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INTRODUCTION: Mesothelioma is a rare, highly aggressive, incurable tumor arising from mesothelial linings in the body. Many drug therapies have been developed to treat this tumor including generalized chemotherapeutic agents, targeted molecular therapies, and immune system modulators. Mouse models including genetically engineered mice and patient-derived xenografts have been instrumental in the discovery and evaluation of such therapies for mesothelioma, but there is a need for improved understanding of the role of inflammation, tumor heterogeneity, mechanisms of carcinogenesis, and the tumor microenvironment. AREAS COVERED: This review is a comprehensive analysis of available mouse models for mesothelioma drug discovery and development. Gaps in our current knowledge of mesothelioma are highlighted, and future directions for mouse model research is considered. EXPERT OPINION: Soon, CRISPR-Cas gene-editing technology will improve our understanding of mesothelioma molecular mechanisms foundational to the discovery and testing of efficacious therapeutic agents. There are at least two areas of methodology development that are likely on this near horizon. One is concerned with better modeling of the role of inflammation associated with mesothelioma. The other is related to the availability of humanized immunocompromised mice harboring patient-derived xenografts. Combining human tumors in an environment with functional human immune cells will enable rapid innovation in immuno-oncology therapeutics.

Mesothelial cells are arranged as a monolayer on covering membranes that invest surfaces of body cavities like the pleura and peritoneum. Primary human mesothelial cell (HMC) cultures are needed for studying mesothelial cell homeostasis and developing disease models, such as wound healing or cancers. Remarkably, there is a paucity of useable HMC lines that are currently available that faithfully recapitulate normal in vivo phenotypic characteristics. Here, we present a strategy to recover HMC from human pleural tissue and to immortalize them for extended in vitro culturing. Human pleural membrane was harvested by minimally invasive surgical techniques. HMC were isolated using a two-step process combining explant cellular outgrowth from biopsy tissue and flow cytometry based on cell surface expression of cadherin-1 and CD71. Cell cultures were generated after lentiviral transfection with human telomerase. The new HMC cultures retain the same phenotypic traits and physiologic features as their in vivo counterparts, yet they can be adapted for short-term or long-term culture in large-scale in vitro experimentation. In particular, we generated a new HMC line harboring a germline mutation in breast cancer type-1-associated protein-1 ( BAP1), a causal tumor suppressor gene, that could be instrumental to malignant mesothelioma research. Patient-specific, normal HMC may serve as novel discovery tools allowing more powerful research models of both normal physiology and disease processes. Our surgically driven approach leads to a limitless resource of novel mesothelial cell cultures.

Malignant pleural mesothelioma (MPM) is an incurable, aggressive neoplasm with distinctive features, including preservation of wild-type p53, irrespective of histologic subtype. We posited that this consistent molecular characteristic represents an underexploited therapeutic target that can be approached by leveraging biologic effects of microRNA (miRNA). The Cancer Genome Atlas was surveyed to identify p53-responsive prognostic miRNA(s) in MPM. Using patient samples, in vitro MPM cell lines, and murine tumor xenograft models, we verified specific gene pathways targeted by these miRNAs, and we examined their therapeutic effects. miR-215-5p is a poor prognosis miRNA downregulated in MPM tissues, which has not been recognized previously. When miR-215-5p was ectopically re-expressed in MPM cells and delivered in vivo to tumor xenografts, it exerted significant cell killing by activating p53 function and inducing apoptosis. The mechanistic basis for this effect is due to combinatorial effects of a positive feedback loop of miR-215-MDM2-p53 signaling, additional mouse double minute 2 (MDM2)-p53 positive feedback loop(s) with other miRNAs such as miR-145-5p, and suppression of diverse gene targets associated with cell cycle dynamics not previously drug treatable in MPM clinical studies. Our results suggest a potential pathophysiologic role for and therapeutic significance of miR-215-5p in MPM.

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 (>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 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.

