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S. Heavey
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-065 - Elucidating the Role of PIM Kinase and Its Therapeutic Potential in NSCLC (ID 4328)
14:30 - 14:30 | Author(s): S. Heavey
- Abstract
Background:
PIM kinases are a family of three serine/threonine kinases: PIM1, PIM2 and PIM3 that have been shown to play a role in tumorigenesis. PIM1 is a downstream effector of oncoproteins ABL and JAK/STAT and regulator of BCL2/BAD and CXCR4. PIM activity is synergistic with the PI3K/Akt/mTOR pro-survival pathway and PIM2 has been shown to phosphorylate translational repressor 4E-BP1 and p70S6 independently of the PI3K pathway. Furthermore a synergism between PIM kinases and c-Myc has been reported. Here we investigate the expression of PIM1/PIM2/PIM3 in NSCLC cell lines and patient matched normal/tissue samples. The effect of a novel combined inhibitor of PI3K/mTOR/PIM kinases (IBL-301) on cell signalling, cell death and proliferation is also examined.
Methods:
PIM1/PIM2/PIM3 expression were examined by Western blot analyses in NSCLC cells (H1975 and H1838). Additionally, the frequencies of PIM1/PIM2/PIM3 expression in NSCLC patient tumour and matched normal adjacent samples (n=31) were investigated. The effectiveness of IBL-301 on cell signalling, cell viability and proliferation were examined by Western blot analysis, cell titre blue and BrdU assay respectively.
Results:
All three PIM isoforms were detected in the lung cancer cell lines tested. Similarly, all three PIM isoforms were expressed across the 31 NSCLC patient tumour and match normal adjacent tissue samples. To investigate this further PIM1 staining of FFPE tumour and match normal tissue from this cohort is currently underway. In two lung cancer cell lines, H1975 and H1838, IBL-301 was found to have a dose dependent effect on proliferation/viability with IC~50~ values in the nanomolar range. Additionally, western blot analyses have indicated that these novel drugs can suppress the phosphorylation of key players in cell signalling pathways linked to tumorigenesis including pAkt, p4E-BP1 and peIF4B.
Conclusion:
This is the first study to investigate the expression of all 3 isoforms of PIM in lung cancer specifically. All 3 isoforms were abundantly expressed across cells lines and patient tumour samples. Observed PIM expression in the immune cells of normal adjacent tissue may indicate a role in inflammation. This finding coupled with the promising in vitro data demonstrate the therapeutic potential of targeting PIM in NSCLC.
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-031 - CCL Chemokines May Play an Important Role in Cisplatin Resistance (ID 4861)
14:30 - 14:30 | Author(s): S. Heavey
- Abstract
Background:
In the absence of a targetable mutation, cisplatin based chemotherapy is the backbone of NSCLC treatment. However, a diverse patient population combined with complex tumour heterogeneity is hampering its’ clinical utility. Although intrinsic and acquired resistance to cisplatin is common, the mechanisms have not yet been fully elucidated. However, some studies have suggested that inflammatory pathways may play a key role in chemo-resistance. The aim of this project is to increase our understanding of inflammatory mediated cisplatin resistance in NSCLC.
Methods:
A number of isogenic cell line models of NSCLC (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) cisplatin resistance were utilised to assess the role of inflammation in chemo-resistance. These included a sensitive parental cell line (PT) and a matched resistant subtype (CisR). The cell lines were screened for NFKB and a number of inflammatory mediators including chemokines and TLRs at the mRNA (RT-PCR/qPCR) and protein level (Western Blot/ELISA). A specific NFKB inhibitor, DHMEQ, and recombinant chemokines were employed to further characterise inflammatory pathways in PT and CisR cells in terms of cisplatin sensitivity, proliferation (BrdU ELISA), cellular viability (Cytell Cell Imaging System) and DNA damage response (Comet). An in vivo study was also completed using DHMEQ alone and in combination with cisplatin.
