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John Ashton
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P1.01 - Advanced NSCLC (Not CME Accredited Session) (ID 933)
- Event: WCLC 2018
- Type: Poster Viewing in the Exhibit Hall
- Track:
- Presentations: 1
- Moderators:
- Coordinates: 9/24/2018, 16:45 - 18:00, Exhibit Hall
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P1.01-05 - Metformin in Combination with Crizotinib for The Treatment of EML4-ALK+ Lung Cancer. (ID 12801)
16:45 - 18:00 | Author(s): John Ashton
- Abstract
Background
Lung cancer accounts for the highest incidence of cancer mortality worldwide, equating to around 2 million deaths each year. Oncogenic receptor tyrosine kinases (RTK) have been discovered to play a role in cancer progression, and can be the target of novel therapeutics. The anaplastic lymphoma kinase receptor (ALK) is an RTK, and as a result of a chromosomal 2 rearrangement, fuses with echinoderm microtubule associated protein like-4 (EML4) forming the oncogenic EML4-ALK receptor. EML4-ALK contributes to 2-7% of all lung cancer cases and currently the first-line treatment is crizotinib, a tyrosine kinase inhibitor. Although crizotinib produces tumor regression, most patients will develop resistance, constituting a major research problem. Novel strategies are being explored and one such treatment is the anti-hyperglycemic, metformin. Epidemiological studies have shown that metformin reduces the risk of cancer and pre-clinical studies are examining this cytotoxic effect. This work aimed to determine the cytotoxicity and mechanism of a crizotinib/metformin combination in vitro in EML4-ALK+ lung cancer.
a9ded1e5ce5d75814730bb4caaf49419 Method
Crizotinib, metformin and the combination were examined using EML4-ALK+ human lung adenocarcinoma cells (H3122), ALK-/KRAF+ human lung adenocarcinoma cells (A549) and crizotinib-resistant (CR-H3122) cells maintained in crizotinib (0.8 µM). Cytotoxicity was tested using the sulforhodmaine B assay and to assess the validity of this assay the MTT assay was used as a comparison. Western blotting examined changes in ALK, p-ALK, mTOR, p-mTOR. Propidium iodine staining and flow cytometry examined changes in the cell cycle in H3122 cells.
4c3880bb027f159e801041b1021e88e8 Result
Crizotinib exhibits greater potency in H3122 cells compared to the ALK- A549 (IC50’s of 0.16 µM vs. 1 µM). Interestingly, the addition of metformin (5 mM) to both cell lines did not change the IC50, however, reduced cell viability by ~30% at lower doses of crizotinib. The MTT assay found the same effect. The IC50 of metformin in H3122 cells was 22 mM, whereas this potency was lost in CR-H3122 cells (only a 50% reduction in cell viability with 50 mM). Mechanistically it was discovered that metformin decreases mTOR phosphorylation, a downstream protein of ALK, but has no effect on p-ALK. Both crizotinib and metformin produced a G1 phase arrest, with the effect being enhanced with the combination.
8eea62084ca7e541d918e823422bd82e Conclusion
Metformin enhances the effect of low dose crizotinib. It appears that metformin acts by targeting the downstream protein, mTOR, and produces a G1 phase arrest, instead of directly inhibiting ALK. The addition of metformin to chemotherapeutics provides a potential treatment for EML4-ALK+ and other lung cancers.
6f8b794f3246b0c1e1780bb4d4d5dc53
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P2.01 - Advanced NSCLC (Not CME Accredited Session) (ID 950)
- Event: WCLC 2018
- Type: Poster Viewing in the Exhibit Hall
- Track:
- Presentations: 1
- Moderators:
- Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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P2.01-89 - Synergistic Cytotoxicity Through MAPK/ERK Pathway and ALK Inhibition in Crizotinib Resistant EML4-ALK-Positive Lung Cancer (ID 11310)
16:45 - 18:00 | Author(s): John Ashton
- Abstract
Background
Anaplastic lymphoma kinase (ALK)-positive lung cancer is an aggressive cancer that most commonly arises through EML4-ALK chromosomal fusion. Crizotinib is the first-line treatment for ALK-positive lung cancer, providing a greater progression free survival and overall response rate than chemotherapy. However, patients invariably develop acquired resistance, usually within a year. Mechanisms of resistance include copy number gain, secondary mutations in ALK, or activation of bypass signalling pathways. The aim of this study was to investigate the combination effect of ALK inhibitor, crizotinib, and MEK inhibitor, selumetinib, in crizotinib resistance ALK-positive lung cancer cells.
a9ded1e5ce5d75814730bb4caaf49419 Method
Cytotoxicity of single and combined treatment of crizotinib and selumetinib in ALK-positive (H3122), ALK-negative (A549) and crizotinib resistance ALK-positive (CR-H3122) cells was determined by Sulforhodamine B assay. Chou-Talay analysis was performed to determine the nature of interaction between the two drugs used in combination treatment. Endogenous expression of p-ALK and p-ERK was determined by western blotting. The effect of combination treatment on cell cycle arrest and apoptosis induction was determined by flow cytometer.
4c3880bb027f159e801041b1021e88e8 Result
Combined treatment with crizotinib and selumetinib had significantly greater cytotoxicity and showed a synergistic effect compared to the single drug treatments in H3122 cells. The combination treatment had no synergistic effect in A549 cells, indicating that the cytotoxic effect was specific to ALK. The most prominent cytotoxic effect of the combination treatment was observed with CR-H3122 cells, with synergistic suppression of cell viability even at low drug concentrations. Endogenous protein expression of p-ALK and p-ERK was decreased by combination of crizotinib and selumetinib. Moreover, combination treatment lead to increased G1 arrest and apoptosis compared to single treatment in H3122 cells.
8eea62084ca7e541d918e823422bd82e Conclusion
Our results showed that combination of crizotinib and selumetinib have potent cytotoxic effects in crizotinib resistant ALK-positive lung cancer. The proposed combination treatment could be a promising therapy for acquired crizotinib resistant ALK-positive lung cancers.
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