Human Malignant Pleural Mesothelioma Cell Lines Research Articles

Abstract 4085: The anti-VISTA x MSLN bispecific antibody-interferon beta mutein fusion protein: A trispecific immunocytokine with potent therapeutic efficacy against malignant pleural mesothelioma

Abstract Background Malignant pleural mesothelioma (MPM) is a rare and highly fatal cancer primarily induced by asbestos exposure. The 5-year overall survival rate for MPM patients is less than 20%. Before the FDA approval of the combination of immune checkpoint inhibitors (Nivolumab + Ipilimumab, CHECKMATE-743), chemotherapy was the only systemic therapy for MPM. However, the predominant histological type of MPM, epithelioid MPM, comprising approximately 70% of cases, showed limited clinical benefits from immune checkpoint inhibitors when compared to non-epithelioid histology. Consequently, there is an unmet medical need to develop a new anti-cancer drug specifically for the treatment of epithelioid MPM. In this study, we discovered that V-domain Ig suppressor of T cell activation (VISTA), an immune checkpoint protein, and Mesothelin (MSLN), a tumor differentiation antigen, are co-expressed in epithelioid MPM. Subsequently, we developed a biomarker-based anti-VISTA x MSLN bispecific antibody (BsAb) and anti-VISTA x MSLN BsAb-IFN-β mutein fusion protein (trispecific immunocytokine) for the treatment of epithelioid MPM. Method We designed and screened various structures of the anti-VISTA x MSLN BsAb. To produce the trispecific immunocytokine, we fused IFN-β mutein (R27T/C17S), which exhibits improved stability, productivity, and pharmacokinetic properties compare to recombinant IFN-β. The in vitro efficacy of the trispecific immunocytokine was tested in human MPM cell lines, and its in vivo efficacy was evaluated in a human MPM xenograft mouse model and a human IFNAR1/2 Knock-in (KI) mouse model. Results We demonstrated a strong positive correlation between the expression levels of VISTA and MSLN in epithelioid MPM. The Anti-VISTA x MSLN BsAb showed improved binding affinity and antibody-dependent cell cytotoxicity (ADCC) activity. The trispecific immunocytokine displays analogous biological activities to both Anti-VISTA x MSLN BsAb and IFN-β mutein. The trispecific immunocytokine exhibited potent in vivo and in vitro anti-cancer activities against epithelioid MPM preclinical models, operating through both direct cytotoxicity and indirect activation of immune cells. Conclusion The trispecific immunocytokine demonstrated robust in vitro and in vivo anti-cancer efficacy in the epithelioid MPM model. Our findings suggest that the trispecific immunocytoine is a promising drug candidate for epithelioid MPM. Citation Format: Hee Geon Park, Hyeon Ju Kim, Myeung Ryun Seo, Ji Eun Park, Tae Won Kim, Jeung Hoo Choi, Jay Park, Kyoung Song, Chan Gyu Lee, Hae Min Jeong, Jun Young Choi, Yeong Jeong Jeon, Yoon La Choi, Sung Youl Hong, Young Kee Shin. The anti-VISTA x MSLN bispecific antibody-interferon beta mutein fusion protein: A trispecific immunocytokine with potent therapeutic efficacy against malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4085.

Expression of CD24 gene in human malignant pleural mesothelioma and its relationship with prognosis

