Malignant Pleural Mesothelioma Cell Research Articles

Anticancer agent α-sulfoquinovosyl-acylpropanediol enhances the radiosensitivity of human malignant mesothelioma in nude mouse models.

Malignant pleural mesothelioma (MPM) is a highly malignant disease that develops after asbestos exposure. Although the number of MPM cases is predicted to increase, no effective standard therapies have been established. The novel radiosensitizer α-sulfoquinovosyl-acylpropanediol (SQAP) enhances the effects of γ-radiation in human lung and prostate cancer cell lines and in animal models. In this study, we explored the radiosensitizing effect of SQAP and its mechanisms in MPM. The human MPM cell lines MSTO-211H and MESO-4 were implanted subcutaneously into the backs and thoracic cavities of immunodeficient KSN/Slc mice, then 2 mg/kg SQAP was intravenously administered with or without irradiation with a total body dose of 8 Gy. In both the orthotopic and ectopic xenograft murine models, the combination of irradiation plus SQAP delayed the implanted human MSTO-211H tumor growth. The analysis of the changes in the relative tumor volume of the MSTO-211H indicated a statistically significant difference after 8 Gy total body combined with 2 mg/kg SQAP, compared to both the untreated control (P = 0.0127) and the radiation treatment alone (P = 0.0171). After the treatment in each case, immunostaining of the harvested tumors revealed decreased cell proliferation, increased apoptosis and normalization of tumor blood vessels in the SQAP- and irradiation-treated group. Furthermore, hypoxia-inducible factor (HIF) 1 mRNA and protein expression were decreased, indicating reoxygenation in this group. In conclusion, SQAP improved hypoxic conditions in tumor tissue and may elicit a radiosensitizing effect in malignant mesothelioma models.

MALAT1 promotes malignant pleural mesothelioma by sponging miR-141-3p.

The aim of this study was to clarify the role of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in proliferation, migration, and invasion of malignant pleural mesothelioma (MPM) cells. The quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression of MALAT1 in MPM cell lines. The effects of MALAT1 and miR-141-3p on the proliferation, migration, and invasion of MPM cells were studied through a series of in vitro cellular experiments. The flow cytometry was utilized to detect the cell apoptosis. The dual‐luciferase reporter assay was employed to explore the binding relationship among MALAT1, miR-141-3p, and YES-associated protein 1 (YAP1). MALAT1 was overexpressed in MPM cell lines, while its knockdown significantly inhibited the cell proliferation, migration, and invasion, and increased the number of MPM cells in the G0/G1 phase. In addition, MALAT1 could directly bind to miR-141-3p and inhibit its expression. YAP1 has been identified as a downstream target of miR-141-3p, and its expression level was inhibited by miR-141-3p. MALAT1 can be used as a competitive endogenous RNA (ceRNA) to regulate the YAP1-Hippo signaling pathway through miR-141-3p, promote the proliferation, migration, and invasion of MPM cells, and provide a new target for the therapy of MPM.

