Text of PC9 GR_6_AZDR_1 EGFR T790M (8%) PC9 GR_6_AZDR_2 EGFR T790M (13%) NRAS gain PC9 GR_6_AZDR_3 EGFR...
PC9 GR_6_AZDR_1 EGFR T790M (8%) PC9 GR_6_AZDR_2 EGFR T790M (13%) NRAS gain PC9 GR_6_AZDR_3 EGFR T790M (12%) NRAS gain PC9 GR_6_AZDR_4 EGFR T790M (13%) PC9 GR_6 EGFR T790M (13%) PC9 GR_1 EGFR T790M (7%) KRAS gain PC9 GR_2 NRAS E63K (22%) PC9 GR_8 EGFR T790M (8%) KRAS gain PC9 GR_3 T790M (11%) PC9 GR_4 T790M (7%) PC9 GR_7 T790M (9%) PC9 GR_5 T790M (7%) Gefitinib PC9 GR_1_AZDR_1 EGFR T790M (4%) KRAS gain PC9 GR_1_AZDR_2 KRAS gain PC9 GR_1_AZDR_3 EGFR T790M (7%) KRAS gain PC9 GR_1_AZDR_4 EGFR T790M (2%) KRAS gain AZD9291 PC9 AZDR_6 NRAS E63K (24%) PC9 AZDR_7 NRAS G12R (2.4%) PC9 AZDR_5 NRAS E63K (21%) PC9 AZDR_4 PC9 AZDR_3 MAPK1 gain CRKL gain PC9 AZDR_1 NRAS gain MAPK1 gain CRKL gain PC9 AZDR_2 NRAS G12V (2.4%) PC9 Parent AZD9291 Afatinib PC9 AR_1 KRAS gain PC9 AR_4 EGFR T790M (8%) PC9 AR_6 EGFR T790M (8%) NRAS gain PC9 WZR_3 KRAS amp WZ4002 PC9 WZR_1 NRAS Q61K* AZD9291 Supplementary Figure S1 A B NCI-H1975 EGFR T790M (78%) NCI-H1975 AZDR_1 EGFR T790M (75%) NCI-H1975 AZDR_2 EGFR T790M (86%) NCI-H1975 AZDR_4 EGFR T790M (92%) NRAS Q61K (6%) NCI-H1975 AZDR_3 EGFR T790M (99%) AZD9291 Supplementary Figure S1. Comparison of genetic alterations across multiple populations resistant to AZD9291 and other EGFR TKIs (A) Genetic alterations detected in DNA from 28 separate PC9 resistant populations Allele frequencies are shown. (B) Genetic alterations detected in DNA from 4 separate NCI-H1975 resistant populations. Allele frequencies are shown
PC9_GR T790M+ populations with a partial response to AZD9291 PC9_GR T790M+ populations with a full response to AZD9291 PC9_GR T790M+ population with no response to AZD9291 Supplementary Figure S2A (i)(ii)(iii)
Supplementary Figure S2B Supplementary Figure S2. Treatment of resistant populations with AZD9291. PC9 gefitinib resistant T790M+ populations were treated with a dose response of AZD9291 and dose response curves plotted to determine IC 50 values. (A) Representative dose response curves indicate varying sensitivity of the cells within the populations to AZD9291 (i) less than 50% of the cells are sensitive to AZD9291 growth inhibition; (ii) almost all of the cells are sensitive to AZD9291 growth inhibition; (iii) none of the cells are sensitive to AZD9291 growth inhibition. (B) Representative dose response curves for the sensitive cells from the partially sensitive populations. IC 50 values for AZD9291 growth inhibition are similar across all sensitive cells.
Supplementary Figure S3 (A)(B) Supplementary Figure S3. Detection and Validation of a novel NRAS E63K mutation. Integrative Genomic Viewer (IGV) screen shots of the NRAS E63K mutation from orthogonal NGS platforms. (Table insert shows read depth and nucleotide counts for each sample.) (A) Life Technologies PGM platform: the E63K variant has an allele frequency of 20 and 23%, respectively for the two resistant samples. (B) Illumina platform: the E63K variant has an allele frequency of 21 and 22%, respectively for the two resistant samples. The parental sample, PC-9, shows no evidence of the NRAS E63K variant on either platform.
