Search

Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into an epithelial-mesenchymal gradient of RMC cells associated with loss of renal epithelial transcription factors TFCP2L1, HOXB9 and MITF and gain of MYC and NFE2L2-associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells.

3D in vitro biological systems are progressively replacing 2D systems to increase the physiological relevance of cellular studies. Microfluidics-based approaches can be powerful tools towards such biomimetic systems, but often require high-end complicated and expensive processes and equipments for microfabrication. Herein, a drug screening platform is proposed, minimizing technicality and manufacturing steps. It provides an alternate way of spheroid generation in droplets in tubes. Droplets microfluidics then elicit multiple droplets merging events at programmable times, to submit sequentially the spheroids to chemotherapy and to reagents for cytotoxicity screening. After a comprehensive study of tumorigenesis within the droplets, the system is validated for drug screening (IC50) with chemotherapies in cancer cell lines as well as cells from patient-derived-xenografts (PDX). As compared to microtiter plates methods, our sytem reduces the initial amout of cell up to 10 times and opens new avenues towards primary tumors drug screening approaches.

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.

Supplementary Table 2 provides a summary (a), list of structural variants (b), list of copy number alterations (c), list of Tier 1 single nucleotide variants (d), indels (e), and purity analyses (f).

Ewing sarcoma is a primary bone tumor initiated by EWSR1–ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples.Significance: Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers. Cancer Discov; 4(11); 1342–53. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 1243

Supplementary Table from Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma

Supplementary Table 1 describes the clinical characteristics of patients.

Supplementary Data from Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma

Supplementary Table 4 provides the precise description of all STAG2 mutations in Ewing sarcoma.

Supplementary Figure 1. Representative CIRCOS plots. Supplementary Figure 2. Scheme of copy number alterations. Indicates the copy number alterations in Ewing sarcoma. Supplementary Figure 3. Mutation spectrum in Ewing sarcoma. Indicates the proportion of each somatic base changes in Ewing sarcoma. Supplementary Figure 4. CDKN2A analysis across cell lines. RT-QPCR and western blot results in cell lines. Supplementary Figure 5. CDKN2A analysis across the extended cohort. QPCR analysis of the CDKN2A locus across the whole series of patients.

Supplementary Table 3. Analysis of the statistical significance of mutations.

Cell line growth curves and PDX data for idasanutlin combination

GEMM ALK inhibitor data

List of supplementary tables and figures.

Supplementary methods, supplementary tables, supplementary figure legends

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc− transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy.Significance:Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659

Cell line and PDX genetics

STAG2 mutation in three cases that show a reduction of STAG2-immunopositive cells at disease recurrence.

GEMM sequencing data

PDTC experimental data

PDX data and clinical data

Ewing sarcoma is characterized by pathognomonic translocations, most frequently fusing EWSR1 with FLI1. An estimated 30% of Ewing sarcoma tumors also display genetic alterations in STAG2, TP53, or CDKN2A (SPC). Numerous attempts to develop relevant Ewing sarcoma models from primary human cells have been unsuccessful in faithfully recapitulating the phenotypic, transcriptomic, and epigenetic features of Ewing sarcoma. In this study, by engineering the t(11;22)(q24;q12) translocation together with a combination of SPC mutations, we generated a wide collection of immortalized cells (EWIma cells) tolerating EWSR1-FLI1 expression from primary mesenchymal stem cells (MSC) derived from a patient with Ewing sarcoma. Within this model, SPC alterations strongly favored Ewing sarcoma oncogenicity. Xenograft experiments with independent EWIma cells induced tumors and metastases in mice, which displayed bona fide features of Ewing sarcoma. EWIma cells presented balanced but also more complex translocation profiles mimicking chromoplexy, which is frequently observed in Ewing sarcoma and other cancers. Collectively, these results demonstrate that bone marrow–derived MSCs are a source of origin for Ewing sarcoma and also provide original experimental models to investigate Ewing sarcomagenesis.Significance:These findings demonstrate that Ewing sarcoma can originate from human bone-marrow–derived mesenchymal stem cells and that recurrent mutations support EWSR1-FLI1 translocation-mediated transformation.

