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S.G. Gray



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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)

      09:30 - 16:30  |  Author(s): S.G. Gray

      • 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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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-013 - SDHB is overexpressed and may be a candidate target for therapeutic intervention in Malignant Pleural Mesothelioma (ID 3287)

      09:30 - 16:30  |  Author(s): S.G. Gray

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura. Asbestos exposure (through inhalation) is the most well established risk factor for mesothelioma. The current standard of care for patients suffering from MPM is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Most patients however, die within 24 months of diagnosis. New therapies are therefore urgently required for this disease. The Succinate Dehydrogenase Complex, Subunit B, IronSulfur Protein (SDHB), is a subunit of the Succinate dehydrogenase or succinate-coenzyme Q reductase (SQR) or respiratory Complex II, and has recently been identified by us as an important element in MPM.

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of SDHB by RT-PCR. Levels were subsequently examined in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies by RT-PCR and western blot. Finally the expression of SDHB in a large cohort of MPM specimens with clinical data was asessed by IHC.

      Results
      Expression of SDHB occurs in all cell lines. Significantly higher expression of SDHB is observed in the malignant tumour material versus benign pleura. There was a trend towards better survival for patients expressing higher levels of SDHB, but this was not statistically significant.

      Conclusion
      SDHB, a key member of oxidative energy metabolism is significantly altered in MPM. This may have important future implications for the management 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 - 16:30  |  Author(s): S.G. Gray

      • 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 - 16:30  |  Author(s): S.G. Gray

      • 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.

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    P3.01 - Poster Session 3 - Cancer Biology (ID 147)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P3.01-013 - KAT5 may be a candidate therapeutic target in Malignant Pleural Mesothelioma (ID 3273)

      09:30 - 16:30  |  Author(s): S.G. Gray

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura. Asbestos exposure (through inhalation) is the most well established risk factor for mesothelioma. The current standard of care for patients suffering from MPM is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Most patients however, die within 24 months of diagnosis. New therapies are therefore urgently required for this disease. Lysine acetyltransferases (KATs) including KAT5 have been linked with the development of cisplatin resistance. This gene 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 KAT5 variants by RT-PCR. Levels were subsequently examined in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies by RT-PCR. The effects of a small molecule inhibitor of KAT5 (MG-149) on cellular proliferation were examined.

      Results
      We show that the expression of KAT5A is dramatically increased in MPM. When separated according to histological subtype, KAT5A was significantly overexpressed in both the the sarcomatoid and biphasic subgroups for all transcript variants. Treatment of cells with the small molecule MG-149 caused significant inhibition of cellular proliferation (p<0.0001). We continue to asess this compound by other methodologies to confirm its potential utility in the treatment of MPM.

      Conclusion
      KAT5, a lysine acetyltransferase associated with cisplatin resistance in cancer is 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.

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      P3.01-014 - Expression and post-translational modifications of AKT isoforms in Malignant pleural Mesothelioma cells (ID 3252)

      09:30 - 16:30  |  Author(s): S.G. Gray

      • Abstract

      Background
      Background: The PI3K/AKT signaling pathway is aberrantly active and has an important biologic impact in malignant pleural mesothelioma (MMe) cell cycle progression and chemo-resistance. Akt consists of three isoforms, Akt1, Akt2, and Akt3. Despite the growing amount of research demonstrating the existence of isoform-specific regulation, many papers still draw generalized conclusions about AKT without focusing on functional specificity of each isoforms. Recent data have clearly demonstrated a role of SIRT1 in the modulation of AKT1 activation and a role of PARP1 as a gatekeeper for SIRT1 activity by limiting NAD+ availability.

      Methods
      Methods: We explored the expression of AKT isoforms in MMe in vivo and in vitro and the balancing between their acetylation and phosphorylation status in human MMe derived cell lines in vitro.

      Results
      Results: We firstly described that MMe tumors in vivo and MMe derived cell lines express both AKT1 and AKT3 isoforms but not AKT2, and their expression results significantly increased in the biphasic histotype. Furthermore, we demonstrated an inverse correlation between AKTs acetylation and phosphorylation modulated by PARP1/SIRT1 activation status. By immunoprecipitation experiments, we evidenced that in basal conditions AKT1 is in part acetylated and in part phosphorylated and became highly phosphorylated and completely de-acetylated upon PARP1 inhibition. Interestingly, AKT1 activation, related to PARP1 inhibition, is unable to modulate pro-survival signals, because the downstream pathway is interrupted at the level of its effector mTOR. Conversely, SIRT1 inhibition or silencing result in a more evident AKT1 and its interactors acetylation.

      Conclusion
      Conclusions: In conclusion, our results clearly show how both PARP1 and SIRT1 affect critical cellular pathways involved in MMe progression and offer a model of a regulatory inter-relationship between these proteins. These data could be helpful for designing new effective therapeutic strategies.

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    P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.05-022 - BMS-777607 as a candidate therapeutic agent in Malignant Pleural Mesothelioma (ID 3272)

      09:30 - 16:30  |  Author(s): S.G. Gray

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Untreated, MPM has a median survival time of 6 months, and most patients die within 24 months of diagnosis. There is an urgent unmet need to identify new therapies for this disease. Receptor tyrosine kinases (RTK) are an area of active research in cancer therapy. The identification of “addicted” signalling networks could rapidly lead to candidate inhibitors (RTKi) that could potentially be uused to target MPM. We have previously identified RON/MST1R as a candidate therapeutic target in MPM. Additional RTKs identified by other studies include Axl and c-MET. The agent BMS-777607 is an orally bioavailable small molecule with the capacity to inhibit c-MET, RON/MST1R, Axl and Tyro3 (at nanomolar concentrations) and has just completed a Phase I/II trial (ClinicalTrials.gov Identifier: NCT00605618). This drug may therefore have applicability in MPM.

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of Tyro3, c-MET, RON/MST1R and Axl by RT-PCR. A cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies were subsequently examined for the expression of these RTKs. The effects of BMS-777607 on MPM cellular proliferation and viability are being assessed.

      Results
      Expression of various RON/MST1R isoforms, c-MET, Tyro3 and Axl were observed in all cell lines. Significantly higher expression of all genes were found in the malignant tumour material versus benign pleura. The effects of BMS-777607 on cellular proliferation/viability are ongoing and the results will be presented at the meeting.

      Conclusion
      BMS-777607 represents a unique compound capable of targeting four RTKs whose expression are significantly elevated in maligant MPM. Given the many indications that these RTKs are associated with “addicted” RTK networks, this compound may therefore have potential clinical utility in the clinical management of MPM.