BMI1 is a target gene of E2F-1 and is strongly expressed in primary neuroblastomas

The oncogene BMI1 encodes a polycomb group transcription factor that is required for embryonic development and self-renewal of stem cells. Despite these important functions little is known about the regulation of BMI1 expression. A cDNA microarray based search for target genes of E2F-1 in neuroblastoma cells expressing a 4-OHT-regulated E2F-1-ER fusion protein identified many hitherto unknown E2F-1 regulated genes. A total of 10% of these genes, including BMI1, encode proteins that function primarily in the regulation of gene expression. The BMI1 promoter contains a putative E2F binding site that was required for the activation of a BMI1 promoter-dependent reporter construct by E2F-1. Chromatin immunoprecipitation revealed 4-OHT-dependent binding of E2F-1-ER and binding of endogenous E2F-1 to the BMI1 promoter in tumor cells. We have previously shown activation of the oncogene MYCN by E2F. Thus, in neuroblastomas deregulated E2F-1 can activate two oncogenes, MYCN and BMI1 that are known to co-operate in tumor formation. Consistent with a role of Bmi1 in neuroblastoma tumorigenesis we found strong Bmi1 expression in primary neuroblastomas. Our results reveal a novel link between E2F and polycomb transcription factors and suggest a role of Bmi1 in neuroblastomas

The histone deacetylase inhibitor SAHA acts in synergism with fenretinide and doxorubicin to control growth of rhabdoid tumor cells

Background: Rhabdoid tumors are highly aggressive malignancies affecting infants and very young children. In many instances these tumors are resistant to conventional type chemotherapy necessitating alternative approaches. Methods: Proliferation assays (MTT), apoptosis (propidium iodide/annexin V) and cell cycle analysis (DAPI), RNA expression microarrays and western blots were used to identify synergism of the HDAC (histone deacetylase) inhibitor SAHA with fenretinide, tamoxifen and doxorubicin in rhabdoidtumor cell lines. Results: HDAC1 and HDAC2 are overexpressed in primary rhabdoid tumors and rhabdoid tumor cell lines. Targeting HDACs in rhabdoid tumors induces cell cycle arrest and apoptosis. On the other hand HDAC inhibition induces deregulated gene programs (MYCC-, RB program and the stem cell program) in rhabdoid tumors. These programs are in general associated with cell cycle progression. Targeting these activated pro-proliferative genes by combined approaches of HDAC-inhibitors plus fenretinide, which inhibits cyclinD1, exhibit strong synergistic effects on induction of apoptosis. Furthermore, HDAC inhibition sensitizes rhabdoid tumor cell lines to cell death induced by chemotherapy. Conclusion: Our data demonstrate that HDAC inhibitor treatment in combination with fenretinide or conventional chemotherapy is a promising tool for the treatment of chemoresistant rhabdoid tumors.<br

ETMR-05: Single-cell transcriptomics of ETMR reveals developmental cellular programs and tumor-pericyte communications in the microenvironment [Abstract]

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are pediatric brain tumors bearing a grim prognosis, despite intensive multimodal therapeutic approaches. Insights into cellular heterogeneity and cellular communication of tumor cells with cells of the tumor microenvironment (TME), by applying single-cell (sc) techniques, potentially identify mechanisms of therapy resistance and target-directed treatment approaches. MATERIAL AND METHODS: To explore ETMR cell diversity, we used single-cell RNA sequencing (scRNA-seq) in human (n=2) and murine ETMR (transgenic mode; n=4) samples, spatial transcriptomics, 2D and 3D cultures (including co-cultures with TME cells), multiplex immunohistochemistry and drug screens. RESULTS: ETMR microenvironment is composed of tumor and non-tumor cell types. The ETMR malignant compartment harbour cells representing distinct transcriptional metaprograms, (NSC-like, NProg-like and Neuroblast-like), mirroring embryonic neurogenic cell states and fuelled by neurogenic pathways (WNT, SHH, Hippo). The ETMR TME is composed of oligodendrocyte and neuronal progenitor cells, neuroblasts, microglia, and pericytes. Tumor-specific ligand-receptor interaction analysis showed enrichment of intercellular communication between NProg-like ETMR cells and pericytes (PC). Functional network analyses reveal ETMR-PC interactions related to stem-cell signalling and extracellular matrix (ECM) organization, involving factors of the WNT, BMP, and CxCl12 networks. Results from ETMR-PC co-culture and spatial transcriptomics pointed to a pivotal role of pericytes in keeping ETMR in a germinal neurogenic state, enriched in stem-cell signalling. Drug screening considering cellular heterogeneity and cellular communication suggested novel therapeutic approaches. CONCLUSION: ETMR demonstrated diversity in the microenvironment, with enrichment of cell-cell communications with pericytes, supporting stem-cell signalling and interfering in the organization of the tumor extracellular matrix. Targeting ETMR-PC interactions might bring new opportunities for target-directed therapy