Malignant pleural mesothelioma (MPM) is an incurable surface neoplasm with peculiar pathobiology. MPM proliferates by using the tyrosine-kinase-Ras pathway. Despite representing an attractive therapeutic target, there are no standard agent(s) specifically inhibiting Ras signaling adopted in clinical settings. We posited that biologic effects of microRNA (miRNA) can disrupt this molecular network. Using patient samples, cell lines, and murine tumor xenograft models, we confirmed specific genes in the Ras pathway are targeted by an MPM-associated miRNA and then examined its therapeutic effects. We verified significant and consistent downregulation of miR-206 in MPM tissues. When miR-206 is ectopically re-expressed in MPM cells and delivered to tumor xenografts in mice, it exerted significant cell killing by suppressing multiple components of the receptor-tyrosine-kinase-Ras-cell-cycle-signaling network; some of which were prognostic when overexpressed and/or have not been druggable. Of note, we validated CDK6 as a novel target of miR-206. Overall, this miR-206-targeting mechanism manifested as induced G1/S cell cycle arrest. In addition, we identified a novel MPM therapeutic combination by adding systemic-route abemaciclib with local-route miR-206, which showed additive efficacy translating to improved survival. Our pre-clinical study suggests a potential pathophysiologic role for, and therapeutic relevance of, miR-206 in MPM.

While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.

Malignant pleural mesothelioma (MPM) is a biologically unusual, highly aggressive cancer that defies current multimodality treatments. Epidemiologic data suggest that this malignancy has not abated despite increasingly strict environmental regulations on asbestos, the putative causative agent for sporadic cases. An incomplete understanding of all the factors mechanistically driving mesothelioma is largely responsible for the current lack of curative treatments. Many approaches have been employed to ascertain the step-by-step molecular events involved in mesothelioma oncogenesis including in vitro , small animal in vivo , and human experimental models; though clearly defined, druggable mechanisms still are elusive. Importantly, the foundation of the latest accepted model of tumor initiation is derived from in vitro systems. A thorough review of in vitro mesothelioma oncogenesis models may suggest further opportunities for discovery.

Introduction/Objectives Mediator is a multi-subunit complex organized into four modules (head, middle, tail and kinase module). It is involved in several steps of PolII-dependent transcription and enhancer–promoter (E–P) interactions. Mutations in different subunits (SU) are associated with cancer and developmental diseases. Mediator's function and structure have been mainly studied in yeast. Earlier studies suggest significant differences with the mammalian complex in terms of composition and structure. Additionally, the chromatin architecture between both species is different, particularly E–P interactions. We aimed to comprehensively characterize the mammalian complex. Materials/Patients and methods We screened for cell essentiality of Mediator subunits, in a murine B-lymphoma cell line, CH12, in embryonic stem cells and in primary CD4T cells, using CRISPR-Cas9 technology. We generated knockout clones (KO) whenever possible in CH12 cells, and a degron-based conditional KO of the essential Mediator's backbone, Med14. We analyzed the impact on gene expression (RNAseq), on PolII and Mediator recruitment at promoters and enhancers (ChIPseq), on chromatin accessibility (ATACseq), on histone modifications (MNase ChIPseq), on chromatin architecture (in situ HiC) and on structure (cryo-EM). Results Depletion of non-essential SUs induces transcriptional deregulation of only a subset of genes, whereas depleting the essential Med14 induces global decrease in transcription associated with a decrease in PolII recruitment. Interestingly, the ablation of the whole tail module is viable. With this tailless model and the Med14-degron we do not observe an impact on chromatin architecture. Preliminary cryo-EM results show a conserved location of common SUs between yeast and mammalian cells. We are able to locate mammalian specific SUs and describe the interactions between the different SUs in the mammalian complex. Conclusions Our study provides new insights into mammalian Mediator structure and a comprehensive view on how Mediator and its tail module are involved in transcription and chromatin architecture. This data is essential for a better understanding of transcriptional regulation and Mediator related diseases.

