Supplementary Tables

Supplementary Table S1. Generation of resistant cell populations.

Method used for generating resistant lines / EGFR inhibitor / Final conc. of EGFR inhibitor (nM) / Full name of resistant cell population / Abbreviated name of resistant cell population
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_1 / PC9 GR_1
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_2 / PC9 GR_2
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_3 / PC9 GR_3
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_4 / PC9 GR_4
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_5 / PC9 GR_5
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_6 / PC9 GR_6
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_7 / PC9 GR_7
Dose escalation / Gefitinib / 1500 / PC9_gefitinib resistant_8 / PC9 GR_8
Dose escalation / Afatinib / 1500 / PC9_afatinib resistant_1 / PC9 AR_1
Dose escalation / Afatinib / 1500 / PC9_afatinib resistant_2 / PC9 AR_2
Dose escalation / Afatinib / 1500 / PC9_afatinib resistant_3 / PC9 AR_3
Dose escalation / Afatinib / 1500 / PC9_afatinib resistant_4 / PC9 AR_4
Dose escalation / Afatinib / 1500 / PC9_afatinib resistant_5 / PC9 AR_5
Dose escalation / Afatinib / 1500 / PC9_afatinib resistant_6 / PC9 AR_6
Dose escalation / WZ4002 / 1500 / PC9_WZ4002 resistant_1 / PC9 WZR_1
Dose escalation / WZ4002 / 1500 / PC9_WZ4002 resistant_2 / PC9 WZR_2
Dose escalation / WZ4002 / 1500 / PC9_WZ4002 resistant_3 / PC9 WZR_3
Dose escalation / WZ4002 / 1500 / PC9_WZ4002 resistant_4 / PC9 WZR_4
Dose escalation / WZ4002 / 1500 / PC9_WZ4002 resistant_5 / PC9 WZR_5
Dose escalation / WZ4002 / 1500 / PC9_WZ4002 resistant_6 / PC9 WZR_6
Dose escalation / WZ4002 / 1500 / PC9_WZ4002 resistant_7 / PC9 WZR_7
Dose escalation / AZD9291 / 160 / PC9_AZD9291 resistant_1 / # PC9 AZDR_1
Dose escalation / AZD9291 / 160 / PC9_AZD9291 resistant_2 / # PC9 AZDR_2
Dose escalation / AZD9291 / 160 / PC9_AZD9291 resistant_3 / # PC9 AZDR_3
Dose escalation / AZD9291 / 160 / PC9_AZD9291 resistant_4 / PC9 AZDR_4
Single dose of compound / AZD9291 / 160 / PC9_AZD9291 resistant_5 / PC9 AZDR_5
Single dose of compound / AZD9291 / 160 / PC9_AZD9291 resistant_6 / PC9 AZDR_6
Single dose of compound / AZD9291 / 160 / PC9_AZD9291 resistant_7 / PC9 AZDR_7
Single dose of compound / AZD9291 / 160 / PC9_AZD9291 resistant_8 / PC9 AZDR_8
Dose escalation / AZD9291 / 1500 / PC9_AZD9291 resistant_9 / PC9 AZDR_9
Dose escalation / AZD9291 / 1500 / PC9_AZD9291 resistant_10 / PC9 AZDR_10
Dose escalation / AZD9291 / 1500 / PC9_AZD9291 resistant_11 / PC9 AZDR_11
Dose escalation / AZD9291 / 1500 / PC9_AZD9291 resistant_12 / PC9 AZDR_12
Dose escalation / WZ4002 / 1500 / NCI-H1975_WZ4002 resistant / NCI-H1975 WZR
Dose escalation / Afatinib / 1500 / NCI-H1975_afatinib resistant / NCI-H1975 AR
Dose escalation / AZD9291 / 1500 / NCI-H1975_AZD9291 resistant_1 / NCI-H1975 AZDR_1
Dose escalation / AZD9291 / 1500 / NCI-H1975_AZD9291 resistant_2 / NCI-H1975 AZDR_2
Dose escalation / AZD9291 / 1500 / NCI-H1975_AZD9291 resistant_3 / NCI-H1975 AZDR_3
Dose escalation / AZD9291 / 1500 / NCI-H1975_AZD9291 resistant_4 / NCI-H1975 AZDR_4
Single dose of compound / AZD9291 / 160 / NCI-H1975_AZD9291 resistant_5 / # NCI-H1975 AZDR_5
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_1_AZD9291 resistant_1 / PC9_GR_1_ AZDR_1
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_1_AZD9291 resistant_2 / PC9_GR_1_ AZDR_2
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_1_AZD9291 resistant_3 / PC9_GR_1_ AZDR_3
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_1_AZD9291 resistant_4 / PC9_GR_1_ AZDR_4
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_6_AZD9291 resistant_1 / PC9_GR_6_ AZDR_1
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_6_AZD9291 resistant_2 / PC9_GR_6_ AZDR_2
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_6_AZD9291 resistant_3 / PC9_GR_6_ AZDR_3
Dose escalation / AZD9291 / 1500 / PC9 gefitinib resistant_6_AZD9291 resistant_4 / PC9_GR_6_ AZDR_4
Dose escalation / WZ4002 / 1500 / PC9 vandetinib resistant_WZ4002 resistant / PC9VR_WZR
Dose escalation / Afatinib / 1500 / PC9 vandetinib resistant_afatinib resistant / PC9VR_AR

