Targeting the EMT transcription factor TWIST1 overcomes resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer

Abstract

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial-mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.

Understanding Lineage Plasticity as a Path to Targeted Therapy Failure in EGFR-Mutant Non-small Cell Lung Cancer

“…Many of them, as discussed earlier, suggested that transition toward mesenchymal phenotype co-occurs with a kinase switch. However, in some cases, bypass activation was not identified, suggesting that EMT itself might be responsible for the development of therapyrefractory disease (Lee A. F. et al, 2017;Song et al, 2018;Yochum et al, 2019).…”

“…Additionally, expression of pro-mesenchymal factor TWIST in EGFR-mutant NSCLC has been linked to induction of EMT and acquired resistance to EGFR TKIs. Some studies suggest that TWIST can inhibit transcriptional activation of pro-apoptotic protein BIM either by directly binding to its promoter, or by inducing ZEB1, which then acts as a repressor of BIM transcription (Song et al, 2018;Yochum et al, 2019). Therefore, BH3 mimetics, a class of drugs that target BCL family of proteins to promote apoptosis, may be beneficial for treating EMTassociated EGFR TKI resistance.…”

Androgen Receptor promotes renal cell carcinoma (RCC) vasculogenic mimicry (VM)viaaltering TWIST1 nonsense-mediated decay through lncRNA-TANAR

“…2E, 2D assay and 2F, 3D assay). Similarly, Harmine (39,40), which is a TWIST1 inhibitor causing its protein degradation Together, results from Fig. 2A-I indicate that AR may function through modulating TWIST1 expression to influence the matrigel-coated 2D and collagen I based 3D VM formation in vitro.…”

“…We found that AR can increase ccRCC VM formation via up-regulating TWIST1. TWIST1 is a basic helix-loop-helix transcriptional factor, which plays important roles in epithelial-mesenchymal transition and VM in diverse types of tumors (39,54,55). Previous literature based on 163 ccRCC clinical samples revealed that elevated levels of TWIST1, which is mainly localized in the cytoplasm of ccRCC cells (98.8%), was closely associated with higher stage, vascular invasion, and poor prognosis in RCC (56).…”

“…1C; R=0.34; P ＜0.001), which is consistent with our in vivo or in vitro data. Furthermore, we treated ccRCC cell with Harmine, a naturally occurring beta-carboline alkaloid widely used herb, as a TWIST1 inhibitor selected through the unbiased screen and validated in the lung cells (39,40), and found that it led to a dramatic degradation of TWIST1 protein at either 10 M or 40 M while also drastically diminished VM formation in ccRCC cells. Thus, targeting TWIST1 signaling might be a promising therapy for ccRCC with high VM formation.…”