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S. Yano
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MA07 - ALK-ROS1 in Advanced NSCLC (ID 385)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Advanced NSCLC
- Presentations: 1
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MA07.10 - HDAC Inhibition Overcomes Crizotinib-Resistance by Mesenchymal-Epithelial Transition (MET) in EML4-ALK Lung Cancer Cells (ID 4367)
12:06 - 12:12 | Author(s): S. Yano
- Abstract
- Presentation
Background:
ALK rearrangement, most commonly EML4-ALK, is detected in approximately 3–7% of non-small cell lung cancer (NSCLC). Crizotinib, an ALK tyrosine kinase inhibitor (TKI), shows dramatic clinical efficacy in ALK-rearranged NSCLC patients. However, almost all patients acquire resistance after only 1 to 2 years. A variety of mechanisms, including ALK-secondary mutations, ALK amplification, and activation of alternative pathway, have been reported to mediate acquired resistance to crizotinib. While epithelial–mesenchymal transition (EMT) was recently reported to be associated with resistance to crizotinib in EML4-ALK lung cancer cells in vitro, the underlying mechanism has not been defined and no optimal therapy to overcome EMT-associated resistance has been identified.
Methods:
We continuously gave crizotinib treatment to SCID mice inoculated with EML4-ALK lung cancer cell line A925L into thoracic cavity and established crizotinib resistant A925LCR cells. After the limiting dilution of A925LCR cells, we obtained several single cell clones. The effects of the HDAC inhibitor quisinostat on the EMT state and the growth of the cells were examined in vitro and in vivo.
Results:
We found that some clones acquired EMT phenotypes, such as spindle shape morphology, expression of EMT-related proteins, and increased cell motility. Interestingly, Histone deacetylase (HDAC) inhibitor, quisinostat, induced mesenchymal-epithelial transition (MET) of A925LCR clones in vitro. Quisinostat reduced ZEB1 expression, induced MET, and thus restored sensitivity to crizotinib. Knockdown of ZEB1 expression in the A925LCR clones by si-RNA also induced MET and restored sensitivity to crizotinib, suggesting that quisinostat-induced MET depends on ZEB-1 suppression. MicroRNA profile analysis revealed that the A925LCR clones expressed significantly lower levels of miR-200 family including miR-200c which targets ZEB1, compared with parental A925L cells. Furthermore, quisinostat recovered miR-200c expression and antago-miR-200c abrogated quisinostat-induced MET in the A925LCR clone cells. These results indicate that quisinostat induced MET by up-regulating miR-200c expression which target ZEB1 and thereby re-sensitizing to crizotinib. In a pleural carcinomatosis model with A925LCR clone cells, quisinostat induced MET and caused remarkable tumor regression during the subsequent crizotinib re-challenge. Furthermore, we analyzed tumor tissue obtained at autopsy from an ALK-rearranged NSCLC patient who acquired resistance to crizotinib. We found that EMT was induced in both primary and metastasis lesions after crizotinib treatment, indicating that EMT is associated with crizotinib resistance in clinical therapy.
Conclusion:
Our findings suggest that EMT is possibly occurred in acquired resistance to crizotinib and intermittent use of HDAC inhibitor could be a novel therapeutic strategy for overcoming EMT-associated crizotinib-resistance in EML4-ALK lung cancer.
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OA08 - Targeted Therapies in Brain Metastases (ID 381)
- Event: WCLC 2016
- Type: Oral Session
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:D. Ball, B. Perin
- Coordinates: 12/05/2016, 16:00 - 17:30, Schubert 1
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OA08.03 - MET Copy Number Gain Associates with Gefitinib Resistance in Leptomeningeal Carcinomatosis of EGFR Mutant Lung Cancer (ID 4364)
16:20 - 16:30 | Author(s): S. Yano
- Abstract
- Presentation
Background:
Central nervous system (CNS) metastasis, such as brain metastasis and leptomeningeal carcinomatosis (LMC), occurs frequently in EGFR mutant lung cancer. EGFR-TKIs are generally effective to CNS metastasis in EGFR mutant lung cancer patients who are naïve to TKI treatment. Nevertheless, progression of CNS lesions are frequently observed during EGFR-TKI treatment. Brain metastases are manageable by concomitant use of EGFR-TKI and local intervention, including whole brain irradiation and stereotactic radiotherapy. There is, however, no established therapy for LMC, which is resistant to first and second generation EGFR-TKIs. Therefore, novel and effective therapies need to be developed for managing LMC in EGFR mutant lung cancer patients who become refractory to these EGFR-TKIs. The purpose of this study is to clarify the mechanism of EGFR-TKI resistance in LMC and establish novel therapeutic strategy.