EGFRm+ and EGFRm+/T790M cell lines were used to generate a panel of EGFR inhibitor resistant models. Cells were chronically treated with escalating concentrations of gefitinib, afatinib, WZ4002 or AZD9291 or a single concentration of AZD9291. Each cell line represents a pool of the resistant colonies that emerged following chronic treatment with EGFR inhibitor. Populations highlighted in yellow were those selected for further phenotypic and genetic analysis. # indicates populations selected for RPPA analysis.

Supplementary Table S2. Small molecule inhibitors.

Compound / Inhibitor target / Reference
Selumetinib AZD6244 (ARRY-142886) / MEK1/2 / Mol Cancer Ther. August 2007 6; 2209
AZD5363 / AKT / Mol Cancer Ther. 2012 (4):873-87
AZD8055 / mTORC1/mTORC2 / Cancer Res. 2010;70(1):288-98
AZD2014 / mTORC1/mTORC2 / Bioorg Med Chem Lett. 2013;23(5):1212-6
GDC-0941 / Pan PI3K / J Med Chem. 2008; 51: 5522–5532
AZ_0012 / AXL / AZ in-house compound
AZ_6592 / MET / AZ in-house compound
AZ_1902 / IGF-1R / AZ in-house compound
BMS-536924 / IGF-1R / Journal of Medicinal Chemistry. 48 5639–5643
AZ_9424 / HDAC / AZ in-house compound
AZD4547 / FGFR / Cancer Res. 2012; 72(8); 2045–56
AZD1152 (HQPA) / Aurora kinase B / Clin Cancer Res. 2007; 13(12):3682-8

Supplementary Table S3. IC50 (µM) values from cell growth inhibition assays comparing compound sensitivity between parental and resistant cell popoulations.

Cells were treated with a dose response of indicated inhibitors alone for parental cells and in the presence of original EGFR inhibitor for resistant populations. Values highlighted in green in resistant populations represent >5 fold increased sensitivity to agent compared to the respective parental cell line. Values highlighted in pink in resistant populations represent >5 fold decreased sensitivity to AZD9291 compared to the respective parental line. Values represent an average of at least 2 independent experiments. Errors in brackets are standard deviation.

Supplementary Table S4. Genetic analysis of resistant cell populations.

