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M. Barr



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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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      MO20.03 - Development and characterization of a panel of GDC-0980 resistant NSCLC cell lines (ID 2798)

      10:30 - 12:00  |  Author(s): M. Barr

      • Abstract
      • Presentation
      • Slides

      Background
      The PI3K-Akt- mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancer due to mutation, amplification, deletion, methylation and post-translational modifications. PI3K pathway activation in NSCLC has been shown by us and others to lead to a more aggressive disease correlating to poor prognosis for patients. Multiple novel agents, targeting different regulators within the pathway are currently under development. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which demonstrated excellent downstream inhibition of the PI3K pathway in vitro, with the strongest effects being observed in lung, breast and prostate cancer cell lines. There are 12 clinical trials ongoing for this drug, with Phase I studies in solid tumours and Phase II studies in endometrial carcinoma, renal cell carcinoma, prostate cancer and breast cancer. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. Multiple mechanisms of resistance to GDC-0980 may develop while a patient is being treated with this drug. The aim of this project is to develop four cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to predict which mechanisms of resistance may occur in patients. This will allow us to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies.

      Methods
      H460, A549, H1975 and SKMES-1 cells were treated with a dose response curve of GDC-0980 and BrdU proliferation assays determined IC50 values for each cell line. Each cell line was then cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Each month, BrdU proliferation assay were carried out in order to track the development of resistance to the drug. When a log fold difference between the parent and resistant IC50s was observed, the cells were deemed to be resistant. Matched parent and resistant cells were then screened for a panel of mutations. Cells lines were also screened for gene alterations using a human cancer drug resistance PCR array. Identified genes of interest were validated at the RNA and protein level by PCR and Western blot, respectively.

      Results
      All four cell lines exhibited a dose-dependent decrease in proliferation when treated with GDC-0980. H1975 cells (adenocarcinoma; PIK3CA mutant) were most sensitive to GDC-0980, however they developed resistance to the drug more rapidly than the other 3 cell lines. Results from mutational analysis and investigation of the gene and protein expression of each of the 4 pairs of parent and resistant cell lines will be presented.

      Conclusion
      While the panel of four NSCLC cell lines all responded well to GDC-0980 treatment initially, resistance to the drug developed rapidly. As such, understanding the mechanisms involved in the development of resistance to this drug will be crucial so that we may design optimal treatment strategies. Specific conclusions regarding the mechanisms of resistance in this panel of cell lines will be drawn based on identified genes and proteins of interest.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P1.01-011 - Targeting the Urokinase Plasminogen Activator (uPA) System to overcome cisplatin resistance in NSCLC (ID 3347)

      09:30 - 16:30  |  Author(s): M. Barr

      • Abstract

      Background
      The urokinase plasminogen activator (uPA) system (uPAS) has been shown to play a significant multifunctional role in tumour progression including angiogenesis, adhesion and migration. Increased levels of urokinase plasminogen activator (uPA) and its receptor uPAR (CD87) strongly correlate with poor prognosis and a poor clinical outcome. It has been shown previously that a subpopulation of uPAR-positive cells in Small Cell Lung Cancer (SCLC) cell lines demonstrate significant drug resistance to traditional chemotherapeutic agents such as cisplatin, 5-fluorouracil (5-FU) and etoposide. The uPAS is regulated by NF-κB which has been shown to be constitutively activated in several cancer types including non-small cell lung cancer (NSCLC). Furthermore, we have shown NF-κB to be involved in the development of resistance to cisplatin in NSCLC. This project focuses on determining the role of the uPA system in the invasive phenotype of cisplatin resistant NSCLC cells.

      Methods
      Expression of NF-κB (p65) in parent and resistant NSCLC cell lines was quantified by qPCR, western blot and high content screening (HCS). The expression profiles of NFκB target genes were quantified using a Roche custom NFκB RTPCR array. Gene “hits” with a fold change >2 between parent and cisplatin resistant cells were validated by qPCR analysis. The upregulation of the urokinase-type plasminogen activator (uPA) in cisplatin resistant cells was determined by western blot. The effect of uPA inhibition on cell migration and invasion, using the monoclonal anti-uPAR antibody ATN-658, is being determined using the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform.