Impact of Tumor Treating Fields (TTFields) on DNA Damage Repair in Mesothelioma

Tumor Treating Fields (TTFields) are low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields delivered to solid tumors noninvasively and locoregionally. TTFields have demonstrated a promising median overall survival in patients with malignant pleural mesothelioma (MPM), an aggressive thoracic cancer with poor prognosis, without increases in systemic toxicity (STELLAR clinical trial). Consequently, TTFields therapy combined with pemetrexed and a platinum-based chemotherapy agent are approved as first line treatment for unresectable MPM in the US and Europe. While efficacy of TTFields for MPM treatment is well-established, the underlying mechanism of action needs further elucidation. This study investigated the TTFields effect on DNA damage and repair in MPM and tested the combination of TTFields with DNA damaging agents such as cisplatin and pemetrexed, in vitro and in vivo.TTFields frequencies ranging from 100-400 kHz were applied to human MPM cell lines (NCI-H2052 and MSTO-211H) to identify the most effective frequency. To detect DNA double strand breaks (DSB), the effect of optimal TTFields frequency on the formation of ɣH2AX foci was examined by fluorescent microscopy. Levels of DNA damage repair related proteins were evaluated via immunoblotting of cell lysates. The combined cytotoxic effect of TTFields with cisplatin or pemetrexed was tested in vitro. Efficacy of TTFields concomitant with both cisplatin and pemetrexed was examined in C57BL/6 mice inoculated subcutaneously with RN-5 cells by measuring tumor volume and DNA damage within the tumor.The optimal TTFields frequency was identified as 150 kHz in both MPM cell lines, displaying significant cytotoxicity and formation of DNA DSB. These effects were accompanied by increased expression of p21 and p27, proteins involved in DNA damage-induced cell cycle arrest, and reduced expression of proteins from the Fanconi Anemia (FA) DNA repair pathway - FANCA, FANCD2, FANCJ, and BRCA1. Co-treatment of TTFields with cisplatin or pemetrexed augmented efficacy versus each treatment alone, with the TTFields-pemetrexed combination displaying an additive effect, and the co-administration of TTFields with cisplatin demonstrating synergistic interaction. In vivo, tumor volume fold change was significantly decreased for the combination of TTFields with chemotherapy (cisplatin + pemetrexed) versus control, while levels of DNA damage within the tumor were increased.Efficacy of TTFields for the treatment of MPM is associated with increased DNA damage, elevated levels of DNA-damage related cell cycle arrest proteins, and reduced expression of FA pathway proteins. This latter effect may account for the synergistic interaction displayed for the TTFields-cisplatin combination, as cisplatin is known to cause DNA damage that requires the FA pathway for repair.

P35.01 YB-1 is a Key Player in Aggressive Behaviour and Chemoresistance in Mesothelioma

Malignant pleural mesothelioma (MPM) is characterised by aggressive growth and frequent resistance to chemotherapy, poor prognosis for patients and limited therapeutic options. To establish potential new therapy targets, a better understanding of the MPM biology underlying these malignant behaviours is crucial. One potential candidate is the multifunctional oncoprotein YB-1, which is often overexpressed in various cancers and associated with aggressiveness, metastasis and poor outcome. YB-1 expression was analysed by qPCR or western blot in cell lines, and in patient material by immunohistochemistry. YB-1 overexpression was achieved by a doxycycline-inducible, retroviral construct, which was stably introduced into MPM cell lines. YB-1 knockdown was performed using YB-1 specific siRNA. For inhibition of YB-1 phosphorylation, the RSK inhibitor BI-D1870 was used. Effects of drug interaction (CI values) were calculated using the CompuSyn software. Cell migration was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. For the zebrafish model, RFP-expressing MPM cells were injected into 48 hour old larvae, imaged after 1 and 2 days and the number of cells in the tail was manually counted. We previously reported that YB-1 is up-regulated in MPM cell lines compared to non-malignant controls. In this study we analysed MPM tissue (n>120) and normal pleura (n=3) specimens for YB-1 expression. While all control samples were negative, tumours showed a heterogeneous YB-1 expression. YB-1 knockdown decreases MPM cell migration and invasion, hence we evaluated the impact of YB-1 overexpression on these phenotypes. MPM cell lines (n=6) which overexpress YB-1 in a doxycycline-inducible manner showed significantly increased cell scattering and migration. Furthermore, YB-1 overexpression lead to significantly higher migratory capacity in vivo using a zebrafish xenograft model. Additionally, when tumour spheroids were co-cultured with endothelial cells, YB-1 overexpression led to a significantly more extensive formation of gaps in the endothelial layer. Finally, since we found that YB-1 knockdown not only decreases cell growth in vitro and in vivo but also decreases the expression of LRP1 and ABCC1, two genes involved in drug resistance, we combined YB-1 knockdown as well as inhibition of YB-1 phosphorylation via an RSK inhibitor with cisplatin chemotherapy. Our data showed highly synergistic combination effects (CI values: 0.1 – 0.5) for several cisplatin doses in combination with either YB-1 siRNA or the RSK inhibitor. In this study we report a deregulated YB-1 expression in MPM tumor specimens compared to non-malignant controls. Our data show an important role of YB-1 in the regulation of cell migration and invasion in vitro and in vivo, which are key characteristics of MPM. Additionally, YB-1 knock down as well as pharmacological inhibition of YB-1 phosphorylation not only reduce MPM cell growth but also sensitise cells to cisplatin chemotherapy. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