Malignant pleural mesothelioma (MPM) is causally associated with exposure to asbestos and accounts for roughly 75% of mesothelioma cancers. It is an aggressive tumor with a median survival time of approximately 12 months. Treatment options are limited, largely palliative, and still based on cisplatin and pemetrexed, a regimen that remains unchanged since 2003. Despite widespread study, the complexities of MPM biology remain poorly described, especially at the point of tumor initiation where development of novel clinical interventional strategies would be expected to yield the most effect. The extended latency period in humans consisting of decades (over 20-30 years) remains a prohibitive barrier to gain accurate, in vivo insights about MPM oncogenic processes. As such, all in vitro MPM models, which are designed to accelerate tumor development as a practical endpoint of study, to date have not yielded clinically translatable molecular targets. Here, we present a novel in vitro model system for exploring the earliest molecular mechanisms contributing to the development of MPM using normal, human mesothelial cell lines (MeT-5a and LP9-hTERT) engineered to harbor constitutively enhanced NFkB signaling activity. Both 3D-sphere and soft agar assays were used to assess cellular transformation status in these induced cells compared to their wild-type counterparts. Heterotopic xenograft implantation studies were performed in NOD scid-gamma (NSG) immunocompromised mice (sub-cutaneous dorsal flank injection) for tumorigenic potential. In all cases, these induced cells (despite the complete absence of asbestos exposure) exhibited a significant malignant-like phenotype. Tumor growth in mouse xenograft models occurred very rapidly, and was marked by extensive vascular recruitment with invasion into surrounding tissues. In addition, these transformed cells expressed typical mesothelioma markers such as Wilms tumor protein (WT1 gene product) as revealed by immunohistofluorescence. This new MPM model system could represent a powerful construct to explore the early molecular events involved in transformation of normal mesothelial cells leading to malignancy. Citation Format: Nathanael D. Pruett, Anand Singh, Nisan Bhattacharyya, Chuong D. Hoang. Oncogenesis modeling in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3118.

Introduction: Since there remains no effective, durable chemotherapy against malignant pleural mesothelioma (MPM), we propose to leverage microRNA(miR)-based approaches that recently reached phase I trial testing. We posit there are many miR with therapeutic potential yet to be identified in MPM. Methods/ Results: Our prior miRNA expression profiling results of MPM (GSE40345) were cross-referenced with a novel p-value minimization analytic technique to identify prognostic miRNA. Both MPM cell lines and tissues (tumors and normal pleura) were assessed using quantitative and functional biologic assays. In silico algorithms identified gene targets that were verified by 3'-UTR luciferase assay.In silico analyses showed underexpression of miR-206 by 12.1-fold in MPM tumors compared to normal pleura. Using our p-value minimization technique, we assessed the clinical impact of this underexpressed miR in TCGA data (n=73 MPM) and noted a significant association to worse survival for patients with lower miR-206 expression (p=0.024). We confirmed that miR-206 was significantly underexpressed in tumor tissues by qRT-PCR analysis of a new, randomly selected MPM cohort (n=41) versus normal pleura (n=14). In a set of established MPM cell lines with low endogenous miR-206 level, ectopic re-expression of miR-206 dramatically suppressed cell proliferation, invasiveness, colony foci formation, and growth in soft-agar. Treatment of non-malignant mesothelial cells MeT-5a and LP9 with ectopic miR-206 showed no untoward effects. In silico gene target predictions for miR-206 were summated across 3 databases and screened by prognostic effect based on Kaplan-Meier results from TCGA data (n=87 MPM). Interestingly, we noted several overexpressed MPM-prognostic genes (p Citation Format: Anand Singh, Roma Pahwa, Li Zhang, Nathanael Pruett, R Taylor Ripley, David S. Schrump, Chuong D. Hoang. miR-206 inhibits pleural mesothelioma by targeting an active KRAS/CDK4/CCND1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 523.

SummaryA key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.

SUMMARY The ‘‘CTCF code’’ hypothesis posits that CTCF pleiotropic functions are driven by recognition of diverse sequences through combinatorial use of its 11 zinc fingers (ZFs). This model, however, is supported by in vitro binding studies of a limited number of sequences. To study CTCF multivalency in vivo, we define ZF binding requirements at � 50,000 genomic sites in primary lymphocytes. We find that CTCF reads sequence diversity through ZF clustering. ZFs 4‐7 anchor CTCF to � 80% of targets containing the core motif. Nonconserved flanking sequences are recognized by ZFs 1‐2 and ZFs 8‐11 clusters, which also stabilize CTCF broadly. Alternatively, ZFs 9‐11 associate with a second phylogenetically conserved upstream motif at � 15% of its sites. Individually, ZFs increase overall binding and chromatin residence time. Unexpectedly, we also uncovered a conserved downstream DNA motif that destabilizes CTCF occupancy. Thus, CTCF associates with a wide array of DNA modules via combinatorial clustering of its 11 ZFs.

Summary The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA + enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.