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Shuta Ohara
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P76 - Targeted Therapy - Clinically Focused - EGFR (ID 253)
- Event: WCLC 2020
- Type: Posters
- Track: Targeted Therapy - Clinically Focused
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P76.71 - RYK Confers Drug Tolerance to Osimertinib in Lung Cancer Cells with EGFR Mutations (ID 3355)
00:00 - 00:00 | Presenting Author(s): Shuta Ohara
- Abstract
Introduction
Emergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance.
Methods
Five EGFR-mutant NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array.
Results
DTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. Next, we compared the phosphorylation levels of EGFR in HCC4006 cells after exposure to 600 nM osimertinib or 60 nM afatinib. 600 nM osimertinib inhibited the phosphorylation of EGFR in HCC4006 cells more than 60 nM afatinib. This was inconsistent with the experiments using PC9 cells (both 600 nM osimertinib and 60 nM afatinib induced drug tolerance in PC9 cells), showing that inhibition of EGFR phosphorylation was similar between 600 nM osimertinib and 60 nM afatinib.To investigate if the suppression level of EGFR phosphorylation was related to the acquisition of a drug-tolerant phenotype, we exposed HCC4006 cells to different concentrations of osimertinib or afatinib. Lower doses of osimertinib (66 nM and 200 nM) failed to induce a drug-tolerant state in HCC4006 cells, although the growth inhibition rate of 66 nM osimertinib for HCC4006 parental cells was identical to that of 600 nM osimertinib. However, higher doses of afatinib (180 nM and 540 nM) induced a drug-tolerant state in HCC4006 cells. We also observed that lower doses of osimertinib and afatinib failed to induce a drug-tolerant state in PC9 and H1975 cells. These results indicated that stronger inhibition of phosphorylated EGFR is necessary to induce DTCs. Next, in the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. This phenomenon was HCC4006 cell line-specific since we observed that PC9 parental cells and PC9-600 nM osim-DTCs were both resistant to RYK knockdown. In addition, cabozantinib, which reportedly inhibit RYK, monotherapy effectively inhibited the proliferation of HCC4006-600 nM osim-DTCs but not HCC4006 parental cells. However, cabozantinib was not active against PC9-600 nM osim-DTCs and PC9 parental cells.
Conclusion
Our study found that the inducibility of DTCs depended on the type of cell line and the drug concentration. It is possible that the optimal dose of EGFR-TKI in each patient may reduce the emergence of DTCs in clinical practice. We also found high expression of RYK was a molecular mechanism of the drug-tolerant state in HCC4006 cells against osimertinib. Further studies are necessary to fully understand the DTCs that are essential for the appropriate primary double-strike therapy for lung cancers with EGFR mutations.
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P86 - Targeted Therapy - Clinically Focused - New Target (ID 263)
- Event: WCLC 2020
- Type: Posters
- Track: Targeted Therapy - Clinically Focused
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P86.05 - In Vitro Validation Study for HER2 Mutations Identified in Secondary Analysis of the LUX-Lung 8 Randomized Clinical Trial (ID 3274)
00:00 - 00:00 | Author(s): Shuta Ohara
- Abstract
Introduction
LUX-Lung 8 demonstrated prolonged overall survival in patients with metastatic squamous cell carcinoma of the lung treated with afatinib compared to those treated with erlotinib. Its secondary analysis suggested that the patients with rare HER2 mutations contributed to the superiority of afatinib, since afatinib but not erlotinib has HER2 inhibitory activity (JAMA Oncol. 2018;4:1189-1197). However, transforming ability of all detected HER2 mutations but one has not been explored.
Methods
We have introduced ten HER2 mutations (Q57R, G152A, S250C, E265K, E395K, P489L, R683L, G815R, R929W and P1037L) into mouse pro-B cell line (Ba/F3). Since Ba/F3 requires interleukin 3 (IL-3) for its growth, IL-3 independent growth of Ba/F3 indicates that transduced mutation has transforming ability. For Ba/F3 cells which acquired IL-3 independent growth, we analyzed the efficacy of six EGFR or pan-ERBB tyrosine kinase inhibitors (TKIs) including afatinib, erlotinib, dacomitinib, neratinib, poziotinib, and osimertinib using growth inhibitory assay. The efficacy of six TKIs was compared using a sensitivity index as IC50 (50% inhibitory concentration) divided by the trough concentration at the recommended dose.
Results
Conclusion
Seven (Q57R, G152A, S250C, E265K, P489L, R683L and P1037L) out of ten Ba/F3 cells expressing HER2 mutations did not grow in the absence of IL-3, indicating that they were passenger or non-pathogenic mutations. On the other hand, three Ba/F3 cells expressing one of E395K, G815R or R929W acquired IL-3 independent growth, suggesting their transforming ability. The sensitivity indices for afatinib were five or more times higher than those of erlotinib in all three Ba/F3 lines (Figure 1). In the secondary analysis of the LUX-Lung 8 trial, patients with E395K and G815R mutations received afatinib, and survived without disease recurrence for 17 months and 6 months, respectively. The patient with R929W mutation received erlotinib, and survived without disease recurrence for only 2 months. Dacomitinib, neratinib, poziotinib, and osimertinib also effectively inhibited the growth of these three Ba/F3 lines.
HER2 E395K, G815R and R929W mutations had transforming ability, and 2nd/3rd generation TKIs, including afatinib, showed higher efficacy compared with erlotinib. These results may support the search of HER2 mutation in lung squamous cell carcinoma patients.