A3 Adenosine and P2X7 Purinergic Receptors as New Targets for an Innovative Pharmacological Therapy of Malignant Pleural Mesothelioma.

Human malignant pleural mesothelioma (MPM) is a rare, but aggressive tumor of the serosal cavities whose 5-year survival rate is 15%. At present, there are no effective therapies for MPM. Although recent findings suggest that A3 adenosine (A3AR) and P2X7 (P2X7R) receptors can be employed as antitumoral pharmacological targets in MPM, their potential role in a combined therapy is currently unknown. The A3AR agonist Cl-IB-MECA and the P2X7 receptor antagonist AZ10606120, as a single compound or in combination, were investigated in vitro for their anti-tumor activities. Assays were carried out in MPM cell lines IST-Mes2 and MPP89 and in primary human normal mesothelial cells (HMCs), as control. Single treatment with Cl-IB-MECA reduced cell proliferation and favored a pro-apoptotic effect in both MPP89 and IST-Mes2 cell lines, whereas AZ10606120 inhibited cell proliferation and induced apoptosis in IST-Mes2, only. The combined treatment with Cl-IB-MECA and AZ10606120 reduced cell proliferation and favored apoptosis in MPP89 and IST-Mes2 cell lines, whereas no synergistic effect was detected. These data cumulatively suggest the absence of a synergistic effect in combined targeting of A3 adenosine and P2X7 receptors of MPM cell lines. This study may stimulate further investigations aimed at determining new combinations of antitumor compounds and more effective therapeutic strategies against MPM.

MA04.06 MicroRNAs Contribute to the Chemotherapy Response of Malignant Pleural Mesothelioma

Although platinum-pemetrexed chemotherapy remains the gold-standard for treatment of malignant pleural mesothelioma (MPM), to date we are still lacking true predictive biomarkers able to identify those patients who truly respond to chemotherapy. Aiming to overcome this problem, we previously performed microRNA expression profiling in tumour tissue of responders and non-responders to induction chemotherapy, which identified candidate predictive microRNAs. Candidates from this profiling are now investigated in vitro for their potential to alter the response of MPM cell lines to cisplatin and pemetrexed.

Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an invasive malignancy that develops in the pleural cavity, and antifolates are used as chemotherapeutics for treating. The majority of antifolates, including pemetrexed (PMX), inhibit enzymes involved in purine and pyrimidine synthesis. MPM patients frequently develop drug resistance in clinical practice, however the associated drug-resistance mechanism is not well understood. This study was aimed to elucidate the mechanism underlying resistance to PMX in MPM cell lines. We found that among the differentially expressed genes associated with drug resistance (determined by RNA sequencing), TYMS expression was higher in the established resistant cell lines than in the parental cell lines. Knocking down TYMS expression significantly reduced drug resistance in the resistant cell lines. Conversely, TYMS overexpression significantly increased drug resistance in the parental cells. Metabolomics analysis revealed that the levels of dTMP were higher in the resistant cell lines than in the parental cell lines; however, resistant cells showed no changes in dTTP levels after PMX treatment. We found that the nucleic acid-biosynthetic pathway is important for predicting the efficacy of PMX in MPM cells. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays suggested that H3K27 acetylation in the 5′-UTR of TYMS may promote its expression in drug-resistant cells. Our findings indicate that the intracellular levels of dTMP are potential biomarkers for the effective treatment of patients with MPM and suggest the importance of regulatory mechanisms of TYMS expression in the disease.

