Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: IC 50 values (nM) for the mutant EGFR-expressing Ba/F3 cells, PC9 cells or MGH121 cells.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) The results of screening the growth-inhibitory activity of 30 drugs in Ba/F3 cells expressing four types of EGFR-del19 with or without T790M or C797S mutations are shown in a heat map. Ba/F3 cells expressing each EGFR mutant were treated with 100 nM of the indicated inhibitors. After 72 h of drug treatment, the cell viability was measured using the CellTiter-Glo assay. Relative cell viability was calculated from each value divided by the DMSO control. Among the inhibitors, only brigatinib and ponatinib were sufficiently efficacious against the triple-mutant EGFR. AZD3463 acted as a weak inhibitor to the triple mutation. ( b ) Growth inhibition assessed by the CellTiter-Glo assay of EGFR-C797S/T790M/del19 (triple-del19)-mutated Ba/F3 cells treated with gefitinib, osimertinib and brigatinib.; N =3. Results are expressed as mean±s.d. IC 50 values were calculated using growth inhibition assay. ( c ) Phosphorylation of EGFR and downstream signals were significantly inhibited by brigatinib in Ba/F3 cells expressing triple-del19 even though afatinib and osimertinib did not suppress at all the EGFR signalling of triple-del19.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Activity Assay, Expressing, Mutagenesis, Glo Assay, Control, Inhibition, Growth Inhibition Assay, Phospho-proteomics

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) Chemical structures of six ALK–TKIs were very similar. ( b , c ) IC 50 values in Ba/F3 cells expressing four mutation types of EGFR-del19 were obtained by treatment with brigatinib, AP26113-analog, AZD3463, TAE684, ceritinib and ASP3026 for 72 h. Those of C797S/T790M/del19 were shown by bar graph ( b ) and those of all mutation types were demonstrated by a table ( c ). The CellTiter-Glo assay was used to measure cell viability. ( d , e ) Ba/F3 cells expressing T790M/del19 ( d ) or C797S/T790M/del19 ( e ) were treated with the indicated concentrations of brigatinib, AP26113 analog, TAE684, ceritinib or ASP3026 for 6 h. Phosphorylation of EGFR and its downstream signals were evaluated by western blotting with the indicated antibodies.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Mutagenesis, Glo Assay, Phospho-proteomics, Western Blot

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a – e ) PC9 (del19) ( a ), PC9-T790M (T790M/del19) ( b ), PC9-triple mutant (C797S/T790M/del19) ( c ), MGH121 parent (T790M/del19) ( d ) and MGH121 resistant-2 (C797S/T790M/del19) ( e ) cells were treated with serially diluted gefitinib, osimertinib and brigatinib for 72 h. Cell viability was measured using the CellTiter-Glo assay.; N =3. Results are expressed as mean±s.d. ( f ) Western blotting of PC9 triple mutant (C797S/T790M/del19) cells indicated that brigatinib and AP26113 analog, but not afatinib or osimertinib, suppressed phosphorylation of EGFR and its downstream signalling. ( g ) Similar results were obtained in MGH121 resistant-2.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Mutagenesis, Glo Assay, Western Blot, Phospho-proteomics

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) The cell growth inhibition of Ba/F3 cells expressing EGFR-C797S/T790M/del19 (EGFR-triple-del19) treated with brigatinib, AP26113-analog, AZD3463 and osimertinib at indicated concentrations combined with or without cetuximab (10 μg ml −1 ) for 72 h assessed by CellTiter-Glo assay. ( b ) Inhibition of EGFR signal pathway in BaF3 EGFR-triple-del19 cells treated with brigatinib+cetuximab (10 μg ml −1 ) for 6 h was evaluated using western blotting. ( c , d ) The cell growth inhibition of PC9 triple-mutant cells ( c ) and MGH121-res2 cells ( d ) treated with brigatinib and osimertinib at indicated concentrations combined with or without cetuximab (10 μg ml −1 ) for 72 h assessed by CellTiter-Glo assay. ( e , f ) Inhibition of EGFR signal pathway in PC9 triple-mutant cells ( e ) and MGH121-res2 cells ( f ) treated with brigatinib+cetuximab (10 μg ml −1 ) for 6 h was evaluated using western blotting.; Results in a , c , e are expressed as mean±s.d. ( N =3). The significance of difference between indicated groups are calculated by Student's t -test (NS; not significant, * P <0.05, ** P <0.01).

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Inhibition, Expressing, Glo Assay, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a , b ) PC9 cells expressing EGFR-C797S/T790M/del19 were subcutaneously implanted into Balb-c nu/nu mice. When the average tumour volume reached ∼200 mm 3 , the mice were randomized into vehicle control or treatment groups (50 mg kg −1 of osimertinib, 75 mg kg −1 of brigatinib, 1 mg per mouse of cetuximab three times a week or 75 mg kg −1 of brigatinib combined with cetuximab administered as previously described) and treated once daily by oral gavage for the indicated period. Tumour volume ( V ) was calculated as 0.5 × length × width 2 , and body weights (B.W.) of mice were measured twice weekly.; N =6. Results are expressed as mean±s.d. The significance of difference between the mean tumour volume of control and of brigatinib on day 7, between brigatinib and brigatinib+cetuximab on day 23, respectively, are calculated by Mann–Whitney U test (** P <0.01). ( c ) Survival periods of mice in each treatment arm were demonstrated using the Kaplan–Meier curve. ( d ) Phosphorylation of EGFR and its downstream signalling in two tumour samples obtained from each group were evaluated using western blotting. ( e , f ) In vivo experiment of PC9 triple-mutant cells following a similar protocol as in , using panitumumab 0.5 mg per mouse two times a week administered peritoneally instead of cetuximab.; N =6. Results are expressed as mean±s.d. The significance of difference between the mean tumour volume of control and of brigatinib on day 16, between brigatinib and brigatinib+panitumumab on day 23, respectively, are calculated by Mann–Whitney U test (** P <0.01). ( g ) A Kaplan–Meier curve of the survival of the mice in each treatment arm. ( h ) Phosphorylation of EGFR and its downstream signalling in two tumour samples obtained from xenografts of PC9-triple mutant cells treated for 8 days with the indicated drugs (brigatinib: 75 mg kg −1 daily, administered orally; panitumumab: 0.5 mg per mouse two times a week, administered peritoneally) were assessed by western blotting with the indicated antibodies.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Control, MANN-WHITNEY, Phospho-proteomics, Western Blot, In Vivo, Mutagenesis

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a , b ) MGH121-res2 expressing EGFR-C797S/T790M/del19 were subcutaneously implanted into SCID-beige mice. When the average tumour volume reached ∼200 mm 3 , the mice were randomized into vehicle control and treatment groups (50 mg kg −1 of osimertinib (po), 75 mg kg −1 of brigatinib (po), 1 mg per mouse of cetuximab two times a week and 75 mg kg −1 of brigatinib combined with cetuximab administered as previously described, respectively) and treated for the indicated period. Tumour volume ( V ) was calculated as 0.5 × length × width 2 , and the body weights (B.W.) of the mice were measured twice weekly. N =6. Results are expressed as mean±s.d. The significance in difference between the mean tumour volume of control and of osimertinib, brigatinib and cetuximab, between cetuximab and brigatinib+cetuximab, respectively, on day 42 are calculated by Mann–Whitney U test (NS: not significant, * P <0.05, ** P <0.01).

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Control, MANN-WHITNEY