with relevance to treatment of mesothelioma. However, the loss of BRCA1 is unknown in mesothelioma. The aim of this study is to determine the functional relationship between BAP1 and BRCA1 and examine their role in vinorelbine resistance in mesothelioma cells. We conducted functional genetic analysis of BAP1 and BRCA1 in two MPM cell lines, MSTO and H2452, the latter carrying an inactivating A95D mutation in the UCH domain of BAP1. BAP1 knockdown was achieved by siRNA transfection, while BRCA1 knockdown was achieved by doxycycline induction of an integrated shRNA. Patient samples were processed for BAP1 and BRCA1 immunohistochemistry from MEDUSA cohort. Loss of BAP1 expression led to reduced expression of BRCA1, whereas knockdown of BRCA1 did not affect BAP1 expression. Treatment with the proteasome inhibitor, MG132, restored BRCA1 expression in the absence of BAP1 indicating that BAP1 contributes to post-translational stabilization of BRCA1 protein and the stabilization of BRCA1 by BAP1 is independent of its de-ubiquitination activity. Knockdown of BAP1 induced SAC deficiency and vinorelbine resistance concurrent with reduced expression of BRCA1 and the SAC component, MAD2L1. We also identified a positive and significant correlation between BAP1 and BRCA1 expressionin patient samples. Our data demonstrates that BAP1 regulates BRCA1 expression through regulating its protein stability. Our findings suggest that BAP1 inactivation dysregulates the spindle assembly checkpoint via BRCA1 dependent and independent mechanisms, conferring resistance to vinorelbine. As such BAP1 may have potential as a predictive biomarker for spindle poisons, to underpin chemotherapy stratification. A hypothesis that would be tested in a multicentre randomised phase II VIM trial.

P1.06-18 MicroRNA Expression Is Linked to Response of Malignant Pleural Mesothelioma to Cisplatin-Pemetrexed Chemotherapy

Treatment of malignant pleural mesothelioma is difficult due to a high intrinsic drug resistance of these tumors. Currently, platinum-based chemotherapy represents the backbone of MPM treatment. However, only approximately 40% of patients respond to this therapy, and true predictors for response have yet to be identified. Towards this end, we here investigate the expression of microRNAs in responders and non-responders to chemotherapy. FFPE tumour samples were available from 32 MPM patients, who showed either partial response (PR, N=21) or progressive disease (PD, N=11) following 3-4 cycles of cisplatin-pemetrexed chemotherapy. RT-qPCR based microRNA profiling was performed on chemo-naïve tissue of 5 PD and 5 PR patients using TaqMan Low Density Arrays (TLDAs, Thermo Fisher), which cover the expression of 754 microRNAs. Candidate microRNAs with differential expression (P≤0.05 Mann-Whitney Test) were then measured in the remaining samples using microRNA-specific RT-qPCR. Expression of these microRNAs was also assessed in post-chemotherapy specimens (obtained during extrapleural pneumonectomy) and compared to that in chemo-naïve samples. In addition, for two candidates, preliminary in vitro experiments investigating the effect of microRNA overexpression (transfection with microRNA mimics) on cell growth were performed. TLDA-based profiling identified 35 microRNA with differential expression between patients with PD and PR following cisplatin-pemetrexed chemotherapy. The majority of these microRNAs showed higher expression in patients who showed no response to therapy. In an initial step, 8 candidates identified from the profiling (miR-145, miR-193a-3p, miR-30a-3p, miR-24, miR-380-5p, miR-494, miR-625-3p, miR-221-3p) were further evaluated in additional 16 PR and 6 PD samples. This confirmed a trend towards differential expression for miR-145 (p=0.08). Interestingly, when comparing expression pre- and post-chemotherapy, levels of miR-145 significantly decreased in patients with PD, while they remained stable in PR. Lack of validation of other microRNAs could be the result of the low number of cases with PD in this preliminary validation set, and additional samples will included. For miR-221-3p and miR-380-5p, preliminary analysis in vitro showed that overexpression in established MPM cell lines results in an increased sensitivity towards cisplatin. Taken together, our data show that several microRNAs show trends towards differential expression between responders and non-responders to chemotherapy. Overall, higher expression appears to be linked to PD under cisplatin-pemetrexed, however further in-depth investigations are required. Furthermore, preliminary in vitro data suggest that altering expression of specific microRNAs has the potential to increase sensitivity of MPM to chemotherapy.