The Role of E-Cadherin and microRNA on FAK Inhibitor Response in Malignant Pleural Mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited effective treatment options. Focal adhesion kinase (FAK) inhibitors have been shown to efficiently suppress MPM cell growth initially, with limited utility in the current clinical setting. In this study, we utilised a large collection of MPM cell lines and MPM tissue samples to study the role of E-cadherin (CDH1) and microRNA on the efficacy of FAK inhibitors in MPM. The immunohistochemistry (IHC) results showed that the majority of MPM FFPE samples exhibited either the absence of, or very low, E-cadherin protein expression in MPM tissue. We showed that MPM cells with high CDH1 mRNA levels exhibited resistance to the FAK inhibitor PND-1186. In summary, MPM cells that did not express CDH1 mRNA were sensitive to PND-1186, and MPM cells that retained CDH1 mRNA were resistant. A cell cycle analysis showed that PND-1186 induced cell cycle disruption by inducing the G2/M arrest of MPM cells. A protein−protein interaction study showed that EGFR is linked to the FAK pathway, and a target scan of the microRNAs revealed that microRNAs (miR-17, miR221, miR-222, miR137, and miR148) interact with EGFR 3′UTR. Transfection of MPM cells with these microRNAs sensitised the CHD1-expressing FAK-inhibitor-resistant MPM cells to the FAK inhibitor.

Statins Decrease Programmed Death-Ligand 1 (PD-L1) by Inhibiting AKT and β-Catenin Signaling.

Retrospective observational studies have reported that statins improve clinical outcomes in patients previously treated with programmed cell death protein 1 (PD-1)-targeting monoclonal antibodies for malignant pleural mesothelioma (MPM) and advanced non-small cell lung cancer (NSCLC). In multiple mouse cancer models, de novo synthesis of mevalonate and cholesterol inhibitors was found to synergize with anti-PD-1 antibody therapy. In the present study, we investigated whether statins affect programmed death-ligand 1 (PD-L1) expression in cancer cells. Four statins, namely simvastatin, atorvastatin, lovastatin, and fluvastatin, decreased PD-L1 expression in melanoma and lung cancer cells. In addition, we found that AKT and β-catenin signaling involved PD-L1 suppression by statins. Our cellular and molecular studies provide inspiring evidence for extending the clinical evaluation of statins for use in combination with immune checkpoint inhibitor-based cancer therapy.

Evidence-based diagnostic performance of novel biomarkers for the diagnosis of malignant mesothelioma in effusion cytology.

Cytology effusions are often the only material available for diagnosing malignant pleural mesothelioma (MPM). However, the cytomorphological features alone are not always diagnostic, and cytology samples preclude an assessment for pleural tissue invasion. Accordingly, immunohistochemical, soluble, and molecular biomarkers have been developed. The aim of this study is to provide quantitative evidence regarding the diagnostic performance of novel biomarkers. To that end, a systematic literature review was performed of articles dealing with a loss of BRCA1-associated protein 1 (BAP1), methylthioadenosine (MTAP), 5-hydroxymethylcitosine (5-hmC), glucose transporter 1 (GLUT1), insulin like-growth factor II messenger RNA-binding protein 3 (IMP3), enhanced zeste homologue 2 (EZH2) staining, cyclin-dependent kinase inhibitor 2A (CDKN2A) homozygous deletion (HD) testing, soluble mesothelin, and microRNA quantification in cytological samples for the diagnosis of MPM versus reactive atypical mesothelial cells. Sensitivity and specificity were extracted, and a meta-analysis was performed. The quality of the studies was assessed with Quality Assessment of Diagnostic Accuracy Studies 2, and the quality of the evidence was evaluated with the Grading of Recommendations Assessment, Development, and Evaluation approach. Seventy-one studies were included. BAP1 loss showed a sensitivity of 0.65 (confidence interval [CI], 0.59-0.71) and a specificity of 0.99 (CI, 0.93-1.00). MTAP loss and p16 HD showed 100% specificity with sensitivities of 0.47 (CI, 0.38-0.57) and 0.62 (CI, 0.53-0.71), respectively. BAP1 loss and CDKN2A HD combined showed maximal specificity and a sensitivity of 0.83 (CI, 0.78-0.89). GLUT1 and IMP3 showed sensitivities of 0.82 (CI, 0.70-0.90) and 0.65 (CI, 0.41-0.90), respectively, with comparable specificity. Mesothelin showed a sensitivity of 0.73 (CI, 0.68-0.77) and a specificity of 0.90 (CI, 0.84-0.93). In conclusion, some of the recently emerging biomarkers are close to 1.00 specificity. Their moderate sensitivity on their own, however, can be significantly improved by the use of 2 biomarkers, such as a combination of BAP1 and CDKN2A with fluorescence in situ hybridization or a combination of BAP1 and MTAP immunohistochemistry.

