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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
MINI35.11 - Mutant ARAF Drives Lung Carcinogenesis Through a Distinct Oncogenic Mechanism (ID 1016)
18:30 - 20:00 | Author(s): M. Imielinski
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.
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.
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.
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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