Abstract
Osteosarcoma (OS) and Ewing sarcoma (ES) are the most common bone cancers in children. They are rare cancers and thus difficult to study due to scarcity of patient material, large genomic ...instability and a wide histological heterogeneity (in OS) or a lack of satisfactory transgenic animal model and availability of preclinical tests (in ES). There is a dire need for new models and novel therapeutic approaches. Although patient-derived xenografts (PDXs) may recapitulate human tumor biology and predict drug response, propagating PDXs in mice limits its use as a drug-testing platform.
We have established and standardized ES and OS spheroid culture and developed a semi-automated drug-screening platform in tumor spheroids. We established several robust techniques for spheroid formation, with clear pathophysiological gradients, but without central necroses at the onset of drug treatment. We performed RNA-seq comparing spheroid transcription profiles to 2D culture and observed dramatic changes in overall expression patterns. We observed upregulation of genes shown to correlate with poor prognosis in OS patients. We saw upregulation of processes associated with regulation of cell migration, negative regulation of proliferation and modulation of the extracellular matrix (ECM). In addition to ES spheroid models, we created bioprinted 3D-models of ES cell lines and of cells obtained from ES PDXs, using extrusion bioprinting techniques (where cells are encapsulated within the cross-linked polymers, thus allowing homogeneous distribution and high cell density). PDX-derived cells were kept in liquid culture and as 3D-bioprinted constructs, while their transcription profiles were compared with the initial PDX. The mevalonate pathway was the most overrepresented in all ES 3D-models, consistent with predominant upregulation of this metabolic pathway integral to tumor growth and progression. After 15 days in 3D-bioprinted culture, we observed pronounced upregulation of genes involved in ECM signaling, suggesting that the construct promoted in vivo-like tumor-ECM interactions, without further promoting main proliferation and cell survival pathways, which was observed in liquid culture. Furthermore, we showed potential for combinatorial treatment with statins and confirmed feasibility of drug testing in patient-derived 3D models.
Finally, as our spheroid models showed upregulation of many processes involved in metastasis (genes associated with invasion, migration, angiogenesis and hypoxia), we focused on lung as the most common site of metastasis in ES and OS patients. We are thus establishing mixed airway organoid/tumoroid cultures, to investigate further the lung metastatic niche, with a goal to provide proof of concept for patient-specific 3D-models of lung metastatic tumors to guide personalized drug selection for patients with advanced disease.
Citation Format: Branka Radic Sarikas, Mathias Ilg, Marica Markovic, Caterina Sturtzel, Eva Scheuringer, Justine Zulini, Martin Metzelder, Florian Halbritter, Martin Distel, Didier Surdez, Olivier Delattre, Aleksandr Ovsianikov, Heinrich Kovar. 3D-models of pediatric bone sarcomas for personalized therapeutic screening 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 6245.
STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter ...to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy.
STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter ...to generate transgenic mouse models that expressed either human STAT5B or STAT5B.sup.N642H in the hematopoietic compartment. While STAT5B -expressing mice lacked a hematopoietic phenotype, the STAT5B.sup.N642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8.sup.+ lymphoma or leukemia, indicating that the STAT5B.sup.N642H mutation drives cancer development. Persistent and enhanced levels of STAT5B.sup.N642H tyrosine phosphorylation in transformed CD8.sup.+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5B.sup.N642H-expressing CD8.sup.+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5B.sup.N642H mutation who respond poorly to conventional chemotherapy.
Abstract
Introduction: Neuroblastoma is the most common solid tumor in infants and arises from progenitor cells during sympathoadrenal development. While the majority of primary tumor cells resembles ...adrenergic neurons, more undifferentiated mesenchymal or neural-crest cell-like phenotypes have been found, especially in pretreated and high-risk cases. In most high-risk neuroblastoma patients, tumor cells have disseminated to the bone marrow.