Identification of novel COX-2 / CYP19A1 axis involved in the mesothelioma pathogenesis opens new therapeutic opportunities

BackgroundBased on previous studies highlighting that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of malignant pleural mesothelioma (MPM), and that aromatase (CYP19A1), an enzyme that plays a key role in estrogen biosynthesis, along with estradiol (E2) were expressed in MPM, this study aimed to investigate the possible interplay between COX-2 and CYP19A1 in the pathogenesis of mesothelioma, as well as the underlying mechanism.MethodsThe interaction between COX-2 and CYP19A1 was first investigated on different MPM lines upon PGE2, and COX-2 inhibitor (rofecoxib) treatment by western blot, RT-PCR. The key regulatory pathways involved in the COX-2 and CYP19A1 axis were further studied in MPM cells, after rofecoxib and exemestane (CYP19A1 inhibitor) treatment in monotherapy and in combination, by cell cycle distribution, western blot, and combination index analysis. To explore the role of COX-2/CYP19A1 axis in 3D preclinical models of MPM cells, we analyzed the effect of combination of COX-2 and CYP19A1 inhibitors in mesosphere formation. Immunohistochemical analysis of MPM mesosphere and specimens was utilized to evaluate the involvement of COX-2 on the CYP19A1 activity and the relationship between E2 and COX-2.ResultsPGE2 or rofecoxib treatment caused in MPM cells an increased or decreased, respectively, CYP19A1 expression at mRNA and protein levels. The effect of rofecoxib and exemestane combination in MPM cell proliferation was synergistic. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for MPM cell lines. Increased expression levels of p53, p21, and p27, downregulation of cyclin D1 and inhibition of Akt activation (pAKT) were also found. The antagonistic effect of rofecoxib and exemestane combination found only in one cell line, was reverted by pretreatment with MK2206, a pAKT inhibitor, indicating pAKT as an actionable mediator in the COX-2-CYP19A1 axis. Reduction of size and sphere-forming efficiency in MPM spheres after treatment with both inhibitor and a decrease in COX-2 and E2 staining was found. Moreover, immunohistochemical analysis of 46 MPM samples showed a significant positive correlation between COX-2 and E2.ConclusionsCollectively, the results highlighted a novel COX-2/CYP19A1 axis in the pathogenesis of MPM that can be pharmacologically targeted, consequently opening up new therapeutic options.

Unsymmetric Cisplatin-Based Pt(IV) Conjugates Containing a PARP-1 Inhibitor Pharmacophore Tested on Malignant Pleural Mesothelioma Cell Lines.