Objective: To investigate the expression of CD24 gene in human malignant pleural mesothelioma (MPM) cells and tissues, and evaluate its relationship with clinicopathological characteristics and clinical prognosis of MPM patients. Methods: In February 2021, UALCAN database was used to analyze the correlation between CD24 gene expression and clinicopathological characteristics in 87 cases of MPM patients. The TIMER 2.0 platform was used to explore the relationship between the expression of CD24 in MPM and tumor immune infiltrating cells. cBioportal online tool was used to analyze the correlation between CD24 and MPM tumor marker gene expression. RT-qPCR was used to analyze the expressions of CD24 gene in human normal pleural mesothelial cell lines LP9 and MPM cell lines NCI-H28 (epithelial type), NCI-H2052 (sarcoma type), and NCI-H2452 (biphasic mixed type). RT-qPCR was performed to detect the expressions of CD24 gene in 18 cases of MPM tissues and matched normal pleural tissues. The expression difference of CD24 protein in normal mesothelial tissue and MPM tissue was analyzed by immunohistochemistry. A Kaplan-Meier model was constructed to explore the influence of CD24 gene expression on the prognosis of MPM patients, and Cox regression analysis of prognostic factors in MPM patients was performed. Results: The CD24 gene expression without TP53 mutation MPM patients was significantly higher than that of patients in TP53 mutation (P<0.05). The expression of CD24 gene in MPM was positively correlated with B cells (r(s)=0.37, P<0.001). The expression of CD24 gene had a positive correlation with the expressions of thrombospondin 2 (THBS2) (r(s)=0.26, P<0.05), and had a negative correlation with the expression of epidermal growth factor containing fibulin like extracellular matrix protein 1 (EFEMP1), mesothelin (MSLN) and calbindin 2 (CALB2) (r(s)=-0.31, -0.52, -0.43, P<0.05). RT-qPCR showed that the expression level of CD24 gene in MPM cells (NCI-H28, NCI-H2052 and NCI-H2452) was significantly higher than that in normal pleural mesothelial LP9 cells. The expression level of CD24 gene in MPM tissues was significantly higher than that in matched normal pleural tissues (P<0.05). Immunohistochemistry showed that the expressions of CD24 protein in epithelial and sarcoma MPM tissues were higher than those of matched normal pleural tissues. Compared with low expression of CD24 gene, MPM patients with high expression of CD24 gene had lower overall survival (HR=2.100, 95%CI: 1.336-3.424, P<0.05) and disease-free survival (HR=1.800, 95%CI: 1.026-2.625, P<0.05). Cox multivariate analysis showed that compared with the biphasic mixed type, the epithelial type was a protective factor for the prognosis of MPM patients (HR=0.321, 95%CI: 0.172-0.623, P<0.001). Compared with low expression of CD24 gene, high expression of CD24 gene was an independent risk factor for the prognosis of MPM patients (HR=2.412, 95%CI: 1.291-4.492, P=0.006) . Conclusion: CD24 gene and protein are highly expressed in MPM tissues, and the high expression of CD24 gene suggests poor prognosis in MPM patients.

CHEMORESISTANCE RELATED TO HYPOXIA ADAPTATION INMESOTHELIOMA CELLS FROM TUMOR SPHEROIDS.

Hypoxia has been noted as a key factor for induction and maintenance of cancer stemness thereby leading to therapy resistance. Three-dimensional (3D) spheroid models demonstrate a heterogeneity of hypoxic regions replicating the in vivo situation within tumors. Utilizing an established 3D spheroid model, we investigated whether extrinsic hypoxia reinforced chemoresistance in malignant pleural mesothelioma (MPM) spheroids. Tumor spheres were generated from Meso-1 (a typical human MPM cell line) cells having high spheroid-forming ability. To induce hypoxia condition, we utilized a hypoxia chamber with regulation of O2and CO2levels. Cell viability was estimated by a WST-8assay. Real-time polymerase chain reaction and Western blot were performed to evaluate the expression at mRNA and protein levels. Compared with cells cultured in the two-dimensional monolayer model, tumor sphere cells showed elevated mRNA levels of cancer stemness markers (CD26, CD44and ABCG2) and protein levels of the stemness and hypoxia adaptation markers (ABCG2, ALDH1A1and HIFs). Correlating with this, 3D spheroid cells were more resistant to permetrexed and topotecan than the two-dimensional cells, indicative of their potential for hypoxic adaptation. Furthermore, significantly stronger resistance to both chemotherapeutic agents was observed in spheroid cells upon hypoxic challenge compared to spheroid cells under normoxia. From the present data, it is concluded that hypoxia adaptation of MPM cells from tumor spheres could enhance their chemoresistance.

