The functional genomic circuitry of human glioblastoma stem cells

Abstract

SummarySuccessful glioblastoma (GBM) therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells to interrogate function of the coding genome, we identify diverse actionable pathways responsible for growth that reveal the gene-essential circuitry of GBM stemness. In particular, we describe the Sox developmental transcription factor family; H3K79 methylation by DOT1L; and ufmylation stress responsiveness programs as essential for GBM stemness. Additionally, we find mechanisms of temozolomide resistance and sensitivity that could lead to combination strategies with this standard of care treatment. By reaching beyond static genome analysis of bulk tumors, with a genome wide functional approach, we dive deep into a broad range of biological processes to provide new understanding of GBM growth and treatment resistance.SignificanceGlioblastoma (GBM) remains an incurable disease despite an increasingly thorough depth of knowledge of the genomic and epigenomic alterations of bulk tumors. Evidence from multiple approaches support that GBM reflects an aberrant developmental hierarchy, with GBM stem cells (GSCs), fueling tumor growth and invasion. The properties of this tumor subpopulation may also in part explain treatment resistance and disease recurrence. Unfortunately, we still have a limited knowledge of the molecular circuitry of these cells and progress has been slow as we have not been able, until recently, to interrogate function at the genome-wide scale. Here, using parallel genome-wide CRISPR-Cas9 screens, we identify the essential genes for GSC growth. Further, by screening in the presence of low and high dose temozolomide, we identify mechanisms of drug resistance and sensitivity. These functional screens in patient derived cells reveal new aspects of GBM biology and identify a diversity of actionable targets such as genes governing stem cell traits, epigenome regulation and the response to stress stimuli.

The DEAD-box helicase DDX56 is a conserved stemness regulator in normal and cancer stem cells

“…The addition of DOX reduced sphere-forming frequency from 20.4% to 5.3% in GSC523_DDX56 after 2 weeks (Figure 3C). The same experiment in GSC564_DDX56 cells did not alter sphere forming, as expected from the screen data in which DDX56 did not score as an essential gene (Figure 3D) (MacLeod et al, 2019). In total, we confirmed that DDX56 was required for population growth and self-renewal in GSC523 but not GSC564 cells.…”

“…In total, 82.5% of all DDX56 + cells co-expressed the NSC marker SOX2, while 43.8% of all SOX2 + cells co-expressed DDX56; 9,564 cells (35.8%) were SOX2 + /DDX56 + , thus supporting frequent DDX56 expression in these stem-like cell populations (Figures S3A-S3C). Several of those GSC lines have been subjected to genome-wide CRISPR screens for gene essentiality (MacLeod et al, 2019). From the screen data, we chose to investigate two GSC lines that demonstrated different essentiality with respect to DDX56: GSC523 (essential) and GSC564 (non-essential).…”

“…GSC523 or GSC564 expressing a constitutively active human codon optimized Cas9 was obtained from others (MacLeod et al, 2019) and its growth was supplemented with 3 mg/mL blasticiden.…”

“…Lentivirus was concentrated with Lenti-X Concentrator and resuspended in DMEM/F12. Lentiviral transduction was performed in a similar manner to (MacLeod et al, 2019). Briefly, 1.5 million GSC523 or GSC564 cells stably expressing Cas9 were transduced with 40 ml of virus in the presence of 0.8 mg/ml polybrene.…”

Halocins, Bacteriocin-Like Antimicrobials Produced by the Archaeal Domain: Occurrence and Phylogenetic Diversity in Halobacteriales

“…This superphylum has been joined by recently proposed phyla: Bathyarchaeota, Geoarchaeota and Lokiarchaeota [18]. Another superphylum, DPANN, was proposed in 2013 and includes Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota phyla (Figure 1) [19]. Finally, the phyla Woesearchaeota and Pacearchaeota, described in 2016, were grouped in the DPANN superphylum (Figure 1).…”