Clustered regularly interspaced short palindromic repeats screens in pediatric tumours: A review

Abstract In recent years, the discovery and development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized and accelerated functional genetic screening in cancer research. In this review, we discuss different methods of executing CRISPR screens, with a focus on pediatric tumour entities. Historically, these tumours were thought to resemble their adult counterparts. However, the new era of genomic research and the extensive use of gene editing has identified pediatric tumours as distinct entities with drastically different development and presentation. Here we provide an overview of CRISPR screens performed in pediatric tumour models and highlight unique considerations for pediatric tumour screens. In particular, the results from CRISPR screens combining treatment with genetic knockouts can push treatment for pediatric patients. We conclude by discussing the potential of CRISPR genetic screening to unravel pediatric tumour biology and identify new treatment options

Signaling pathways and mesenchymal transition in pediatric high-grade glioma

Pediatric high-grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal types of cancer in children. In recent years, it has become evident that these tumors are driven by epigenetic events, mainly mutations involving genes encoding Histone 3, setting them apart from their adult counterparts. These tumors are exceptionally resistant to chemotherapy and respond only temporarily to radiotherapy. Moreover, their delicate location and diffuse growth pattern make complete surgical resection impossible. In many other forms of cancer, chemo- and radioresistance, in combination with a diffuse, invasive phenotype, are associated with a transcriptional program termed the epithelial-to-mesenchymal transition (EMT). Activation of this program allows cancer cells to survive individually, invade surrounding tissues and metastasize. It also enables them to survive exposure to cytotoxic therapy, including chemotherapeutic drugs and radiation. We here suggest that EMT plays an important, yet poorly understood role in the biology and therapy resistance of pHGG and DIPG. This review summarizes the current knowledge on the major signal transduction pathways and transcription factors involved in the epithelial-to-mesenchymal transition in cancer in general and in pediatric HGG and DIPG in particular. Despite the fact that the mesenchymal transition has not yet been specifically studied in pHGG and DIPG, activation of pathways and high levels of transcription factors involved in EMT have been described. We conclude that the mesenchymal transition is likely to be an important element of the biology of pHGG and DIPG and warrants further investigation for the development of novel therapeutics

An efficient method for the transduction of primary pediatric glioma neurospheres

Pediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are rare, but rapidly fatal malignancies of the central nervous system (CNS), and the leading cause of cancer-related death in children. Besides the scarcity of available biological material for research, the study of these diseases has been hampered by methodological problems. One of the major obstacles is the difficulty with which these cells can be genetically modified by conventional laboratory methods, such as lentiviral transduction. As a result, only very few successful stable modifications have been reported. As pHGG and DIPG cells are most often cultured as neurospheres, and therefore retain stem cell-like characteristics, we hypothesized that this culture method is also responsible for their resistance to transduction. We therefore developed a protocol in which pHGG and DIPG cells are temporarily forced to form an adherent monolayer by exposure to serum, to create a window-of-opportunity for lentiviral transduction. We here demonstrate that this protocol reliably and reproducibly introduces stable genetic modifications in primary DIPG and pHGG cells. • Short-term serum exposure enables lentiviral transduction of primary pediatric glioma neurospheres

Defining tumor-associated vascular heterogeneity in pediatric high-grade and diffuse midline gliomas

The blood–brain barrier (BBB) plays important roles in brain tumor pathogenesis and treatment response, yet our understanding of its function and heterogeneity within or across brain tumor types remains poorly characterized. Here we analyze the neurovascular unit (NVU) of pediatric high-grade glioma (pHGG) and diffuse midline glioma (DMG) using patient derived xenografts and natively forming glioma mouse models. We show tumor-associated vascular differences between these glioma subtypes, and parallels between PDX and mouse model systems, with DMG models maintaining a more normal vascular architecture, BBB function and endothelial transcriptional program relative to pHGG models. Unlike prior work in angiogenic brain tumors, we find that expression of secreted Wnt antagonists do not alter the tumor-associated vascular phenotype in DMG tumor models. Together, these findings highlight vascular heterogeneity between pHGG and DMG and differences in their response to alterations in developmental BBB signals that may participate in driving these pathological differences

Celastrol-induced degradation of FANCD2 sensitizes pediatric high-grade gliomas to the DNA-crosslinking agent carboplatin

