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É.F. Dockry



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    MO19 - Lung Cancer Immunobiology (ID 91)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO19.01 - Epigenetic Targeting of CD1d increases anti-tumour iNKT activity in Non-Small Cell Lung Cancer (ID 3373)

      10:30 - 12:00  |  Author(s): É.F. Dockry

      • Abstract
      • Presentation
      • Slides

      Background
      Immunotherapy is now accepted as the fourth most important modality for malignant tumours, and is currently being applied in the treatment of non-small cell lung cancer (NSCLC). CD1d, a MHC class-like molecule, presents glycolipids to natural killer T (NKT) cells. In doing so, CD1D contributes to their anti-tumour activity. CD1d is expressed in certain tumour types, and there is increasing evidence that CD1d acts as a target for NKT-mediated cell killing, however most human and mouse solid tumours are CD1d-negative. Recently evidence has indicated that CD1d expression is epigenetically regulated through HDAC1/2 and Spl.

      Methods
      CD1d levels were examined at the mRNA level by RT-PCR. To determine whether CD1d expression is subject to epigenetic regulation, non-small cell lung cancer (NSCLC) cell lines were treated with various epigenetic modifying agents. The A549 (adenocarcinoma) and SK-MES-1 (Squamous cell carcinoma) cell lines were treated with HDACi, (a) SAHA at a dose of 5 µM for 24 h. Treatments with other epigenetic modifyng agents such as trichostatin A (TSA) and DNA methyltransferase inhibitors are currenly ongoing. iNKT cells were co-cultured wiht NSCLC cells to examine their antitumourigenic activity using a CD107a externalised assay.

      Results
      Using RT-PCR it was possible to confirm that SAHA significantly induced CD1d expression in both A549 and SKMES cell lines (p ≤ <0.005). RT-PCR was also performed on A549 and SKMES cell lines that had been allowed to recover, following treatment with SAHA. A549 cells showed a statistically significant induction in recovered SAHA-treated cells (p < 0.05). Using flow cytometry the cytotoxic T-lymphocytes (CTL) activity of iNKTs was measured. While there was a slight induction of CD107a-bearing iNKT cells, it is believed that treatment with HDACi will increase the anti-tumour activity of these cells. This work is ongoing at present.

      Conclusion
      CD1d expression is significantly induced using the HDACi, SAHA. This induction is present in A549 cells treated with SAHA and allowed to recover; indicating that SAHA may possibly be used to increase the anti-tumourigenic activity of iNKT cells. These results may have important consequences for treating patients with combined epigenetic targeting agents and immunotherapy.

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    P2.01 - Poster Session 2 - Cancer Biology (ID 145)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.01-011 - The IL-23 family may be a novel therapeutic target in non small cell lung cancer (ID 3304)

      09:30 - 16:30  |  Author(s): É.F. Dockry

      • Abstract

      Background
      IL-23A is a member of the IL-6 super-family and is composed of covalently bound IL-12p40 and IL-23p19 (IL-23A) subunits. The functional IL-23R is composed of the IL-23R and IL-12Rβ1 subunits and once activated can promote the STAT3 signalling pathway. IL-23A plays a key role in chronic inflammation through the promotion of IL-17 production by T helper 17 cells (Th 17). Stimulation of IL-23A associated pathways produces pro and anti oncogenic effects which are cell type dependant. We have previously identified IL-23A axis as deregulated in NSCLC and sought to examine the family in greater detail.

      Methods
      The expression of IL-23A/IL-23R was examined in a series of resected fresh frozen NSCLC tumours and in a panel of normal (HBEC) and NSCLC cell lines. Epigenetic regulation of IL-23A/IL-23R was determined in NSCLC cell lines using histone deacetylase (HDi) and DNA methyltransferase (DNMTi) inhibitors. A ChIP assay was performed to investigate the direct effect of Trichostatin A (TSA) on the IL-23A/IL-23R promoter regions. Additionally, the effect of Gemcitabine on IL-23A/IL-23R expression was examined. Finally, the effect of recombinant IL-23 treatment and Apilimod (STA 5326) (a small molecule which blocks the expression of IL-23 and IL-12) on NSCLC cell line proliferation was examined. IL-23 expression was also studied in a panel of NSCLC cisplatin sensitive and resistant cell lines. Furthermore due to its role in immune regulation, we are currently studying the effect of IL-23 on innate immune cell function.

      Results
      IL-23A expression was significantly elevated in a cohort of NSCLC patient tumour samples (p<0.05). Differential IL-23R expression was evident in a panel of NSCLC normal/tumour matched pairs (n=20), with no expression in 25%, reduced levels in 20% and increased levels in 55% of tumour samples compared with matched normal. In addition, IL-23A/IL-23R was found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation in the A549 cell line (p<0.05), with associated chromatin remodelling at both promoters. Gemcitabine, also induced IL-23A/IL-23R expression in this cell line (p<0.05). Furthermore, treatment with recombinant IL-23 resulted in increased cellular proliferation in the A549 cell line, while Apilimod reduced cellular proliferation. Preliminary results indicate that IL-23 can potentially affect the function of innate γδ T cells. In a panel of cisplatin resistant and sensitive cell lines, IL-23A was up-regulated in 3/5 resistant cell lines. Work is ongoing to assess the IL-23R expression in CisR and Parent cells lines and the effect of co-treating cells with HDi and Apilimod and to determine whether these compounds can re-sensitise cells to cisplatin therapy.

      Conclusion
      Based on our results the IL-23A/IL-23R axis is dysregulated in NSCLC. The IL-23 family is subject to epigenetic regulation through HDAC modifications and CpG island methylation. Gemcitabine treatment also affected the expression of this family. Recombinant IL-23 was pro-proliferative in NSCLC and blocking IL-23 with Apilimod reduced the proliferative capacity of the cells. Levels of IL-23A are differentially expressed between cisplatin sensitive and resistant cell lines. Apilimod may be a novel therapeutic drug in the treatment of NSCLC.