Immunostimulatory oncolytic activity of coxsackievirus A11 in human malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive solid cancer with a poor prognosis, whereas coxsackievirus A11 (CVA11) is a potential oncolytic virus for cancer treatment. We here investigated the oncolytic activity of CVA11 with human MPM cell lines. CVA11 infection was cytotoxic in all six MPM cell lines examined and showed no or minimal cytotoxicity toward normal human normal cell lines. MPM cells with a higher surface level of intercellular adhesion molecule-1 (ICAM-1) expression tended to be more susceptible to CVA11-induced cytotoxicity, and a neutralizing antibody to ICAM-1 attenuated such cytotoxicity. CVA11 infection activated signaling by Akt and extracellular signal-regulated kinase (ERK) pathways, and inhibitors of such signaling also abrogated CVA11-mediated cytotoxicity. Furthermore, CVA11 infection-triggered multiple modes of tumor cell death including apoptosis, pyroptosis, and necroptosis, and such death was accompanied by the release or exposure of the proinflammatory cytokine interleukin-1β and damage-associated molecular patterns such as calreticulin, high-mobility group box-1, annexin A1, and heat shock protein 70, which are hallmarks of immunogenic cell death. Notably, in vivo treatment of human MPM xenografts with intratumoral CVA11 injection resulted in significant suppression of tumor growth in SCID mice, and all mice infected with CVA11 showed no significant change in body weight. Our findings collectively suggest that the oncolytic activity of CVA11 for MPM is dependent on ICAM-1 as a virus receptor, as well as on Akt and ERK signaling, and that oncolytic virotherapy with CVA11 is a promising treatment modality with immunostimulatory activity for human MPM.

Possible Therapeutic Utility of anti-Cell Adhesion Molecule 1 Antibodies for Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly aggressive malignant tumor, and the effective therapeutic drugs are limited. Thus, the establishment of novel therapeutic method is desired. Considerable proportion of MPMs are shown to express cell adhesion molecule 1 (CADM1), and to use CADM1 to bind to and proliferate on the pleural mesothelial surface, suggesting that CADM1 is a possible therapeutic target. Here, anti-CADM1 ectodomain chicken monoclonal antibodies, 3E1 and 9D2, were examined for their possible therapeutic utility. The full-length form of CADM1 was expressed in eight out of twelve human MPM cell lines. MPM cell lines were cultured on a confluent monolayer of mesothelial MeT-5A cells in the presence of 9D2, the neutralizing antibody. 9D2 suppressed the cell growth of CADM1-positive MPM cells with the loss and aggregation of CADM1 molecules on the MPM cell membrane, but not of CADM1-negative MPM cells. Co-addition of 3E1, lacking the neutralizing action, enhanced the growth-suppressive effect of 9D2. The two antibodies were tested as drug delivery vectors. 3E1 was converted into a humanized antibody (h3E1) and conjugated with monomethyl auristatin E (MMAE), a tubulin polymerization inhibitor. When the resulting h3E1–MMAE antibody-drug conjugate (ADC) was added to the standard cultures of CADM1-positive MPM cells, it suppressed the cell growth in a dose-dependent manner. Co-addition of 9D2 enhanced the growth-suppressive effect of h3E1–MMAE ADC. Anti-CADM1 ectodomain antibodies were suggested to serve as both antibody drugs and drug vectors in the treatment of MPM.

Tumor treating fields affect mesothelioma cell proliferation by exerting histotype-dependent cell cycle checkpoint activations and transcriptional modulations

Although clinical antitumor activity of Tumor Treating Fields (TTFields) has been reported in malignant pleural mesothelioma (MPM) patients, the mechanisms behind the different selectivity displayed by the various MPM histotypes to this physical therapy has not been elucidated yet. Taking advantage of the development of well characterized human MPM cell lines derived from pleural effusion and/or lavages of patients’ thoracic cavity, we investigated the biological effects of TTFields against these cells, representative of epithelioid, biphasic, and sarcomatoid histotypes. Growth inhibition and cell cycle perturbations caused by TTFields were investigated side by side with RNA-Seq analyses at different exposure times to identify pathways involved in cell response to treatment. We observed significant differences of response to TTFields among the cell lines. Cell cycle analysis revealed that the most sensitive cells (epithelioid CD473) were blocked in G2M phase followed by formation of polyploid cells. The least sensitive cells (sarcomatoid CD60) were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptosis was present in all samples, but while epithelioid cell death was already observed during the first 24 h of treatment, sarcomatoid cells needed longer times before they engaged apoptotic pathways. RNA-Seq experiments demonstrated that TTFields induced a transcriptional response already detectable at early time points (8 h). The number of differentially expressed genes was higher in CD473 than in CD60 cells, involving several pathways, such as those pertinent to cell cycle checkpoints, DNA repair, and histone modifications. Our data provide further support to the notion that the antitumor effects of TTFields are not simply related to a non-specific reaction to a physical stimulus, but are dependent on the biological background of the cells and the particular sensitivity to TTFields observed in epithelioid MPM cells is associated with a higher transcriptional activity than that observed in sarcomatoid models.

