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S. Wennstedt



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    P1.01 - Poster Session 1 - Cancer Biology (ID 143)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.01-012 - The KDM6 Lysine Demethylases are candidate therapeutic targets in Malignant Pleural Mesothelioma (ID 3282)

      12:04 - 12:18  |  Author(s): S. Wennstedt

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is a rare cancer affecting the pleura and is commonly caused by prior exposure to asbestos. Treatment of MPM is difficult with limited options. The current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed), yet most patients die within 24 months of diagnosis. There is therfore an unmet need to identify new therapeutic approaches for the treatment of MPM. Lysine Demethylases (KDMs) represent novel targets for the treatment of cancer. Overexpression of many KDMs occurs in many cancers, and these proteins play important roles in tumorigenesis. One such family, the KDM6/JMJD3 family, was investigated for changes in expression in MPM and to determine if this family could represent a novel candidate target(s) for intervention in MPM.

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KDM6 family members (KDM6A/UTX and KDM6B/JMJD3) by RT-PCR. mRNA levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. The effects of a small molecule inhibitor of KDM6B/JMJD3, (GSK-J4) on cellular proliferation and gene expression were examined.

      Results
      We show that the expression of the KDM6 family is detectable in all cell lines across our panel of cell lines. In primary tumours however, the expression of KDM6A/UTX was very significantly (p<0.001) and KDM6B/JMJD3 was significantly elevated (p<0.05) in malignant MPM compared to benign pleura. When separated across histological subtype KDM6A/UTX was most significantly elevated in the Sarcomatoid subtype (p<0.001), while only KDM6B/JMJD3 was significantly elevated (p<0.05) in the Biphasic subset. Treatment of REN/ NCI-H226 cells with the KDM6B/JMJD3 inhibitor GSK-J4 caused significant inhibition of cellular proliferation, with the REN cell line being more sensitive than NCI-H226. The effects of GSK-J4 on gene expression were examined on a panel of genes associated with Tumor-Invasion/Metastasis and on pro-inflammatory cytokines.

      Conclusion
      The KDM6 family of lysine demethylases are significantly altered in MPM. A small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. We continue to assess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM.

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P1.05-022 - BET Bromodomains: Are they a potential therapeutic targets in Malignant Pleural Mesothelioma? (ID 3269)

      09:30 - 09:30  |  Author(s): S. Wennstedt

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive cancer affecting the pleura. Treatment options are limited and most patients die within 24 months of diagnosis. The recommended first line chemotherapy for MPM is a combination of cisplatin/pemetrexed (or alternatively Raltitrexed), and there is no recommeneded second-line therapy. As such new therapeutic approaches are required for the management of MPM. Bromodomain and extra terminal domain (BET) proteins function as epigenetic signaling factors that associate with acetylated histones to facilitate the transcription of target genes. Various inhibitors targeting the activity of BET proteins have been developed and have shown potent antiproliferative effects in hematological cancers, and more recently been studied for in vitro efficacy in lung adenocarcinoma cell lines (1). We examined the expression of various members of the BET in MPM and assessed the effects of one of these inhibitors (JQ-1) to determine if this family could represent a novel candidate target(s) for therapeutic intervention in MPM.

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of BRD2 and BRD4 by RT-PCR. mRNA levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. The expression of a known target of the BET inhibitor JQ1, oncogenic transcription factor FOSL1 was also examined. The effects of a small molecule inhibitor of BET proteins (JQ-1) on cellular proliferation was examined (BrdU ELISA).

      Results
      We show that the expression of the BRD2 and BRD4 variants are detectable in all cell lines across our panel of cell lines. In primary tumours however, the expression of BRD2 was very significantly downregulated (p=0.0006). BRD4 comprises 2 transcript variants, a long variant (BRD4L – Refseq NM_058243.2), and a short variant (BRD4S – Refseq NM_014299.2). BRD4L was not significantly affected in malignant MPM compared to benign pleura, whereas BRD4S was significantly elevated in the tumours compared to the benign pleura (p<0.05). When separated across histological subtype BRD2 was significantly decreased across all histological subtypes (p=0.0009). FOSL1 a candidate target of JQ1 (1) was found to be significantly elevated in malignant MPM compared to benign pleura (p<0.05). Treatment of REN/ NCI-H226 cells with JQ1 caused significant inhibition of cellular proliferation, with NCI-H226 being more sensitive than REN to this compound.

      Conclusion
      The BET domain proteins are altered in MPM, and a small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. We continue to asess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM. Reference: 1. Lockwood WW et al., (2012). Proc Natl Acad Sci U S A. 109(47):19408-13.

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      P1.05-023 - The KDM4/JMJD2 Lysine Demethylases are candidate therapeutic targets in Malignant Pleural Mesothelioma (ID 3278)

      09:30 - 09:30  |  Author(s): S. Wennstedt

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura associated with exposure to asbestos. Treatment options are limited, and the current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Despite this treatment option, almost all patients die within 24 months of diagnosis. Therefore, new therapeutic options are urgently required for the treatment of MPM. Lysine Demethylases (KDMs) represent novel targets for the treatment of cancer. Overexpression of KDMs are common in many cancers, and play important roles in tumorigenesis. The jumonji (JMJ) family of lysine demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. One such family, the KDM4/JMJD2 family, may therefore be altered in MPM and could represent a novel candidate target for intervention

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KDM4 family members by RT-PCR. mRNA levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. The effects of a small molecule inhibitor of KDM4A/JMJD2A, 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde or PA) on cellular proliferation and gene expression were examined.

      Results
      We show that the expression of the KDM4 family is ubiquitously expressed across our panel of cell lines. In primary tumours however, the expression of KDM4 members KDM4A, KDM4B and KDM4C were significantly elevated in malignant MPM compared to benign pleura. Treatment of REN/ NCI-H226 cells with the small molecule PA caused significant inhibition of cellular proliferation (p<0.0001). We continue to asess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM.

      Conclusion
      The KDM4/JMJD2 family of lysine demethylases are significantly altered in MPM. A small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. Targeting this protein may have important future implications for the management of MPM.