Cisplatin is widely employed as a first-line chemotherapeutic agent for many solid tumors, including malignant pleural mesothelioma (MPM). However, its clinical use is limited by heavy side effects and acquired resistance, the latter being mainly related to enhanced DNA repair. Many clinical trials using combinations of platinum drugs and PARP-1 inhibitors (PARPis) have been carried out, with the hope that such combinations might lead to improved therapeutic efficacy against tumors. Here, the synthesis and efficacy in reducing MPM cell viability of four cisplatin-based Pt(IV) prodrugs containing the PARPi 3-aminobenzamide (3-ABA) fragment are described. The most promising conjugate is more effective than cisplatin or cisplatin/3-ABA combination, administered in equimolar doses, in inhibiting PARP-1 activity and inducing apoptosis in BRCA1/2 wild type MPM cells, grown as monolayer or as multicellular spheroids.

Phytochemicals in Malignant Pleural Mesothelioma Treatment-Review on the Current Trends of Therapies.

Malignant pleural mesothelioma (MPM) is a rare but highly aggressive tumor of pleura arising in response to asbestos fibers exposure. MPM is frequently diagnosed in the advanced stage of the disease and causes poor prognostic outcomes. From the clinical perspective, MPM is resistant to conventional treatment, thus challenging the therapeutic options. There is still demand for improvement and sensitization of MPM cells to therapy in light of intensive clinical studies on chemotherapeutic drugs, including immuno-modulatory and targeted therapies. One way is looking for natural sources, whole plants, and extracts whose ingredients, especially polyphenols, have potential anticancer properties. This comprehensive review summarizes the current studies on natural compounds and plant extracts in developing new treatment strategies for MPM.

Abstract 1064: Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines

Abstract Despite the increasing knowledge of the biology of malignant pleural mesothelioma (MPM), patients with this diagnosis still have a poor prognosis with a 5-year survival rate of lower than 10%. The effectiveness demonstrated by Tumor Treating Fields (TTFields) in combination with chemotherapy in preclinical experiments and in MPM patients (NCT02397928) affirms TTFields as a promising therapeutic tool. Knowledge of the mechanism of action of TTFields and its interaction with chemotherapeutic agents may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients' thoracic cavity, before administration of therapeutic treatment, obtaining six cell lines from the 3 types of MPM cells: epithelioid CD473 and CD484, sarcomatoid CD60 and CD432, and biphasic CD487 and CD491. RNAseq analysis revealed that the transcriptional profiles of CD473 and CD60 cells were comparable with those derived from patient biopsies of the same histotype, representing clinically relevant MPM models. Antiproliferative effects induced by TTFields alone (1.12 V/cm; 150 KHz; for 96h duration) were studied in these MPM cell lines. Cell survival and cell cycle perturbations analyses during and after treatment were performed. Consistent with clinical observations, epithelioid MPM cells demonstrated greater sensitivity to TTFields than biphasic and sarcomatoid MPM cells. Cell cycle analysis revealed that the former cell type was blocked in G2M phase and was followed by formation of polyploid cells; while the latter cell types were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptotic cells were present in all treated samples, but while epithelioid and biphasic cell death was already observed during the first 24h of treatment, sarcomatoid cells needed to be treated for at least 24h before starting apoptotic pathways. The antiproliferative effect observed in epithelioid cells was persistent even after treatment cessation, whereas biphasic and sarcomatoid cells were able to reinitiate growth upon TTFields cessation. RNAseq experiments were performed in CD473 and CD60 cells at different times during TTFields application to explore transcriptional modifications that may explicate these varied MPM cell responses. Preliminary data suggested that TTFields induced a transcriptional response already detectable at earlier time points of treatment (8h; maximum effect at 24h). The number of differentially expressed genes was higher in CD473 relative to CD60 cells (1951 in CD473 and 336 in CD60 at 24h), involving several pathways, that need to be further investigated. These data demonstrate that TTFields induce specific effects on cell proliferation in varied subsets of mesothelioma cells and provide a mechanistic rationale for future therapies and combinations with other anticancer drugs for MPM treatment. Citation Format: Monica Lupi, Federica Mirimao, Nicolò Panini, Greta Piazza, Lara Paracchini, Laura Mannarino, Sergio Marchini, Federica Grosso, Serena Penpa, Antonio Maconi, Giovanni L. Ceresoli, Maurizio D'Incalci. Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines [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 1064.