Picropodophyllin inhibits the growth of pemetrexed-resistant malignant pleural mesothelioma via microtubule inhibition and IGF-1R-, caspase-independent pathways.

BackgroundAcquired pemetrexed resistance leads to treatment interruption in patients with malignant pleural mesothelioma (MPM). Insulin-like growth factor-I receptor (IGF-1R) inhibitor is a candidate for treating pemetrexed-naïve MPM. However, the efficacy of cytotoxic and targeted drugs in acquired-pemetrexed resistant MPM is unclear. We explored anticancer drugs, including the IGF-1R inhibitor picropodophyllin, against acquired pemetrexed-resistant MPMs.MethodsAcquired pemetrexed-resistant human MPM cell lines, named as H2452/PEM and 211H/PEM after their parental lines H2452 and 211H, respectively, were established by exposure to pemetrexed in vitro. Picropodophyllin and siRNA for IGF-1R knockdown were used to evaluate the efficacy of IGF-1R inhibition. Immunofluorescence was used to evaluate microtubule localization. The efficacy of picropodophyllin was evaluated in 3-dimensional MPM models.ResultsThe acquired pemetrexed-resistant MPM lines retained their resistance after the removal of culture treatment. IGF-1R levels in H2452/PEM cells were higher than those in H2452 cells but not in 211H/PEM cells compared to the respective parental line. Picropodophyllin induced sub-G1 arrest in H2452/PEM cells but induced G2/M phase arrest in 211H/PEM cells, leading to caspase-independent cell death in the two acquired pemetrexed-resistant MPM lines. Although picropodophyllin inhibited phosphorylation of IGF-1R, specific inhibition of IGF-1R by RNA interference did not reduce the viability of pemetrexed-resistant MPM lines. Additionally, picropodophyllin reduced the viability of both IGF-1R knockdown pemetrexed-resistant MPM cells. Picropodophyllin was cytotoxic in acquired-pemetrexed-resistant MPM lines because of inhibition of microtubule formation and induction of aberrant mitosis. Moreover, combination treatment with picropodophyllin and vinorelbine synergistically affected the pemetrexed-resistant MPM lines but not the parental lines. Furthermore, we observed a similar efficacy of picropodophyllin in 3-dimensional pemetrexed-resistant MPM models.ConclusionsPicropodophyllin may offer novel therapeutic properties for treating acquired pemetrexed-resistant MPM. Targeting tubulin may be an important strategy in the treatment of MPM after the discontinuation of pemetrexed.

Cisplatin-Chelated Iminodiacetic Acid-Conjugated Hyaluronic Acid Nanogels for the Treatment of Malignant Pleural Mesothelioma in Mice.

Malignant pleural mesothelioma (MPM) is one of the intractable cancers that require a more effective therapeutic strategy for clinical practice. Hyaluronic acid (HA) nanogels were prepared by the chelation of cisplatin (CDDP) with different molecular weights of iminodiacetic acid-conjugated hyaluronic acid (HA-IDA). The sizes of the 100, 850, and 2000 kDa HA nanogels were 33, 43, and 44 nm, respectively. MSTO-211H, a human MPM cell line, was more effective in taking up all three HA nanogels compared to AB22, a mouse MPM cell line. In addition, the 850 kDa HA nanogel showed higher anticancer activity against AB22 and MSTO-211H than 100 and 2000 kDa HA nanogels. Furthermore, all the HA nanogels showed a milder cytotoxic effect on normal Met-5A mesothelial cells compared to that exhibited by free CDDP. Finally, the 850 kDa HA nanogel was administrated intrapleurally into both the MSTO-211H xenograft and AB22 allograft mouse models of MPM using an injectable HA-based hydrogel. HA nanogels showed a significant therapeutic effect in both the xenograft and allograft models.

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.

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.

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.