Results:
A number of NFKB targets and responsive pathways are deregulated in CisR cells compared with their matched sensitive PT cell line. Amongst others, CCL2 and CCL5 were altered across all NSCLC subtypes. Preliminary data suggests that DHMEQ enhances cisplatin sensitivity in both PT and CisR cells, conversely recombinant chemokines elicit a protective effect. Additionally, DHMEQ treatment resulted in opposite affects on CCL2 and CCL5 mRNA levels in the PT and CisR cell lines. This may reflect an alternative pathway hierarchy within the cells. Further characterisation is ongoing assessing chemokine specific inhibitors. Although, in vivo data suggests a trend of decreased tumour growth in the DHMEQ cohorts compared with vehicle control, the data was not significant. However, tumour samples appeared more necrotic with DHMEQ and are currently being characterised using IHC for necrosis and proliferation.
Conclusion:
Targeting chemokines downstream of NFKB may provide a means to overcome inflammatory mediated acquired and intrinsic NSCLC chemo-resistance. Given the increased significance of immuno-oncology agents to harness the body’s own immune system in the fight against cancer, these agents may also prove fruitful in re-sensitising patients to chemotherapy.
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P3.02c - Poster Session with Presenters Present (ID 472)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.02c-010 - Resistance Mechanisms to PI3K-mTOR Inhibition in NSCLC (ID 5355)
14:30 - 14:30 | Author(s): S. Heavey
- Abstract
Background:
Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality globally, having a 5 year survival rate of less than 15%. PI3K-mTOR signalling has been implicated in various hallmarks of cancer and this pathway is often dysregulated in NSCLC. Efforts to therapeutically target the PI3K-mTOR pathway have been hindered by emerging drug resistance. In this study, mechanisms of drug resistance were investigated in a H1975 cell line model of acquired resistance, following chronic exposure to a phase II PI3K-mTOR inhibitor (GDC-0980), Additionally, short term exposure of BEZ235 (another phase II PI3K-mTOR inhibitor) and IBL-301 (a novel PIM/PI3K/mTOR inhibitor) were investigated for effects on cell viability/proliferation and downstream signalling pathways.
Methods:
Alterations to the mRNA expression profile of GDC-0980 acquired resistant H1975 cells versus matched parent cells were examined using an 84-gene IL-6/STAT3 signalling-specific profiler array. Subsequently, selected genes were validated by qPCR. The effectiveness of BEZ235 and IBL-301 on cell viability of two lung cancer cell lines (H1975 and H1838) following 72 hour treatment were investigated by Cell Titre Blue. pAkt and p4E-BP1 expression were examined by Western blot analyses following treatment with BEZ235 and IBL-301 at 3, 6 and 24 hours.
Results:
Thirty candidate gene alterations were identified from the array profile and six genes were chosen for validation by qPCR (n=3). The pro-proliferative and pro-metabolic regulator mTOR and the anti-apoptotic protein BCL-2 were increased in GDC-0980 resistant cells (p<0.05 and p<0.001). Similarly, TNF-α and its receptor co-stimulatory molecule CD40 were increased (p<0.05 and p<0.01). Furthermore, the cell cycle inhibitor, CDKN1, and JAK-signalling blocker, SOCS1 were downregulated (both p<0.01) in GDC-0980 resistant cells. BEZ235 and IBL-301 had a dose-dependent effect on the viability of NSCLC cell lines with respective IC~50~ values of 9.42nM and 136.55nM in H1975 cells and 103.35nM and 159.27nM in H1838 cells. Treatments of 250nM BEZ235 or IBL-301 inhibited pAKt at all time points in the lung cancer cell lines. BEZ235 blocked translation repressor protein (p4E-BP1) across all 3 cell lines and time points while IBL-301 treatment resulted in a reduction in p4E-BP1 at 24 hours.
Conclusion:
This study highlights a number of genes involved in IL-6/STAT3 signalling that may contribute to PI3K-mTOR inhibitor resistance. BEZ235 and IBL-301 both decrease cell viability and inhibit PI3K pathway signalling and cap-dependent translation in NSCLC cell lines that warrant further investigation.
