Abstract PO-067: The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer
Authors: Lafargue Audrey , Wang Hailun , Chettiar Sivarajan T. , Gajula Rajendra P. , Smack Caleb , Siddiqui Ismaeel , Taparra Kekoa , Lam Christine , Carrieri Francesca , Nugent Katriana , Zachara Natasha , Tran Phuoc T. 
Published: April 2021

Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer mortality. Although therapeutic advances have been made, resistance to treatments remain high and the overall survival is still dismal. The high expression of the transcription factor TWIST1 strongly correlates with invasive and metastatic cancers, and is generally attributed to the epithelial-to-mesenchymal transition phenotype. We have demonstrated that TWIST1 can antagonize the induction of fail-safe programs as oncogene (KrasG12D)-induced senescence (OIS) in primary NSCLC tumor. OIS suppression by TWIST1 required increased global O-GlcNAcylation, which perhaps can also impact DNA repair and radiation response. As TWIST1 is essential for development, deciphering the critical domains and downstream transcriptional targets required for pro-tumorigenicity and radioresistance may allow the identification of new therapeutic strategies by targeting TWIST1. We created a transactivation-null TWIST1 mutant, by mutation of phenylalanine 191 to glycine, genetically engineered mouse model (GEMM) utilizing the tetracycline-inducible gene expression system. In these GEMMs, doxycycline treatment allows a concomitant induction of KrasG12D oncogene (R) with TWIST1 (T) or with TWIST1F191G mutant (F) expression, specifically in the lung epithelium directed by CCSP promoter-rtTA (C) transgene. CRT mice presented a more aggressive tumor progression and a shorter survival (median= 15.6 weeks) compared to CR (31 weeks). TWIST1F191G expression in CRF abrogates these effects (26.7 weeks) suggesting that the TWIST1 transactivation domain is required for TWIST1-dependent accelerated tumorigenesis. CRT mice, HBEC, and H460 cells overexpressing TWIST1 showed radiation resistance. A second KrasG12D lung tumor GEMM with induction of TWIST1 prior to 15Gy lung tumor irradiation showed lung tumor stasis compared to regression without TWIST1 expression. Histological analysis showed a strong expression of TWIST1 in CRT lungs while CRF showed a progressive loss over time suggesting that TWIST1F191G was non-functional and conferred no selective advantage. CRT mice also had lung tumors with higher proliferation (by Ki67), reduced apoptosis (by cleaved caspase3) and a decreased cell cycle arrest (by p16) compared to CR lung tumors. In comparison, CRF mice lung tumors did not show any change in cell death but showed increased p16 cell cycle arrest marker suggesting that the transactivation domain of TWIST1 is important for the suppression of OIS. We are exploring similar in vitro phenotypes using a primary immortalized HBEC cell lines co-transfected with HRasG12V oncogene and TWIST1 versus TWIST1 transactivation mutant. In future work, we are investigating the role of the TWIST1 transactivation domain in the induction of O-GlcNAcylation and the stabilization and/or activation of critical targets for OIS suppression and radiation resistance, with the goal of identifying new therapeutic targets and radiosensitizers.

Citation Format: Audrey Lafargue, Hailun Wang, Sivarajan T. Chettiar, Rajendra P. Gajula, Caleb Smack, Ismaeel Siddiqui, Kekoa Taparra, Christine Lam, Francesca Carrieri, Katriana Nugent, Natasha Zachara, Phuoc T. Tran. The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-067.