      Results
      Gene expression data, from the NFκB target gene array identified a panel of genes including; PLAU (gene for uPA), RIPK and NLRP12 amongst others that were over-expressed in H460 cisplatin resistant cell lines compared to the isogenic parent cell line. uPA overexpression at the protein level was confirmed in a panel of cisplatin resistant cells compared to parent cell lines. The effect of ATN-658 on the inhibition of cell migration and invasion in cisplatin sensitive and resistant cell lines will be presented.

      Conclusion
      Overexpression of uPA across a panel of cisplatin resistant NSCLC cell lines highlights its significance as a marker of resistance. Targeting the uPA system may be exploited in cisplatin resistant NSCLC to inhibit cell migration and invasion.

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      P1.01-016 - Targeting NF-κB regulated pathways to overcome cisplatin resistance in non small cell lung cancer (ID 3270)

      09:30 - 16:30  |  Author(s): M. Barr

      • Abstract

      Background
      Cisplatin based doublet chemotherapy is the mainstay of non small cell lung cancer (NSCLC) treatment with an initial objective response rate of approximately 40-50%. However, intrinsic and acquired resistance to cisplatin constitutes a major clinical obstacle in lung cancer management and has yet to be fully understood. Inflammatory mediators may play an important role in the development of cisplatin resistance, such as those regulated by NF-κB. We have previously demonstrated that levels of NF-κB are increased in cisplatin resistant cells compared with sensitive Parent cells. We are currently assessing a number of NF-κB regulated targets in cisplatin resistant cell line models, using DHMEQ, a specific NF-κB inhibitor. DHMEQ treatment results in greater cell death in the cisplatin resistant cells compared with Parent. This study will elucidate the efficacy of DHMEQ to overcome cisplatin resistance and identify novel targets within the NF-κB pathway that may improve therapeutic strategies for NSCLC patients.

      Methods
      NF-κB downstream targets and signalling mediators were examined using NF-κB signalling and target pathway qPCR arrays (168 genes) in the H460 CisR and Parent cell line model. Targets identified are currently undergoing validation using qPCR and western blot. Biological and functional relevance of these targets in the development of cisplatin resistance will be examined further using DHMEQ and siRNA knockdown strategies. In addition, a xenograft murine model will be utilised to assess the effect of DHMEQ alone and in combination with cisplatin on tumour growth in vivo.

      Results
      Data from qPCR arrays have demonstrated that a number of genes are differentially regulated between the CisR and Parent cell lines. These include genes which activate the NF-κB signalling cascade (TLR3, TLR4), regulators of the pathway (BIRC3, CASP1), transcription factors (Myc) and NF-κB responsive genes (TNF, CXCL8). A number of these genes will be modulated to determine their involvement in cisplatin resistance. In addition, DHMEQ is being used in combination studies to determine, whether it can re-sensitise cells to cisplatin therapy. At present a dosing study is ongoing to establish the effect of DHMEQ on xenograft tumours derived from Parent and CisR cells. The results of which will be presented.

      Conclusion
      Preliminary data indicates that NF-κB and a number of its downstream targets are deregulated in cisplatin resistant cells. This project aims to validate the role of these NF-κB regulated genes in cisplatin resistant NSCLC. It will also determine whether DHMEQ may be a novel targeted agent for the treatment of NSCLC. The data obtained in this study will ultimately benefit patients by providing insights into novel druggable targets and new clinical strategies to re-sensitise patients to cisplatin therapy.

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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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      P2.01-016 - Targeting the PI3K-mTOR-NFκB pathway to overcome cisplatin resistance in NSCLC. (ID 2788)

      09:30 - 16:30  |  Author(s): M. Barr

      • Abstract

      Background
      Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality in the Western world with a poor overall 5 year survival of <15%. The most effective systemic chemotherapy for NSCLC is cisplatin-based combination treatment. However, chemoresistance is a major therapeutic problem and understanding the mechanisms involved is critical to the development of new therapeutic intervention strategies. The PI3K pathway plays an important role in NSCLC and we and others have shown increased PI3K signaling to be associated with a more aggressive disease with poor prognosis. Several proteins in this pathway have been indicated as potential mediators of cisplatin resistance in other cancers, and our group has previously identified the PI3K-activated transcription factor NFκB as a key player in this setting. In this study, targeted inhibition of three strategic points of the PI3K pathway was carried out with the aim of overcoming acquired resistance to cisplatin in these cell lines.

