Abstract P10: B-cell lymphoma CRISPR screens: NSD2 loss confers EZH2 inhibition resistance, while AEBP2 is a specific genetic dependency
Authors: Daniel I Angelov, James Nolan, Grainne Holland, David Reck, Darragh Nimmo, Craig Monger, Elisabeth Vandenberghe, Adrian P Bracken
Journal: Blood Cancer Discovery (2024)

Abstract Introduction The characterization of the mutational landscape of human cancers over the past 30 years has resulted in the rise of targeted cancer therapies. This era of precision oncology aims to enable cancer patients to receive therapies that are specific, efficacious, and result in fewer toxicities. A major challenge however is the emergence of drug resistance. Predicting resistance mechanisms using in vitro models can help to plan rational clinical trials using combination approaches and avoiding treating specific subtypes of cancers that are unlikely to respond. Here, we apply CRISPR screens in non-Hodgkin lymphoma (NHL) to predict resistance mechanisms and genetic synergies to EZH2 inhibitor (EZH2i) treatment. Materials and Methods Genome-wide CRISPR-Cas9 screens were performed in EZH2 ‘change-of-function’ mutant and EZH2 wild-type patient-derived NHL cell lines, treated with either EZH2i or vehicle control. Validatory cell competition assays were then performed using individual CRISPR sgRNAs targeting specific genes of interest, including AEBP2 and NSD2. These sgRNAs were tagged with a green fluorescence protein enabling the identification of the knock-out population using flow cytometry. An additional CRISPR screen using a PRC2 ‘tiled’ approach was also performed in the NHL cell lines to elucidate the essential domains within PRC2 proteins. Clonal knock out cells were generated using CRISPR and CUT&amp;RUN experiments were performed for histone post-translational modifications in the knock-out cells to understand the mechanisms of resistance. Lastly, Quant-seq experiments were undertaken in the clonal knock out cells to characterise the transcriptional changes underlying the mechanism of resistance and sensitization observed with specific genetic knock outs. Results and Discussion We identified the NSD2 gene as a ‘resistor’ gene to EZH2i treatment, as sgRNAs targeting NSD2 were more abundant in the EZH2i treated cells compared to control cells. Conversely, the PRC2 accessory component AEBP2 was identified as a ‘sensitizer’ gene in this context. Cell competition assays using several independent sgRNAs to target the NSD2 and AEBP2 genes validated these findings. The EZH2i resistance phenotype was replicated using an orthogonal approach of pharmacological NSD2 degradation. These results point to the possibility of acquired loss of function mutations in NSD2 resulting in resistance to EZH2i in vivo. Finally, we explored the changes in transcriptional programs and H3K36 and H3K27 methylations across the genome. We propose a model for how NSD2 and AEBP2 loss confer resistance and sensitivity, respectively, to EZH2i in NHL cells. Conclusion We identified NSD2 loss as a potential mechanism by which NHL cells can acquire resistance to therapeutic EZH2i. We propose a potential solution of combining EZH2i treatment with targeted inhibition of AEBP2, thus preventing the occurrence of EZH2i resistance. Our work suggests that pharmacological inhibitors or degraders of AEBP2 should be prioritised for development. Citation Format: Daniel I Angelov, James Nolan, Grainne Holland, David Reck, Darragh Nimmo, Craig Monger, Elisabeth Vandenberghe, Adrian P Bracken. B-cell lymphoma CRISPR screens: NSD2 loss confers EZH2 inhibition resistance, while AEBP2 is a specific genetic dependency [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P10.