1. TBP
200
LC2/ad
MGH134
MGH134 CCDC6-RET
MGH134 EV
CCDC6-RET
PC9
PC9 CCDC6-RET
PC9 EV
1000
1500
Supplementary Figure 1. Detection of the CCDC6-RET fusion gene. CCDC6-RET fusion gene or internal control TBP transcripts from LC2/ad cells and PC9CCDC6-RET or MGH134CCDC6-RET cells were amplified by RT-PCR.
Supplemental

2. A
pERK
pAKT
pRET
MGH134 EV
MGH134 CCDC6-RET
Osimertinib
Cabozantinib
BLU-667 +
pEGFR
EGFR
ERK
AKT
Actin
RET B
Supplementary Figure 2. Combined EGFR and RET inhibitors suppresses survival signaling and resensitizes MGH134CCDC6-RET cells. A, MGH134EV and MGH134CCDC6-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, MGH134EV and MGH134CCDC6-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

3. A
PC9 EV
PC9 CCDC6-RET
Osimertinib
Afatinib
Cabozantinib +
pEGFR
pERK
pAKT
EGFR
ERK
AKT
Actin
pRET
RET B
Supplementary Figure 3. Combined EGFR and RET inhibitors can overcome CCDC6-RET acquired resistance. A, PC9EV and PC9CCDC6-RET cells were treated with 100 nM afatinib, 1 μM osimertinib, cabozantinib or combinations for 6 hours and harvested for western blot analysis. The arrow indicates phospho-RET band. B, PC9EV and PC9CCDC6-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

4. A
Erlotinib
Carbo/Pem
ASP8273
Osi
LUL bx: EGFR del19
EGFR T790M
LUL bx: EGFR del19
Liver bx: EGFR del19
PCBP2-BRAF fusion
6 mo.
BRAF
Kinase domain
BRAF (Exons 9-18)
PCBP2 (Exons 2-13)
(Exon 2) 2
(Exon 6) 1
(Exon 13)
(Exon 18)
E13 c.1244
E9 c.1366 B C D
200
1000
1500
2000
PC9
MGH845-1R
TBP
PCBP2-BRAF 1 2
Primer
Tyr 790
Supplementary Figure 4. Generation of MGH845-1 patient- derived cell line harboring the PCBP2-BRAF fusion gene. A, Treatment history of patient MGH845. The cell line was generated from the post-osimertinib liver biopsy, which had "lost" EGFR T790M. B, Gene structure of the PCBP2-BRAF fusion. Primer pairs (1 and 2) used for PCR validation of fusion in panel C are denoted by arrows. C, PCBP2-BRAF fusion or control TBP transcripts from MGH845-1 were amplified by RT-PCR. D, The MGH845-1 cell line was confirmed to be T790 wild-type.
Supplemental

5. pBRAF
MGH845-1
siSCR
siBRAF #1
siBRAF #2
PC9
Actin
PCBP2-BRAF
WT BRAF A B C
Supplementary Figure 5. Knock-down of BRAF or pharmacologic inhibition of MEK resensitizes MGH845-1 cells to osimertinib. A, MGH845-1 cells were transfected with negative control siRNA or BRAF siRNAs. Knock-down of BRAF protein was confirmed by phospho-BRAF immunoblotting. PC9 which harbor only wild-type BRAF are shown for comparison. B, after siRNA transfection, cells were treated with or without 1 μM osimertinib for five days and cell viability was determined. Data shown are normalized to untreated siSCR cells are the mean ± s.e.m. of independent biological replicates C, MGH845-1 cells were treated with osimertinib, dabrafenib (DAB; pan-RAF inhibitor), LXH254 (pan-RAF inhibitor), or trametinib (TRA; MEK inhibitor) with or without 1 μM osimertinib for 96 hours and cell viability was determined.
Supplemental