Effectiveness and Safety of Tumor Treating Fields (150 kHz) plus Cisplatin or Pemetrexed Combination in Mesothelioma: in Vitro and in Vivo Findings

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer linked to asbestos exposure with a poor prognosis and limited treatment options. Tumor Treating Fields (TTFields) are a noninvasive, antineoplastic treatment delivered locoregionally to the tumor bed via low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. TTFields are FDA-approved as a first line therapy with pemetrexed plus platinum-based agent for unresectable MPM. TTFields with this combination was evaluated in the phase 2 STELLAR clinical trial (Ceresoli et al, Lancet Oncology 2019) in patients with unresectable MPM, demonstrating a promising median overall survival of ∼18.2 months (vs ∼12 months; historical control) without increases in systemic toxicity. Utilizing in vitro and in vivo models, we investigated the effects of TTFields alone and in combination with cisplatin or pemetrexed in MPM. Using the inovitroTM system, cells from human MPM cell lines (NCI-H2052 and MSTO-211H) were treated at various frequencies of TTFields for 72 hours. The combined treatment of TTFields with cisplatin or pemetrexed was tested by applying TTFields at the 150 kHz optimal frequency with various concentrations of cisplatin (80–5120 nM) or pemetrexed (2–4096 nM). Cell counts, clonogenic potential, and apoptosis induction were determined. TTFields (150 kHz) were applied to Fisher rats with IL-45 intrapleural tumors cells for 8 days and to C57BL/6 mice with RN5 S.C. tumors for 7 days. Tumor volume was determined using MRI. To determine treatment safety, TTFields (2–3 V/cm) were applied to the torsos of Sprague-Dawley rats at 150 kHz for 2 weeks and all major internal organs were histologically compared. The optimal TTFields frequency was 150 kHz in both NCI-H2052 and MSTO-211H human MPM cell lines. TTFields (150 kHz; 1.1 V/cm) application for 72 hours led to a 45-51% reduction in cell counts and a 46-64% additional decrease in clonogenic potential. TTFields plus cisplatin or pemetrexed significantly reduced the cell count, the clonogenic potential and induced apoptosis versus each treatment alone (P <0.0001). Tumor volume was significantly decreased in the TTFields plus pemetrexed plus cisplatin combination group versus the control group in both tumor models. Histological analysis of major internal organs showed no increase in pathological findings when TTFields were applied to the rat torso. These in vitro and in vivo data demonstrate that the optimal TTFields frequency to inhibit MPM is 150 kHz. TTFields alone showed effectiveness and safety, and combined with cisplatin or pemetrexed demonstrated enhanced effectiveness with no increased safety concerns in the torso. Findings were consistent with efficacy and safety findings in the STELLAR trial.

High Oncolytic Activity of a Double-Deleted Vaccinia Virus Copenhagen Strain against Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a cancer of the pleura that lacks efficient treatment. Oncolytic immunotherapy using oncolytic vaccinia virus (VV) may represent an alternative therapeutic approach for the treatment of this malignancy. Here, we studied the oncolytic activity of VV thymidine kinase (TK)-ribonucleotide reductase (RR)-/green fluorescent protein (GFP) against MPM. This virus is a VV from the Copenhagen strain that is deleted of two genes encoding the TK (J2R) and the RR (I4L) and that express the GFP. First, we show in vitro that VVTK-RR-/GFP efficiently infects and kills the twenty-two human MPM cell lines used in this study. We also show that the virus replicates in all eight tested MPM cell lines, however, with approximately a 10-fold difference in the amplification level from one cell line to another. Then, we studied the therapeutic efficiency of VVTK-RR-/GFP in non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice that bear peritoneal human MPM tumors. One intraperitoneal infection of VVTK-RR-/GFP reduces the tumor burden and significantly increases mice survival compared to untreated animals. Thus, VVTK-RR- may be a promising oncolytic virus (OV) for the oncolytic immunotherapy of MPM.

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9-18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced. This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

Abstract 3086: Discovery of a first-in-class TEAD inhibitor which directly inhibits YAP/TAZ-TEAD protein-protein interaction and shows a potent anti-tumor effect in malignant pleural mesothelioma

