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K.L. Bennewith



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    MA 05 - Immuno-Oncology: Novel Biomarker Candidates (ID 658)

    • Event: WCLC 2017
    • Type: Mini Oral
    • Track: Immunology and Immunotherapy
    • Presentations: 1
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      MA 05.12 - Oncogenic Drivers Induce Production of CCL5 to Recruit Regulatory T-Cells Early in Lung Cancer Progression (ID 10289)

      17:00 - 17:05  |  Author(s): K.L. Bennewith

      • Abstract
      • Presentation
      • Slides

      Background:
      Lung cancer development is driven by the expression of mutant oncogenes, with EGFR and KRAS the most frequent in lung adenocarcinoma. However, these mutations alone are not sufficient for tumorigenesis suggesting additional factors influence tumour development and progression, including the balance of anti-tumour immune effector cells and pro-tumorigenic immune suppressor cells. Tumour cells can evade immune surveillance by producing cytokines to recruit immune modulatory cells that promote an immune suppressive environment, such as regulatory T cells (Tregs). We hypothesized that oncogene signaling regulates the production of cytokines by tumour cells in order to recruit immune suppressive cells and promote lung tumour development.

      Method:
      We used CIBERSORT to quantify 22 immune cell types in over 300 human lung adenocarcinoma and 100 matched normal lung tissues, and validated findings with immunohistochemistry. Cells expressing doxycycline inducible EGFR[L858R] and KRAS[G12V]were analyzed for cytokine production using a multiplex assay (LUMINEX). EGFR (Afatinib) and MEK (Trametinib) inhibitors were used in lung cancer cell lines harbouring EGFR or KRAS mutations and cytokine production was quantified using ELISA. Conditioned media from EGFR[L858R] and KRAS[G12V] expressing cells were used in a trans-well assay to determine if secreted cytokines could induce Treg migration. Transgenic mouse models of lung adenocarcinoma and bronchoalveolar lavage (BAL) from patients with and without lung cancer were used to assess CCL5 and Tregs in vivo.

      Result:
      Treg cells were significantly enriched in lung tumours and not normal tissue. CCL5 production is increased rapidly upon oncogene induction and subsequent transformation of normal cells and is dependent on sustained ERK signaling for continued expression. Conditioned medium from EGFR[L858R] expressing cells increased Treg migration, which was mitigated by an anti-CCL5 antibody. Transgenic mice expressing EGFR[L858R ]or KRAS[G12D] in the lung epithelium recruited Tregs to the lung upon tumor induction. Assessment of CCL5 in BAL from patients with and without lung cancer is currently in progress.

      Conclusion:
      Oncogene driven ERK signaling may regulate expression of CCL5 from lung tumour cells, and oncogene induced CCL5 production stimulates Treg migration ex vivo. These data suggest CCL5-mediated Treg recruitment to lung tumours may occur in early stages of lung tumour development and that targeted inhibition of CCL5 or ERK signaling may represent therapeutic strategies to block recruitment of immunosuppressive Tregs by lung tumours.

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    P3.02 - Biology/Pathology (ID 620)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P3.02-090 - Hypoxia-Induced Modifications of the Small Non-Coding RNA Transcriptome Delineates Risk of Recurrence in Early-Stage Lung Adenocarcinoma (ID 8400)

      09:30 - 09:30  |  Author(s): K.L. Bennewith

      • Abstract

      Background:
      Hypoxia is central to neoplastic diseases. It has been associated with reduced survival in several tumor types, including NSCLC. A spectrum of poor outcome suggests a multifactorial modulation of the hypoxic response. Piwi-interacting RNAs (piRNAs) are small non-coding RNAs (sncRNAs) with a pivotal role in genomic stability and epigenetic regulation of gene expression. Changes in piRNA expression have been recently found to be deregulated during tumor progression, and responsive to tumor microenvironment conditions. Here, we investigate if hypoxia alters the piRNA transcriptome in human lungs, and assessed whether these alterations are indicative of outcome in patients with hypoxic lung tumours.

      Method:
      Tumors from lung and other six organs (TCGA) were classified according to their oxygenation state using signatures derived from established hypoxia-associated gene expression changes. By investigating >3000 piRNA transcriptomes, we generated a baseline of hypoxia-related changes which were further validated in a panel of cell lines exposed to hypoxia in vitro (16h at 1% O~2~). In lung tumors (n=1,018), we identified the most robust hypoxia-related changes between hypoxic and non-hypoxic groups by including piRNAs that: 1) ≥ 10 RPKM median expression, 2) > 2-fold change in median expression, and 3) a significant p value following a Mann-Whitney U test. A piRNA-based score (piSco) was generated by grouping HR-piRNAs expression, weighted by coefficients from a Cox proportional hazard model.

      Result:
      Overall, piRNA expression is selectively deregulated by hypoxia. In vitro tumour models recapitulate HR-piRNAs expression patterns. Seventy-one HR-piRNAs were identified, showing statistically significant differences in expression between hypoxic and non-hypoxic tumours. Of these, 13 were exclusively deregulated in NSCLC tumors, showing a remarkable subtype specificity between adenocarcinomas and squamous cell carcinomas. We next investigated if the HR-piRNA based score (piSco) was associated with NSCLC patient outcome. We found that piSco classify patients with hypoxic lung LUAD and LUSC as low or high risk of RFS. Remarkably, piSco was also able to classify Stage I LUAD patients into the same categories above (p = 0.0051).

      Conclusion:
      This study reveals the influence of the tumour microenvironment on DNA-level regulatory mechanisms with important implications for predicting recurrence in patients with hypoxic lung tumours. Our data encourage further exploration of HR-piRNAs as clinical tools for evaluating the likelihood of tumour recurrence, and as a mean to identify patients that would most benefit from adjuvant therapies and/or therapies designed to target hypoxic tumour cells.