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Supplementary Table 1

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

Supplementary Figure legends and methods

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.

Background: The mechanisms by which stress hormones impact triple-negative breast cancer (TNBC) etiology and treatment are unclear. We have previously shown that stress hormones, cortisol, and catecholamines induce rapid DNA damage and impact DNA repair in NIH 3T3 fibroblasts. This study investigates whether stress hormones increase DNA damage in breast cancer cells and if this impacts drug efficacy. Methods: We first screened a panel of 39 breast cancer cell lines for expression of adrenergic and glucocorticoid receptors and examined if stress hormones induce DNA damage and alter cell cycle regulation in vitro. A TNBC xenograft model was used to assess the impact of restraint stress on tumour growth and chemosensitivity to paclitaxel. Results: We found that stress hormones induced DNA damage, phosphorylation of ATR, which was accompanied by an up-regulation of the G1 cell kinase inhibitor p21 and a cell cycle halt of TNBCs in the G1 phase. p21 knockdown abrogated G1 arrest by stress hormones. We also demonstrated that stress significantly decreased efficacy of paclitaxel. Conclusion: We describe a novel mechanism through which stress hormones can induce drug resistance to paclitaxel, which may have profound implications for treating drug resistance in patients with TNBC. Fil: Reeder, A.. University of Pittsburgh; Estados Unidos Fil: Attar, M.. University of Pittsburgh; Estados Unidos Fil: Nazario, L.. University of Pittsburgh; Estados Unidos Fil: Bathula, C.. University of Pittsburgh; Estados Unidos Fil: Zhang, A.. University of Pittsburgh; Estados Unidos Fil: Hochbaum, Daniel. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Roy, E.. University of Pittsburgh; Estados Unidos Fil: Cooper, K. L.. University of Pittsburgh; Estados Unidos Fil: Oesterreich, S.. University of Pittsburgh; Estados Unidos Fil: Davidson, N. E.. University of Pittsburgh; Estados Unidos Fil: Neumann, C. A.. University of Pittsburgh; Estados Unidos Fil: Flint, M. S.. University of Pittsburgh; Estados Unidos. University of Brighton; Reino Unido

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.

The c-Met (MET) receptor and its ligand, hepatocyte growth factor (HGF), have been shown to mediate epithelial-mesenchymal transition (EMT), proliferation, invasion, motility, and angiogenesis. The HGF/MET pathway is frequently altered in non-small cell lung cancer (NSCLC) and has emerged as a targetable oncogenic driver, as patients with MET amplification and/or mutations have demonstrated marked responses to the MET tyrosine kinase inhibitor (TKI), crizotinib. However, long-term efficacy of MET TKIs is limited as acquired resistance is inevitable and almost half of patients with MET alterations fail to respond to MET TKIs. HGF overexpression has been identified as a mechanism of resistance to both MET and EGFR TKIs in MET altered and EGFR mutant NSCLC. Furthermore, MET amplification has been implicated in EGFR TKI resistance. However, the mechanism(s) by which the HGF-MET pathway causes resistance are poorly understood. We have previously shown that the EMT-transcription factor, TWIST1, is required for MET-driven NSCLC. Here, we investigated the requirement of TWIST1 in HGF-mediated resistance to MET and EGFR TKIs and the role of TWIST1 in de novo and acquired resistance to MET and EGFR TKIs. We found that HGF treatment induced EMT in NSCLC cell lines and increased TWIST1 protein expression through a post-translational mechanism. We demonstrated that targeting TWIST1 pharmacologically with the TWIST1 inhibitor, harmine, overcame HGF-mediated resistance to both MET and EGFR TKIs in MET and EGFR-driven NSCLC. This suggests that TWIST1 is specifically required for HGF-mediated resistance to targeted therapies. We also found that TWIST1 is overexpressed in a subset of MET and EGFR altered cell lines and TWIST1 overexpression was sufficient to cause resistance to MET and EGFR TKIs. In MET-driven and EGFR mutant cell lines that express TWIST1 and are resistant to targeted therapies, we demonstrated that harmine treatment resensitized resistant cells to MET and EGFR TKIs, respectively. To investigate the role of TWIST1 overexpression in Hgf-driven lung cancer, we utilized a CCSP-Hgf (CH) mouse model that constitutively overexpresses Hgf in the lung and develops crizotinib-sensitive tumors following treatment with the tobacco carcinogen, nicotine-derived nitrosamine ketone (NNK). We demonstrated that the Twist1 overexpressing CTH (CCSP-rtTA/Twist1-tetO-luc/CCSP-Hgf) mice developed significantly larger tumors in response to NNK as compared to CH and CCSP-rtTA/Twist1-tetO-luc (CT) mice. In summary, we established that HGF-regulated TWIST1 expression and that TWIST1 expression is required for resistance to MET and EGFR TKIs in the presence and absence of HGF overexpression. These studies suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome HGF-MET-driven resistance in EGFR mutant NSCLC as well as MET TKI resistance in MET-driven NSCLC. Citation Format: Zachary A. Yochum, Suman Chatterjee, Eric H. Huang, Deena M. Maurer, Myriam A. Attar, Sanja Dacic, Laura P. Stabile, Timothy F. Burns. TWIST1 is a key mediator of HGF-MET-driven resistance to targeted therapies in EGFR mutant and MET-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5889.

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising

The ARF-GEF ARNO promotes motility by activating ARF6 and a subsequent downstream activation of Rac. ARNO is shown to associate with the Rac GEF Dock180 via its coiled-coil domain. Knockdown of scaffold proteins that bind ARNO disrupts the formation of this complex and disrupts ARF-to-Rac signaling., ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein–protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.

Background The transition of epithelial cells from their normal non-motile state to a motile one requires the coordinated action of a number of small GTPases. We have previously shown that epithelial cell migration is stimulated by the coordinated activation of Arf and Rac GTPases. This crosstalk depends upon the assembly of a multi-protein complex that contains the Arf-activating protein cytohesin 2/ARNO and the Rac activating protein Dock180. Two scaffolding proteins that bind directly to cytohesin 2 organize this complex. Results We now have found that Rac activation in response to hepatocyte growth factor (HGF) requires cytohesin 2 and Dock180. GRASP/Tamalin is one of the scaffolds that builds the complex containing cytohesin 2 and Dock180. We determine here that the Ala/Pro rich region of GRASP directly interacts with the SH3 domain of Dock180. By binding to both cytohesin 2/ARNO and Dock180, GRASP bridges the guanine nucleotide exchange factors (GEFs) that activate Arf and Rac, thereby promoting Arf-to-Rac signaling. Furthermore, we find that knockdown of GRASP impairs hepatocyte growth factor (HGF)-stimulated Rac activation and HGF-stimulated epithelial migration. Conclusions GRASP binds directly both cytohesin 2 and Dock180 to coordinate their activities, and by doing so promotes crosstalk between Arf and Rac.