Abstract Objective: Hippo signaling pathway is known to regulate organ development. In Hippo signaling pathway, YAP or TAZ works as a transcriptional co-activator and forms a transcriptional complex with TEAD. In several cancers, upstream factors in Hippo pathway are inactivated by genetic alterations. When the upstream factors are inactivated, TEAD is activated and forms a complex with YAP/TAZ resulting in enhancement of cell proliferation, drug resistance and so on. In the activation process, S-palmitoylation of TEAD is necessary for binding to YAP/TAZ. Malignant pleural mesothelioma (MPM) is one of cancer types which have genetic alterations in Hippo pathway genes. Although YAP/TAZ-TEAD inhibitor should be an ideal drug for MPM therapy, there are only a few reports about YAP/TAZ-TEAD inhibitor and the efficacy and selectivity are not sufficient. In this study, we succeeded to synthesize a small molecule TEAD inhibitor, K-975, and evaluated its mechanism of action and anti-tumor effect against MPM. Materials/methods: Inhibitory activity of K-975 on YAP/TAZ-TEAD protein-protein interaction (PPI) was evaluated in surface plasmon resonance (SPR) and co-immunoprecipitation assay. The effect of K-975 on palmitoylation status of TEAD was also evaluated. The three-dimensional structure of YAP-binding domain of TEAD1 in complex with K-975 was determined by X-ray crystallography. Anti-tumor effect of K-975 was evaluated by using MPM cell lines. Furthermore, using a derivative of K-975, 2 week-toxicity studies in rats and monkeys were performed. Results: K-975 inhibited YAP-TEAD and TAZ-TEAD PPI in NCI-H226 cells, a human MPM cell line. Also, K-975 inhibited palmitoylation of TEAD. The crystal structure revealed that K-975 directly bound to cysteine residue in YAP-binding domain of TEAD1. This cysteine residue is highly conserved in TEAD family and known as a site of S-palmitoylation. K-975 inhibited the cell proliferation of NCI-H226 with GI50 of about 20 nmol/L. K-975 also induced a change of gene expressions similar to that induced by YAP knockdown. In vivo experiments, K-975 strongly suppressed the tumor growth in several s.c. xenograft models and showed a significant survival benefit in an orthotopic xenograft model. However, 2 week-toxicity studies of a K-975 derivative with optimized bioavailability showed some pathological findings which suggested the renal toxicity. Conclusion: We synthesized a first-in-class drug which directly binds to TEAD protein and inhibits YAP/TAZ-TEAD PPI. K-975 showed a strong anti-tumor effect in pre-clinical MPM models. Although the renal toxicity might cause some difficulty in clinical use, we believe that a K-975 derivative has a possibility to become an effective drug candidate for MPM therapy. Citation Format: Ayumi Kaneda, Toshihiro Seike, Takeshi Uemori, Kensuke Myojo, Kensuke Aida, Tomohiro Danjo, Takahiro Nakajima, Daisuke Yamaguchi, Tomoko Hamada, Yoshiro Tsuji, Kaori Hamaguchi, Mai Yasunaga, Nobumasa Otsubo, Hideyuki Onodera, Yoichi Nishiya, Michihiko Suzuki, Junichi Saito, Toshihiko Ishii, Ryuichiro Nakai. Discovery of a first-in-class TEAD inhibitor which directly inhibits YAP/TAZ-TEAD protein-protein interaction and shows a potent anti-tumor effect in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3086.

ADAM10 mediates malignant pleural mesothelioma invasiveness

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options and treatment efficiency. Even if the latency period between asbestos exposure, the main risk factor, and mesothelioma development is very long, the local invasion of mesothelioma is very rapid leading to a mean survival of one year after diagnosis. ADAM10 (A Disintegrin And Metalloprotease) sheddase targets membrane-bound substrates and its overexpression is associated with progression in several cancers. However, nothing is known about ADAM10 implication in MPM. In this study, we demonstrated higher ADAM10 expression levels in human MPM as compared to control pleural samples and in human MPM cell line. This ADAM10 overexpression was also observed in murine MPM samples. Two mouse mesothelioma cell lines were used in this study including one primary cell line obtained by repeated asbestos fibre injections. We show, in vitro, that ADAM10 targeting through shRNA and pharmacological (GI254023X) approaches reduced drastically mesothelioma cell migration and invasion, as well as for human mesothelioma cells treated with siRNA targeting ADAM10. Moreover, ADAM10 downregulation in murine mesothelioma cells significantly impairs MPM progression in vivo after intrapleural cell injection. We also demonstrate that ADAM10 sheddase downregulation decreases the production of a soluble N-cadherin fragment through membrane N-cadherin, which stimulated mesothelioma cell migration. Taken together, we demonstrate that ADAM10 is overexpressed in MPM and takes part to MPM progression through the generation of N-cadherin fragment that stimulates mesothelioma cell migration. ADAM10 inhibition is worth considering as a therapeutic perspective in mesothelioma context.

Nintedanib Is Active in Malignant Pleural Mesothelioma Cell Models and Inhibits Angiogenesis and Tumor Growth In Vivo.