Abstract 1186: Efficacy of Tumor Treating Fields (TTFields) in mesothelioma is associated with reduced capacity for DNA damage repair

Abstract Introduction: Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer with a poor prognosis and limited treatment options. Tumor Treating Fields (TTFields) are a noninvasive, locoregionally, antineoplastic treatment, delivering low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields, that has demonstrated a promising median overall survival in patients with MPM without increases in systemic toxicity (STELLAR clinical trial). Accordingly, TTFields with pemetrexed and a platinum-based chemotherapy agent received FDA-approval as first line therapy for MPM. While efficacy of TTFields for MPM treatment is well-established, the underlying mechanism of action needs further elucidation. Methods: Human MPM cell lines (NCI-H2052 and MSTO-211H) were treated using various TTFields frequencies to assess the most effective frequency. The effect of optimal frequency TTFields on levels of DNA double strand breaks (DSB) was examined by fluorescent microscopy detection of γH2AX foci, and the levels of DNA damage repair proteins was evaluated by immunoblotting. The combined cytotoxic effect of TTFields with cisplatin or pemetrexed was tested in vitro, and efficacy of TTFields in combination with both chemotherapeutic agents was examined in C57BL/6 mice injected subcutaneously with RN-5 cells, by measuring tumor volume and through detection for DNA damage within the tumor. Results: The optimal TTFields frequency in both MPM cell lines was 150 kHz, demonstrating significant cytotoxicity and increases in formation of DNA DSB. These effects were associated with reduced expression of proteins from the Fanconi Anemia (FA) repair pathway for DNA repair - FANCA, FANCD2, FANCJ, and BRCA1. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each treatment alone, with an additive effect shown by the TTFields-pemetrexed combination, and a tendency towards synergism displayed for TTFields-cisplatin co-administration. In animal models, tumor volume fold increase was significantly decreased for co-treatment with TTFields and chemotherapy (cisplatin + pemetrexed) versus the control, showing also increased DNA damage within the tumor bed in comparison to control or chemotherapy alone. Conclusions: The results presented here demonstrate that the efficacy of TTFields for treatment of MPM is associated with reduced expression of FA pathway proteins and increased DNA DSB. This effect may account for the synergistic effect seen for TTFields-cisplatin co-treatment, as cisplatin is known to cause DNA damage that requires the FA pathway for repair. This research provides further insights on the mechanism of action of TTFields in MPM, a treatment already approved against this malignancy. Citation Format: Helena Mumblat, Antonia Martinez, Ori Braten, Mijal Munster, Eyal Dor-On, Rosa S. Schneiderman, Yaara Porat, Tali Voloshin, Shiri Davidi, Roni Blatt, Anna Shteingauz, Catherine Tempel-Brami, Einav Zeevi, Carolina Lajterer, Yuval Shmueli, Shiri Danilov, Adi Haber, Moshe Giladi, Adrian Kinzel, Uri Weinberg, Yoram Palti. Efficacy of Tumor Treating Fields (TTFields) in mesothelioma is associated with reduced capacity for DNA damage repair [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 1186.

Cellular cannibalism and consequent thrombocytopenia in a patient with bone marrow metastasis of malignant pleural mesothelioma: A case report.

Cellular cannibalism is a tumor activity where a cell is engulfed by another cell. This process promotes tumor cell survival under unfavorable conditions. The current report describes an extremely rare case of thrombocytopenia resulting from cellular cannibalism in a patient with bone marrow metastasis due to malignant pleural mesothelioma (MPM). A 77-year-old male presented with hemothorax and thrombocytopenia. He was diagnosed with MPM of the sarcomatoid cell type. However, his disease progressed rapidly and he died 11 days after admission. Bone marrow aspiration revealed metastatic MPM cells that had engulfed other blood cells. Accordingly, the observed thrombocytopenia was attributed to cellular cannibalism by metastatic MPM tumor cells. To the best of our knowledge, this is the first reported case of thrombocytopenia due to cellular cannibalism in a patient with this type of malignancy (MPM). The results suggested that although MPM rarely metastasizes to the bone marrow, bone marrow aspiration could be useful in such cases.