Single-cell transcriptomics identifies potential cells of origin of MYC rhabdoid tumors

Rhabdoid tumors (RT) are rare and highly aggressive pediatric neoplasms. Their epigenetically-driven intertumoral heterogeneity is well described; however, the cellular origin of RT remains an enigma. Here, we establish and characterize different genetically engineered mouse models driven under the control of distinct promoters and being active in early progenitor cell types with diverse embryonic onsets. From all models only Sox2-positive progenitor cells give rise to murine RT. Using single-cell analyses, we identify distinct cells of origin for the SHH and MYC subgroups of RT, rooting in early stages of embryogenesis. Intra- and extracranial MYC tumors harbor common genetic programs and potentially originate from fetal primordial germ cells (PGCs). Using PGC specific Smarcb1 knockout mouse models we validate that MYC RT originate from these progenitor cells. We uncover an epigenetic imbalance in MYC tumors compared to PGCs being sustained by epigenetically-driven subpopulations. Importantly, treatments with the DNA demethylating agent decitabine successfully impair tumor growth in vitro and in vivo. In summary, our work sheds light on the origin of RT and supports the clinical relevance of DNA methyltransferase inhibitors against this disease

Improved 6-year overall survival in AT/RT - results of the registry study Rhabdoid 2007

Atypical teratoid rhabdoid tumors (AT/RT) are characterized by mutations and subsequent inactivation of SMARCB1 (INI1, hSNF5), a predilection for very young children and an unfavorable outcome. The European Registry for rhabdoid tumors (EU-RHAB) was established to generate a common European database and to establish a standardized treatment regimen as the basis for phase I/II trials. Thus, genetic analyses, neuropathologic and radiologic diagnoses, and a consensus treatment regimen were prospectively evaluated. From 2005 to 2009, 31 patients with AT/RT from four countries were recruited into the registry study Rhabdoid 2007 and treated with systemic and intraventricular chemotherapy. Eight patients received high-dose chemotherapy, 23 radiotherapy, and 17 maintenance therapy. Reference evaluations were performed in 64% (genetic analyses, FISH, MLPA, sequencing) up to 97% (neuropathology, INI1 stain). Germ-line mutations (GLM) were detected in 6/21 patients. Prolonged overall survival was associated with age above 3years, radiotherapy and achievement of a complete remission. 6-year overall and event-free survival rates were 46% (+/- 0.10) and 45% (+/- 0.09), respectively. Serious adverse events and one treatment-related death due to insufficiency of a ventriculo peritoneal shunt (VP-shunt) and consecutive herniation were noted. Acquisition of standardized data including reference diagnosis and a standard treatment schedule improved data quality along with a survival benefit. Treatment was feasible with significant but manageable toxicity. Although our analysis is biased due to heterogeneous adherence to therapy, EU-RHAB provides the best available basis for phase I/II clinical trials

Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target

Infants and newborns with Atypical Teratoid Rhabdoid Tumors (ATRT) and Extracranial Malignant Rhabdoid Tumors (eMRT) in the EU-RHAB registry: a unique and challenging population

