Neural G0: a quiescent-like state found in neuroepithelial-derived cells and glioma.

Single-cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA-seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent-like state in neuroepithelial-derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non-dividing neural progenitors. Putative glioblastoma stem-like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down-regulation of quiescence-associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent-like state found in neuroepithelial-derived cells and gliomas.

FBXO42 activity is required to prevent mitotic arrest, spindle assembly checkpoint activation and lethality in glioblastoma and other cancers.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. To identify genes differentially required for the viability of GBM stem-like cells (GSCs), we performed functional genomic lethality screens comparing GSCs and control human neural stem cells. Among top-scoring hits in a subset of GBM cells was the F-box-containing gene FBXO42, which was also predicted to be essential in ∼15% of cell lines derived from a broad range of cancers. Mechanistic studies revealed that, in sensitive cells, FBXO42 activity prevents chromosome alignment defects, mitotic cell cycle arrest and cell death. The cell cycle arrest, but not the cell death, triggered by FBXO42 inactivation could be suppressed by brief exposure to a chemical inhibitor of Mps1, a key spindle assembly checkpoint (SAC) kinase. FBXO42's cancer-essential function requires its F-box and Kelch domains, which are necessary for FBXO42's substrate recognition and targeting by SCF (SKP1-CUL1-F-box protein) ubiquitin ligase complex. However, none of FBXO42's previously proposed targets, including ING4, p53 and RBPJ, were responsible for the observed phenotypes. Instead, our results suggest that FBOX42 alters the activity of one or more proteins that perturb chromosome-microtubule dynamics in cancer cells, which in turn leads to induction of the SAC and cell death.

Functional genomic analysis of adult and pediatric brain tumor isolates.

Background: Adult and pediatric tumors display stark differences in their mutation spectra and chromosome alterations. Here, we attempted to identify common and unique gene dependencies and their associated biomarkers among adult and pediatric tumor isolates using functional genetic lethal screens and computational modeling. Methods: We performed CRISRP-Cas9 lethality screens in two adult glioblastoma (GBM) tumor isolates and five pediatric brain tumor isolates representing atypical teratoid rhabdoid tumors (ATRT), diffuse intrinsic pontine glioma, GBM, and medulloblastoma. We then integrated the screen results with machine learning-based gene-dependency models generated from data from >900 cancer cell lines. Results: We found that >50% of candidate dependencies of 280 identified were shared between adult GBM tumors and individual pediatric tumor isolates. 68% of screen hits were found as nodes in our network models, along with shared and tumor-specific predictors of gene dependencies. We investigated network predictors associated with ADAR, EFR3A, FGFR1 (pediatric-specific), and SMARCC2 (ATRT-specific) gene dependency among our tumor isolates. Conclusions: The results suggest that, despite harboring disparate genomic signatures, adult and pediatric tumor isolates share a preponderance of genetic dependences. Further, combining data from primary brain tumor lethality screens with large cancer cell line datasets produced valuable insights into biomarkers of gene dependency, even for rare cancers. Importance of the Study: Our results demonstrate that large cancer cell lines data sets can be computationally mined to identify known and novel gene dependency relationships in adult and pediatric human brain tumor isolates. Gene dependency networks and lethality screen results represent a key resource for neuro-oncology and cancer research communities. We also highlight some of the challenges and limitations of this approach.

Efficient Multi-Allelic Genome Editing of Primary Cell Cultures via CRISPR-Cas9 Ribonucleoprotein Nucleofection.

CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR-Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl-3'phosphorothioate-modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion-deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem-like cells. © 2020 Wiley Periodicals LLC Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion-deletion (indel) mutation and genomic deletion generation in primary cell cultures.

A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures.

BACKGROUND: CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells. AIMS: To create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient-derived glioblastoma stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments. METHODS AND RESULTS: We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl 3'phosphorothioate-modified sgRNAs and purified Cas9 protein. Insertion-deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss-of-function experiments. Using Western blotting, we observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. Furthermore, by using RNA-seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in glioblastoma, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities. CONCLUSION: Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.

Selective Translation of Cell Fate Regulators Mediates Tolerance to Broad Oncogenic Stress.

Human skin tolerates a surprisingly high burden of oncogenic lesions. Although adult epidermis can suppress the expansion of individual mutant clones, the mechanisms behind tolerance to oncogene activation across broader regions of tissue are unclear. Here, we uncover a dynamic translational mechanism that coordinates oncogenic HRAS-induced hyperproliferation with loss of progenitor self-renewal to restrain aberrant growth and tumorigenesis. We identify translation initiator eIF2B5 as a central co-regulator of HRAS proliferation and cell fate choice. By coupling in vivo ribosome profiling with genetic screening, we provide direct evidence that oncogene-induced loss of progenitor self-renewal is driven by eIF2B5-mediated translation of ubiquitination genes. Ubiquitin ligase FBXO32 specifically inhibits epidermal renewal without affecting overall proliferation, thus restraining HRAS-driven tumorigenesis while maintaining normal tissue growth. Thus, oncogene-driven translation is not necessarily inherently tumor promoting but instead can manage widespread oncogenic stress by steering progenitor fate to prolong normal tissue growth.

Professional Bereavement Photography in the Setting of Perinatal Loss: A Qualitative Analysis.

Perinatal loss, including fetal and infant death, is a devastating experience for parents, resulting in long-term adverse physical and psychosocial outcomes. However, little is known about what services might best support grieving parents. We aimed to understand the role of professional bereavement photography in assisting the grieving process of parents who have lost a fetus or infant, by examining the perspectives of bereaved parents, professional photographers, and health care professionals. Twenty semistructured interviews were conducted, and interview transcripts were analyzed using modified grounded theory. Twenty-three individuals participated, including 6 bereaved parents, 8 photographers, and 9 health care professionals. Analyses generated 5 major themes describing ways in which the photographs were valuable to parents: validation of the experience, permission to share, creation of a permanent and tangible legacy, creation of positive memories, and moving forward after the loss. Hospitals should consider incorporation of professional bereavement photography services into palliative care and bereavement programs.