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.
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This work was supported by the following funding sources: HW has received research funding from the Uniting Against Lung Cancer Foundation. TFB was supported for this project from a V Foundation Scholar Award, American Lung Association Award (LCD 257864), Sidney Kimmel Foundation (SKF-15-099) and a Doris Duke Charitable Foundation Clinical Scientist Award (2015097), a Pittsburgh LUNG SPORE CDA P50CA090440 and generous support from the We Wish Foundation. This project used the Hillman Cancer Center Animal, Flow Cytometry and Biostatistics Facilities that are supported in part by award P30CA047904. ZAY has received research funding for this project from the Howard Hughes Medical Institute Medical Fellowship, T32GM008421-22 and a 1F30CA213765-01. PTT has received research funding for this project from the Nesbitt Family; Movember-Prostate Cancer Foundation; Uniting Against Lung Cancer Foundation; American Lung Association (LCD-339465); Commonwealth Foundation; Sidney Kimmel Foundation (SKF-13-021); ACS (122688-RSG-12-196-01-TBG); DoD (W81XWH-13-1-0182); NIH/NCI (R01CA166348). We Laura Stabile, PhD, James G. Herman, MD, and Frank P. Vendetti PhD at the University of Pittsburgh for the discussion, advice regarding experimental design and supply of reagents when applicable. In addition, we would like to acknowledge the helpful discussion and comments provided by the Burns, Rudin, and Tran laboratory members.
Conflict of interest
The authors declare that they have no conflict of interest.
These authors contributed equally: Zachary A. Yochum, Jessica Cades and Hailun Wang.
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Yochum, Z.A., Cades, J., Wang, H. et al. Targeting the EMT transcription factor TWIST1 overcomes resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer. Oncogene 38, 656–670 (2019). https://doi.org/10.1038/s41388-018-0482-y
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