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A. Eramo

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    MO20 - Preclinical Therapeutic Models II (ID 93)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO20.06 - Histone deacetylase inhibition downregulates thymidylate synthase (TS) expression and enhances pemetrexed-induced cytotoxicity in NSCLC models (ID 2010)

      10:30 - 12:00  |  Author(s): A. Eramo

      • Abstract
      • Presentation
      • Slides

      Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed (PEM), a multi-target folate antagonist, has demonstrated targeted efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS, one of PEM’s molecular targets) expression. Recently, TS expression has been found to be regulated by histone acetylation status, thus raising the interesting hypothesis that histone deacetylase inhibitors (HDACi) may sensitize NSCLC cells to PEM cytotoxicity.

      Molecular and functional effects of single and combined HDAC inhibition and PEM exposure were assessed in NSCLC cell lines (A549, H1299, H1650, Calu-1) and patient-derived lung cancer stem cells (L-CSC). Pharmacologic interactions were assessed by conservative isobologram analysis using the Chou-Talalay method and the Calcusyn software. TS expression was studied by WB analysis and real-time PCR. Apoposis induction was assessed by flow cytometry and WB. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells, detection of acidic vesicle organelles (AVO) formation and WB. In vivo experiments were conducted in xenograft models established by i.m. injection of NSCLC cells into 6-8 week-old male athymic mice (nu/nu).

      In NSCLC cell lines and L-CSC, the HDACi ITF2357 dose-dependently inhibited cell growth (IC~50~: <1-20 mM), induced histone H3 acetylation, and downregulated TS expression at the mRNA and protein levels. Combined HDAC inhibition and PEM exposure was then tested using three different administration schedules: simultaneous exposure to both drugs, ITF2357 followed by PEM, and the reverse sequence. Simultaneous PEM/ITF2357 treatment resulted in antagonistic growth inhibitory interactions (combination index – CI >1) in all cell lines tested, while ITF2357 followed by PEM had additive effects in A549 cells and slightly synergistic effects in H1299 and Calu-1 cells; conversely, PEM followed by ITF2357 had strikingly synergistic effects (CI <<1) in all NSCLC cell lines, as well as in the L-CSC143. Most notably, only the ITF2357 followed by PEM sequence synergistically induced apoptosis, resulting in approximately 50% Annexin V-positive cells; apoptosis was only partially rescued by caspase inhibition by z-VAD-fmk, which led us to investigate autophagy as an alternative mechanism of combination-induced cell death. Indeed, ITF2357, and to a significantly greater extent PEM followed by ITF2357, induced autophagy as evidenced by AVO formation, LC3BII processing, p62 downregulation, and Beclin1 induction. Most importantly, autophagy induction was instrumental to the cytotoxic interaction between PEM and ITF2357, as Beclin1 silencing by shRNA completely reversed their growth inhibitory synergism and prevented both autophagy and apoptosis induction. The synergistic cytotoxic interaction between PEM and ITF2357 was at least partly due to ITF2357 ability to prevent PEM-induced TS upregulation, as TS silencing by siRNA further enhanced apoptosis induction by single and combined PEM/ITF2357 exposure. Finally, both H1650 and H1299 xenografts had a robust response to sequential PEM/ITF2357 administration in vivo, resulting in an approximately doubled mice survival in the H1650 model.

      Overall, our data indicate that HDAC inhibition by ITF2357 downregulates TS expression and synergistically potentiates apoptosis and autophagy induction following PEM exposure, supporting the clinical investigation of sequential PEM/ITF2357 schedules for the treatment of advanced NSCLC.

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