Hypoxia and HIFs in Ewing sarcoma: new perspectives on a multi-facetted relationship.

Hypoxia develops during the growth of solid tumors and influences tumoral activity in multiple ways. Low oxygen tension is also present in the bone microenvironment where Ewing sarcoma (EwS) - a highly aggressive pediatric cancer - mainly arises. Hypoxia inducible factor 1 subunit alpha (HIF-1-a) is the principal molecular mediator of the hypoxic response in cancer whereas EWSR1::FLI1 constitutes the oncogenic driver of EwS. Interaction of the two proteins has been shown in EwS. Although a growing body of studies investigated hypoxia and HIFs in EwS, their precise role for EwS pathophysiology is not clarified to date. This review summarizes and structures recent findings demonstrating that hypoxia and HIFs play a role in EwS at multiple levels. We propose to view hypoxia and HIFs as independent protagonists in the story of EwS and give a perspective on their potential clinical relevance as prognostic markers and therapeutic targets in EwS treatment.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma.

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically 'cold'.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Integrative clinical transcriptome analysis reveals TMPRSS2-ERG dependency of prognostic biomarkers in prostate adenocarcinoma.

In prostate adenocarcinoma (PCa), distinction between indolent and aggressive disease is challenging. Around 50% of PCa are characterized by TMPRSS2-ERG (T2E)-fusion oncoproteins defining two molecular subtypes (T2E-positive/negative). However, current prognostic tests do not differ between both molecular subtypes, which might affect outcome prediction. To investigate gene-signatures associated with metastasis in T2E-positive and T2E-negative PCa independently, we integrated tumor transcriptomes and clinicopathological data of two cohorts (total n = 783), and analyzed metastasis-associated gene-signatures regarding the T2E-status. Here, we show that the prognostic value of biomarkers in PCa critically depends on the T2E-status. Using gene-set enrichment analyses, we uncovered that metastatic T2E-positive and T2E-negative PCa are characterized by distinct gene-signatures. In addition, by testing genes shared by several functional gene-signatures for their association with event-free survival in a validation cohort (n = 272), we identified five genes (ASPN, BGN, COL1A1, RRM2 and TYMS)-three of which are included in commercially available prognostic tests-whose high expression was significantly associated with worse outcome exclusively in T2E-negative PCa. Among these genes, RRM2 and TYMS were validated by immunohistochemistry in another validation cohort (n = 135), and several of them proved to add prognostic information to current clinicopathological predictors, such as Gleason score, exclusively for T2E-negative patients. No prognostic biomarkers were identified exclusively for T2E-positive tumors. Collectively, our study discovers that the T2E-status, which is per se not a strong prognostic biomarker, crucially determines the prognostic value of other biomarkers. Our data suggest that the molecular subtype needs to be considered when applying prognostic biomarkers for outcome prediction in PCa.

Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies.

While sarcomas account for approximately 1% of malignant tumors of adults, they are particularly more common in children and adolescents affected by cancer. In contrast to malignancies that occur in later stages of life, childhood tumors, including sarcoma, are characterized by a striking paucity of somatic mutations. However, entity-defining fusion oncogenes acting as the main oncogenic driver mutations are frequently found in pediatric bone and soft-tissue sarcomas such as Ewing sarcoma (EWSR1-FLI1), alveolar rhabdomyosarcoma (PAX3/7-FOXO1), and synovial sarcoma (SS18-SSX1/2/4). Since strong oncogene-dependency has been demonstrated in these entities, direct pharmacological targeting of these fusion oncogenes has been excessively attempted, thus far, with limited success. Despite apparent challenges, our increasing understanding of the neomorphic features of these fusion oncogenes in conjunction with rapid technological advances will likely enable the development of new strategies to therapeutically exploit these neomorphic features and to ultimately turn the "undruggable" into first-line target structures. In this review, we provide a broad overview of the current literature on targeting neomorphic features of fusion oncogenes found in Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma, and give a perspective for future developments. Graphical abstract Scheme depicting the different targeting strategies of fusion oncogenes in pediatric fusion-driven sarcomas. Fusion oncogenes can be targeted on their DNA level (1), RNA level (2), protein level (3), and by targeting downstream functions and interaction partners (4).

A comparative view on the expression patterns of PD-L1 and PD-1 in soft tissue sarcomas.

