ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

The Future of Antibody Drug Conjugation by Comparing Various Methods of Site-Specific Conjugation.

The field of oncology is continuously seeking to find effective treatment therapies with limited side effects. Antibody-drug conjugates (ADCs) are a promising class of cancer therapies that have been shown to be effective with limited side effects. Although promising, these therapies experience shortcomings, such as the stability and reproducibility of current conjugation methods. Historically, ADCs have been produced by stochastic conjugation methods; however, new methods of site-specific conjugation have evolved to mitigate current ADC shortcomings. In this article, we highlight the success of ADCs as well as some of their challenges. We also highlight the shortcomings of stochastic conjugation and explore the various site-specific conjugation methods and their advantages over stochastic conjugation.

EWSR1::ATF1 Orchestrates the Clear Cell Sarcoma Transcriptome in Human Tumors and a Mouse Genetic Model.

Clear cell sarcoma (CCS) is a rare, aggressive malignancy that most frequently arises in the soft tissues of the extremities. It is defined and driven by expression of one member of a family of related translocation-generated fusion oncogenes, the most common of which is EWSR1::ATF1. The EWSR1::ATF1 fusion oncoprotein reprograms transcription. However, the binding distribution of EWSR1::ATF1 across the genome and its target genes remain unclear. Here, we interrogated the genomic distribution of V5-tagged EWSR1::ATF1 in tumors it had induced upon expression in mice that also recapitulated the transcriptome of human CCS. ChIP-sequencing of V5-EWSR1::ATF1 identified previously unreported motifs including the AP1 motif and motif comprised of TGA repeats that resemble GGAA-repeating microsatellites bound by EWSR1::FLI1 in Ewing sarcoma. ChIP-sequencing of H3K27ac identified super enhancers in the mouse model and human contexts of CCS, which showed a shared super enhancer structure that associates with activated genes.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Natural Products and Small Molecules Targeting Cellular Ceramide Metabolism to Enhance Apoptosis in Cancer Cells.

Molecular targeting strategies have been used for years in order to control cancer progression and are often based on targeting various enzymes involved in metabolic pathways. Keeping this in mind, it is essential to determine the role of each enzyme in a particular metabolic pathway. In this review, we provide in-depth information on various enzymes such as ceramidase, sphingosine kinase, sphingomyelin synthase, dihydroceramide desaturase, and ceramide synthase which are associated with various types of cancers. We also discuss the physicochemical properties of well-studied inhibitors with natural product origins and their related structures in terms of these enzymes. Targeting ceramide metabolism exhibited promising mono- and combination therapies at preclinical stages in preventing cancer progression and cemented the significance of sphingolipid metabolism in cancer treatments. Targeting ceramide-metabolizing enzymes will help medicinal chemists design potent and selective small molecules for treating cancer progression at various levels.

Formulation, Characterization, and In Vitro/In Vivo Efficacy Studies of a Novel Liposomal Drug Delivery System of Amphiphilic Jaspine B for Treatment of Synovial Sarcoma.

Sphingomyelin is a cell membrane sphingolipid that is upregulated in synovial sarcoma (SS). Jaspine B has been shown to inhibit sphingomyelin synthase, which synthesizes sphingomyelin from ceramide, a critical signal transducer; however, jaspine B's low bioavailability limits its application as a promising treatment option. To address this shortcoming, we used microfluidics to develop a liposomal delivery system with increased anticancer efficacy. The nano-liposome size was determined by transmission electron microscopy. The jaspine B liposome was tested for its tumor inhibitory efficacy compared to plain jaspine B in in vitro and in vivo studies. The human SS cell line was tested for cell viability using varying jaspine B concentrations. In a mouse model of SS, tumor growth suppression was evaluated during four weeks of treatment (3 times/week). The results show that jaspine B was successfully formulated in the liposomes with a size ranging from 127.5 ± 61.2 nm. The MTT assay and animal study results indicate that jaspine B liposomes dose-dependently lowers cell viability in the SS cell line and effectively suppresses tumor cell growth in the SS animal model. The novel liposome drug delivery system addresses jaspine B's low bioavailability issues and improves its therapeutic efficacy.

