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L.H. Araujo
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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T.A. Boyle, M.G. Kris
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 2c-3c
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MINI35.11 - Mutant ARAF Drives Lung Carcinogenesis Through a Distinct Oncogenic Mechanism (ID 1016)
19:25 - 19:30 | Author(s): L.H. Araujo
- Abstract
- Presentation
Background:
We recently identified a novel somatic mutation in ARAF in a lung adenocarcinoma from a patient that demonstrated a remarkable response to sorafenib. The S214C lies in a negative regulatory domain of ARAF, distinct from the catalytic domain mutations commonly found in BRAF. The aim herein was to characterize the biochemical and functional aspects of ARAF S214C.
Methods:
ARAF constructs were generated and ectopically expressed in an immortalized bronchial epithelial cell line (BEAS-2B). We evaluated the acquisition of anchorage independence, MEK activation, and cell morphology. COS7 cells were used for co-immunoprecipitation (IP) and kinase assays.
Results:
Cells expressing ARAF S214C substantially increased soft agar colony formation relative to vector, wild-type, kinase-dead (D429A), and double-mutant (S214C+D429A) variants. Accordingly, ARAF S214C cells exhibited increased phospho-MEK levels, suggesting that the transforming potential is dependent on its kinase activity. Interestingly, ARAF S214C cells acquired an elongated, fibroblast-like shape, characteristic of MEK-active cells, whereas none of other variants presented this morphology. We also demonstrated that cells expressing ARAF S214C with an additional RAS-binding domain mutation (R52L) or dimerization interface mutation (R362H) lacked MEK activation, showing that RAS binding and RAF-RAF dimerization are essential for activity. To elucidate the role of BRAF and RAF1 as dimerization partners of ARAF S214C, we performed knockdowns of BRAF, RAF1, or both. ARAF S214C-induced MEK activation was not reversed by the BRAF knockdown, however both RAF1 and double knockdowns (BRAF and RAF1) led to loss of MEK activation, suggesting that RAF1 is required. Subsequently, COS7 cells were co-transfected with tagged constructs of ARAF and either BRAF or RAF1, followed by co-IP. We showed that mutant ARAF presents a higher rate of dimerization than wild-type ARAF in the presence of sorafenib. Importantly, sorafenib-induced heterodimers lacked kinase activity, compatible with the clinical response reported.
Conclusion:
ARAF S214C demonstrates the in vitro features of a driver oncogene, and also a distinct mechanism of action. This oncogenic process can be successfully suppressed by RAF inhibitors like sorafenib, and could represent a new target for personalized therapy in advanced lung adenocarcinoma. Figure 1 Figure: Summary of the ARAF S214C oncogenic mechanism.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-018 - Whole Transcriptome Analysis of EGFR Wildtype Non-Small Cell Lung Cancer Patients with Clinical Benefit from Erlotinib (ID 2357)
09:30 - 09:30 | Author(s): L.H. Araujo
- Abstract
Background:
Despite the success of targeted assays of EGFR mutations in defining the non-small cell lung cancer patients who benefit from EGFR-tyrosine kinase inhibition, there still remains a significant portion of patients whose tumors do not harbor EGFR mutations, yet achieve clinical benefit (progression-free survival > 6 months) from erlotinib treatment. We apply whole transcriptome sequencing (RNAseq) to discover expression and mutation changes associated with erlotinib response.
Methods:
We report the results of 108 stage IV non-small cell lung cancer patients treated with first line erlotinib. The primary endpoint assessed was progression-free survival (PFS), to which erlotinib has already shown to be beneficial when compared to placebo. Furthermore, RNAseq was performed on 73 tumors from 29 (40%) males and 44 (60%) females. The RNAseq samples were processed to obtain mutation and expression data.
Results:
108 patients were followed for PFS, 7 of which declined to be followed, 2 came off erlotinib due to toxicity, 3 died before completion of the first cycle of erlotinib, 5 were ineligible, and 2 have not had tumor recurrence to date. The remaining 92 patients had a mean PFS of 4.71 months (±1.03 months, 95% CI). No patients experienced a complete response, and 14 of 92 (15%) patients had a partial response. Of the tumors analyzed via RNAseq, 7 harbored EGFR mutations, including a complex exon 18 deletion in a patient with a partial response to erlotinib. 14 of 64 (22%) patients without EGFR mutations showed clinical benefit from erlotinib, none of which harbored other known actionable mutations. These EGFR wildtype tumors did not exhibit mutations in other known oncogenes in lung cancer. We hypothesize that they are addicted to EGFR signaling through other means than overactive kinase activity caused by activating mutations. Figure 1
Conclusion:
We present results from a clinical trial of first line erlotinib in stage IV non-small cell lung cancer. We show that there is a significant cohort of EGFR wildtype patients who receive clinical benefit from erlotinib and present preliminary data of their mutation status.