Soft tissue sarcomas (STSs) are heterogeneous cancers associated with poor prognosis due to high rates of local recurrence and metastasis. The programmed death receptor ligand 1 (PD-L1) is expressed in several cancers. PD-L1 interacts with its receptor, PD-1, on the surface of tumor-infiltrating lymphocytes (TILs), thereby attenuating anti-cancer immune response. Immune checkpoint inhibitors targeting this interaction have been established as effective anti-cancer drugs. However, studies on the PD-L1 and PD-1 expression status in STS are commonly limited by small sample size, analysis of single STS subtypes, or lack of combinatorial marker assessment. To overcome these limitations, we evaluated the expression patterns of intratumoral PD-L1, the number of TILs, their PD-1 expression, and associations with clinicopathological parameters in a large and comprehensive cohort of 225 samples comprising six STS subtypes. We found that nearly all STS subtypes showed PD-L1 expression on the tumor cells, albeit with a broad range of positivity across subtypes (50% angiosarcomas to 3% synovial sarcomas). Co-expression and correlation analyses uncovered that PD-L1 expression was associated with more PD-1-positive TILs (P < 0.001), higher tumor grading (P = 0.016), and worse patients' 5-year overall survival (P = 0.028). The results were in line with several publications on single STS subtypes, especially when comparing findings for STS with low and high mutational burden. In sum, the substantial portion of PD-L1 positivity, the co-occurrence of PD-1-positive TILs, and the association of PD-L1 with unfavorable clinical outcome provide rationales for immune checkpoint inhibition in patients with PD-L1-positive STS.

Functional genomics identifies AMPD2 as a new prognostic marker for undifferentiated pleomorphic sarcoma.

Soft-tissue sarcomas are rare, heterogeneous, and often aggressive mesenchymal cancers. Many of them are associated with poor outcome, partially because biomarkers that can identify high-risk patients are lacking. Studies on sarcomas are often limited by small sample-sizes rendering the identification of biomarkers difficult when focusing on individual cohorts. However, the increasing number of publicly available 'omics' data opens inroads to overcome this obstacle. Here, we combine transcriptome analyses, immunohistochemistry, and functional assays to show that high adenosine monophosphate deaminase 2 (AMPD2) is a robust prognostic biomarker for worse outcome in undifferentiated pleomorphic sarcoma (UPS). Gene expression and survival data for UPS from two independent studies were subjected to survival association-testing. Genes, whose high expression was significantly correlated with worse outcome in both cohorts, were considered as biomarker candidates. The best candidate, AMPD2, was validated in a tissue microarray. Analysis of DNA copy-number data and matched transcriptomes indicated that high AMPD2 expression is significantly correlated with gains at the AMPD2 locus. Gene set enrichment analyses of AMPD2 co-expressed genes in both transcriptome datasets suggested that AMPD2-high UPS are enriched in tumorigenic signatures. Consistently, knockdown of AMPD2 by RNA interference in an UPS cell line inhibited proliferation in vitro and tumorigenicity in vivo. Collectively, we provide evidence that AMPD2 may serve as a biomarker for outcome prediction in UPS. Our study exemplifies how the integration of 'omics' data, immunohistochemistry, and functional experiments can identify novel biomarkers even in a rare sarcoma, which may serve as a blueprint for biomarker identification for other rare cancers.

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-κB signaling.

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.

Small round cell sarcomas.

Undifferentiated small round cell sarcomas (SRCSs) of bone and soft tissue comprise a heterogeneous group of highly aggressive tumours associated with a poor prognosis, especially in metastatic disease. SRCS entities mainly occur in the third decade of life and can exhibit striking disparities regarding preferentially affected sex and tumour localization. SRCSs comprise new entities defined by specific genetic abnormalities, namely EWSR1-non-ETS fusions, CIC-rearrangements or BCOR genetic alterations, as well as EWSR1-ETS fusions in the prototypic SRCS Ewing sarcoma. These gene fusions mainly encode aberrant oncogenic transcription factors that massively rewire the transcriptome and epigenome of the as yet unknown cell or cells of origin. Additional mutations or copy number variants are rare at diagnosis and, depending on the tumour entity, may involve TP53, CDKN2A and others. Histologically, these lesions consist of small round cells expressing variable levels of CD99 and specific marker proteins, including cyclin B3, ETV4, WT1, NKX3-1 and aggrecan, depending on the entity. Besides locoregional treatment that should follow standard protocols for sarcoma management, (neo)adjuvant treatment is as yet ill-defined but generally follows that of Ewing sarcoma and is associated with adverse effects that might compromise quality of life. Emerging studies on the molecular mechanisms of SRCSs and the development of genetically engineered animal models hold promise for improvements in early detection, disease monitoring, treatment-related toxicity, overall survival and quality of life.

