Genome-wide pooled CRISPR screening in neurospheres.

Spheroid culture systems have allowed in vitro propagation of cells unable to grow in canonical cell culturing conditions, and may capture cellular contexts that model tumor growth better than current model systems. The insights gleaned from genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening of thousands of cancer cell lines grown in conventional culture conditions illustrate the value of such CRISPR pooled screens. It is clear that similar genome-wide CRISPR screens of three-dimensional spheroid cultures will be important for future biological discovery. Here, we present a protocol for genome-wide CRISPR screening of three-dimensional neurospheres. While many in-depth protocols and discussions have been published for more typical cell lines, few detailed protocols are currently available in the literature for genome-wide screening in spheroidal cell lines. For those who want to screen such cell lines, and particularly neurospheres, we provide a step-by-step description of assay development tests to be performed before screening, as well as for the screen itself. We highlight considerations of variables that make these screens distinct from, or similar to, typical nonspheroid cell lines throughout. Finally, we illustrate typical outcomes of neurosphere genome-wide screens, and how neurosphere screens typically produce slightly more heterogeneous signal distributions than more canonical cancer cell lines. Completion of this entire protocol will take 8-12 weeks from the initial assay development tests to deconvolution of the sequencing data.

BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell-like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. SIGNIFICANCE: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1-BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma. See related commentary by Beytagh and Weiss, p. 2730. See related article by Mo et al., p. 2906.

A phenotypic screen for compounds that reverse cAMP-mediated suppression of T cell functions.

The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z' was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls.

Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells.

The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions. We developed CERES, a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy number-specific effect. In our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this data set. We found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sgRNA libraries. We further demonstrate the utility of this collection of screens, after CERES correction, for identifying cancer-type-specific vulnerabilities.