Navigating the genomic instability mine field of osteosarcoma to better understand implications of non-coding RNAs.

Osteosarcoma is one of the most genomically complex cancers and as result, it has been difficult to assign genomic aberrations that contribute to disease progression and patient outcome consistently across samples. One potential source for correlating osteosarcoma and genomic biomarkers is within the non-coding regions of RNA that are differentially expressed. However, it is unsurprising that a cancer classification that is fraught with genomic instability is likely to have numerous studies correlating non-coding RNA expression and function have been published on the subject. This review undertakes the formidable task of evaluating the published literature of noncoding RNAs in osteosarcoma. This is not the first review on this topic and will certainly not be the last. The review is organized with an introduction into osteosarcoma and the epigenetic control of gene expression before reviewing the molecular function and expression of long non-coding RNAs, circular RNAs, and short non-coding RNAs such as microRNAs, piwi RNAs, and short-interfering RNAs. The review concludes with a review of the literature and how the biology of non-coding RNAs can be used therapeutically to treat cancers, especially osteosarcoma. We conclude that non-coding RNA expression and function in osteosarcoma is equally complex to understanding the expression differences and function of coding RNA and proteins; however, with the added lens of both coding and non-coding genomic sequence, researchers can begin to identify the patterns that consistently associate with aggressive osteosarcoma.

Oncostatin M Receptor as a Therapeutic Target for Radioimmune Therapy in Synovial Sarcoma.

Synovial sarcoma (SS) is a pediatric muscle cancer that primarily affects adolescents and young adults and has few treatment options. Complicating the treatment of synovial sarcoma is the low mutational burden of SS. Inflammatory pathways have been identified as being upregulated in some SS, leading to the discovery of upregulated oncostatin M receptor (OSMR). It was found that OSMR is upregulated in SS by RNAseq analysis and quantitative PCR, highlighting its potential in the treatment of SS. Also, OSMR is upregulated in mouse models for synovial sarcoma as demonstrated by western blot and immunohistochemistry, and the protein is present in both primary and metastatic sites of disease. Using a radioimmune therapy drug model, targeted therapy was synthesized for use in OSMR expressing SS and it was demonstrated that this drug is stable, while capable of efficient OSMR binding and isotope capture. Finally, this antibody conjugate exhibited ideal pharmacokinetics and targeted sites of disease in our mouse model and was taken up in both primary and metastatic diseased tissue. This suggests OSMR as an ideal target for therapy and this radioimmune therapy provides a novel treatment option for a disease with few therapy choices.

The Impact of Hempseed Consumption on Bone Parameters and Body Composition in Growing Female C57BL/6 Mice.

Optimizing peak bone mass is critical to healthy aging. Beyond the established roles of dietary minerals and protein on bone integrity, fatty acids and polyphenols modify bone structure. This study investigated the effect of a diet containing hempseeds (HS), which are rich in polyunsaturated fatty acids and polyphenols, on bone mineral density, bone cell populations and body composition. Groups (n = 8 each) of female C57BL/6 mice were fed one of three diets (15% HS by weight; 5% HS; 0% HS (control)) from age 5 to 30 weeks. In vivo whole-body composition and bone mineral density and content were measured every 4 weeks using dual-energy X-ray absorptiometry. Ex vivo humeri cell populations in the epiphyseal plate region were determined by sectioning the bone longitudinally, mounting the sections on slides and staining with tartrate-resistant acid phosphatase and alkaline phosphatase stain to identify osteoclasts and osteoblasts, respectively. Mixed models with repeated measures across experimental weeks showed that neither body weight nor body weight gain across weeks differed among groups yet mice fed the 15% HS diet consumed significantly more food and more kilocalories per g body weight gained than those fed the 5% HS and control diets (p < 0.0001). Across weeks, fat mass was significantly higher in the 5% HS versus the control group (p = 0.02). At the end point, whole-body bone mineral content was significantly higher in the control compared to the 5% HS group (p = 0.02). Humeri from both HS groups displayed significantly lower osteoblast densities compared to the control group (p < 0.0001). No relationship was seen between osteoblast density and body composition measurements. These data invite closer examination of bone cell activity and microarchitecture to determine the effect of habitual HS consumption on bone integrity.

