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Abstract 3312: A CRISPR-Cas9 screen in meningioma reveals GCN2/ATF4 is a novel target that enhances the response to radiation


 Purpose: Meningioma is a common primary brain tumor in adults and accounts for ~ 36% of all adult brain tumors. Therapy for patients with meningioma is individualized and comprised of a combination of surgery and/or radiotherapy. Chemotherapy is not effective and there is no approved targeted treatment. Therefore, novel therapeutic approaches are warranted. We used CRISPR screening-based technology to uncover novel mechanisms of radiation resistance that may be therapeutically relevant in meningioma.
 Methods: A patient-derived meningioma cell line with TRAF7/KLF4 mutations (Tm77) was transduced with a Human Kinome CRISPR KnockOut (KO) library containing 3,052 RNA guides (sgRNA) targeting 763 kinase genes involved in cancer therapy. Following radiation, cells were collected (400x coverage) and analyzed using next-generation sequencing (NGS). MAGeCK analysis was used to evaluate sgRNA enrichment and depletion. Radiation survival was determined by clonogenic analysis. Kinase activity and downstream signaling were assessed by phospho-blot analysis. Cell cycle analysis was performed by flow cytometry and DNA damage was assessed by gH2AX immunofluorescence.
 Results: Analysis of NGS results shows that after radiation ATM and DNA-PK sgRNAs were depleted (p<0.001; FDR<0.001), suggesting that the inhibition of either induces radiosensitivity. These genes, together with ATR, are involved in the DNA damage response induced by external stress such as radiation. ATM (AZD1930) and ATR (AZD6738) inhibitors were used to test the screening results. While AZD1390 reduces ATM signaling, gH2AX foci, and radiation survival with a dose enhancement ratio (DER) at 2Gy of 2; AZD6738 did not induce any changes in signaling, gH2AX foci, or radiosensitivity. These results indicate that inhibition of ATM can regulate radiation response in meningioma and thus validated the screening results. The CRISPR-Cas9 KO screen also identified a potential novel druggable target, GCN2. In the NGS analysis, GCN2 sgRNAs were enriched (p<0.001; FDR<0.005), and in agreement with this finding GCN2-KO enhanced radiation survival with a DER at 2Gy of 1.4. In contrast, halofuginone, a natural product that activates GCN2 reduced radiation survival in Tm77 with a DER at 2Gy of 1.25 through activation of ATF4 and cell cycle arrest. These effects were not observed in GCN2-KO cell lines. In addition, the proteasome inhibitor, bortezomib, also activates ATF4 signaling, causes cell cycle arrest, and radiosensitization with a DER at 2Gy of 1.3.
 Conclusion: CRISPR-Cas9 KO screening provides a novel and unbiased approach to identify cellular targets that modify the response to radiation. These results suggests that the activation of ATF4 radiosensitizes meningioma. The evidence provided in this study supports further basic and preclinical research on approaches for activating GCN2 in combination with radiation therapy in meningioma.
 Citation Format: Marta Baro, Murat Gunel, Jennifer Moliterno, Joseph N. Contessa. A CRISPR-Cas9 screen in meningioma reveals GCN2/ATF4 is a novel target that enhances the response to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3312.