Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells

Chad M Toledo; Yu Ding; Pia Hoellerbauer; Ryan J Davis; Ryan Basom; Emily J Girard; Eunjee Lee; Philip Corrin; Traver Hart; Hamid Bolouri; Jerry Davison; Qing Zhang; Justin Hardcastle; Bruce J Aronow; Christopher L Plaisier; Nitin S Baliga; Jason Moffat; Qi Lin; Xiao-Nan Li; Do-Hyun Nam; Jeongwu Lee; Steven M Pollard; Jun Zhu; Jeffery J Delrow; Bruce E Clurman; James M Olson; Patrick J Paddison

doi:10.1016/j.celrep.2015.11.021

Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells

Cell Rep. 2015 Dec 22;13(11):2425-2439. doi: 10.1016/j.celrep.2015.11.021. Epub 2015 Dec 7.

Authors

Chad M Toledo¹, Yu Ding², Pia Hoellerbauer¹, Ryan J Davis³, Ryan Basom⁴, Emily J Girard⁵, Eunjee Lee⁶, Philip Corrin², Traver Hart⁷, Hamid Bolouri², Jerry Davison⁴, Qing Zhang⁴, Justin Hardcastle², Bruce J Aronow⁸, Christopher L Plaisier⁹, Nitin S Baliga⁹, Jason Moffat⁷, Qi Lin¹⁰, Xiao-Nan Li¹⁰, Do-Hyun Nam¹¹, Jeongwu Lee¹², Steven M Pollard¹³, Jun Zhu⁶, Jeffery J Delrow⁴, Bruce E Clurman¹⁴, James M Olson¹⁵, Patrick J Paddison¹⁶

Affiliations

¹ Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA.
² Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
³ Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA; Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
⁴ Genomics and Bioinformatics Shared Resources, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
⁵ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
⁶ Department of Genetics and Genomic Sciences, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁷ Department of Molecular Genetics, University of Toronto and Donnelly Centre, Toronto, ON M5S3E1, Canada; Canadian Institute for Advanced Research, Toronto, ON M5G1Z8, Canada.
⁸ Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
⁹ Institute for Systems Biology, Seattle, WA 98109, USA.
¹⁰ Brain Tumor Program, Texas Children's Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.
¹¹ Institute for Refractory Cancer Research, Samsung Medical Center, Seoul 135-710, Korea.
¹² Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44192, USA.
¹³ Edinburgh CRUK Cancer Research Centre and MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh EH16 4UU, UK.
¹⁴ Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
¹⁵ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. Electronic address: [email protected].
¹⁶ Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA. Electronic address: [email protected].

Abstract

To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

Keywords: CRISPR-Cas9; Glioblastoma; Myt1; PKMYT1; WEE1; cancer therapeutics; functional genomics; gene editing.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CDC2 Protein Kinase / antagonists & inhibitors
CDC2 Protein Kinase / metabolism
CRISPR-Cas Systems / genetics*
Cell Cycle Proteins / antagonists & inhibitors
Cell Cycle Proteins / genetics*
Cell Cycle Proteins / metabolism
Cell Survival / drug effects
Cyclin B / metabolism
ErbB Receptors / metabolism
Gene Library
Genome, Human*
Glioblastoma / metabolism
Glioblastoma / pathology
Humans
Membrane Proteins / antagonists & inhibitors
Membrane Proteins / genetics*
Membrane Proteins / metabolism
Microscopy, Video
Mitosis
Neoplastic Stem Cells / cytology
Neoplastic Stem Cells / metabolism*
Nuclear Proteins / antagonists & inhibitors
Nuclear Proteins / genetics*
Nuclear Proteins / metabolism
Phosphorylation
Protein Serine-Threonine Kinases / antagonists & inhibitors
Protein Serine-Threonine Kinases / genetics*
Protein Serine-Threonine Kinases / metabolism
Protein-Tyrosine Kinases / antagonists & inhibitors
Protein-Tyrosine Kinases / genetics*
Protein-Tyrosine Kinases / metabolism
Proto-Oncogene Proteins c-akt / metabolism
Pyrazoles / pharmacology
Pyrimidines / pharmacology
Pyrimidinones
RNA Interference
Time-Lapse Imaging
Tumor Suppressor Protein p53 / genetics
Tumor Suppressor Protein p53 / metabolism

Substances

Cell Cycle Proteins
Cyclin B
Membrane Proteins
Nuclear Proteins
Pyrazoles
Pyrimidines
Pyrimidinones
Tumor Suppressor Protein p53
EGFR protein, human
ErbB Receptors
Protein-Tyrosine Kinases
WEE1 protein, human
PKMYT1 protein, human
Protein Serine-Threonine Kinases
Proto-Oncogene Proteins c-akt
CDC2 Protein Kinase
adavosertib

Abstract

Publication types

MeSH terms

Substances

Grants and funding