Circling back to epigenetic regulation in osteosarcoma: Comment on 'Hsa_circ_0088212-mediated miR-520h/APOA1 axis inhibits the osteosarcoma progression' by ' Hao Peng'.

Osteosarcoma is a genetically complex cancer, thus there are increasing efforts to identify biomarkers and regulators within the epigenome that can better predict patient outcomes and provide new therapeutic targets. In this commentary, we have evaluated the work of Peng and colleagues on the identification and function of the signaling axis circ_0088212/miR_520 h/APOA1 in osteosarcoma. We provide the context of how novel it is to demonstrate the anti-tumor functions of APOA1 and not just correlative gene expression with patient outcome. We further postulate why some studies involving circRNAs in osteosarcoma contradict one another. We conclude that the study performed by Peng and colleagues was performed with enough rigor to give confidence that circ_0088212 is a bona fide tumor suppressor in osteosarcoma.

Tissue Engineering Through 3D Bioprinting to Recreate and Study Bone Disease.

The applications of 3D bioprinting are becoming more commonplace. Since the advent of tissue engineering, bone has received much attention for the ability to engineer normal bone for tissue engraftment or replacement. While there are still debates on what materials comprise the most durable and natural replacement of normal tissue, little attention is given to recreating diseased states within the bone. With a better understanding of the cellular pathophysiology associated with the more common bone diseases, these diseases can be scaled down to a more throughput way to test therapies that can reverse the cellular pathophysiology. In this review, we will discuss the potential of 3D bioprinting of bone tissue in the following disease states: osteoporosis, Paget's disease, heterotopic ossification, osteosarcoma, osteogenesis imperfecta, and rickets disease. The development of these 3D bioprinted models will allow for the advancement of novel therapy testing resulting in possible relief to these chronic diseases.

The clear cell sarcoma functional genomic landscape.

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP-induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.

A Role for SMARCB1 in Synovial Sarcomagenesis Reveals That SS18-SSX Induces Canonical BAF Destruction.

Reduced protein levels of SMARCB1 (also known as BAF47, INI1, SNF5) have long been observed in synovial sarcoma. Here, we show that combined Smarcb1 genetic loss with SS18-SSX expression in mice synergized to produce aggressive tumors with histomorphology, transcriptomes, and genome-wide BAF-family complex distributions distinct from SS18-SSX alone, indicating a defining role for SMARCB1 in synovial sarcoma. Smarcb1 silencing alone in mesenchyme modeled epithelioid sarcomagenesis. In mouse and human synovial sarcoma cells, SMARCB1 was identified within PBAF and canonical BAF (CBAF) complexes, coincorporated with SS18-SSX in the latter. Recombinant expression of CBAF components in human cells reconstituted CBAF subcomplexes that contained equal levels of SMARCB1 regardless of SS18 or SS18-SSX inclusion. In vivo, SS18-SSX expression led to whole-complex CBAF degradation, rendering increases in the relative prevalence of other BAF-family subtypes, PBAF and GBAF complexes, over time. Thus, SS18-SSX alters BAF subtypes levels/balance and genome distribution, driving synovial sarcomagenesis. SIGNIFICANCE: The protein level of BAF component SMARCB1 is reduced in synovial sarcoma but plays a defining role, incorporating into PBAF and SS18-SSX-containing canonical BAF complexes. Reduced levels of SMARCB1 derive from whole-complex degradation of canonical BAF driven by SS18-SSX, with relative increases in the abundance of other BAF-family subtypes.See related commentary by Maxwell and Hargreaves, p. 2375.This article is highlighted in the In This Issue feature, p. 2355.

