Abstract 634: Tuba-seq and CRISPR/Cas9 analysis of TBX2 subfamily genes: Exploring contextual tumor suppression in lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAD) is a multifaceted and genetically diverse cancer, shaped by a myriad of oncogenic and tumor suppressive events. Through recent clinical and in vitro investigations, we have discovered that the TBX2 subfamily of genes, encompassing TBX2, TBX3, TBX4, and TBX5, are linked to LUAD progression. Specifically, we have observed marked downregulation of their expression in clinical LUAD specimens, and reduced growth in LUAD cell lines when the subfamily is re-activated- hinting at a potential tumor suppressive function. Paradoxically, the role of TBX2 subfamily genes in other cancer types remains enigmatic, with varying reports of both oncogenic and tumor suppressive effects. In this study, we harnessed the power of Tumor Barcoding with Ultradeep Barcode Sequencing technology (Tuba-seq) and CRISPR/Cas9-mediated genome editing to systematically investigate the effects of knocking-out Tbx genes and associated partners EGR1, CHD2, A20 and ATF3 in LUAD preclinical models. We investigated their effects in both normal lung epithelium and Ras-driven lung tumors via in situ gene editing and assessed tumor burden via histology and targeted DNA sequencing of tumor barcodes. TBX2 subfamily gene losses appear to have a profound effect on tumor initiation and early growth, which somewhat attenuates with progression. In Ras-driven tumors, we found that these genes are moderately tumor suppressive when measuring mean growth rate, although these effects (sans Egr1 loss) are more pronounced at 6 weeks of growth than at 20 weeks. Strikingly, however, we observed an unprecedented increase in total tumor burden when deleting these genes in genomically-normal (Kras w. t. ) cells, including 4-20+ fold increase in Tbx2, Tbx3, Tbx4, and Tnfaip3-deficient cell lineages 20-week following transduction. Increases in total lineage burden of this magnitude are typically only seen for frequently-deleted hallmark tumor suppressor genes such as Tp53, Stk11, or Pten. This increased burden, however, seems to primarily affect early-stage progression, as no appreciable increases in mean lineage size relative to w. t. lineages are observed after 40 weeks of growth. Our study reveals the context-specific functions of the TBX2 subfamily while introducing the first in vivo model for TBX2-driven LUADs. This model facilitates the development of targeted therapeutic strategies and encourages accurate tumor modeling across various progression stages and diverse genetic backgrounds. Citation Format: Athar Khalil, Jaff Maltas, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher McFarland. Tuba-seq and CRISPR/Cas9 analysis of TBX2 subfamily genes: Exploring contextual tumor suppression in lung adenocarcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 634.