Figure 4. T55delinsRT mutation in MEK1 is responsible for resistance to vemurafenib in M032R4.

1. Somatic mutations present in each of these metastases, revealing that mutations were either shared by the pre-treatment tumor and all metastases, by some metastases, or only by single metastases.
2. A 3-bp in-frame insertion in MEK1 (T55delinsRT) was identified in M032R4. This mutation was located at the base of the α-helix at the N-terminal site of MEK1.
3. Validation of T55delinsRT was done by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1^WT were used.
4. Transfection of pQXCIP-GFP, MEK1-WT, or MEK1-T55delinsRT into HEK293T cells showed that this mutation induces p-ERK and p-RSK.
5. Validation of expression of pQXCIP-GFP, MEK1^WT, or MEK1^T55delinsRT in A375 melanoma cells by immunoblotting.
6. Dose–response curves for A375 melanoma cells expressing GFP, MEK1^WT, or MEK1^T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.
7. Colony formation assays with A375 melanoma cells, infected with GFP, MEK1^WT, or MEK1^T55delinsRT-encoding lentivirus, and treated with indicated doses and inhibitors.
8. Treatment of A375 MEK1^T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).

Source data are available online for this figure.