Methods:
We examined EGFR mutations, including T790M gatekeeper mutation, in 32 re-biopsy specimens from 12 LMC and 20 extracranial lesions (e.c., lung metastasis and malignant pleural effusions) of EGFR mutant lung cancer patients who became refractory to EGFR-TKI treatment. To clarify molecular mechanisms of acquired EGFR-TKI resistance in LMC, we utilized in vivo imaging model of LMC with EGFR mutant lung cancer cell line PC-9/ffluc and induced acquired resistance to gefitinib by continuous oral treatment.
Results:
We found that all 32 re-biopsy specimens had the same baseline EGFR mutations and that T790M was less frequent in LMC specimens than extracranial specimens (8% vs 55%). Compared with subcutaneous tumors, T790M was less frequent in LMC which acquired resistance to gefitnib. We further established PC-9/LMC-GR cells from the gefitinib-resistant LMC model and found that PC-9/LMC-GR cells were intermediately resistant to gefitinib and osimertinib (3[rd] generation EGFR-TKI). While EGFR-T790M was negative, MET copy number gain associated MET activation was involved in the gefitinib resistance in PC-9/LMC-GR cells. Moreover, combined use of EGFR-TKI and crizotinib, having inhibitory activity against MET, dramatically regressed LMC which already acquired resistance to gefitinib or osimertinib.
Conclusion:
These findings suggest that combined use of MET inhibitors may be promising for controlling LMC which acquires resistance to EGFR-TKIs including osimertinib.
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P1.03 - Poster Session with Presenters Present (ID 455)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Radiology/Staging/Screening
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.03-032 - In vivo Imaging Models for Preclinical Screening of Molecular Targeted Drugs against Brain Metastasis (ID 4433)
14:30 - 14:30 | Author(s): S. Yano
- Abstract
Background:
Background: Molecular targeted drugs are generally effective on tumors with driver oncogene, including EGFR, ALK, and NTRK1. However, patients with these oncogenes frequently experience progression of brain metastasis during the targeted drug treatment. Thus, it is essential to establish more effective treatment for controlling brain metastasis.
Methods:
Methods: We established in vivo imaging brain tumor models by intracranial inoculation of human cancer cell lines, such as lung adenocarcinoma H1975 cells with EGFR-L858R and T790M mutations, HGF-dependent gastric cancer NUGC4 cells, and TPM3-NTRK1-fusion gene positive colorectal cancer KM12SM cells, in SCID mice. We investigated the activity of several molecular targeted drugs on cell proliferation of these cell lines in vitro. In addition, we evaluated the efficacy of these drugs on brain tumor models, comparing with extracranial tumor models.
Results:
Results: In vitro conditions, H1975 cells were sensitive to the 3[rd] generation EGFR inhibitor, osimertinib. HGF stimulated proliferation of gastric cancer NUGC4 cells, and the HGF-induced proliferation was inhibited by crizotinib, which has anti-MET activity, in a dose-dependent manner. KM12SM cells, which are the highly liver metastatic variant derived from TPM3-NTRK1 fusion gene positive colon cancer KM12C cells. KM12SM were sensitive to TRK-A inhibitors, crizotinib and entrectinib. In H1975-cell in vivo models, osimertinib (25mg/kg) inhibited the progression of both brain tumors and subcutaneous tumors. In NUGC4-cell in vivo models, crizotinib (50mg/kg) delayed the progression of brain tumors as well as peritoneal carcinomatosis, and prolonged the survival of the tumor bearing mice. In KM12SM-cell in vivo models, we evaluated the effect of crizotinib (50mg/kg) or entrectinib (15mg/kg) in the brain tumor model and liver metastasis model. Crizotinib treatment slightly delayed the progression of brain tumors but failed to prolong the survival of the recipient mice. Entrectinib treatment more discernibly delayed the progression of brain tumors and did prolong the survival. These results indicate that the effect of targeted drugs against brain tumors can be different from that against extracranial tumors.
Conclusion:
Conclusion: Our in vivo imaging brain tumor models may be useful for preclinical drug screening against brain metastasis.
