Twist1-induced suppression of oncogene-induced senescence in non-small cell lung cancer requires the transactivation domain of Twist1

Non-small cell lung carcinoma (NSCLC) is a major cause of cancer mortality. High expression of the epithelial-to-mesenchymal transition transcription factor (EMT-TF) Twist1 is strongly associated with metastatic cancers and with treatment resistance. Twist1 can also upregulate O-GlcNAcylation to suppress fail-safe programs such as KrasG12D oncogene-induced senescence (OIS) that accelerates NSCLC tumorigenesis. We wanted to decipher the critical domains and transcriptional targets required for Twist1 acceleration of lung tumorigenicity. We created a novel genetically-engineered mouse model for autochthonous lung cancer through lung epithelial expression of KrasG12D oncogene (CR) concomitantly with Twist1wt (CRT) or a Twist1F191G transactivation-null mutant (CRF191G). Compared to CR and CRF191G, CRT mice had shorter tumor-free survival and more aggressive tumors histologically. CRT lung tumors also showed higher proliferation and lower cell-cycle arrest suggesting that the Twist1 transactivation-domain is important for OIS suppression. Supporting these data, we observed in non-cancer human bronchial epithelial cells (HBECs) that the co-expression of human TWIST1wt enhanced tumorigenic/invasive programs and could suppress HRasG12V-induced senescence while co-expressing TWIST1F187G transactivation-null mutant could not. TWIST1wt co-expression with HRasG12V in HBECs differentially modulated MYC downstream transcriptional programs. Finally, OIS induction in HBEC-HRasG12V-TWIST1wt was rescued by O-GlcNAcylation inhibition or by treatment with a novel MYC inhibitor MYCi975. Altogether, these results indicate that the Twist1 transactivation domain is required for Twist1-dependent acceleration of lung tumorigenesis via MYC and nominate MYCi975 as a means to activate latent OIS programs. MYC targeting strategies could limit pro-tumorigenic programs and serve as a therapeutic for TWIST1-overexpressing NSCLCs. Structure-functions studies of TWIST1 domains may allow for more precise targeting of pro-oncogenic downstream transcriptional programs/targets involved in NSCLC aggressiveness. TWIST1 is a pleiotropic basic helix-loop-helix (bHLH) transcription factor that can activate or repress E-box-regulated target genes. TWIST1 contains four main domains: (1) an amino-terminal Twist domain, (2) a glycine-rich region domain, (3) a bHLH domain responsible for DNA binding and dimerization, and (4) a highly conserved carboxy-terminal Twist-box domain responsible for transactivation of gene targets. In the present study, we set out to determine whether the transactivation function of TWIST1 was important for the acceleration of tumorigenesis in NSCLC, and we developed and characterized a novel genetically-engineered mouse model (GEMM) of KrasG12D-driven lung tumors that also overexpresses mouse Twist1wt or Twist1 transactivation-null point mutant (phenylalanine 191 to glycine - F191G). We complemented these GEMM experiments with in vitro human bronchial epithelial cell (HBEC) models co-overexpressing HRasG12V along with human TWIST1wt or TWIST1 transactivation-null point mutant (phenylalanine 187 to glycine - F187G). We used RNA sequencing analyses on HBEC expressing or not Twist1wt or Twist1F87G and co-expressing HRasG12V-EGFP or EGFP to determine differentially expressed genes and programs between the different conditions.