Problem Statement and Aim: However, the bone marrow, as a key regulator of hematopoietic and mesenchymal stem cell quiescence, is a frequent site of dissemination not only of neuroblastoma but also of other solid cancers such as breast and prostate cancer. Still, comprehensive single-cell analyses of bone marrow metastases, i.e. disseminated tumor cells (DTCs) and cells of their microenvironment, have not yet been undertaken. Herein, we aimed to capture tumor heterogeneity and investigate microenvironmental changes in a solid cancer with bone marrow involvement.
Methods: To that end, we applied a multi-omics data mining approach to define a multiplex imaging panel and designed DeepFLEX, a computational pipeline for subsequent multiplex image analysis. Thereby, we obtained a single-cell map of over 35,000 DTCs and cells of their microenvironment in the metastatic bone marrow niche. In addition, we independently profiled the transcriptome of 38 patients with and without bone marrow metastasis.
Results: We revealed vast diversity among DTCs and suggest that FAIM2 can act as a complementary marker to capture DTC heterogeneity. However, DTCs in this study mainly expressed markers of the adrenergic lineage, such as GD2, but did not show a mesenchymal phenotype. Interestingly, we demonstrate that malignant bone marrow infiltration is associated with an inflammatory response and at the same time the presence of immuno-suppressive cell types, most significantly an immature neutrophil/granulocyte-myeloid derived suppressor-like cell type.
Conclusion: Our findings demonstrate a high level of heterogeneity among DTCs and suggest that the latter shape the bone marrow microenvironment, warranting deeper investigations of spatio-temporal dynamics at the single-cell level and their clinical relevance.
Citation Format: Daria Lazic, Florian Kromp, Fikret Rifatbegovic, Peter Repiscak, Filip Mivalt, Florian Halbritter, Marie Bernkopf, Andrea Bileck, Marek Ussowicz, Inge M. Ambros, Peter F. Ambros, Christopher Gerner, Ruth Ladenstein, Christian Ostalecki, Sabine Taschner-Mandl. Mapping bone marrow metastasis in neuroblastoma by deep multiplex imaging and transcriptomics 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 977.
Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are ...frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3 encoding Ikaros and Aiolos have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NK cells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of proinflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS-mediated epigenetic and transcriptional dysregulation.
•Germline heterozygous mutations in IKZF2 cause a novel syndrome associated with immunodeficiency and profound immuno-dysregulation.•Germline heterozyogous mutations in IKZF2 disrupt interaction with the NuRD complex.
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Abstract
Early childhood malignancies are driven by sparse genetic aberrations in oncogenes that often co-occur with large copy number variants (CNVs). The combination of these mutations is thought ...to transform developmentally pliant embryonic cells to initiate tumorigenesis. However, the mechanistic interactions between CNVs, oncogenes, and differentiation have not been systematically studied due to several obstacles: (i) CNVs cannot be engineered efficiently yet; (ii) transient embryonic progenitors are absent in full-grown tumors; and (iii) inter-species differences in lineage specification limit the applicability of animal models.
To overcome these challenges, we used isogenic human embryonic stem cell (hESC) lines carrying gains of chromosome 17q/1q, which are prevalent in the embryonal tumor neuroblastoma (NB). We differentiated these cells toward trunk neural crest (NC) and their sympathoadrenal derivatives, the putative cells-of-origin of NB, and performed single-cell RNA sequencing and cell-biological assays at key differentiation stages. We found that CNVs impaired the specification of sympathoadrenal cell types and instead potentiated early Schwann-cell-precursor-like phenotypes. Additional overexpression of the oncogene MYCN (which is frequently amplified together with CNVs in high-risk NB tumors) exacerbated these differentiation defects, enabled tumourigenic cell proliferation, and generated cell states in vitro that transcriptionally resembled NB tumor cells. Finally, using epigenome analysis, we connected these states to a stepwise disruption of gene-regulatory networks centered on developmental transcription factors.
Together, our results chart a mechanistic route to NB tumorigenesis and provide a general framework for the CNV-driven initiation of embryonal tumors, in which CNVs ‘prime’ embryonic cells for oncogenic transformation. The tumor-like cells in our model may serve as proxies to experimentally test therapeutic interventions during tumorigenesis.
