Supplementary Figure 1: FGF2 treatment induces branching morphogenesis of primary epithelial cells in 3D culture. Supplementary Figure 2: Basal Expression Levels of TWIST1 in KRAS mutant and MET Amplified/Mutant NSCLC Cell Lines. Supplementary Figure 3: Harmine treatment induces Oncogene-Induced Senescence (OIS) in EGFR and MET-mutant NSCLC cell lines. Supplementary Figure 4: Harmine induces apoptosis in oncogene-driven NSCLC cell lines. Supplementary Figure 5: Harmine treatment promotes TWIST1 degradation and decreases TWIST1 protein stability. Supplementary Figure 6: Silencing of E2A induces apoptosis and phenocopies silencing of TWIST1. Supplementary Figure 7: Overexpression of TWIST1 or its binding partner, E2A, rescues harmine induced growth inhibition. Supplementary Figure 8: Treatment with harmine decreases tumor growth in a KRAS mutant Patient-Derived Xenograph (PDX) model and degrades Twist1 and induces apoptosis in transgenic mouse model of Kras mutant lung cancer. Supplementary Table 1: Rank list of compounds from Connectivity mapping (CMAP) analysis Supplementary Table 2: List of primers used for qRT-PCR Supplementary Table 3: List of primers used for Taqman qRT-PCR Supplementary Table 4: List of antibodies used in current study Supplementary Table 5: Sequences for TWIST1/TCF3 shRNA (5’ â€" 3’) in pKLO.1 Supplementary Table 6: ORFs obtained from Johns Hopkins University HiT Center Supplementary Table 7: Source of Plasmids utilized
Funding
American Lung Association
Sidney Kimmel Foundation
Doris Duke Charitable Foundation Clinical Scientist Award
Howard Hughes Medical Institute
American Cancer Society
NIH
ARTICLE ABSTRACT
TWIST1, an epithelial–mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non–small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical–bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a harmala alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764–76. ©2017 AACR.