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Pooled genetic screens to identify vulnerabilities in TERT-promoter-mutant glioblastoma

Oncogene volume 42, pages 3274–3286 (2023)Cite this article

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Abstract

Pooled genetic screens represent a powerful approach to identify vulnerabilities in cancer. Here we used pooled CRISPR/Cas9-based approaches to identify vulnerabilities associated with telomerase reverse transcriptase (TERT) promoter mutations (TPMs) found in >80% of glioblastomas. We first developed a platform to detect perturbations that cause long-term growth defects in a TPM-mutated glioblastoma cell line. However, we could not detect dependencies on either TERT itself or on an E-twenty six transcription (ETS) factor known to activate TPMs. To explore this finding, we cataloged TPM status for 441 cell lines and correlated this with genome-wide screening data. We found that TPM status was not associated with differential dependency on TERT, but that E-twenty six (ETS) transcription factors represent key dependencies in both TPM+ and TPM- lines. Further, we found that TPMs are associated with expression of gene programs regulated by a wide array of ETS-factors in both cell lines and primary glioblastoma tissues. This work contributes a unique TPM cell line reagent, establishes TPM status for many deeply-profiled cell lines, and catalogs TPM-associated vulnerabilities. The results highlight challenges in executing genetic screens to detect TPM-specific vulnerabilities, and suggest redundancy in the genetic network that regulates TPM function with therapeutic implications.

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Fig. 1: Generation of TPM + GBM cell line with inducible, exogenous TERT expression.
Fig. 2: Mini pooled screen to identify genetic dependencies that can be rescued by exogenous TERT expression.
Fig. 3: TERT dependency scores in DepMap lines.
Fig. 4: GABP subunit dependency scores in DepMap cell lines.
Fig. 5: Expression patterns associated with TPM status in GBM cell lines.
Fig. 6: Expression patterns associated with TPM status in 500 primary GBM tumors.

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Data availability

The genetic screen data datasets generated during and/or analyzed during the current study are available in the supplemental materials and DepMap database (https://depmap.org/portal/). The datasets generated during and/or analyzed during the current study are also available from the corresponding author on reasonable request. The primary glioblastoma data that support the findings of this study are available from TEMPUS and ASCO but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of TEMPUS and ASCO.

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Acknowledgements

We thank the Tempus/ASCO collaborative for access to their primary tumor databanks and their kind partnership. We thank David G. Kirsch for helpful critical feedback on the project. We thank sources of funding including NCI K08256045 Mentored Clinician Scientist Development Award, U19CA264385 Glioblastoma Therapeutics Network and developmental funds from the Cancer Center Support Grant P30CA014236 to ZJR, and Fund to Retain Clinician Scientists at Duke from the Doris Duke Foundation to ZJR, and support for ZJR career development including the ChadTough Defeat DIPG Foundation, the SoSo Strong Foundation, the Pediatric Brain Tumor Foundation, the St. Baldrick’s Foundation, NCI P50CA190991 Duke SPORE in Brain Cancer developmental funds to ZJR. KJT was supported by a scholarship from the Amgen Foundation, Banneker/Key Scholarship, and Churchill Scholarship.

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Authors and Affiliations

  1. Department of Radiation Oncology, Duke University School of Medicine, Durham, NC, 27710, USA

    Kevin J. Tu, Connor E. Stewart, Peter G. Hendrickson, Joshua A. Regal & Zachary J. Reitman

  2. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 21044, USA

    Kevin J. Tu

  3. Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Kevin J. Tu

  4. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, 27710, USA

    So Young Kim

  5. Duke Cancer Institute, Duke University School of Medicine, Durham, NC, 27710, USA

    So Young Kim, David M. Ashley, Matthew S. Waitkus & Zachary J. Reitman

  6. Department of Neurosurgery, Duke University School of Medicine, Durham, NC, 27710, USA

    David M. Ashley, Matthew S. Waitkus & Zachary J. Reitman

  7. The Preston Robert Tisch Brain Tumor Center at Duke, Durham, NC, 27710, USA

    David M. Ashley, Matthew S. Waitkus & Zachary J. Reitman

  8. Department of Pathology, Duke University School of Medicine, Durham, NC, 27710, USA

    Zachary J. Reitman

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Contributions

ZJR, CES, and KJT conceived the study. CES and ZJR carried out tissue culture experiments. KJT, ZJR, CES, and PGH performed bioinformatic analyses. SYK designed CRISPR/Cas9 plasmids and assisted in the design of CRISPR/Cas9 experiments and data analysis. JAR, MSW, and DMA contributed reagents and assisted in experimental design and analysis. ZJR and KJT drafted the manuscript, with all other authors also participating.

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Correspondence to Zachary J. Reitman.

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Competing interests

ZJR is listed as an inventor for intellectual property related to genetic testing for TERT and other alterations in brain tumors that is managed by Duke Office of Licensing and Ventures and has been licensed to Genetron Health. The other authors have no conflicts of interest to report.

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Tu, K.J., Stewart, C.E., Hendrickson, P.G. et al. Pooled genetic screens to identify vulnerabilities in TERT-promoter-mutant glioblastoma. Oncogene 42, 3274–3286 (2023). https://doi.org/10.1038/s41388-023-02845-w

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