      Methods
      A panel of cisplatin resistant cell lines was previously generated in our laboratory through prolonged exposure to the drug. Expression of PI3K pathway related genes was compared between H460 parent (H460PT) and H460 cisplatin resistant (H460CR) cells using a PI3K pathway SABiosciences RTPCR array. Identified genes of interested were further investigated via PCR and Western blot in these cells as well as A549 parent (A549PT) and A549 cisplatin resistant (A549CR) cells. Three strategic points of the pathway were inhibited using GDC-0980, a dual PI3K-mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFkB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors on the parent & cisplatin resistant cell lines both with and without cisplatin were assessed by BrdU proliferation assay and multiparameter apoptosis assay (High Content Analysis).

      Results
      One of the most notable targets to emerge from the PI3K pathway RTPCR array screen was NFKBIA; the gene which codes for NFκB inhibitor IκBα. This gene was shown to be 12 fold overexpressed in H460CR compared to H460PT. This finding was validated at both the RNA and protein level by PCR and Western blot. NFκB was also found to be overexpressed in cisplatin resistant cells compared to their respective parent cells. Inhibition of NFκB by DHMEQ led to significantly improved inhibition of proliferation and induction of apoptosis in cisplatin resistant cells compared to parent cells. Preliminary data indicates that inhibition of PI3K and mTOR by GDC-0980 did not offer as significant a benefit as inhibition of NFκB in the cisplatin resistance setting, though further data from combination studies will be presented.

      Conclusion
      We conclude that the PI3K pathway plays an important role in resistance to cisplatin in NSCLC, particularly when signaling proceeds through the transcription factor NFκB. Targeting this pathway may be of benefit in re-sensitizing cisplatin resistant tumours to the drug.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.01-012 - Co-targeting the PI3K and MEK pathways in NSCLC: an in vitro evaluation and mutation prevalence in an Irish patient cohort. (ID 2794)

      09:30 - 16:30  |  Author(s): M. Barr

      • Abstract

      Background
      PI3K pathway activation in NSCLC has been shown by us and others to lead to a more aggressive disease correlating to poor prognosis for patients. Unfortunately, the success of PI3K targeted inhibition has been hampered by a high rate of innate and acquired resistance. Mutations in KRAS and B-RAF, ERK hyperactivation as well as extensive PI3K-MEK pathway cross-talk allow the MEK pathway to provide a bypass track. Preclinical studies demonstrate a rationale for a PI3K-MEK co-targeted treatment strategy which may provide a more effective response. A Phase I clinical trial is underway investigating the combination of GDC-0941, a pan-PI3K inhibitor, with GDC-0973, a MEK inhibitor. GDC-0980 is a dual PI3K-mTOR inhibitor which may offer improved pathway inhibition compared to GDC-0941. No data has been published to date on the combination of GDC-0980 and GDC-0973, which we believe may offer improved overall inhibition of survival signaling in NSCLC cells. We aim to elucidate the role of mutation status in response to this co-targeted inhibition approach in vitro, as well as investigating the frequency of PI3K and MEK pathway mutations in a well characterized Irish NSCLC patient cohort.

      Methods
      The effects of GDC-0941, GDC-0980 and GDC-0973 on proliferation and apoptosis in a panel of four NSCLC cell lines were analysed by BrdU Assay and HCA Apoptosis Assay, respectively. The four cell lines investigated were H460 (adenocarcinoma, PIK3CA mutant & KRAS mutant), A549 (adenocarcinoma, PIK3CA wild type & KRAS mutant), H1975 (adenocarcinoma, PIK3CA mutant, KRAS wild type & EGFR TKI resistant) and SKMES-1 (squamous cell carcinoma, PIK3CA wild type & KRAS mutant). Further investigation involved expression analysis of pAkt, pGSK-3β, pp70S6K, pS6RP, ERK and pERK in cell lines treated with each inhibitor alone or in combination using Mesoscale technology and Western blot. DNA was extracted from 120 NSCLC patient tissue samples, and screened for 547 mutations in 59 genes (including PI3K and MEK pathway members) using the Sequenom.