Rural Community Perceptions and Interests in Pharmacogenomics.

Pharmacogenomics testing is a rapidly expanding field with increasing importance to individualized patient care. However, it remains unclear whether the general public in rural areas would be willing to engage in this service. The objective of this survey was to determine rural community-dwelling members' perceptions of pharmacogenomics. A questionnaire was developed consisting of five Likert-style questions on knowledge and perceptions of pharmacogenomics, a single multiple-choice question on cost of testing, and a free-response question. Two cohorts received the same questionnaire: attendees at a university-sponsored health fair and patients presenting to two independent community pharmacies in southeastern Idaho. While both showed positive reception to the implementation and value of pharmacogenomics, those at the health fair were more in favor of pharmacogenomics, suggesting a need for greater outreach and education to the general public. The findings suggest that interest of rural community-dwelling individuals may be amenable to the expansion of pharmacogenomics testing.

Underlying Ossification Phenotype in a Murine Model of Metastatic Synovial Sarcoma.

Synovial sarcoma, an uncommon cancer, typically affects young adults. Survival rates range from 36% to 76%, decreasing significantly when metastases are present. Synovial sarcomas form in soft tissues, often near bones, with about 10% demonstrating ossification in the tumor. The literature is inconclusive on whether the presence of ossification portends a worse prognosis. To this end, we analyzed our genetic mouse models of synovial sarcoma to determine the extent of ossification in the tumors and its relationship with morbidity. We noted higher ossification within our metastatic mouse model of synovial sarcoma. Not only did we observe ossification within the tumors at a frequency of 7%, but an even higher frequency, 72%, of bone reactivity was detected by radiography. An enrichment of bone development genes was associated with primary tumors, even in the absence of an ossification phenotype. In spite of the ossification being intricately linked with the metastatic model, the presence of ossification was not associated with a faster or worse morbidity in the mice. Our conclusion is that both metastasis and ossification are dependent on time, but that they are independent of one another.

Quantifying Fluorescently Labeled Ceramide Levels in Human Sarcoma Cell Lines in Response to a Sphingomyelin Synthase Inhibitor.

Sphingolipid metabolism is an important process in sustaining the growth needs of rapidly dividing cancer cells. Enzymes that synthesize sphingolipids have become attractive targets in cancer pharmacology. Ceramide is a precursor for synthesizing sphingolipids such as sphingomyelin, sphingosine-1-phosphate, and glucosylceramide. Sphingomyelin synthase (SMS) is the enzyme that transfers a phosphatidylcholine to ceramide to generate sphingomyelin. To test the inhibition of SMS, scientists assess the buildup of ceramide in the cell, which is cytotoxic. Because ceramide is a small lipid molecule, there are limited tools like antibodies to detect its presence. Alternatively, designated machines for small-molecule separation coupled with mass spectrometry detection can be used; however, these can be cost-prohibitive. We used a commercially available NBD-ceramide to apply to human cancer cell lines in the presence or absence of a known SMS inhibitor, jaspine B. After short incubation times, we were able to collect cell lysates and using solvent extraction methods, run the cellular material on a thin-layer chromatography plate to determine the levels of intact fluorescently labeled ceramide. Brighter fluorescence on the TLC plate correlated to greater SMS inhibition. Small molecules can then be screened quantifiably to determine the biological impact of inhibiting the sphingolipid metabolism pathways involving ceramide.

Epigenetic Changes at the Birc5 Promoter Induced by YM155 in Synovial Sarcoma.

