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M. Meyerson
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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): M. Meyerson
- 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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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-039 - Characterization of RNA Splicing Factor Mutations in Lung Adenocarcinoma (ID 2949)
09:30 - 09:30 | Author(s): M. Meyerson
- Abstract
Background:
Large-scale genomic surveys of lung adenocarcinoma have revealed unexpected mutations in RNA splicing factors such as U2AF1 and RBM10, and it remains unknown how changes in splicing are involved in promoting cancer. Somatic alterations in the RNA-binding protein RBM10 occur at a frequency of approximately 7% and consist predominantly of loss-of-function mutations. In this study, we sought to investigate the functional impact of RBM10 mutations in lung cancer.
Methods:
RBM10 mutant non-small cell lung cancer (NSCLC) cell lines were identified by analysis of Cancer Cell Line Encyclopedia gene expression data and Sanger sequencing of RBM10 coding exons. Ectopic expression of wildtype RBM10 or a control protein (BFP) was induced using the tetracycline-regulatory system. RNA–sequencing and JuncBASE software were used to identify differentially spliced transcripts between RBM10 wildtype and mutant cells. Changes in individual splicing events were validated by RT-PCR.
Results:
We have identified several NSCLC cell lines harboring loss-of-function mutations in RBM10. Restoring expression of wildtype RBM10 in these RBM10-mutant cell lines resulted in significant growth suppression and inhibition of anchorage-independent growth. These phenotypic effects were associated with a variety of splicing changes and expression of wildtype RBM10 frequently increased skipping of cassette exons. Expression of RBM10 variants with either deletion of an RNA recognition motif (RRM), or containing a cancer-associated missense mutation in the RRM, were significantly diminished in their ability to promote exon skipping and suppress cellular proliferation.
Conclusion:
Our results suggest that RBM10 functions as a novel tumor suppressor in lung adenocarcinoma through its effects on RNA splicing. Further work is needed to better understand how changes in specific splicing events may be directly contributing to lung tumorigenesis.
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PLEN 03 - Science Drives Lung Cancer Advances (ID 52)
- Event: WCLC 2015
- Type: Plenary
- Track: Plenary
- Presentations: 1
- Moderators:T. Mitsudomi, T. Mok
- Coordinates: 9/09/2015, 08:15 - 09:45, Plenary Hall (Bellco Theatre)
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PLEN03.01 - Lung Cancer Genomes - Adenocarcinoma (ID 2043)
08:20 - 08:35 | Author(s): M. Meyerson
- Abstract
- Presentation
Abstract not provided
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