Gene expression profiling of homologous recombination repair pathway indicates susceptibility for olaparib treatment in malignant pleural mesothelioma in vitro

BackgroundMalignant pleural mesothelioma (MPM) is a tumour arising from pleural cavities with poor prognosis. Multimodality treatment with pemetrexed combined with cisplatin shows unsatisfying response-rates of 40%. The reasons for the rather poor efficacy of chemotherapeutic treatment are largely unknown. However, it is conceivable that DNA repair mechanisms lead to an impaired therapy response. We hypothesize a major role of homologous recombination (HR) for genome stability and survival of this tumour. Therefore, we analysed genes compiled under the term “BRCAness”. An inhibition of this pathway with olaparib might abrogate this effect and induce apoptosis.MethodsWe investigated the response of three MPM cell lines and lung fibroblasts serving as a control to treatment with pemetrexed, cisplatin and olaparib. Furthermore, we aimed to find possible correlations between response and gene expression patterns associated with BRCAness phenotype. Therefore, 91 clinical MPM samples were digitally screened for gene expression patterns of HR members.ResultsA BRCAness-dependent increase of apoptosis and senescence during olaparib-based treatment of BRCA-associated-protein 1 (BAP1)-mutated cell lines was observed. The gene expression pattern identified could be found in approx. 10% of patient samples. Against this background, patients could be grouped according to their defects in the HR system. Gene expression levels of Aurora Kinase A (AURKA), RAD50 as well as DNA damage-binding protein 2 (DDB2) could be identified as prognostic markers in MPM.ConclusionsDefects in HR compiled under the term BRCAness are a common event in MPM. The present data can lead to a better understanding of the underlaying cellular mechanisms and leave the door wide open for new therapeutic approaches for this severe disease with infaust prognosis. Response to Poly (ADP-ribose)-Polymerase (PARP)-Inhibition could be demonstrated in the BAP1-mutated NCI-H2452 cells, especially when combined with cisplatin. Thus, this combination therapy might be effective for up to 2/3 of patients, promising to enhance patients’ clinical management and outcome.

Pemetrexed-loaded nanoparticles targeted to malignant pleural mesothelioma cells: an in vitro study.

PurposeMalignant pleural mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis. Its incidence is steadily increasing due to widespread asbestos exposure. There is still no effective therapy for MPM. Pemetrexed (Pe) is one of the few chemotherapeutic agents approved for advanced-stage disease, although the objective response to the drug is limited. The use of gold nanoparticles (GNPs) as a drug delivery system promises several advantages, including specific targeting of malignant cells, with increased intracellular drug accumulation and reduced systemic toxicity, and, in the case of MPM, direct treatment administration into the pleural space. This study aims at exploring CD146 as a potential MPM cell-specific target for engineered Pe-loaded GNPs and to assess their effectiveness in inhibiting MPM cell line growth.MethodsMPM cell lines and primary cultures obtained by pleural effusions from MPM patients were assayed for CD146 expression by flow cytometry. Internalization by MPM cell lines of fluorescent dye-marked GNPs decorated with a monoclonal anti CD146 coated GNPs (GNP-HC) was proven by confocal microscopy. The effects of anti CD146 coated GNPs loaded with Pe (GNP-HCPe) on MPM cell lines were evaluated by cell cycle (flow cytometry), viability (MTT test), clonogenic capacity (soft agar assay), ROS production (electric paramagnetic resonance), motility (wound healing assay), and apoptosis (flow cytometry).ResultsGNP-HC were selectively uptaken by MPM cells within 1 hour. MPM cell lines were blocked in the S cell cycle phase in the presence of GNP-HCPe. Both cell viability and motility were significantly affected by nanoparticle treatment compared to Pe. Apoptotic rate and ROS production were significantly higher in the presence of nanoparticles. Clonogenic capacity was completely inhibited following nanoparticle internalization.ConclusionGNP-HCPe treatment displays in vitro antineoplastic action and is more effective than Pe alone in inhibiting MPM cell line malignant phenotype. The innovative use of specifically targeted GNPs opens the perspective of local intrapleural administration to avoid normal cell toxicity and enhance chemotherapy efficacy.