Fusion protein-driven IGF-IR/PI3K/AKT signals deregulate Hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 in myxoid liposarcoma.

Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.

ABCA6 affects the malignancy of Ewing sarcoma cells via cholesterol-guided inhibition of the IGF1R/AKT/MDM2 axis.

PURPOSE: The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. METHODS: The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. RESULTS: We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. CONCLUSIONS: Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs.

Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1.

Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Analysis of Migration and Invasion in Ewing Sarcoma.

The metastasis is a complex, well-orchestrated process, which includes migration from the primary tumor and invasion into secondary locations as main features. In Ewing sarcoma, metastasis is the main determinant of malignancy, with ~30% of patients presenting with metastatic disease at diagnosis. Therefore, analyzing migration and invasion in different experimental settings in vitro is key to understanding this disease. Among the variety of possible techniques to study migration, this chapter described the methods of wound healing (migration in 2D) and transwell (migration through a porous membrane in response to a given stimulus). Additionally, this chapter includes a variation of the transwell protocol that allows for the analysis of cell invasion through a gel matrix in response to stimulus.

Drug Screening by Resazurin Colorimetry in Ewing Sarcoma.

In Ewing sarcoma (EwS), development of new therapeutic strategies is crucial in order to refine treatment and improve patient survival, especially in metastatic or recurrent disease stages. Thus, preclinical drug screening is a key issue in EwS research. As especially in such drug screening assays, the cell viability aspect of cell proliferation is important, resazurin colorimetry shall be reviewed here as a fast, high-throughput method with automated readout to efficiently screen for potency of drugs via measurement of cell viability.

Tumor Growth Analysis of Ewing Sarcoma Cell Lines Using Subcutaneous Xenografts in Mice.

Subcutaneous murine xenograft models are one of the most commonly used in vivo experimental methods in the cancer research field. Due to the lack of appropriate animal models for Ewing sarcoma, subcutaneous murine xenograft models currently offer the simplest way to investigate antineoplastic effects of therapeutics or biological functions of target genes in vivo. In order to properly carry out tumor growth analysis via subcutaneous xenografts of Ewing sarcoma cells many factors should be taken into account beforehand at the planning phase of experiments. Therefore, in this chapter we describe in detail a widely used procedure for subcutaneous injection in mice, focusing on the specific characteristics of Ewing sarcoma cell lines.

Ewing Sarcoma-Specific (Re)expression Models.

Gene expression and knockdown systems are powerful tools to study the function of single genes and their pathway interaction. Plasmid transfection and viral transduction have revolutionized the field of molecular biology and paved the ground for various gene-editing strategies such as TALEN, zinc finger nucleases, and ultimately CRISPR. In Ewing sarcoma (EwS), almost as many genes are repressed by the expression of EWSR1-FLI1 as are upregulated by the fusion oncogene. Here we present a useful point-to-point protocol for the generation of transgene expression systems in EwS that allow (conditional) reexpression of a gene of interest. We provide an extensive instruction on molecular cloning, plasmid generation, viral transduction, and expression validation. Finally, we address common problems and highlight potential pitfalls, which can easily be avoided by thoughtful guidance.

Therapeutic targeting of the PLK1-PRC1-axis triggers cell death in genomically silent childhood cancer.

Chromosomal instability (CIN) is a hallmark of cancer1. Yet, many childhood cancers, such as Ewing sarcoma (EwS), feature remarkably 'silent' genomes with minimal CIN2. Here, we show in the EwS model how uncoupling of mitosis and cytokinesis via targeting protein regulator of cytokinesis 1 (PRC1) or its activating polo-like kinase 1 (PLK1) can be employed to induce fatal genomic instability and tumor regression. We find that the EwS-specific oncogenic transcription factor EWSR1-FLI1 hijacks PRC1, which physiologically safeguards controlled cell division, through binding to a proximal enhancer-like GGAA-microsatellite, thereby promoting tumor growth and poor clinical outcome. Via integration of transcriptome-profiling and functional in vitro and in vivo experiments including CRISPR-mediated enhancer editing, we discover that high PRC1 expression creates a therapeutic vulnerability toward PLK1 inhibition that can repress even chemo-resistant EwS cells by triggering mitotic catastrophe.Collectively, our results exemplify how aberrant PRC1 activation by a dominant oncogene can confer malignancy but provide opportunities for targeted therapy, and identify PRC1 expression as an important determinant to predict the efficacy of PLK1 inhibitors being used in clinical trials.

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.