Background: Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death during childhood. Due to their diffuse growth characteristics, chemoresistance and location behind the blood-brain barrier (BBB), the prognosis of pHGG has barely improved in the past decades. As such, there is a dire need for new therapies that circumvent those difficulties. Since aberrant expression of DNA damage-response associated Fanconi anemia proteins play a central role in the onset and therapy resistance of many cancers, we here investigated if FANCD2 depletion could sensitize pHGG to additional DNA damage. Methods: We determined the capacity of celastrol, a BBB-penetrable compound that degrades FANCD2, to sensitize glioma cells to the archetypical DNA-crosslinking agent carboplatin in vitro in seven patient-derived pHGG models. In addition, we tested this drug combination in vivo in a patient-derived orthotopic pHGG xenograft model. Underlying mechanisms to drug response were investigated using mRNA expression profiling, western blotting, immunofluorescence, FANCD2 knockdown and DNA fiber assays. Findings: FANCD2 is overexpressed in HGGs and depletion of FANCD2 by celastrol synergises with carboplatin to induce cytotoxicity. Combination therapy prolongs survival of pHGG-bearing mice over monotherapy and control groups in vivo (P<0.05). In addition, our results suggest that celastrol treatment stalls ongoing replication forks, causing sensitivity to DNA-crosslinking in FANCD2-dependent glioma cells. Interpretation: Our results show that depletion of FANCD2 acts as a chemo-sensitizing strategy in pHGG. Combination therapy using celastrol and carboplatin might serve as a clinically relevant strategy for the treatment of pHGG. Funding: This study was funded by a grant from the Children Cancer-Free Foundation (KIKA, project 210). The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Development of transient radioresistance during fractionated irradiation in vitro

Background and purpose: Effective combination treatments with fractionated radiotherapy rely on a proper understanding of the dynamic responses that occur during treatment. We explored the effect of clinical fractionated radiotherapy on the development and timing of radioresistance in tumor cells. Methods and materials: Different colon (HT29/HCT116/COLO320/SW480/RKO) and high-grade astrocytoma (D384/U-251MG) cancer cell lines were treated for 6 weeks with daily fractions of 2 Gy, 5 days per week. Clonogenic survival was determined throughout the treatment period. In addition, the radiosensitivity of irradiated and non-irradiated was compared. Finally, the effect of different dose fractions on the development of radioresistance was determined. Results: All cell lines developed radioresistance within 2–3 weeks during fractionated radiotherapy. This was characterized by the occurrence of a steady state phase of clonogenic survival. In U-251MG cells this was accompanied by increased cell senescence and stemness. After recovering from six weeks of treatment, the radiosensitivity of fractionally irradiated and non-irradiated cells was similar. Including transient radioresistance, described as (α/β)−( d +1), as a factor in the classic LQ model resulted in a perfect fit with the experimental data observed during fractionated radiotherapy. This was confirmed when different dose fractions were applied. Conclusions: Fractionated irradiation of cancer cells in vitro following clinical radiation schedules induces a reversible radioresistance response. This adaptive response can be included in the LQ model as a function of the dose fraction and the alpha/beta-ratio of a given cell line. These findings warrant further investigation of the mechanisms and clinical relevance of adaptive radioresistance

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen metho

AURKA and PLK1 inhibition selectively and synergistically block cell cycle progression in diffuse midline glioma

Diffuse midline gliomas (DMG) are highly malignant incurable pediatric brain tumors. In this study, we show that Aurora kinase A (AURKA) is overexpressed in DMG and can be used as a therapeutic target. Additionally, AURKA inhibition combined with CRISPR/Cas9 screening in DMG cells, revealed polo-like kinase 1 (PLK1) as a synergistic target with AURKA. Using a panel of patient-derived DMG culture models, we demonstrate that treatment with volasertib, a clinically relevant and selective PLK1 inhibitor, synergizes with different AURKA inhibitors, supporting the CRISPR screen results. Mechanistically, our results show that combined loss of PLK1 and AURKA causes a G2/M cell cycle arrest which blocks vital parts of DNA-damage repair and induces apoptosis, solely in DMG cells. Altogether, our findings highlight the importance of AURKA and PLK1 for DMG propagation and demonstrate the potential of concurrently targeting these proteins as a therapeutic strategy for these devastating pediatric brain tumors

Gemcitabine therapeutically disrupts essential SIRT1-mediated p53 repression in Atypical Teratoid/Rhabdoid Tumors

Background:Atypical Teratoid/Rhabdoid Tumors (ATRT) are highly malignant embryonal tumors of the central nervous system with a dismal prognosis. Despite recent advances in understanding the molecular characteristics and subclasses of these tumors, effective therapeutic options remain scarce.Methods:In this study, we developed and validated a novel patient-derived ATRT culture and xenograft model, which we used alongside a panel of other primary ATRT models for large-scale drug discovery assays. The identified hits were mechanistically and therapeutically investigated using an array of molecular assays and two orthotopic xenograft murine models.Results: We found that ATRT are selectively sensitive to the nucleoside analogue gemcitabine, with additional efficacy in Sonic Hedgehog (SHH)-subtype ATRT. Gene expression profiles and protein analyses indicated that gemcitabine treatment causes degradation of Sirtuin 1 (SIRT1), resulting in cell death through activation of NF-kB and p53. Furthermore, we discovered that gemcitabine-induced loss of SIRT1 results in a nucleus-to-cytoplasm translocation of the SHH signaling activator GLI2, explaining the additional gemcitabine sensitivity in SHH-subtype ATRT. Treatment of SHH-subgroup ATRT xenograft-bearing mice with gemcitabine resulted in a &gt;30% increase in median survival (p&lt;0.005, log-rank test) and yielded long-term survivors in two independent patient-derived xenograft models.Conclusions:These findings demonstrate that ATRT are highly sensitive to gemcitabine treatment, and we propose that gemcitabine may form part of a future multimodal treatment strategy for ATRT