Inhibition of Human Malignant Pleural Mesothelioma Growth by Mesenchymal Stromal Cells.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. Methods: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. Results: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. Conclusions: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.

Therapeutic Synergy in Esophageal Cancer and Mesothelioma Is Predicted by Dynamic BH3 Profiling.

Approximately 20,000 patients per year are diagnosed with esophageal adenocarcinoma (EAC) and malignant pleural mesothelioma (MPM); fewer than 20% survive 5 years. Effective therapeutic strategies are limited although patients receive a combination of chemotherapeutics. These tumors harbor thousands of mutations that contribute to tumor development. Downstream of oncogenic driving mutations, altered tumor mitochondria promote resistance to apoptosis. Dynamic Bcl-2 homology-3 profiling (DBP) is a functional assay of live cells that identifies the mitochondrial proteins responsible for resistance to apoptosis. We hypothesized that DBP will predict which protein to target to overcome resistance thereby enhancing combinatorial therapy.DBP predicted that targeting either Mcl-1 or Bcl-xL increases the efficacy of the chemotherapeutic agent, cisplatin, whereas targeting Bcl-2 does not. We performed these assays by treating EAC and MPM cells with a combination of Bcl-2 homology-3 (BH3) mimetics and cisplatin. Following treatments, we performed efficacy assessments including apoptosis assays, IC50 calculations, and generation of a combinatorial index. DBP confirmed that targeting mitochondria with BH3 mimetics alters the threshold of apoptosis. These apoptotic effects were abolished when the mitochondrial pathway was disrupted. We validated our findings by developing knockdown models of antiapoptotic proteins Mcl-1, Bcl-xL, and the mitochondrial effector proteins Bax/Bak. Knockdown of Mcl-1 or Bcl-xL recapitulated the results of BH3 mimetics. In addition, we report an approach for BH3 profiling directly from patient tumor samples. We demonstrate that the DBP assay on living tumor cells measures the dynamic changes of resistance mechanisms, assesses response to combinatorial therapy, and provides results in a clinically feasible time frame.

Prospective real-world study on the pharmacokinetics of pembrolizumab in patients with solid tumors

BackgroundDosing schemes of pembrolizumab (anti-programmed cell death protein 1 monoclonal antibody) are solely based on pharmacokinetic (PK) modelling derived from phase I–III trials. The current study aimed to determine factors...

A novel BH3-mimetic, AZD0466, targeting BCL-XL and BCL-2 is effective in pre-clinical models of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive cancer with treatment limited to Cisplatin and Pemetrexed chemotherapy. Recently, we showed that drugs targeting the BCL-2-regulated apoptosis pathway could kill MPM cell lines in vitro, and control tumor growth in vivo. These studies showed BCL-XL was the dominant pro-survival BCL-2 family member correlating with its high-level expression in cells and patient tumor samples. In this study we show another inhibitor, AZD4320 that targets BCL-XL (and BCL-2), can also potently kill MPM tumor cells in vitro (EC50 values in the 200 nM range) and this effect is enhanced by co-inhibition of MCL-1 using AZD5991. Moreover, we show that a novel nanoparticle, AZD0466, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer, was as effective as standard-of-care chemotherapy, Cisplatin, at inhibiting tumor growth in mouse xenograft studies, and this effect was enhanced when both drugs were combined. Critically, the degree of thrombocytopenia, an on-target toxicity associated with BCL-XL inhibition, was significantly reduced throughout the treatment period compared to other BCL-XL-targeting BH3-mimetics. These pre-clinical findings provide a rationale for the future clinical evaluation for novel BH3-mimetic formulations in MPM, and indeed, other solid tumor types dependent on BCL-XL.