MGH 134 CCDC 6 RET MGH 134 EV

MGH 134 CCDC 6 -RET MGH 134 EV MGH 134 LC 2/ad PC 9 EV PC 9 CCDC 6 -RET 1500 CCDC 6 -RET 1000 TBP 200 Supplementary Figure 1. Detection of the CCDC 6 -RET fusion gene or internal control TBP transcripts from LC 2/ad cells and PC 9 CCDC 6 -RET or MGH 134 CCDC 6 -RET cells were amplified by RTPCR. Supplemental

A MGH 134 EV + + + + MGH 134 CCDC 6 -RET + + Osimertinib Cabozantinib + + + BLU-667 p. EGFR p. RET p. AKT p. ERK Actin B Supplementary Figure 2. Combined EGFR and RET inhibitors suppresses survival signaling and resensitizes MGH 134 CCDC 6 -RET cells. A, MGH 134 EV and MGH 134 CCDC 6 RET cells were treated with 1μM osimertinib, cabozantinib, BLU-667 or combinations for 6 hours and harvested for western blot analysis. The arrow indicates phospho-RET band. B, MGH 134 EV and MGH 134 CCDC 6 -RET cells were treated with cabozantinib or BLU-667, or osimertinib with or without 1 μM RET inhibitor (cabozantinib, BLU-667) for 72 hours and cell viability was determined. Data are shown as a percentage of vehicle treated control and are the mean ± s. e. m of three independent biological replicates. Supplemental

A PC 9 EV + + PC 9 CCDC 6 -RET + + + + Afatinib + Osimertinib + + + Cabozantinib p. EGFR p. RET p. AKT p. ERK Actin B Supplementary Figure 3. Combined EGFR and RET inhibitors can overcome CCDC 6 -RET acquired resistance. A, PC 9 EV and PC 9 CCDC 6 -RET cells were treated with 100 n. M afatinib, 1 μM osimertinib, cabozantinib or combinations for 6 hours and harvested for western blot analysis. The arrow indicates phospho-RET band. B, PC 9 EV and PC 9 CCDC 6 -RET cells were treated with cabozantinib, or EGFR-TKIs (afatinib, osimertinib) with or without 1 μM cabozantinib (CAB) and cell viability was determined after 72 hours. The same cabozantinib data is replotted in both panels for comparison purposes. Data are shown as a percentage of vehicle treated control and are the mean ± s. e. m of three independent biological replicates. Supplemental

A Liver bx: EGFR del 19 PCBP 2 -BRAF fusion LUL bx: EGFR del 19 EGFR T 790 M LUL bx: EGFR del 19 Erlotinib Carbo/Pem ASP 8273 Osi 6 mo. B BRAF (Exons 9 -18) PCBP 2 (Exons 2 -13) E 13 c. 1244 BRAFdomain Kinase E 9 c. 1366 1 2 (Exon 6) 1 (Exon 13) (Exon 2) C PC 9 Primer 2000 1500 1000 2 (Exon 18) 1 2 MGH 845 -1 R 1 D Tyr 790 2 PCBP 2 -BRAF TBP Supplementary Figure 4. Generation of MGH 845 -1 patient- derived cell line harboring the PCBP 2 -BRAF fusion gene. A, Treatment history of patient MGH 845. The cell line was generated from the post-osimertinib liver biopsy, which had "lost" EGFR T 790 M. B, Gene structure of the PCBP 2 -BRAF fusion. Primer pairs (1 and 2) used for PCR validation of fusion in panel C are denoted by arrows. C, PCBP 2 -BRAF fusion or control TBP transcripts from MGH 845 -1 were amplified by RT-PCR. D, The MGH 845 -1 cell line was confirmed to be T 790 wild-type. Supplemental

si. BRAF #2 si. BRAF #1 PC 9 p. BRAF si. SCR MGH 845 -1 A WT BRAF PCBP 2 -BRAF Actin B C Supplementary Figure 5. Knock-down of BRAF or pharmacologic inhibition of MEK resensitizes MGH 845 -1 cells to osimertinib. A, MGH 845 -1 cells were transfected with negative control si. RNA or BRAF si. RNAs. Knock-down of BRAF protein was confirmed by phospho-BRAF immunoblotting. PC 9 which harbor only wild-type BRAF are shown for comparison. B, after si. RNA transfection, cells were treated with or without 1 μM osimertinib for five days and cell viability was determined. Data shown are normalized to untreated si. SCR cells are the mean ± s. e. m. of independent biological replicates C, MGH 845 -1 cells were treated with osimertinib, dabrafenib (DAB; pan-RAF inhibitor), LXH 254 (pan-RAF inhibitor), or trametinib (TRA; MEK inhibitor) with or without 1 μM osimertinib for 96 hours and cell viability was determined. Supplemental