EGFR inhibitor and resistant concentration / Resistant population / EGFR T790M Status (Allele Freq) / Other alterations (Allele Freq or CNV) / Sequencing / aCGH / SnapShot / Sanger Sequencing / Western blot
GeneRead Lung/Illumina / PGM / WES/Illumina
Parental / PC9 / Not Detected / Not Detected / X / X / X / X / X
NCI H1975 / T790M (78%) / Not Detected / X / X
gefitinib resistant (EGFRm+ setting) / PC9 GR_1 / EGFR T790M (7%) / KRAS gain (5.43) / X / X / X / X / X
PC9 GR_ 2 / Not Detected / NRAS E63K (22%) / X / X / X
PC9 GR_3 / EGFR T790M (11%) / Not Detected / X / X
PC9 GR_4 / EGFR T790M (7%) / Not Detected / X / X
PC9 GR_5 / EGFR T790M (7%) / Not Detected / X / X
PC9 GR_6 / EGFR T790M (13%) / Not Detected / X / X / X / X
PC9 GR_7 / EGFR T790M (9%) / Not Detected / X / X
PC9 GR_8 / EGFR T790M (8%) / KRAS gain (7.06) / X / X / X / X
afatinib resistant (EGFRm+ setting) / PC9 AR_1 / Not Detected / KRAS gain (24.6) / X / X
PC9 AR_4 / EGFR T790M (8%) / Not Detected / X / X / X
PC9 AR_6 / EGFR T790M (8%) / NRAS gain (4.23) / X / X
PC9 WZ4002 resistant (EGFRm+ setting) / PC9 WZR_1 / Not Detected / NRAS Q61K / X / X
PC9 WZR_3 / Not Detected / KRAS gain (2.64) / X / X
AZD9291 resistant (EGFRm+ setting) / PC9 AZDR_1 / Not Detected / NRAS gain (2.5), MAPK1 (1.97), CRKL (1.84) / X / X / X
PC9 AZDR_2 / Not Detected / NRAS G12V (2.4%) / X / X / X
PC9 AZDR_3 / Not Detected / MAPK1(3.8), CRKL (3.46) / X / X / X
PC9 AZDR_4 / Not Detected / Not Detected / X / X / X
PC9 AZDR_5 / Not Detected / NRAS E63K (21%) / X / X / X
PC9 AZDR_6 / Not Detected / NRAS E63K (24%) / X
PC9 AZDR_7 / Not Detected / NRAS G12R (20%) / X
AZD9291 resistant (EGFRm+/T790M setting) / PC9 GR_1_AZDR_1 / T790M (4%) / KRAS gain (6.23) / X / X / X
PC9 GR_1_AZDR_2 / Not Detected / KRAS gain (5.66) / X / X / X
PC9 GR_1_AZDR_3 / T790M (7%) / KRAS gain (4.44) / X / X / X
PC9 GR_1_AZDR_4 / T790M (2%) / KRAS gain (5.46) / X / X / X
PC9 GR_6_AZDR_1 / EGFR T790M# (8%) / Not Detected / X / X / X
PC9 GR_6_AZDR_2 / EGFR T790M# (13%) / NRAS gain (2.4) / X / X / X
PC9 GR_6_AZDR_3 / EGFR T790M# (12%) / NRAS gain (3.68) / X / X / X
PC9 GR_6_AZDR_4 / EGFR T790M# (13%) / Not Detected / X / X / X
NCI-H1975 AZDR_1 / EGFR T790M* (75%) / Not Detected / X / X
NCI-H1975 AZDR_2 / EGFR T790M* (86%) / Not Detected / X / X
NCI-H1975 AZDR_3 / EGFR T790M* (99%) / Not Detected / X / X
NCI-H1975 AZDR_4 / EGFR T790M* (92%) / NRAS Q61K (6%) / X / X

DNA from 32 separate populations with acquired resistance to different EGFR inhibitors was analysed for gene mutation and/or gene copy number across a panel of cancer associated genes. Data represents genetic alterations detected in the resistant populations. Variants validated across at least 2 different assay platforms are indicated. Next generation sequencing, Sanger sequencing, comparative genomic hybridisation (CGH), SnaPshot mutation analysis and / or Western blot analysis were used as assay platforms to detect and validate genetic modifications. Bracketted values represent either gain as fold change relative to respective parental cells or percent allele frequency for mutations .

Supplementary Table S5. RPPA analysis of phosphorylated and total protein levels in PC9 and NCI-H1975 AZD9291 resistant populations compared to respective parental cells.

Lysates from AZD9291 resistant and parental cells were prepared and analysed by RPPA. Phsphorylated and total protein levels in resistant cells were compared to levels in corresponding parental cells.

Submitted as an excel file.