Abstract 21: TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma
Authors: Yochum Zachary A. , Cades Jessica A. , Mazacurati Lucia , Chatley Sarah , Tran Phuoc T. , Burns Timothy F. 
Published: August 2015

Abstract
A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. The most common oncogene driver mutation is mutant KRAS for which no effective therapies exist. In addition, acquired resistance to currently available targeted therapies for oncogene driver dependent disease is inevitable. Our lab has demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified NSCLC can induce either oncogene induced senescence or apoptosis. The fact that a subset of oncogene dependent NSCLC undergo apoptosis following TWIST1 inhibition suggests that these cells are potentially “addicted” to TWIST1 and might be more vulnerable to TWIST1 inhibitors. 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 TWIST1 levels via degradation. Given that TWIST1 is rarely expressed post-natally, therapies targeting TWIST1 may have minimal toxicities.
In the current study, we examine the key TWIST1 functions, target genes and apoptotic pathways that are required for suppression of apoptosis. We found that genetic or pharmacological (harmine) inhibition of TWIST1 resulted in apoptosis in several oncogenic driver dependent cell lines. TWIST1 inhibition resulted in cleavage of caspase 3, 8, 9, and PARP. TWIST1 inhibition resulted in increased levels of Bid, Bim, and TNFRSF10B, 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 Bid. Overexpression of Bcl-2 or c-FLIP resulted in partial abrogation of apoptosis following TWIST1 silencing. These findings suggest that the intrinsic and extrinsic pathways are important for TWIST1 mediated suppression of apoptosis. Preliminary gene expression analysis of NSCLC cells following TWIST1 silencing has identified multiple candidate target genes in these apoptotic pathways. In addition, structure/functional analysis of TWIST1 suggests that nuclear localization, homo- and heterodimerization and proper phosphorylation of TWIST1 are necessary for suppression of apoptosis. Remarkably, TWIST inhibition with harmine treatment decreased tumor growth in our mouse model of KrasG12D/Twist1 NSCLC as well as decreased TWIST1 expression and induced apoptosis.
In summary, we found that TWIST1 was required for suppression of apoptosis in several oncogenic driver dependent cell lines. Furthermore, the apoptosis observed after TWIST1 inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through c-FLIP and Bim. Our studies will establish the molecular pathways that are required for suppression of apoptosis with the ultimate goal of identifying predictive biomarkers of response to TWIST1 inhibitors.
Citation Format: Zachary A. Yochum, Jessica A. Cades, Lucia Mazacurati, Sarah Chatley, Phuoc T. Tran, Timothy F. Burns. TWIST1 is required for suppression of apoptosis in oncogene driven non-small cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 21. doi:10.1158/1538-7445.AM2015-21