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Acquired BRAF Rearrangements Induce Secondary Resistance to EGFR therapy in EGFR-Mutated Lung Cancers Morana Vojnic, MD, Daisuke Kubota, MD, PhD, Christopher Kurzatkowski, BSc, Michael Offin, MD, Ken Suzawa, MD, PhD, Ryma Benayed, PhD, Adam J. Schoenfeld, MD, Andrew J. Plodkowski, MD, John T. Poirier, PhD, Charles M. Rudin, MD, PhD, Mark G. Kris, MD, Neal X. Rosen, MD, Helena A. Yu, MD, Gregory J. Riely, MD, PhD, Maria E. Arcila, MD, Romel Somwar, PhD, Marc Ladanyi, MD Journal of Thoracic Oncology Volume 14, Issue 5, Pages 802-815 (May 2019) DOI: 10.1016/j.jtho.2018.12.038 Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 Clinical information for patients with resistance to EGFR tyrosine kinase inhibitor and BRAF fusions. (A) Disease course of patient 2, who was found to have acquired resistance to osimertinib and acylglycerol kinase gene (AGK)/BRAF fusion. (B) Disease course of patient 4, who was found to have acquired resistance to erlotinib and progressive mediastinal disease with AGK/BRAF fusion. Journal of Thoracic Oncology 2019 14, 802-815DOI: (10.1016/j.jtho.2018.12.038) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Generation and characterization of CRISPR/Cas9 acylglycerol kinase gene (AGK)/BRAF cell lines. (A) clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering protocol scheme. (B) Expression of AGK/BRAF. (C). Sanger sequencing of polymerase chain reaction (PCR) product confirming fusion in cell line of AGK exon 2 with exon 8 of BRAF. (D) H1975 and PC9 cells expressing AGK/BRAF were treated with 0.5 osimertinib for 1 hour, whole-cell extracts were prepared, and then lysates were subjected to immunoblotting. The results represent three independent experiments. (E) The indicated cell lines were plated at a density 7500/well in 96-well plates and treated with osimertinib for 96 hours; the concentration that inhibits 50% (IC50) values for growth inhibition are shown in the Table (F). (G and H) Cells were plated at a density of 150,000 cells/well in six-well plates and then infected 24 hours later with lentiviruses harboring the indicated short hairpin RNAs (shRNAs) and then selected with puromycin 48 hours later. BRAF mRNA levels was determined by quantitative reverse-transcriptase PCR (G) or cells were replated in osimertinib and growth assessed 96 hours later (H). The IC50 values for growth inhibition were determined by nonlinear regression analysis by using Graphpad Prism software (GraphPad Software). The results represent the mean plus or minus SD of two experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; gDNA, guide DNA; p, phosphorylated; STAT3, signal transducer and activator of transcription 3; PJA2, praja ring finger ubiquitin ligase 2; -RT, control reaction without reverse transcriptase. Journal of Thoracic Oncology 2019 14, 802-815DOI: (10.1016/j.jtho.2018.12.038) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Generation and characterization of CRISPR/Cas9 acylglycerol kinase gene (AGK)/BRAF cell lines. (A) clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering protocol scheme. (B) Expression of AGK/BRAF. (C). Sanger sequencing of polymerase chain reaction (PCR) product confirming fusion in cell line of AGK exon 2 with exon 8 of BRAF. (D) H1975 and PC9 cells expressing AGK/BRAF were treated with 0.5 osimertinib for 1 hour, whole-cell extracts were prepared, and then lysates were subjected to immunoblotting. The results represent three independent experiments. (E) The indicated cell lines were plated at a density 7500/well in 96-well plates and treated with osimertinib for 96 hours; the concentration that inhibits 50% (IC50) values for growth inhibition are shown in the Table (F). (G and H) Cells were plated at a density of 150,000 cells/well in six-well plates and then infected 24 hours later with lentiviruses harboring the indicated short hairpin RNAs (shRNAs) and then selected with puromycin 48 hours later. BRAF mRNA levels was determined by quantitative reverse-transcriptase PCR (G) or cells were replated in osimertinib and growth assessed 96 hours later (H). The IC50 values for growth inhibition were determined by nonlinear regression analysis by using Graphpad Prism software (GraphPad Software). The results represent the mean plus or minus SD of two experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; gDNA, guide DNA; p, phosphorylated; STAT3, signal transducer and activator of transcription 3; PJA2, praja ring finger ubiquitin ligase 2; -RT, control reaction without reverse transcriptase. Journal of Thoracic Oncology 2019 14, 802-815DOI: (10.1016/j.jtho.2018.12.038) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Generation and characterization of CRISPR/Cas9 acylglycerol kinase gene (AGK)/BRAF cell lines. (A) clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering protocol scheme. (B) Expression