Purpose: Malignant pleural mesothelioma (MPM) is an aggressive thoracic tumor type with limited treatment options and poor prognosis. The angiokinase inhibitor nintedanib has shown promising activity in the LUME-Meso phase II MPM trial and thus is currently being evaluated in the confirmatory LUME-Meso phase III trial. However, the anti-MPM potential of nintedanib has not been studied in the preclinical setting.Experimental Design: We have examined the antineoplastic activity of nintedanib in various in vitro and in vivo models of human MPM.Results: Nintedanib's target receptors were (co)expressed in all the 20 investigated human MPM cell lines. Nintedanib inhibited MPM cell growth in both short- and long-term viability assays. Reduced MPM cell proliferation and migration and the inhibition of Erk1/2 phosphorylation were also observed upon nintedanib treatment in vitro Additive effects on cell viability were detected when nintedanib was combined with cisplatin, a drug routinely used for systemic MPM therapy. In an orthotopic mouse model of human MPM, survival of animals receiving nintedanib per os showed a favorable trend, but no significant benefit. Nintedanib significantly reduced tumor burden and vascularization and prolonged the survival of mice when it was administered intraperitoneally. Importantly, unlike bevacizumab, nintedanib demonstrated significant in vivo antivascular and antitumor potential independently of baseline VEGF-A levels.Conclusions: Nintedanib exerts significant antitumor activity in MPM both in vitro and in vivo These data provide preclinical support for the concept of LUME-Meso trials evaluating nintedanib in patients with unresectable MPM. Clin Cancer Res; 24(15); 3729-40. ©2018 AACR.

Inhibition of yes-associated protein down-regulates PD-L1 (CD274) expression in human malignant pleural mesothelioma.

Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70, P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.

Resveratrol induces cell cycle arrest and apoptosis in epithelioid malignant pleural mesothelioma cells

Abstract Background Resveratrol is a natural anti-carcinogenic polyphenol. Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis. In this study, we investigated the effects of resveratrol on epithelioid MPM. Material and methods Human epithelioid MPM cell line (NCI-H2452) was exposed to resveratrol (5–200 μM) for 24 or 48 h. Cell viability was assessed by WST-1 assay. Flow cytometry analyses were performed to evaluate the effects of resveratrol on cell cycle distribution and apoptosis. Western blot analysis was used to determine protein expression levels of antioxidant enzymes, cyclin D1 and p53. Reactive oxygen species (ROS) were measured using H2DCFDA. Results Resveratrol reduced cell viability of the cells in a concentration and time dependent manner. After treatment, the cells accumulated in G0/G1 phase and the percentage of cells in G2/M phase was reduced. Resveratrol decreased cyclin D1 and increased p53 expression in cell lysates. Treated cells exhibited increased apoptotic activity. ROS were elevated with resveratrol treatment, but there was no change in the expression of superoxide dismutase (SOD)-1, SOD-2 and glutathione peroxidase. Conclusion Our results revealed that resveratrol exhibits anti-cell viability effect on epithelioid MPM cells by inducing cell cycle arrest and apoptosis. Resveratrol may become a potential therapeutic agent for epithelioid MPM.

Turning back the Wheel: Inducing Mesenchymal to Epithelial Transition via Wilms Tumor 1 Knockdown in Human Mesothelioma Cell Lines to Influence Proliferation, Invasiveness, and Chemotaxis.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that arises from the surface of the pleura and is associated with a history of asbestos exposure. The tumor is characterized by a strong local invasiveness and a poor response to any single modality therapy. Therefore clinical outcome of patients with MPM is poor and median survival time of untreated patients with MPM is 7months from initial diagnosis. The Wilms Tumor Protein 1 (WT1) is a transcription factor which is highly expressed by MPM and is involved in cellular development and survival. We evaluated the role of WT1 in two human MPM cell lines (MSTO and H2052) expressing high levels of WT1. We performed a knockdown of WT1 using siRNA. Knockdown of WT1 was confirmed by Westernblotting. After knockdown of WT1 we investigated the effect on proliferation, chemoresistance, chemotaxis and migration. We could demonstrate that knockdown of WT1 suppresses chemoresistance in both cell lines compared with control (scrambled siRNA). Additionally, WT1 knockdown reduces proliferation, chemotaxis and invasiveness of mesothelioma cell lines. WT1 reduces malignancy of malignant mesothelioma cell lines and might be a new molecular target in mesothelioma therapy. Further investigations are needed to discover the mechanisms of chemoresistance depending on WT1.