Abstract C023: Development of isogenic models of marginal zone lymphoma for therapeutic target discovery in TET2-deficient disease

Abstract Introduction. The family of ten-eleven translocation dioxygenases (TETs) regulates dioxygenase activity-dependent DNA demethylation and dioxygenase activity-independent histone modification, controlling the dynamic differentiation and lineage commitment of hematopoietic stem/progenitor cells, regulating the access of key transcription factors to the enhancers of target genes. TET2 somatic mutations, recurrent in marginal zone lymphoma (MZL) as well as in other hematological cancers, are associated with abnormal epigenetic landscape changes. To uncover the role of mutated (mut) TET2, we performed CRISPR editing, pharmacological screening, omics analyses, and in vivo experiments in MZL models. Methods. We used two bona fide MZL cell lines, VL51 and HC1. Genetic editing was done with CRISPR/Cas9. Monoclonal cell lines were obtained from isogenic populations by limiting dilution. In vitro and in vivo proliferation of edited cells was analyzed using Incucyte live cell imaging and xenograft models in NSG mice. Single-cell RNA sequencing (scRNA-Seq), bulk RNA-Seq, and DNA methylation profiling were also performed on edited cells. Additionally, the anti-proliferation effects of TETi76 (Guan et al., 2020) and a custom library of 474 anti-cancer compounds were assessed in edited cells. Results. Using CRISPR/Cas9, we reverted the HC1 cell line from mut-TET2 to wild-type (wt) and inserted the same mutation (P594X) in TET2 wt VL51. In isogenic models, mut-TET2 cells had increased proliferation in vitro and in vivo. In vivo, mice with mut-TET2 VL51 xenografts also showed a trend for poorer survival compared to those with wt xenografts. The TET inhibitor TETi76 was more active in mut-TET2 cells (HC1 and VL51 with CRISPR-generated TET2 mutation). Bulk RNA-Seq and scRNA-Seq showed differences at the transcriptome level between TET2 wt and TET2 edited cells, including immune signaling pathways, PI3K signaling, and MYC targets. Integration of RNA-Seq with DNA methylation data revealed that differential gene expression was associated with differential methylation of the corresponding specific gene promoter regions. Pharmacological screening in wt and mut-TET2 monoclonal cells showed that several drugs, such as pictilisib, rapamycin, romidepsin, tucidinostat, and umbralisib, were more active in mut-TET2 cells, and their targets largely agreed with what was observed at the transcriptome level. Conclusions. Mut-TET2 MZL cell lines generated by CRISPR gene editing exhibited different growth profiles and altered transcriptomes and DNA methylation patterns compared to their wt counterparts. Further, mut-TET2 conferred increased sensitivity to the TET inhibitor TETi76 and to multiple targeted agents, particularly those targeting PI3K, MTOR, and histone deacetylases. Our findings provide novel insights into the role of mut-TET2 in lymphomagenesis and reveal potential drug-targetable pathways for lymphomas with mut-TET2. Citation Format: Fangwen Zhang, Luciano Cascione, Sara Napoli, Chiara Tarantelli, Elisa Civanelli, Giulio Sartori, Francesca Guidetti, Alberto J. Arribas, Filippo Spriano, Anand Tiwari, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Babal K. Jha, Manel Esteller, Afua Adjeiwaa. Mensah, Francesco Bertoni. Development of isogenic models of marginal zone lymphoma for therapeutic target discovery in TET2-deficient disease [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C023.