SIMPLE SUMMARY: Malignant rhabdoid tumors (MRT) are deadly tumors that predominantly affect infants and young children. Even when considering the generally young age of these patients, the treatment of infants below the age of six months represents a particular challenge due to the vulnerability of this patient population. The aim of our retrospective study was to assess the available information on prognostic factors, genetics, toxicity of treatment and long-term outcomes of MRT. We confirmed that, in a cohort of homogenously treated infants with MRT, significant predictors of outcome were female sex, localized stage, absence of a GLM and maintenance therapy, and these significantly favorably influence prognosis. Stratification-based biomarker-driven tailored trials may be a key option to improve survival rates. ABSTRACT: Introduction: Malignant rhabdoid tumors (MRT) predominantly affect infants and young children. Patients below six months of age represent a particularly therapeutically challenging group. Toxicity to developing organ sites limits intensity of treatment. Information on prognostic factors, genetics, toxicity of treatment and long-term outcomes is sparse. Methods: Clinical, genetic, and treatment data of 100 patients (aged below 6 months at diagnosis) from 13 European countries were analyzed (2005–2020). Tumors and matching blood samples were examined for SMARCB1 mutations using FISH, MLPA and Sanger sequencing. DNA methylation subgroups (ATRT-TYR, ATRT-SHH, and ATRT-MYC) were determined using 450 k / 850 k-profiling. Results: A total of 45 patients presented with ATRT, 29 with extracranial, extrarenal (eMRT) and 9 with renal rhabdoid tumors (RTK). Seventeen patients demonstrated synchronous tumors (SYN). Metastases (M+) were present in 27% (26/97) at diagnosis. A germline mutation (GLM) was detected in 55% (47/86). DNA methylation subgrouping was available in 50% (31 / 62) with ATRT or SYN; for eMRT, methylation-based subgrouping was not performed. The 5-year overall (OS) and event free survival (EFS) rates were 23.5 ± 4.6% and 19 ± 4.1%, respectively. Male sex (11 ± 5% vs. 35.8 ± 7.4%), M+ stage (6.1 ± 5.4% vs. 36.2 ± 7.4%), presence of SYN (7.1 ± 6.9% vs. 26.6 ± 5.3%) and GLM (7.7 ± 4.2% vs. 45.7 ± 8.6%) were significant prognostic factors for 5-year OS. Molecular subgrouping and survival analyses confirm a previously described survival advantage for ATRT-TYR. In an adjusted multivariate model, clinical factors that favorably influence the prognosis were female sex, localized stage, absence of a GLM and maintenance therapy. Conclusions: In this cohort of homogenously treated infants with MRT, significant predictors of outcome were sex, M-stage, GLM and maintenance therapy. We confirm the need to stratify which patient groups benefit from multimodal treatment, and which need novel therapeutic strategies. Biomarker-driven tailored trials may be a key option

"BMI-1 ist ein direktes Zielgen von E2F-1 in primären Neuroblastomen"