of AGK/BRAF. (C). Sanger sequencing of polymerase chain reaction (PCR) product confirming fusion in cell line of AGK exon 2 with exon 8 of BRAF. (D) H1975 and PC9 cells expressing AGK/BRAF were treated with 0.5 osimertinib for 1 hour, whole-cell extracts were prepared, and then lysates were subjected to immunoblotting. The results represent three independent experiments. (E) The indicated cell lines were plated at a density 7500/well in 96-well plates and treated with osimertinib for 96 hours; the concentration that inhibits 50% (IC50) values for growth inhibition are shown in the Table (F). (G and H) Cells were plated at a density of 150,000 cells/well in six-well plates and then infected 24 hours later with lentiviruses harboring the indicated short hairpin RNAs (shRNAs) and then selected with puromycin 48 hours later. BRAF mRNA levels was determined by quantitative reverse-transcriptase PCR (G) or cells were replated in osimertinib and growth assessed 96 hours later (H). The IC50 values for growth inhibition were determined by nonlinear regression analysis by using Graphpad Prism software (GraphPad Software). The results represent the mean plus or minus SD of two experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; gDNA, guide DNA; p, phosphorylated; STAT3, signal transducer and activator of transcription 3; PJA2, praja ring finger ubiquitin ligase 2; -RT, control reaction without reverse transcriptase. Journal of Thoracic Oncology 2019 14, 802-815DOI: (10.1016/j.jtho.2018.12.038) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 Characterization of an EGFR-mutant cell line with praja ring finger ubiquitin ligase 2 gene (PJA2)/BRAF fusion. (A) Expression of PJA2/BRAF in a patient-derived cell line, MSK-LX138cl, and xenograft tissue. (B) Sanger sequencing of the polymerase chain reaction product, confirming a fusion between PJA2 exon 7 and BRAF exon 11. (C) MSK-LX138cl cells were treated with 0.5 μM osimertinib for 1 hour, whole-cell extracts were prepared, and then lysates were subjected to immunoblotting. The results represent 3 independent experiments. (D–E) MSK-LX138cl cells were plated at a density 7500/well in 96-well plates and treated with EGFR (D) or BRAF (E) inhibitors. The concentration that inhibits 50% (IC50) values for growth inhibition were determined by nonlinear regression analysis using Graphpad Prism software (GraphPad Software). (F) A quantity of 50,000 MSK-LX138cl cells/well were plated in 96-well plates, and the next day they were treated with 0.1 or 1 μM of the indicated agents or a combination of osimertinib and trametinib. Caspase 3/7 activity was determined 48 hours later. The results represent the mean plus or minus SD of two experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; cDNA, complementary DNA. Journal of Thoracic Oncology 2019 14, 802-815DOI: (10.1016/j.jtho.2018.12.038) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Combined inhibition of MEK and EGFR is effective in reducing growth of cells with EGFR mutation. (A) Cells were plated at a density of 7500 cells/well in 96-well plate and treated with vemurafenib or dabrafenib for 96 hours, after which growth was determined. The concentration that inhibits 50% (IC50) values for growth inhibition are shown (right). (B) MSK-LX138cl cells were plated at a density of 7500 cells/well in 96-well plates and treated with trametinib (0–1 μM) and osimertinib (0–1μM) for 96 hours. Growth inhibition was then determined and the combination index was calculated by using CompuSyn software. Data represent the mean value of growth inhibition ratio at each concentration of the drugs in two independent experiments. Dot plot indicates the combination index and fraction affected (inhibition ratio) of various drug concentrations. AGK, acylglycerol kinase. Journal of Thoracic Oncology 2019 14, 802-815DOI: (10.1016/j.jtho.2018.12.038) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 5 The pan-RAF inhibitor LY3009120 effectively inhibits growth of EGFR-mutant cell lines with BRAF fusions. (A) Cells were plated in 96-well plates and treated with LY3009120, after which growth was determined (left). The concentration that inhibits 50% (IC50) values for growth inhibition were determined by nonlinear regression analysis by using GraphPad Prism software (GraphPad Software) (right). (B) MSK-LX138cl cells were treated with the indicated concentration of LY3009120 or vemurafenib for 1 hour, and then whole-cell extracts were prepared and immunoblotted for the indicated proteins. The results are representative of two independent experiments. (C) Cells were treated with 0.1 or 1 μM of the indicated compounds or osimertinib/trametinib combinations, and caspase 3/7 activity was determined 48 hours later. Journal of Thoracic Oncology 2019 14, 802-815DOI: (10.1016/j.jtho.2018.12.038) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions