Model Of Malignant Pleural Mesothelioma 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.

Treatment with tumor-treating fields (TTFields) suppresses intercellular tunneling nanotube formation in vitro and upregulates immuno-oncologic biomarkers in vivo in malignant mesothelioma.

Disruption of intercellular communication within tumors is emerging as a novel potential strategy for cancer-directed therapy. Tumor-Treating Fields (TTFields) therapy is a treatment modality that has itself emerged over the past decade in active clinical use for patients with glioblastoma and malignant mesothelioma, based on the principle of using low-intensity alternating electric fields to disrupt microtubules in cancer cells undergoing mitosis. There is a need to identify other cellular and molecular effects of this treatment approach that could explain reported increased overall survival when TTFields are added to standard systemic agents. Tunneling nanotube (TNTs) are cell-contact-dependent filamentous-actin-based cellular protrusions that can connect two or more cells at long-range. They are upregulated in cancer, facilitating cell growth, differentiation, and in the case of invasive cancer phenotypes, a more chemoresistant phenotype. To determine whether TNTs present a potential therapeutic target for TTFields, we applied TTFields to malignant pleural mesothelioma (MPM) cells forming TNTs in vitro. TTFields at 1.0 V/cm significantly suppressed TNT formation in biphasic subtype MPM, but not sarcomatoid MPM, independent of effects on cell number. TTFields did not significantly affect function of TNTs assessed by measuring intercellular transport of mitochondrial cargo via intact TNTs. We further leveraged a spatial transcriptomic approach to characterize TTFields-induced changes to molecular profiles in vivo using an animal model of MPM. We discovered TTFields induced upregulation of immuno-oncologic biomarkers with simultaneous downregulation of pathways associated with cell hyperproliferation, invasion, and other critical regulators of oncogenic growth. Several molecular classes and pathways coincide with markers that we and others have found to be differentially expressed in cancer cell TNTs, including MPM specifically. We visualized short TNTs in the dense stromatous tumor material selected as regions of interest for spatial genomic assessment. Superimposing these regions of interest from spatial genomics over the plane of TNT clusters imaged in intact tissue is a new method that we designate Spatial Profiling of Tunneling nanoTubes (SPOTT). In sum, these results position TNTs as potential therapeutic targets for TTFields-directed cancer treatment strategies. We also identified the ability of TTFields to remodel the tumor microenvironment landscape at the molecular level, thereby presenting a potential novel strategy for converting tumors at the cellular level from 'cold' to 'hot' for potential response to immunotherapeutic drugs.

Asbestos accelerates disease onset in a genetic model of malignant pleural mesothelioma.

Hypothesis: Asbestos-driven inflammation contributes to malignant pleural mesothelioma beyond the acquisition of rate-limiting mutations. Methods: Genetically modified conditional allelic mice that were previously shown to develop mesothelioma in the absence of exposure to asbestos were induced with lentiviral vector expressing Cre recombinase with and without intrapleural injection of amosite asbestos and monitored until symptoms required euthanasia. Resulting tumours were examined histologically and by immunohistochemistry for expression of lineage markers and immune cell infiltration. Results: Injection of asbestos dramatically accelerated disease onset and end-stage tumour burden. Tumours developed in the presence of asbestos showed increased macrophage infiltration. Pharmacological suppression of macrophages in mice with established tumours failed to extend survival or to enhance response to chemotherapy. Conclusion: Asbestos-driven inflammation contributes to the severity of mesothelioma beyond the acquisition of rate-limiting mutations, however, targeted suppression of macrophages in established epithelioid mesothelioma showed no therapeutic benefit.

Sequential multi-parametric MRI in assessment of the histological subtype and features in the malignant pleural mesothelioma xenografts

ObjectiveIt is still a challenge to find a noninvasive technique to distinguish the histological subtypes of malignant pleural mesothelioma (MPM) and characterize the development of related histological features. We investigated the potential value of multiparametric MRI in the assessment of the histological subtype and development of histologic features in the MPM xenograft model. MethodsMPM xenograft models were developed by injecting tumour cells into the right axillary space of nude mice. The T1, T2, R2*, T2*, apparent diffusion coefficient (ADC), true diffusion coefficient (D), pseudo diffusion coefficient (D*), and perfusion fraction (f) at 14 d, 28 d, and 42 d were measured and compared between the epithelial and biphasic MPM. Correlations between multiparametric MRI parameters and histologic features, including necrotic fraction (NF) and microvessel density (MVD), were analysed. ResultsThis study found that T2, T2* and IVIM-DWI parameters can reflect the spatial and temporal heterogeneity of MPM. Compared to the epithelial MPM, T2 and T2* were higher and ADC, D, D*, and f were lower in the biphasic MPM (P < 0.05). MRI parameters were different in different stages of epithelial and biphasic MPM. Moderate correlations were found between ADC and tumor volume and NF in the epithelial MPM, and there was a correlation between f and tumor volume and NF and MVD in the two groups. ConclusionMRI parameters changed with tumor progression in a xenograft model of MPM. MRI parameters may provide useful biomarkers for evaluating the histological subtype and histological features development of MPM.

The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.

EP07.01-024 Preclinical Investigation of Immune Checkpoint Blockade and Anti-Angiogenic Therapy in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a fatal cancer type that affects the membranes lining the lungs and is causally associated with asbestos exposure. Despite the effectiveness of conventional anti-cancer treatment, its prognosis remains very poor. We previously demonstrated a significant survival benefit for PD-L1 blockade in preclinical models of MPM. Data from preclinical studies in other tumour models show that anti-PD-L1 and anti-angiogenic therapy can enhance each other’s efficacy, thereby supporting the investigation of combined blockade of PD-L1 and VEGFR2 in MPM.

Abstract 2037: Spatial omic changes of malignant pleural mesothelioma following treatment using tumor-treating fields

Abstract Background: Application of alternating-electric fields as cancer-directed therapy known as Tumor-Treating Fields (TTFields) has been shown to be effective by exerting dipole alignment forces on polar microtubule subunits and dielectrophoretic forces in the cytokinetic furrow. This effect results in disruption of mitosis at the cellular level, and at the clinical level with significantly prolonged overall survival of patients with glioblastoma and malignant pleural mesothelioma (MPM). The molecular alterations that occur at the genomic and transcriptomic levels following TTFields treatment are unknown. We applied a spatial omics approach to elucidate spatial intratumoral effects of TTFields at the molecular level in regions of interest (ROI) in a mouse model of MPM. Methods: Eight Balb/C mice were injected with AB1 MPM cells until sizable tumors were observed (2-3 weeks). TTFields were applied, with heat sham used as a negative control. After 14 days (6 days of treatment, 2 days of rest, then 6 more days of treatment) tumors were resected, fixed and paraffin embedded. Following the Nanostring GeoMx protocol, thin sections of all eight tumors were placed on a slide, and incubated with both Ki-67 antibodies, and a GeoMx Mouse Cancer Transcriptome Atlas RNA probe set. Using Ki-67 staining as a guide, 12 ROIs were selected across each tumor to capture intratumoral heterogeneity, including the core and periphery of each tumor The DSP barcoded RNA probes were cleaved, sequenced and analyzed. We compared differential expression of subset gene classes of TTFields vs heat sham treated tumors; results were further stratified into Ki-67 high and low subsets for each sample. Results were assessed by Gene Set Enrichment. Results: Sham-treated tumors grew to over 300 mm3; TTFields-treated tumors averaged 100 mm3, confirming anti-tumor effect. Gene Set Enrichment Analysis uncovered upregulation of genes associated with interferon-alpha and -gamma responses in TTFields-treated tumors, which also displayed downregulation of pathway components associated with glycolysis, mTOR signaling, oxidative phosphorylation, cell invasion, hypoxia, and TNF-alpha signaling. Spatial analysis detected a heterogeneous response and differential expression between different portions of tumors. Conclusions: TTFields application induces clear patterns of differential expression in the transcriptome of treated tumors, including an increasingly immune-stimulated tumor microenvironment that also affects molecular pathways critical to cellular proliferation and invasion. These findings point the way toward improved understanding of timing and sequences of TTFields in relation to systemic cytotoxic, targeted, and immune modulation forms of therapeutic strategies. Our finding of upregulated immune response implicates TTFields as a potential synergistic tactic when coupled with immunotherapeutic approaches. Citation Format: Emil Lou, Katherine Ladner, Kerem Wainer-Katsir, Karina Deniz, Yaara Porat, Boris Brant, Shiri Davidi, Yuki Padmanabhan, Phillip Wong, Amrinder Nain, Clifford J. Steer, Moshe Giladi. Spatial omic changes of malignant pleural mesothelioma following treatment using tumor-treating fields [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2037.

Abstract 5470: Inhibition of facilitates chromatin transcription complex attenuates mesothelioma growth

Abstract Introduction: Malignant pleural mesothelioma (MPM) is an aggressive incurable cancer usually associated with asbestos exposure. MPM has unique molecular features such as wild-type p53 (&amp;gt;90% regardless of histology) and aberrantly activated NF-κB. A diverse spectrum of anti-MPM therapeutic agents have been trialed, but clinically effective ones remain elusive. Herein, we propose to use a novel small molecule (CBL0137) that simultaneously suppresses NF-κB and activates tumor suppressor p53 via targeting FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone critical for DNA repair in cancers. Methods: We employed in-silico, in-vitro and in-vivo MPM models and patient tumor specimens to characterize CBL0137 efficacy in MPM. CBL0137 effects were assessed in a panel of MPM cell lines by proliferation, colony foci formation, flow cytometry, and cell cycle assays. Drug-specific mechanisms were verified at transcript and protein levels using appropriate molecular assays. Both monotherapy and combinatorial regimens were tested to evaluate the optimal deployment of CBL0137 in comparison to several controls including standard chemotherapy. Immunodeficient (NSG) mice with subcutaneous and orthotopic MPM tumor grafts were used to evaluate the anti-tumor efficacy of CBL0137. Kaplan-Meier and log-rank test were applied for survival outcomes. Results: We observed that transcript and protein levels of SSRP1 and SPT16, subunits of the FACT complex, were significantly upregulated in our collection of MPM cell lines and specimens compared to normal. The TCGA database revealed that overexpression of these FACT subunits was associated with poor survival. As determined by western blot and luciferase-reporter assay, CBL0137 simultaneously inhibited NF-κB and activated p53 in MPM cells. CBL0137 treatment significantly (p&amp;lt;0.05) suppressed MPM cells proliferation, invasiveness, colony foci formation, and increased the number of apoptotic cells. Interestingly, in combination with cisplatin, CBL0137 exhibited additive anti-tumor activity in much lower dose ranges compared to monotherapy. Additionally, CBL0137 synergized with different classes of agents like microRNA-215, which triggers activation of p53 via regulation of MDM2 at transcript level. In vivo, CBL0137 treatment significantly suppressed MPM tumor growth in subcutaneous and orthotopic xenograft models compared to control. Kaplan-Meier analysis showed that CBL0137 treatment improved overall survival. Conclusions: MPM is dependent on FACT for tumor progression since higher levels of SSRP1 and SPT16 are associated with worse prognosis. This MPM vulnerability can be leveraged to identify novel therapeutic paradigms. Targeting this MPM dependency by CBL0137 (dual effects on NF-κB and p53) alone or in various combinations with different classes of drugs holds great promise for improved outcomes regardless of histotype. Citation Format: Anand Singh, Nathanael Pruett, Roma Pahwa, Sudheer K. Gara, Shivani Dixit, Agnes Choi, David S. Schrump, Chuong D. Hoang. Inhibition of facilitates chromatin transcription complex attenuates mesothelioma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5470.

Abstract 51: Multiomic profiling of patient-derived xenografts established from patients with malignant pleural mesothelioma proposes pathways associated with poor prognosis

Abstract Background: Despite recent treatment advances, malignant pleural mesothelioma (MPM) is an aggressive, recalcitrant malignancy. Currently, histologic subtype (epithelioid/non-epithelioid/biphasic) is the primary prognostic factor; other potential biomarkers to guide therapeutic strategies remain elusive. Even with multimodality therapies, recurrence is high in early-stage disease. In the unresectable/metastatic setting, there are only two FDA approved regimens, both in the first line setting: cisplatin/pemetrexed and ipilimumab/nivolumab. Unfortunately, most who respond to first line treatment experience disease progression within a year. A few established MPM cell lines, with inherent limitations, provide minimal preclinical insight. The relative lack of model systems that accurately reflect MPM tumorigenesis is a barrier to therapeutic and diagnostic advances in MPM. Methods: We developed a diverse library of 22 extensively annotated patient-derived xenograft (PDX) models from 22 patients with MPM. Multi-omic analyses including, targeted tumor next-generation sequencing by MSK-IMPACT, RNA-sequencing, and immunohistochemistry was performed. We deconvoluted the mutational landscapes, global expression profiles, and molecular subtypes of these MPM models and further compared the PDXs to MPM clinical specimens, including matched PDX and primary tumor pairs. Results: The mutational landscapes of PDX models strongly correlated with paired tumor samples. There were some differences in CDKN2A/B mutations and relative enrichment of NF2 with fewer BAP1 alterations, the significance of which is being investigated. When compared by histological subtype, we observed an upregulation of genes involved in NOTCH and EMT signaling in the epithelioid models. Models derived from patients with shorter overall survival or poor response to platinum doublet had higher expression of WNT/β-catenin signaling, hedgehog pathway, and epithelial-mesenchymal transition signaling as well as downregulation of immune-activation pathways, including type I and II interferon signaling and inflammatory response pathways. Conclusions: This library of MPM PDXs, the largest to date, effectively mimics human disease and provides unprecedented insight into the genomic, transcriptomic, and protein landscape of MPM. These PDX models will inform future clinical investigations and provide an important new preclinical resource. Citation Format: Michael Offin, Jennifer L. Sauter, Jacklyn V. Egger, Elisa deStanchina, John T. Poirier, Marjorie G. Zauderer, Charles Rudin, Triparna Sen. Multiomic profiling of patient-derived xenografts established from patients with malignant pleural mesothelioma proposes pathways associated with poor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 51.

Photodynamique therapy relieves tumor vascular anergy and promotes immune cell trafficking in an orthotopic mouse model of malignant pleural mesothelioma

Abstract Objective Malignant pleural mesothelioma (MPM) is a deadly disease with limited treatment options. Recently, dual immune checkpoint inhibition therapy (ICI) showed improved patient survival. However, only a fraction of patients were responsive to immunotherapy. One potential mechanism of MPM resistance to ICIs could be their endothelial anergy that hampers leukocyte trafficking to the tumor bulk. Here, we hypothesized that vascular-targeted low dose photodynamic therapy (L-PDT), treatment of MPM could relieve tumor endothelial anergy and improve immunotherapy efficacy. Methods Using an orthotopic syngeneic MPM murine model (AB12 cells injected in the pleura of BALB/c mice), we determined the impact of L-PDT on the endothelial expression of E-Selectin, a key molecule involved in leukocyte diapedesis by immunohistochemistry. Furthermore, to confirm the role of E-selectin, we determined the extravasation of effector T cells (CD8+/CD4+) by immunostaining in L-PDT treated tumors in the presence or absence of an E-selectin blocking antibody. Finally, we assessed tumor growth/survival of our MPM murine model treated with L-PDT alone or combined to ICIs. Results L-PDT pre-treatment enhanced MPM endothelial E-Selectin expression in vivo. The latter was associated with increased CD4+ and CD8+ lymphocyte infiltration of MPM following L-PDT which did not occur after E-Selectin blockade. Also, L-PDT pre-treatment of MPM influenced favorably tumor control, mouse survival and the impact of ICIs compared to controls. Conclusion L-PDT pre-treatment relieves endothelial anergy in MPM which improves antitumor immunity and response to ICI. This approach could constitute a promising pre-treatment option, in combination with ICIs, for the management of this deadly disease.

The Prembion® pre-biotic improves the impact of anti-CTLA4 immune checkpoint inhibitor in a murine model of malignant pleural mesothelioma

Abstract Objective Immune checkpoint inhibition (ICI) therapy has revolutionized the outcome of certain cancers such as malignant pleural mesothelioma (MPM). However, patient responsiveness to this treatment remains unpredictable. Recently, a role for the gut microbiota composition has emerged for patients to generate a robust immune response against their tumors, following immunotherapy. Here, we studied the impact of Prembion®, a pre-biotic and modulator of the gut microbiota, on tumor control and lymphocyte infiltration in a murine MPM model treated by ICI. Methods Prembion® (diluted into drinking water) was administrated to BALBc mice for 14 days. These animals were then inoculated orthotopically with a syngeneic MPM cell line (AB12-luc cells injected in the pleura) and followed by bioluminescence imaging. We determined the tumor growth and mouse survival in different groups: untreated control, Prembion®, IgG control, anti-PDL-1, anti-CTLA4, Prembion®+anti-PDL-1 and Prembion®+anti-CTLA4. A correlation between tumor response/animal survival and MPM infiltration with CD8+ lymphocytes was also performed by immunohistochemistry. Results Prembion® was well tolerated and did not affect animal weight or activity. Interestingly, Prembion® was as effective as anti-PDL1 and anti-CTLA4 monotherapy on tumor control, prolonging survival by 4.0 ± 1.1 days compared to controls (p&amp;lt;0.05). Moreover Prembion® potentiated anti-CTLA4 efficacy with a significant improvement in mouse survival of the Prembion®+anti-CTLA4 compared to controls (3.6 ± 1.1 days, p&amp;lt;0.05). Additionally, this finding correlated with enhanced MPM infiltration by CD8+ lymphocytes compared to controls (p&amp;lt;0.05). Conclusion Prembion® positively regulated the adaptive immune response against MPM and helped to improve the impact of anti-CTLA4 ICI on MPM. Further work focusing on the gut microbiome changes induced by Prembion® are ongoing to better understand the mechanisms involved.

A Community-Driven, Openly Accessible Molecular Pathway Integrating Knowledge on Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly aggressive malignancy mainly triggered by exposure to asbestos and characterized by complex biology. A significant body of knowledge has been generated over the decades by the research community which has improved our understanding of the disease toward prevention, diagnostic opportunities and new treatments. Omics technologies are opening for additional levels of information and hypotheses. Given the growing complexity and technological spread of biological knowledge in MPM, there is an increasing need for an integrating tool that may allow scientists to access the information and analyze data in a simple and interactive way. We envisioned that a platform to capture this widespread and fast-growing body of knowledge in a machine-readable and simple visual format together with tools for automated large-scale data analysis could be an important support for the work of the general scientist in MPM and for the community to share, critically discuss, distribute and eventually advance scientific results. Toward this goal, with the support of experts in the field and informed by existing literature, we have developed the first version of a molecular pathway model of MPM in the biological pathway database WikiPathways. This provides a visual and interactive overview of interactions and connections between the most central genes, proteins and molecular pathways known to be involved or altered in MPM. Currently, 455 unique genes and 247 interactions are included, derived after stringent manual curation of an initial 39 literature references. The pathway model provides a directly employable research tool with links to common databases and repositories for the exploration and the analysis of omics data. The resource is publicly available in the WikiPathways database (Wikipathways : WP5087) and continues to be under development and curation by the community, enabling the scientists in MPM to actively participate in the prioritization of shared biological knowledge.

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.

KRAS signaling in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos‐exposed individuals and rapidly leads to death. MPM harbors loss‐of‐function mutations in BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here, we show that a proportion of human MPM harbor point mutations, copy number alterations, and overexpression of KRAS with or without TP53 changes. These are likely pathogenic, since ectopic expression of mutant KRAS G12D in the pleural mesothelium of conditional mice causes epithelioid MPM and cooperates with TP53 deletion to drive a more aggressive disease form with biphasic features and pleural effusions. Murine MPM cell lines derived from these tumors carry the initiating KRAS G12D lesions, secondary Bap1 alterations, and human MPM‐like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS alterations alone or in accomplice with TP53 alterations likely play an important and underestimated role in a proportion of patients with MPM, which warrants further exploration.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell modelsevaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice. Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of 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.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM)

BackgroundMalignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM.MethodsKDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways.ResultsLevels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth.ConclusionsThe results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

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.

Inhibitors of the Transcription Factor STAT3 Decrease Growth and Induce Immune Response Genes in Models of Malignant Pleural Mesothelioma (MPM).

Simple SummaryMalignant pleural mesothelioma (MPM) is characterized by the lack of effective long-term treatments and highly prevalent drug resistance. The paucity of potential therapeutic targets has led to dismal prognosis. We have examined the functional role of the signal transducer and activator of transcription 3 (STAT3) transcription factor in MPM. Even though highly specific STAT3 inhibitors have not yet come to fruition, we performed experiments targeting STAT3 expression and subsequently supported these experiments with small molecule drugs that were previously validated to target STAT3-dependent activation mechanisms. These drugs are United States Food and Drug Administration (FDA)-approved and showed efficacy in preclinical models of MPM at concentrations that can safely be achieved in humans. We also identified genes that strongly support the essential role of STAT3 in cell growth and are consistent with a role of STAT3 in immune suppression. Overall, the results establish a central role for STAT3 in tumor growth and encourage further expedient development of STAT3 pathway inhibitors for clinical use.Malignant pleural mesothelioma (MPM) is an aggressive cancer defined by loss-of-function mutations with few therapeutic options. We examined the contribution of the transcription factor Signal transducer and activator of transcription 3 (STAT3) to cell growth and gene expression in preclinical models of MPM. STAT3 is activated in a variety of tumors and is thought to be required for the maintenance of cancer stem cells. Targeting STAT3 using specific small hairpin RNAs (shRNAs) or with the pharmacologic inhibitors atovaquone or pyrimethamine efficiently reduced cell growth in established cell lines and primary-derived lines while showing minimal effects in nontransformed LP9 mesothelial cells. Moreover, atovaquone significantly reduced viability and tumor growth in microfluidic cultures of primary MPM as well as in an in vivo xenotransplant model. Biological changes were linked to modulation of gene expression associated with STAT3 signaling, including cell cycle progression and altered p53 response. Reflecting the role of STAT3 in inducing localized immune suppression, using both atovaquone and pyrimethamine resulted in the modulation of immunoregulatory genes predicted to enhance an immune response, including upregulation of ICOSLG (Inducible T-Cell Costimulator Ligand or B7H2). Thus, our data strongly support a role for STAT3 inhibitors as anti-MPM therapeutics.

Patient-derived malignant pleural mesothelioma cell cultures: a tool to advance biomarker-driven treatments

Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived...