Abstract 2020: Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer

Abstract

Abstract Lung cancer is the leading cause of cancer death in the United States and in the world. Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1, in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce either oncogene induced senescence or apoptosis. However, the key functions of TWIST1 that are required for its transcriptional activity in cancer are unknown. In the current study, we engineered domain-specific mutations in TWIST1 to determine the impact of altered DNA binding, dimerization and post-translational modifications on its transcriptional activity by luciferase reporter assays. Testing the promoter activity at three TWIST1-regulated loci involved in tumorigenesis, YBX1, SNAI2, and FLIP, we found that DNA binding and nuclear localization were uniformly required for TWIST1 transcriptional function. However, phosphorylation, TWIST1 box function, and ability to form homo- versus hetero- dimers impacted TWIST1 activity in a locus-specific manner. Previous studies have demonstrated that TWIST1 dimerizes with the E2A proteins, E12 and E47. We have shown that silencing of E2A phenocopies loss of TWIST1 in NSCLC and that formation of the TWIST1-E2A heterodimer results in a reciprocal stabilization of the binding partner. Therefore, we sought to determine how differential dimerization by TWIST1 might modulate tumorigenic gene expression. We utilized tethered TWIST1 dimers with either TWIST1 or the E2A proteins to form TWIST1-TWIST1 (homodimer), TWIST1-E12 or -E47 (heterodimers), and tested their transcriptional activities at several TWIST1-regulated promoters. We found that TWIST1-E2A heterodimers could enhance TWIST1 transcriptional activity compared to that of the TWIST-TWIST homodimer. In addition, we determined that the TWIST-E2A heterodimer is degraded by harmine, a harmala alkaloid that we have shown degrades TWIST1, whereas the TWIST-TWIST homodimer is resistant to harmine degradation. This suggests that the TWIST1-E2A heterodimer is the key target of harmine. Furthermore, we have found that overexpression of the TWIST1, E2A proteins, or the TWIST1-E2A heterodimer is able to partially rescue harmine induced cytotoxicity in KRAS mutant NSCLC. Taken together, these data suggest that E2A is essential for TWIST1 mediated tumorigenesis and that targeting of the TWIST1-E2A axis may be an effective therapeutic strategy against oncogene-driven NSCLC. Citation Format: Susheel K. Khetarpal. Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2020.