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L. Mazzacurati

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    MINI 21 - Novel Targets (ID 133)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI21.12 - Identification of a First in Class TWIST1 Inhibitor with Activity in KRAS Mutant NSCLC (ID 1616)

      16:45 - 18:15  |  Author(s): L. Mazzacurati

      • Abstract
      • Presentation
      • Slides

      Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that the basic helix-loop-helix transcription factor, Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in mouse models, and that inhibition of Twist1 in murine models and KRAS mutant NSCLC cell lines led to oncogene-induced senescence (OIS) and in some cases, apoptosis. Therefore, targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact.

      We used gene expression profiles from KRAS mutant human NSCLC cell lines following shRNA-mediated TWIST1 knockdown to perform connectivity map (CMAP) analysis to identify pharmacologic inhibitors of TWIST1. Growth inhibition was determined through the colony formation and MTS assays. Apoptosis (cl-PARP, active anti-C3) and OIS (SA-β-Gal) was assessed. Genetic (shRNA) and pharmacologic inhibition of the TWIST1-E2A pathway was performed. Lung tumor burden as well as levels of TWIST1 protein, apoptosis and proliferation were measured after treatment with harmine in the CCSP-rtTA/tetO-KrasG12D/Twist1-tetO7-luc(CRT) mice.

      We found that several of our CMAP compounds had significant growth inhibitory effects in NSCLC cell lines. Interestingly, a family of related harmala alkaloids including harmine ranked highly in our CMAP analysis. We observed that harmine could inhibit growth in KRAS mutant NSCLC cell lines through the induction of OIS or apoptosis and phenocopied genetic inhibition of TWIST1. Remarkably, harmine treatment led to TWIST1 protein degradation as well as degradation of its binding partners, the E2A proteins, E12/E47. Furthermore, the growth inhibitory effects of the harmala alkaloids correlated with the ability to degrade TWIST1 and were independent of its ability to inhibit the DYRK kinases. In addition, we demonstrated that heterodimer formation of TWIST1/E12/E47 resulted in a reciprocal stabilization of each binding partner and that E12/E47 are required for TWIST1 mediated suppression of OIS and apoptosis. Importantly, we found that harmine preferential targets the TWIST1-E12 heterodimer for degradation and the growth inhibitory effects of harmine are in due in at least part to the ability to inhibit the TWIST1/E12/E47 heterodimer as overexpression of the E2A proteins can suppress harmine induced cytotoxicity. Finally, we have demonstrated that harmine treatment lead to Twist1 protein degradation and tumor growth inhibition in our Kras[G12D]/Twist1 murine model of lung adenocarcinoma. We are currently testing and designing structure analogs of the initial candidate agents to develop more specific and potent inhibitors of TWIST1.

      We have identified a novel TWIST1 inhibitor harmine that induces degradation of TWIST1 and its binding partners, E12/E47 and inhibits the growth of KRAS mutant NSCLC both in vitro and in vivo. Therefore, we believe that targeting the TWIST1-E2A pathway would be an effective therapeutic strategy. Since TWIST1 is essential not only for KRAS mutant NSCLC but more broadly for oncogene driven NSCLC, the development of this novel class of TWIST1 inhibitors could have a significant clinical impact.

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