Assessment of signaling pathway inhibitors and identification of predictive biomarkers in malignant pleural mesothelioma

ObjectivesMalignant pleural mesothelioma (MPM) is an aggressive tumor with limited therapeutic options, requiring the development of efficient targeted therapies based on molecular phenotype of the tumor and to identify predictive biomarkers of the response. Materials and methodsThe effect of inhibitors was investigated by cell viability assessment on primary MPM cell lines established in our laboratory from patient tumors, well characterized at the molecular level. Effects on apoptosis, cell proliferation and viability on MPM growing in multicellular spheroid were also assessed for verteporfin. Gene and protein expression, and gene knockdown by RNA interference were used to define mechanism of inhibition and specific predictive biomarkers. ResultsAnti-tumor effect of eight major signaling pathways inhibitors involved in mesothelial carcinogenesis was investigated. Three inhibitors were more efficient than cisplatin, the drug used as first-line chemotherapy in patients with MPM: verteporfin, a putative YAP inhibitor, defactinib, a FAK inhibitor and NSC668394, an Ezrin inhibitor. Verteporfin, the most efficient inhibitor, induced cell proliferation arrest and cell death, and is effective on 3D spheroid multicellular model. Verteporfin sensitivity was YAP-independent and related to molecular classification of the tumors. Biomarkers based on gene expression were identified to predict accurately sensitivity to these three inhibitors. ConclusionOur study shows that drug screening on well-characterized MPM cells allows for the identification of novel potential therapeutic strategies and defining specific biomarkers predictive of the drug response.

JQ1, a BET Inhibitor, Synergizes with Cisplatin and Induces Apoptosis in Highly Chemoresistant Malignant Pleural Mesothelioma Cells.

Malignant Pleural Mesothelioma (MPM) is an asbestos-associated tumor with poor prognosis and few therapeutic options. JQ1, a selective antagonist of BRD4, modulates transcription of oncogenes, including MPM chemoresistance-associated c-Myc and Fra-1. We investigated if JQ1 could enhance the efficacy of cisplatin against MPM. The antiproliferative activity of cisplatin in combination with JQ1 was assessed on MPM cell lines representative of the cellular phenotypes of this tumor (epithelioid, sarcomatoid and biphasic), and on one cisplatin resistant sub-line. The combination schedule was optimized adopting a 3Dspheroid model. Drug combination effects were correlated with cell cycle distribution and senescence- associated β-galactosidase positive cells. The expression of c-Myc and Fra-1 proteins and some apoptosis markers was assessed by immunoblotting and RT-qPCR. DNA damage and repair were evaluated by means of alkaline comet assay. JQ1 in combination with cisplatin elicited additive or synergistic (superadditive) antiproliferative effects on MPM cells, depending on the cell line. The combination showed tumor regression on the 3D-spheroid model. It induced increased apoptosis, along with decreased c-Myc and, sometimes, Fra-1 expression. JQ1 decreased cisplatin-induced DNA breaks in all MPM cells and increased senescence even in less proficient cells, thus enhancing the DNA Damage Response (DDR). The superadditive effect is due to c-Myc repression. The consequent DDR enhancement triggers to apoptosis induction and/or permanent growth arrest (senescence), depending on the MPM cellular context, leading to tumor regression. Thus, the pharmacological modulation of BET activity could represent a promising tool for future MPM therapy.