YM155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of survivin (gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is a rare soft-tissue malignancy with high protein expression of survivin. We investigated whether YM155 would be a viable therapeutic option to treat synovial sarcoma. YM155 therapy was applied to human synovial sarcoma cell lines and to a genetically engineered mouse model of synovial sarcoma. We discovered that YM155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM155 by looking at the change of lysine modifications of the histone tails that were within 250 base pairs of the Birc5 promoter. Using chromatin immunoprecipitation (ChIP)-qPCR, we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM155 treatment. H3K27me3 and H3K27ac increased, but the net result was decreased Birc5/survivin expression. Furthermore, the combination of molecular events resulted in caspase 3/7/8 upregulation and death of the sarcoma cells.

Sarcoma-The standard-bearer in cancer discovery.

Sarcoma is a rare tumor type that occurs most frequently in connective tissue. Despite its uncommon occurrence, sarcoma research has provided the means for groundbreaking research that has advanced our understanding of general cancer mechanisms. It is through sarcoma research that the pioneering efforts of cancer immunotherapy were explored, that we understand the inherent genetic nature of cancer mutations, and that we appreciate the subclassification of general cancer types to make more accurate prognoses. This review explores the brief history of sarcoma research and what sarcomas can still teach us about the future of cancer research, especially in regard to novel immunotherapy targets, the role of epigenetics in disease progression and chemoresistance, and the benefits of more focused clinical trials.

Paracrine osteoprotegerin and ß-catenin stabilization support synovial sarcomagenesis in periosteal cells.

Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma that is often discovered during adolescence and young adulthood. Despite the name, synovial sarcoma does not typically arise from a synoviocyte but instead arises in close proximity to bones. Previous work demonstrated that mice expressing the characteristic SS18-SSX fusion oncogene in myogenic factor 5-expressing (Myf5-expressing) cells develop fully penetrant sarcomagenesis, suggesting skeletal muscle progenitor cell origin. However, Myf5 is not restricted to committed myoblasts in embryos but is also expressed in multipotent mesenchymal progenitors. Here, we demonstrated that human SS and mouse tumors arising from SS18-SSX expression in the embryonic, but not postnatal, Myf5 lineage share an anatomic location that is frequently adjacent to bone. Additionally, we showed that SS can originate from periosteal cells expressing SS18-SSX alone and from preosteoblasts expressing the fusion oncogene accompanied by the added stabilization of ß-catenin, which is a common secondary change in SS. Expression and secretion of the osteoclastogenesis inhibitory factor osteoprotegerin enabled early growth of SS18-SSX2-transformed cells, indicating a paracrine link between the bone and synovial sarcomagenesis. These findings explain the skeletal contact frequently observed in human SS and may provide alternate means of enabling SS18-SSX-driven oncogenesis in cells as differentiated as preosteoblasts.

Death by HDAC Inhibition in Synovial Sarcoma Cells.

Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting the causative SS18-SSX fusion oncoprotein are currently available. Histone deacetylase (HDAC) inhibition has been shown in previous studies to disrupt the synovial sarcoma oncoprotein complex, resulting in apoptosis. To understand the molecular effects of HDAC inhibition, RNA-seq transcriptome analysis was undertaken in six human synovial sarcoma cell lines. HDAC inhibition induced pathways of cell-cycle arrest, neuronal differentiation, and response to oxygen-containing species, effects also observed in other cancers treated with this class of drugs. More specific to synovial sarcoma, polycomb group targets were reactivated, including tumor suppressor CDKN2A, and proapoptotic transcriptional patterns were induced. Functional analyses revealed that ROS-mediated FOXO activation and proapoptotic factors BIK, BIM, and BMF were important to apoptosis induction following HDAC inhibition in synovial sarcoma. HDAC inhibitor pathway activation results in apoptosis and decreased tumor burden following a 7-day quisinostat treatment in the Ptenfl/fl;hSS2 mouse model of synovial sarcoma. This study provides mechanistic support for a particular susceptibility of synovial sarcoma to HDAC inhibition as a means of clinical treatment. Mol Cancer Ther; 16(12); 2656-67. ©2017 AACR.