Abstract 3573: TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer
Authors: Yochum Zachary A. , Khetarpal Susheel , Burns Timothy F. 
Published: July 2016

Abstract
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. In addition, acquired resistance to currently available targeted therapies is inevitable. 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 apoptosis, which suggests that a subset of oncogene dependent NSCLC are potentially “addicted” to TWIST1. Importantly, we have identified the harmala alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease levels of TWIST1 and its dimerization partners, the E2A proteins, via degradation.
We examined the target genes and pathways required for suppression of apoptosis by TWIST1 and E2A. Genetic or pharmacological (harmine) inhibition of TWIST1 or E2A resulted in apoptosis in several oncogenic driver dependent cell lines. Additionally, treatment with a pan-caspase inhibitor resulted in rescue of growth inhibition following TWIST1 or E2A silencing or harmine treatment. This suggests that apoptosis is the mechanism of growth inhibition following TWIST1 inhibition. TWIST1 or E2A inhibition resulted in increased levels of Bid, Bim, and DR5, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bim and Bid. c-FLIP appears to be a direct transcriptional target of TWIST1 as TWIST1 overexpression leads to transactivation of the c-FLIP promoter and is dependent on the ability of TWIST1 to bind DNA. Interestingly, the TWIST1-E2A heterodimer results in greater promoter transactivation when compared to the TWIST1 homodimer. Furthermore, knockdown of Bim, overexpression of Bcl-2, or overexpression of c-FLIPs resulted in partial rescue of growth inhibition and apoptosis following TWIST1 silencing. However, only knockdown of Bim or Bcl-2 overexpression was able to rescue apoptosis following harmine treatment. This suggests that apoptosis following harmine treatment only requires the intrinsic machinery, while specifically silencing TWIST1 also engages the extrinsic pathway.
In summary, we found that the apoptosis observed after TWIST1/E2A inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through its novel target genes, c-FLIP and Bim. Our studies will establish the target genes of TWIST1 that are required for suppression of apoptosis with the ultimate goal of identifying biomarkers of response to TWIST1 inhibitors. We also aim to determine if TWIST1, through its apoptotic target genes, modulates response to targeted therapies or standard chemotherapies.
Citation Format: Zachary A. Yochum, Susheel Khetarpal, Timothy F. Burns. TWIST1/E2A signaling axis suppresses apoptosis in oncogene 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 3573.