Abstract C159: Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme
Authors: Toledo Chad M. , Ding Yu , Hoellerbauer Pia , Davis Ryan J. , Basom Ryan , Girard Emily J. , Lee Eunjee , Corrin Philip , Bolouri Hamid , Davison Jerry , Zhang Qing , Nam Do-Hyun , Lee Jeongwu , Pollard Steven M. , Zhu Jun , Delrow Jeffery J. , Clurman Bruce E. , Olson James M. , Paddison Patrick J. 
Published: May 2016

Precision oncology is currently based on the notion that genomic analysis of descriptive molecular signatures from patient tumors will lead to actionable therapeutic targets following the patient's arrival into the clinic. However, this approach has failed to deliver new effective treatments for glioblastoma multiforme (GBM), which is the most common and aggressive form of brain cancer; and thus, ∼90% of adult GBM patients receiving standard of care therapies continue to die within 2 years of diagnosis. In addition, this approach has neglected the importance for using functional genetics in precision oncology. To identify new therapeutic targets for GBM, we applied functional genetics to perform lethal genome-wide CRISPR-Cas9 knockout screens in patient-derived GBM stem-like cells (GSCs) and also non-transformed human neural stem cells (NSCs), non-neoplastic tissue of origin controls.

Here, we present our latest findings from these screens, which include multiple novel GBM-specific lethal genes that were validated by both in vitro and in vivo preclinical studies. Knockout of GBM-specific lethal genes, including the WEE1-like kinase, PKMYT1/Myt1, lead to lethality in GSCs, but not NSCs. Focused mechanistic studies revealed that PKMYT1 acts redundantly with WEE1 to phosphorylate CDK1-Y15 and to promote timely completion of mitosis in NSCs, but that this redundancy is lost in most GBM isolates and in NSCs harboring activated alleles of EGFR and AKT1, which are commonly altered signaling pathways in GBM. Moreover, PKMYT1 depletion in GSCs and genetically altered NSCs requiring PKMYT1 lead to cytokinesis failure and cell death during mitosis.

In addition to lethal genes, genes promoting in vitro expansion of NSCs upon knockout were examined. For this category, we validated multiple genes that are candidate tumor suppressors and involved in: the negative regulation of Hippo signaling, TP53 signaling, epigenetic regulation, promoting neural development, and other cellular functions. Knockout of these genes caused shortened cell cycle transit times and drastic growth advantages in NSCs, and in the case of CREBBP knockout, caused precious entry into S-phase and deregulation of cell cycle gene expression. The identification of these potential tumor suppressors here reveal new genetic drivers in glioma/GBM, which contributes to a growing body of work that will help redefine GBM gene signatures.

Furthermore, we found that the molecular signatures of pathways and genes commonly altered in GBM are not ideal GBM therapeutic targets since most of these targets failed to score as GBM-specific lethal hits in our knockout screens and likely are non-essential or essential in both the GSCs and NSCs. Nonetheless, these common GBM alterations give rise to cancer-specific vulnerabilities, which then lead to genes, such as PKMYT1, that are required to overcome functional impairments in cancer cells. Taken together, we demonstrated here that genomics and functional genetics are equally important for precision oncology, as the combination of both tools can identify genetically altered genes, cancer-specific therapeutic targets, and tumor suppressors. These results are part of a rapidly growing body of work by the science community that will one day allow oncologists to tailor therapies for each patient based upon his or her tumor genetic profile and characteristics.

Citation Format: Chad M. Toledo, Yu Ding, Pia Hoellerbauer, Ryan J. Davis, Ryan Basom, Emily J. Girard, Eunjee Lee, Philip Corrin, Hamid Bolouri, Jerry Davison, Qing Zhang, Do-Hyun Nam, Jeongwu Lee, Steven M. Pollard, Jun Zhu, Jeffery J. Delrow, Bruce E. Clurman, James M. Olson, Patrick J. Paddison. Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C159.