      Results
      GDC-0941 and GDC-0980 treatment induced dose-dependent anti-proliferative and pro-apoptotic responses across all four NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. Protein expression analysis showed that GDC-0980 & GDC-0973 combination treatment induced significantly improved phosphoprotein inhibition compared to treatment with either inhibitor alone in cell lines harbouring PIK3CA mutations, while in one cell line bearing WT PIK3CA (SKMES-1), combination treatment actually increased pathway signalling. NSCLC patient mutational profiling data will be presented.

      Conclusion
      This research underpins the importance of mutation status in sensitivity to targeted therapies. While combination treatment approaches may be beneficial in certain molecular subtypes, in others they may be detrimental. In the era of personalised medicine, patient genotyping is crucial to improve patient survival and reduce toxicities.

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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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      P3.05-021 - Targeting HDACs to overcome cisplatin resistance in malignant plural mesothelioma (ID 3207)

      09:30 - 16:30  |  Author(s): M. Barr

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive cancer of the mesothelial cells and is becoming a worldwide health burden. Progress in the treatment of MPM remains difficult, underscored by the fact that no single treatment option has proven particularly effective and chemo-resistance is a common problem. However, epigenetic modifiers, such as histone deacetylase inhibitors (HDi) have emerged as a novel class of anti-cancer agent and are currently undergoing clinical trials in numerous cancers. There is also evidence to suggest that HDAC expression may play a role in the development of chemo-resistance. We investigated the levels of HDACs in MPM cell lines, patient samples and determined the effect of HDi on MPM cisplatin sensitive and resistant cell lines.

      Methods
      A panel of MPM cell lines (n=16) was screened for the expression of HDAC11, Class I (HDAC1/2/3/8) and Class II (HDAC4/5/6/7/9/10) HDACs at the mRNA level (RT-PCR) and at the protein level (Western Blot). The HDAC expression profile was determined in an isogenic cell line model consisting of a cisplatin sensitive (Parent) and resistant (CisR) MPM cell line, P31. In addition, HDAC expression was examined in panel of twenty patient samples (benign/biphasic/sarcomatoid/epithelial). Also, MPM TMAs were stained by imummo-histochemistry for HDAC5 expression. Furthermore proliferation assays (BrdU ELISA) were performed to determine the effect of two HDi on the p31 cell lines. The HDi used were Vorinostat (pan HDAC inhibitor) and 19i (selective HDAC5 inhibitor).

      Results
      HDAC11, Class I and II HDACs were detected to varying degrees at the mRNA and protein level within our cell line panel. In the P31 isogenic parent/cisplatin resistant, HDAC protein expression was decreased (HDAC2/3/4/5/7) in the CisR compared with the Parent. HDAC5 was significantly reduced at both the protein and the mRNA level (p<0.05). The expression of HDACs 1/2/3/5 were increased in the MPM patient samples (n=15) compared with benign (n=5) (HDAC2/3/5, p<0.05). HDAC5 staining in a cohort of MPM samples, demonstrated no significant association between survival and a low HDAC5 score. However females had a greater median survival than their male counterparts. Vorinostat and 19i significantly reduced the proliferative capacity of the p31 Parent and CisR. SAHA was more effective in the Parent cells compared with CisR. The effect of 19i was similar in both cell lines.

      Conclusion
      Altered HDAC expression was observed in an isogenic Parent and CisR MPM cell model. Work ongoing in non-small cell lung cancer (NSCLC) has also demonstrated significantly reduced HDAC5 levels in CisR compared with Parent. This may suggest that a reduction in HDAC expression is involved in cisplatin resistance. Reduced levels of HDAC5, in an MPM TMA, were not associated with survival. It should be noted, however that patient numbers were small and biphasic and sarcomatoid sub-types had the lowest expression levels of HDAC5. We are currently increasing our patient numbers for an additional IHC study. HDi significantly reduced the proliferative capacity of CisR and Parent MPM cells. HDAC levels may represent an important biomarker in stratifying patients for future epigenetic targeted therapies in MPM.