SupReferences+SupMAt&Met+Suptables+SupFig

Supplemental Materials and Methods

The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS.Significance:These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.

PDF file - 102K, EWSR1-FLI1 expression upon Enzastaurin treatment

PDF file - 114K, Rescuing shRNAs Inhibition of PRKCB

PDF file - 94K, Stable A673-shPRKCB463_B5M3 cell line

PDF file - 75K, H3K4 trimethylation upon PRKCA inhibition

XLS file - 3.4MB, Expression profiles analyses and primer table

PDF file - 66K, Combined effect of TNFa with Enzastaurin on Ewing cell growth

PDF file - 122K, PRKC isoforms expression

PDF file - 196K

PDF file - 94K, EWSR1-FLI1 transcriptional influence on PRKCA and PRKCB

Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient’s tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Paediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This new PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and new treatments development in advanced pediatric malignancies.

BackgroundALKactivating mutations are identified in approximately 10% of newly diagnosed neuroblastomas andALKamplifications in a further 1-2% of cases. Lorlatinib, a third generation ALK inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data has suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway.AimsTo study the preclinical activity of ALK inhibitors alone and in combination with chemotherapy or idasanutlin.MethodsWe compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin and vincristine)in vivodosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient derived xenografts (PDX).ResultsLorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in theALK-amplified PDX model with the highest ALK expression. In this PDX lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth.ConclusionOur study suggests that in neuroblastoma, high ALK expression could be associated with response to lorlatinib and either chemotherapy or idasanutlin. The synergy between MDM2 inhibition and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.STATEMENT OF TRANSLATIONAL RELEVANCENeuroblastoma is a pediatric tumor of the developing sympathetic nervous system. Around 50% of high-risk neuroblastoma patients are curable. Mutations or amplification of Anaplastic Lymphoma Kinase (ALK) have emerged as a marker with which to further risk-stratify patients. The ALK inhibitor lorlatinib will soon be used alongside chemotherapy in upfront treatment of high-risk patients with ALK-aberrant disease. In this preclinical study, we used a panel ofALKaberrant neuroblastoma models to evaluate ALK inhibitors focusing on lorlatinib in combination with conventional chemotherapy and the small molecule MDM2 inhibitor idasanutlin. In both approaches we found synergy in models with high basal ALK expression without MAPK pathway alterations. We conclude that in neuroblastoma the level of ALK expression could be an additional biomarker predictive of clinical response to ALK inhibitors.

PURPOSE Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/ 7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

The advent of dose intensified interval compressed therapy has improved event-free survival for patients with localized Ewing sarcoma (EwS) to 78% at 5 years. However, nearly a quarter of patients with localized tumors and 60-80% of patients with metastatic tumors suffer relapse and die of disease. In addition, those who survive are often left with debilitating late effects. Clinical features aside from stage have proven inadequate to meaningfully classify patients for risk-stratified therapy. Therefore, there is a critical need to develop approaches to risk stratify patients with EwS based on molecular features. Over the past decade, new technology has enabled the study of multiple molecular biomarkers in EwS. Preliminary evidence requiring validation supports copy number changes, and loss of function mutations in tumor suppressor genes as biomarkers of outcome in EwS. Initial studies of circulating tumor DNA demonstrated that diagnostic ctDNA burden and ctDNA clearance during induction are also associated with outcome. In addition, fusion partner should be a pre-requisite for enrollment on EwS clinical trials, and the fusion type and structure require further study to determine prognostic impact. These emerging biomarkers represent a new horizon in our understanding of disease risk and will enable future efforts to develop risk-adapted treatment.

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

Cancer represents a leading cause of death by disease in childhood. Pediatric tumors exhibit a high intertumoral heterogeneity, as different tumor types and subtypes have emerged with peculiar molecular and clinical features; however, compared to cancer in adults, pediatric tumors are rare and mostly present with lower mutational burden. The lack of specific therapeutic options represents the main current challenge; systematic, multi-disciplinary approaches are required to accelerate drug development and ultimately to find cures for all children with cancer. The EU funded “Innovative Therapies for Children with Cancer–Pediatric Preclinical Proof-of-Concept Project” (ITCC-P4; www.itccp4.eu) consortium consists of a public-private partnership including academic and industrial partners with the goal of developing a large-scale platform comprising >400 patient-derived xenograft (PDX) models representing high-risk pediatric cancers. Currently, this collection of PDX models includes the most common types of pediatric tumors, such as leukemia (n=28), bone and soft-tissue sarcomas (n=154), CNS tumors (n=96) and neuroblastomas (n=38), as well as other rare childhood cancers, such as hepatoblastomas (n=20) and malignant rhabdoid tumors (n=18); PDX models have been generated either from primary (n=206) or relapse (n=118) disease. In order to: a) investigate the biology of the pediatric PDX models in a high-throughput and systematic fashion, b) assess whether they accurately reflect the molecular features of the corresponding primary tumor and, c) identify potential new suitable biomarkers, we performed a comprehensive molecular characterization (whole-exome and low-coverage whole-genome sequencing; DNA methylation profiling; RNAseq and gene expression profiling) of the PDX models, as well as their matching human tumors and germline samples. These data contributed to the stratification of the PDX models based on their mutational status and emerging molecular vulnerabilities to inform in vivo drug testing in all these PDX models. This proof-of-concept drug testing has been conducted defining, for each group of models, a panel of single compounds (SOC n=3; novel targeted therapies, n=6) or combinations (with each other or with chemo- or radiotherapy). All processed molecular and drug-testing data are collected in the consortium´s centralized data repository (https://r2.amc.nl) allowing data downstream analysis, visualization and interpretation. Taken together, the ITCC-P4 sustainable platform represents a validated and powerful tool to investigate the biology of pediatric cancer based on the establishment, characterization and preclinical testing of pediatric cancer PDX models, ultimately envisaged to contribute the development of innovative therapeutic options for childhood cancer patients. Citation Format: Aniello Federico, Apurva Gopisetty, Didier Surdez, Yasmine Iddir, Alexandra Saint-Charles, Justyna Wierzbinska, Andreas Schlicker, Richard Volckmann, Danny Zwijnenburg, Sara Colombetti, Olaf Heidenreich, Fatima Iradier, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Christina Guttke, Maureen M. Hattersley, Frédéric Colland, Ashley Strougo, María José Guillén, Louis Chesler, Chris Jones, Maria Eugénia Marques da Costa, Katia Scotlandi, Massimo Moro, Beat Schäfer, Marco Wachtel, Johannes Gojo, Walter Berger, Ángel Montero Carcaboso, Dennis Gürgen, Jens Hoffmann, Emilie Indersie, Stefano Cairo, Julia Schueler, Nicole Huebener, Johannes H. Schulte, Jan J. Molenaar, Birgit Geoerger, David J. Shields, Hubert N. Caron, Gilles Vassal, Lou F. Stancato, Lou F. Stancato, Stefan M. Pfister, Natalie Jäger, Jan Koster, Marcel Kool, Gudrun Schleiermacher. The ITCC-P4 sustainable platform of fully characterized PDXs supports the preclinical proof-of-concept drug testing of high-risk pediatric tumor models. [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 3571.

Advancements in state-of-the-art molecular profiling techniques have resulted in better understanding of pediatric cancers and driver events. It has become apparent that pediatric cancers are significantly more heterogeneous than previously thought as evidenced by the number of novel entities and subtypes that have been identified with distinct molecular and clinical characteristics. For most of these newly recognized entities there are extremely limited treatment options available. The ITCC-P4 consortium is an international collaboration between several European academic centers and pharmaceutical companies, with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers, their tumors and matching controls and to use the PDX models for in vivo testing of novel mechanism-of-action based treatments. Currently, 251 models are fully characterized, including 182 brain and 69 non-brain PDX models, representing 112 primary models, 92 relapse, 42 metastasis and 4 progressions under treatment models. Using low coverage whole-genome and whole exome sequencing, somatic mutation calling, DNA copy number and methylation analysis we aim to define genetic features in our PDX models and estimate the molecular fidelity of PDX models compared to their patient tumor. Based on DNA methylation profiling we identified 43 different tumor subgroups within 18 cancer entities. Mutational landscape analysis identified key somatic and germline oncogenic drivers. Ependymoma PDX models displayed the C11orf95-RELA fusion event, YAP1, C11orf95 and RELA structural variants. Medulloblastoma models were driven by MYCN, TP53, GLI2, SUFU and PTEN. High-grade glioma samples showed TP53, ATRX, MYCN and PIK3CA somatic SNVs, along with focal deletions in CDKN2A in chromosome 9. Neuroblastoma models were enriched for ALK SNVs and/or MYCN focal amplification, ATRX SNVs and CDKN2A/B deletions. Tumor mutational burden across entities and copy number analysis was performed to identify allele-specific copy number detection in tumor-normal pairs. Large chromosomal aberrations (deletions, duplications) detected in the PDX models were concurrent with molecular alterations frequently observed in each tumor type -isochromosome 17 was detected in 5 medulloblastoma models, while deletion of chromosome arm 1p or gain of parts of 17q in neuroblastomas which correlate with tumor progression. We observe clonal evolution of somatic variants not only in certain PDX-tumor pairs but also between disease states. The multi-omics approach in this study provides insight into the mutational landscape and patterns of the PDX models thus providing an overview of molecular mechanisms facilitating the identification and prioritization of oncogenic drivers and potential biomarkers for optimal treatment therapies. Citation Format: Apurva Gopisetty, Aniello Federico, Didier Surdez, Yasmine Iddir, Sakina Zaidi, Alexandra Saint-Charles, Joshua Waterfall, Elnaz Saberi-Ansari, Justyna Wierzbinska, Andreas Schlicker, Norman Mack, Benjamin Schwalm, Christopher Previti, Lena Weiser, Ivo Buchhalter, Anna-Lisa Böttcher, Martin Sill, Robert Autry, Frank Estermann, David Jones, Richard Volckmann, Danny Zwijnenburg, Angelika Eggert, Olaf Heidenreich, Fatima Iradier, Irmela Jeremias, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Petra Hamerlik, Maureen Hattersley, Olaf Witt, Louis Chesler, Alan Mackay, Johannes Gojo, Stefano Cairo, Julia Schueler, Johannes Schulte, Birgit Geoerger, Jan J. Molenaar, David J. Shields, Hubert N. Caron, Gilles Vassal, Louis F. Stancato, Stefan M. Pfister, Natalie Jaeger, Jan Koster, Marcel Kool, Gudrun Schleiermacher. ITCC-P4: Genomic profiling and analyses of pediatric patient tumor and patient-derived xenograft (PDX) models for high throughput in vivo testing [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 234.

Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that have not been functionally validated for their transformation capacity and biological activity. An example is the new fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implemented a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We found that in our transgenic zebrafish and mouse allograft models, VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle. Zebrafish and mouse VGLL2-NCOA2 tumors are consistent with the human disease histologically and transcriptionally, and express diagnostic rhabdomyosarcoma markers. We found a shared molecular feature of these tumors was a suppression of myogenic differentiation pathways and reactivation of developmental programs. To identify impacted developmental programs, we transcriptionally clustered zebrafish VGLL2-NCOA2 tumors with the trajectory of early zebrafish development. A subset of VGLL2-NCOA2 zebrafish tumors cluster with embryonic somitogenesis and pinpoint VGLL2-NCOA2 developmental targets, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse allograft, and patient tumors, ARF6 is highly expressed and is absent from mature skeletal muscle. ARF6 knockout in our C2C12-VGLL2-NCOA2 cell culture model suppresses VGLL2-NCOA2 soft agar colony formation capacity, suggesting a genetic cooperating event in the disease. Our data indicate that VGLL2-NCOA2 is an oncogene which leverages developmental programs for tumorigenesis, and that the reactivation or persistence of ARF6 could represent a therapeutic opportunity. Citation Format: Sarah Watson, Collette A. LaVigne, Lin Xu, Didier Surdez, Joanna Cyrta, Delia Calderon, Matthew V. Cannon, Matthew R. Kent, Katherine M. Silvius, Jack P. Kucinski, Emma N. Harrison, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James F. Amatruda, Genevieve C. Kendall. VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis. [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 3525.

