Lung adenocarcinoma (LUAD) is a complex cancer driven by diverse combinations of oncogenic and tumor suppressive events. The TBX2 subfamily genes, including TBX2, TBX3, TBX4, and TBX5, are known to play a crucial role in lung development as master transcriptional regulators. Our previous clinical and in vitro studies found that their expression is significantly suppressed in LUADs, suggesting a potential tumor suppressor role through modulating tumor methylation patterns in early tumorigenesis. However, the role of TBX2 subfamily genes in other cancer types is paradoxical, with both oncogenic and tumor suppressive effects being reported. Herein, we employed the Tumor Barcoding with Ultradeep Sequencing (Tuba-seq) and CRISPR/Cas9-mediated genome editing technologies to investigate the putative tumor suppressive role of TBX2 genes functionally and quantitatively and their downstream targets in genetically engineered mouse models of LUAD. Lung tumors were initiated by intratracheal intubation with pooled lentiviral-Cre vectors targeting each gene independently with two different sgRNAs per gene. To enable the unique identification of each tumor and its corresponding sgRNA, we utilized Lenti-sgRNA/Cre vectors harboring barcodes specific to each targeted gene. Initial assessment of tumor burden was carried out through fluorescence microscopy, lung weight measurements, and histological analysis. To simultaneously determine the number of neoplastic cells corresponding to each gene knockout, the barcoded region was amplified from genomic DNA extracted from bulk tumor-bearing lung samples and sequenced. Quantification of tumor sizes was carried at 6 and 20 weeks after tumor initiation. Hyperplasia, adenomas, and/or early adenocarcinomas within the lungs were detected among different genetic backgrounds and throughout the study time intervals. Specifically, the inactivation of Tbx2, Tbx3, Tbx4, Tbx5, and Cdh2, increased tumor initiation and growth in the oncogene-negative mouse model (n=8). However, strikingly, inactivation of Tbx3, Tbx4, and Cdh2 suppressed tumorigenesis in a KRAS-driven genetic background while Tbx2, Tbx5, and Tnfaip3, consistently exhibited tumor suppressive effects (n=9). Further investigation is needed to understand these newly discovered interactions between the TBX2 subfamily critical developmental pathway and Ras signaling in modulating lung cancer progression. These insights emphasize the importance of considering tumor suppressor phenotypic heterogeneity and their context-dependent roles when developing targeted therapeutic approaches for lung adenocarcinoma and other cancers.

Citation Format: Athar Khalil, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher Mcfarland. In vivo TuBa-seq growth profiling identifies a differential role of the Tbx2 subfamily in oncogene-negative versus Kras-driven lung cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A177.