Supplementary Figure S4 PC9 GR_1, PC9 GR_8, PC9 WZR_3 and PC9 AR_1 KRAS gain of copy number PC9 AR_6, PC9 GR_6_AZDR_2 and PC9 GR_6_AZDR_3 NRAS gain of copy number Green values represent resistant populations that are >5 fold more sensitive to selumetinib compared to the parental PC9 cells. Red box highlights selumetinib sensitive resistant populations with variable levels of phosphorylated ERK. Purple box highlights selumetinib refractory resistant populations with variable levels of phosphorylated ERK. Supplementary Figure S4. Lysates were prepared from parental PC9 and resistant populations analysed for levels of total and phosphorylated ERK, NRAS and KRAS by western blot. The data shown is representative of 2 separate analyses. 0.220.190.170.110.140.090.230.89 PC9 NRAS tERK KRAS pERK PC9 AZDR_1PC9 AZDR_3PC9 AZDR_2 PC9 AZDR_4 PC9 AZDR_5 PC9 AZDR_6PC9 AZDR_7PC9 AZDR_8PC9 WZR_1PC9 WZR_3PC9 AR_1PC9 AR_4PC9 AR_6PC9PC9 GR_4_AZDR_1PC9 GR_6_AZDR_2H1975PC9 GR_6_AZDR_3PC9 GR_6_AZDR_4 H1975 AZDR_1H1975 AZDR_2H1975 AZDR_3H1975 AZDR_4H1975 AZDR_5 PC9 PC9 GR_1PC9 GR_2PC9 GR_3PC9 GR_4PC9 GR_5PC9 GR_6PC9 GR_7PC9 GR_8 6.950.252.38 1.42.7 1.63 Selumetinib IC 50 M PC9 GR_1_AZDR_1PC9 GR_1_AZDR_2PC9 GR_1_AZDR_3PC9 GR_1_AZDR_4 0.02 6.950.280.540.130.73 4.945.46 3.6 6.75 3.53.4 0.15 10 0.05 7.326.957.240.62 6.23.7 8.77 7.42.5
NRAS-GTP NRAS Total RAS-GTP Total RAS PC9 AZD9291160nM + -+-+-+ KRAS-GTP KRAS - PC9 AZDR_5 NRAS E63K PC9 GR_2 NRAS E63K PC9 WZR_1 NRAS Q61K Supplementary Figure S5 Supplementary Figure S5. The novel NRAS E63K mutation is an activating mutation that when expressed enhances resistance to cell growth inhibition by gefitinib or AZD9291 in EGFRm cell lines. (A) Resistant populations were cultured in media without EGFR inhibitor for 5 days prior to carrying out the assay. Lysates were prepared from parental and resistant cells serum starved overnight and treated for 6 hours +/- 160nM AZD9291. RAS activity was measured using RAS GTPase-specific pulldown assays. (B) (i) Lysates from PC9 cells transfected (electroporation) with pcDNA 3.1+ control and NRAS variant DNA constructs for 48 hours were immunoblotted for indicated proteins. (ii) Lysates were prepared from PC9 cells transfected (Fugene 6 reagent) with pcDNA 3.1+ control and NRAS variant DNA constructs for 48 hours. RAS activity was measured using RAS GTPase-specific pulldown assays.. (C) EGFRm cells were transfected (Fugene 6 reagent) every 96 hours with indicated DNA constructs and cultured in media containing 160nM AZD9291 over a period of 12 days. A C pERK1/2 Vinculin NRAS ERK untransfectedpcDNA 3.1+ controlpcDNA 3.1+Q61K NRAS pcDNA 3.1+ WT NRAS pcDNA 3.1+ E63K NRAS B (i) untransfected pcDNA 3.1+ controlpcDNA 3.1+Q61K NRAS pcDNA 3.1+ WT NRAS pcDNA 3.1+ E63K NRAS NRAS-GTP NRAS Vinculin (ii)
Supplementary Figure S6 A B Supplementary Figure S6. In vitro combination of AZD9291 with selumetinib induces more profound phenotype inhibition. (A) HCC827 and (B) NCI-H820 cells were chronically treated for 51 days with; DMSO, AZD9291, selumetinib or a combination of both AZD9291 with selumetinib. Fold increase in cell number was monitored over time.