Zusammenfassung Das Neuroblastom ist der dritthäufigste maligne Tumor des Kindesalters (9% aller Tumore dieser Altersgruppe). Klinisch und molekularbiologisch weist das Neuroblastom eine große Heterogenität auf. Die Prognose der Patienten hängt im Wesentlichen vom Zeitpunkt der Diagnosestellung und der Amplifikation des MYCN-Onkogens ab. In Neuroblastomen wird MYCN durch E2F-1 reguliert. E2F-1 gehört zur E2F-Genfamilie, die bis zu 5% aller Gene reguliert. E2F-1 wird in Neuroblastomen v.a. zwei genetischen Programmen zugeordnet: 1. Induktion der S-Phase und DNA-Reparatur 2. Induktion von Apoptose Durch Microarray-Analysen im eigenen Labor wurde gezeigt, dass E2F-1 das Polycombgruppen-Gen BMI1 induziert. Die Interaktion zwischen E2F- und Polycombproteinen ist aus vielen anderen Tumoren bekannt. Polycombproteine lagern sich im Wesentlichen zu zwei unterschiedlichen Komplexen zusammen: 1. PcGi=initiating complex besteht aus Eed (embryonic ectoderm development), Enx1/EzH2 und Enx2/EzH1. 2. PcGm=maintenance complex enthält: PSC (posterior sex combs), PH (polyhomeotic), PC Polcomb, RING. In humanen Zellen besteht dieser Komplex auch aus Bmi1, Mel18, Mph1, M33, Mpc2. Die beiden Komplexe haben bei der Regulation der HOX-Gene, die bei der Differenzierung eine Rolle spielen, eine redundante Funktion. Bei der Proliferation der hämatopoetischen Stammzellen verhalten sich die beiden Komplexe allerdings antagonistisch: der EED/EZH-Komplex inhibiert, während der Bmi1-enthaltende Komplex die Proliferation der hämatopoetischen Stammzellen stimuliert. Es gibt mehrere Hinweise, dass BMI1 als Onkogen in verschiedenen Tumoren agiert: 1. In Lymphomen kooperiert BMI1 mit CMYC. BMI1 hemmt hier den INK4a/ARF-Lokus und hemmt dadurch die p53-abhängige Induktion der Apoptose durch Myc. 2. In Mausembryofibroblasten verlangsamt die Überexpression von BMI1 die Seneszenz. BMI1 reprimiert auch hier den INK4a/ARF-Genort. 3. BMI1 wird in vielen Tumoren überexprimiert gefunden: Mantelzelllymphomen, nicht-kleinzelligen Bronchial-Karzinomen, Kolonkarzinomen, Multiplen Myelomen und Medulloblastomen. In Neuroblastomen ist die Rolle, die Bmi1 in der Tumorenstehung spielt weitgehend ungeklärt. Bmi1 wird in primären Neuroblastomen stark exprimiert. Ich habe in der vorliegenden Arbeit die Regulation von Bmi1 durch E2F-1 und die Rolle von Bmi1 für die Funktion von E2F-1 im Neuroblastom untersucht. Ich habe zunächst gezeigt, dass die Induktion von Bmi1 durch E2F-1 auch auf Proteinebene stattfindet. Ich habe anschließend nachgewiesen, dass die Regulation von Bmi1 durch E2F-1 direkt ist. Durch Reporterassays konnte ich die E2F-1-Bindungsstelle im Bmi1-Promotor lokalisieren. Des Weiteren habe ich Zellklone hergestellt, bei denen durch RNA-Interferenz bzw. Überexpression eines dominant negativen Allels die Funktion von Bmi1 gehemmt werden kann. Erste Experimente deuten darauf hin, dass die Hemmung von Bmi1 zu einem Wachstumsarrest führt. Es zeigte sich in den Klonen, in denen Bmi1 herunterreguliert vorlag, kein Effekt auf S-Phase-Induktion und Apoptose. Eine mögliche Erklärung für den Wachstumsarrest der Neuroblastomzellen bei Repression von Bmi1 ist der Verlust des Potentials der Zelle zur Selbsterneuerung. In weiteren Experimenten wurde überprüft, ob Bmi1 in Neuroblastomen an der Regulation des MYCN-Gens beteiligt ist. In einem Luciferaseassay zeigte sich dabei, dass Bmi1 MYCN nicht induziert. Bmi1 stabilisiert und induziert in Brustepithelzellen hTERT. hTERT ist der regulierende Anteil der Telomerase und liegt in vielen Zellen nicht exprimiert vor. Die Expression von hTERT führt zur Induktion der Telomerase und damit zur Stabilisierung der Telomerlänge. Telomere verkürzen sich in humanen Körperzellen im Laufe mehrerer Zellteilungen. Durch die Telomerverkürzung kann Seneszenz in den Zellen eingeleitet werden. Durch die Stabilisierung der Telomerlänge wird die Seneszenzeinleitung verhindert und ein wichtiger Schritt in Richtung Tumorentstehung begangen. Durch eine PCR im Echtzeitgerät in 1A3-Zellen konnte ich zeigen, dass BMI-1 im transienten Ansatz in Neuroblastomzellen hTERT nicht induziert. Zusammenfassend konnte ich durch meine Arbeit folgende wesentliche Punkte zeigen: 1.) E2F-1 reguliert Bmi1 in Neuroblastomzellen sowohl auf RNA-, als auch auf Proteinebene. 2.) Die Regulation von Bmi1 durch E2F-1 erfolgt auf direktem Weg. 3.) Die Induktion von Bmi1 durch E2F-1 erfolgt durch eine einzelne E2F-1-Bindungsstelle im proximalen Promotor des BMI1-Gens. 4.) Die funktionelle Bedeutung von Bmi1 in Neuroblastomen v.a. in Hinblick auf die Zellzyklusregulation bleibt zunächst nicht geklärt. Nach den Ergebnissen meiner Arbeit, führt der Verlust von Bmi1 zwar zum Wachstumsarrest von Neuroblastomzellen, hat aber weder Einfluss auf die Regulation von S-Phase, noch auf die Induktion von Apoptose