Citation Format: Ingrid M. Saldana-Guerrero, Luis F. Montano-Gutierrez, Christoph Hafemeister, Dylan Stavish, Lisa E. Shaw, Irfete S. Fetahu, Andrea Wenninger-Weinzierl, Caterina Sturtzel, Celine Souilhol, Sophia Tarelli, Mohamed R. Shoeb, Marie Bernkopf, Polyxeni Bozatzi, Maria Guarini, Eva Bozsaky, Michelle C. Buri, Eva M. Putz, Peter W. Andrews, Ivana Barbaric, Helen E. Bryant, Martin Distel, Sabine Taschner-Mandl, Matthias Farlik, Anestis Tsakiridis, Florian Halbritter. A stem cell model dissects detrimental effects of neuroblastoma-linked chromosomal aberrations on cell differentiation during neural crest development. abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3542.
Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a ...powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-X
inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-X
, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-X
inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-X
targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.
Embryonic stem cells (ESCs) are a special type of cell marked by two key properties: The capacity to create an unlimited number of identical copies of themselves (self-renewal) and the ability to ...give rise to differentiated progeny that can contribute to all tissues of the adult body (pluripotency). Decades of past research have identified many of the genetic determinants of the state of these cells, such as the transcription factors Pou5f1, Sox2 and Nanog. Many other transcription factors and, more recently, epigenetic determinants like histone modifications, have been implicated in the establishment, maintenance and loss of pluripotent stem cell identity. The study of these regulators has been boosted by technological advances in the field of high-throughput sequencing (HTS) that have made it possible to investigate the binding and modification of many proteins on a genome-wide level, resulting in an explosion of the amount of genomic data available to researchers. The challenge is now to effectively use these data and to integrate the manifold measurements into coherent and intelligible models that will actually help to better understand the way in which gene expression in stem cells is regulated to maintain their precarious identity. In this thesis, I first explore the potential of HTS by describing two pilot studies using the technology to investigate global differences in the transcriptional profiles of different cell populations. In both cases, I was able to identify a number of promising candidates that mark and, possibly, explain the phenotypic and functional differences between the cells studied. The pilot studies highlighted a strong requirement for specialised software to deal with the analysis of HTS data. I have developed GeneProf, a powerful computational framework for the integrated analysis of functional genomics experiments. This software platform solves many recurring data analysis challenges and streamlines, simplifies and standardises data analysis work flows promoting transparent and reproducible methodologies. The software offers a graphical, user-friendly interface and integrates expert knowledge to guide researchers through the analysis process. All primary analysis results are supplemented with a range of informative plots and summaries that ease the interpretation of the results. Behind the scenes, computationally demanding tasks are handled remotely on a distributed network of high-performance computers, removing rate-limiting requirements on local hardware set-up. A flexible and modular software design lays the foundations for a scalable and extensible framework that will be expanded to address an even wider range of data analysis tasks in future. Using GeneProf, billions of data points from over a hundred published studies have been re-analysed. The results of these analyses are stored in an web-accessible database as part of the GeneProf system, building up an accessible resource for all life scientists. All results, together with details about the analysis procedures used, can be browsed and examined in detail and all final and intermediate results are available and can instantly be reused and compared with new findings. In an attempt to elucidate the regulatory mechanisms of ESCs, I use this knowledge base to identify high-confidence candidate genes relevant to stem cell characteristics by comparing the transcriptional profiles of ESCs with those of other cell types. Doing so, I describe 229 genes with highly ESC-specific transcription. I then integrate the expression data for these ES-specific genes with genome-wide transcription factor binding and histone modification data. After investigating the global characteristics of these "regulatory inputs", I employ machine learning methods to first cluster subgroups of genes with ESC-specific expression patterns and then to define a "regulatory code" that marks one of the subgroups based on their regulatory signatures. The tightly co-regulated core cluster of genes identified in this analysis contains many known members of the transcriptional circuitry of ESCs and a number of novel candidates that I deem worthy of further investigations thanks to their similarity to their better known counterparts. Integrating these candidates and the regulatory code that drives them into our models of the workings of ESCs might eventually help to refine the ways in which we derive, culture and manipulate these cells - with all its prospective benefits to research and medicine.
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