Advancements in state-of-the-art molecular profiling techniques has resulted in better understanding of pediatric cancers and their drivers. Conversely, it also became apparent that pediatric cancers are much more heterogeneous than previously thought. Many new types and subtypes of pediatric cancers have been identified with distinct molecular and clinical characteristics. However, for most newly recognized entities there is no specific treatment available yet. The ITCC-P4 consortium is a collaboration between many academic centers across Europe and several pharmaceutical companies involved in preclinical testing, with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers and to use them for in vivo testing of novel mechanism-of-action based treatments. Currently, 340 models are fully established, including 87 brain and 253 non-brain tumor models, together representing different tumor types both from primary (113) and relapsed (92)/metastatic disease (42). 252 of these models have been fully molecularly characterized, representing 18 pediatric cancer entities and 43 different subtypes. Using low coverage whole-genome and whole exome sequencing, somatic mutation calling, DNA copy number, transcriptome analysis and methylation profiling we have observed that the molecular profile of most PDX models closely mimics their original tumors. Clonal evolution of somatic variants was only observed in some PDX-tumor pairs or so between disease states. Somatic copy number variant analysis highlights specific alterations for instance MYB, MYC, MYCN, NTRK3, PTEN loss differently distributed between PDX-patient tumor pairs in high-grade gliomas. Overall, our results show that we have established >250 PDX models of solid pediatric cancers, that well represents the disease spectrum and that is currently being used for in vivo testing of standard of care drugs and targeted small molecules. Treatment responses will be directly linked to molecular data to identify potential biomarkers for prioritization or deprioritization of individual, patient-specific specific drugs.

Acyl-coenzyme-A-binding protein (ACBP), also known as a diazepam-binding inhibitor (DBI), is a potent stimulator of appetite and lipogenesis. Bioinformatic analyses combined with systematic screens revealed that peroxisome proliferator-activated receptor gamma (PPARγ) is the transcription factor that best explains the ACBP/DBI upregulation in metabolically active organs including the liver and adipose tissue. The PPARγ agonist rosiglitazone-induced ACBP/DBI upregulation, as well as weight gain, that could be prevented by knockout of Acbp/Dbi in mice. Moreover, liver-specific knockdown of Pparg prevented the high-fat diet (HFD)-induced upregulation of circulating ACBP/DBI levels and reduced body weight gain. Conversely, knockout of Acbp/Dbi prevented the HFD-induced upregulation of PPARγ. Notably, a single amino acid substitution (F77I) in the γ2 subunit of gamma-aminobutyric acid A receptor (GABA

The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS. Significance: These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.

Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into three RMC cell states along an epithelial-mesenchymal gradient by loss of renal epithelial transcription factors TFCP2L1, HOXB9 and MITF and gain of MYC and NFE2L2-associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells.

BackgroundHigh-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies.MethodsWe combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays.ResultsWe show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes.ConclusionsOur study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment.

Acyl-coenzyme-A-binding protein (ACBP), also known as a diazepam-binding inhibitor (DBI), is a potent stimulator of appetite and lipogenesis. Bioinformatic analyses combined with systematic screens revealed that peroxisome proliferator-activated receptor gamma (PPARγ) is the transcription factor that best explains the ACBP/DBI upregulation in metabolically active organs including the liver and adipose tissue. The PPARγ agonist rosiglitazone-induced ACBP/DBI upregulation, as well as weight gain, that could be prevented by knockout of Acbp/Dbi in mice. Moreover, liver-specific knockdown of Pparg prevented the high-fat diet (HFD)-induced upregulation of circulating ACBP/DBI levels and reduced body weight gain. Conversely, knockout of Acbp/Dbi prevented the HFD-induced upregulation of PPARγ. Notably, a single amino acid substitution (F77I) in the γ2 subunit of gamma-aminobutyric acid A receptor (GABAAR), which abolishes ACBP/DBI binding to this receptor, prevented the HFD-induced weight gain, as well as the HFD-induced upregulation of ACBP/DBI, GABAAR γ2, and PPARγ. Based on these results, we postulate the existence of an obesogenic feedforward loop relying on ACBP/DBI, GABAAR, and PPARγ. Interruption of this vicious cycle, at any level, indistinguishably mitigates HFD-induced weight gain, hepatosteatosis, and hyperglycemia.