In Vivo Multiplexed Modeling Reveals Diverse Roles of the TBX2 Subfamily and Egr1 in Ras -Driven Lung Adenocarcinoma

The TBX2 subfamily of T-box transcription factors (including Tbx2 , Tbx3 , Tbx4 , Tbx5 ) plays an essential role in lung development. Downregulation of these genes in human Lung adenocarcinoma (LUAD) suggests that these genes may be tumor suppressive, however because downregulation appears to occur primarily via epigenetic change, it remains unclear if these changes causally drive tumor progression or are merely the consequence of upstream events. Herein, we developed the first multiplexed mouse model to study the impact of TBX2 subfamily loss, alongside associated signaling genes Egr1 , Chd2 , Tnfaip3a , and Atf3 , in Ras -driven lung cancer. Using TuBa-seq, a high-throughput tumor-barcoding system, we quantified the growth effects of these knockouts during early and late tumorigenesis. Chd2 loss consistently suppressed tumor progression, while Tbx2 loss exhibited stage-dependent effects. Notably, Egr1 emerged as a potent tumor suppressor, with its knockout increasing tumor size (∼5x) at 20 weeks, surpassing Rb1 loss. Transcriptomic analyses of Egr1 -deficient tumors suggested immune dysregulation, including heightened inflammation and potential markers of T cell exhaustion in the tumor microenvironment. These findings indicate that Egr1 may play a role in suppressing tumor growth through modulating immune dynamics, offering new insights into the interplay between tumor progression and immune regulation in LUAD.