Decreased glutathione biosynthesis contributes to EGFR T790M-driven. erlotinib resistance in non-small cell lung cancer

Transcription

1 SUPPLEMEARY DATA Decreased glutathione iosynthesis contriutes to EGFR T79M-driven erlotini resistance in non-small cell lung cancer Hongde Li #, William Stokes #, Emily Chater #, Rajat Roy #, Elza de Bruin, Yili Hu, Zhigang Liu, Egert F. Smit &, Guus J.J.E. Heynen, Julian Downward, Michael J. Seckl, Yulan Wang,Huiru Tang, Olivier E. Pardo State Key Laoratory of Genetic Engineering, Ministry of Education Key Laoratory of Contemporary Anthropology, Collaorative Innovation Centre for Genetics and Development, Shanghai International Centre for Molecular Phenomics, Metaonomics and Systems Biology Laoratory, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, 2438, China. Key Laoratory of Magnetic Resonance in Biological Systems, National Centre for Magnetic Resonance in Wuhan, State Key Laoratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 437, China. Division of Cancer, Department of Surgery and Cancer, Imperial College, Hammersmith Hospital, Du Cane Road, London W2 NN, UK. Signal Transduction laoratory, CRUK London Research Institute, London, UK. & Dept. Pulmonary Diseases, VU University Medical Centre and Section of molecular carcinogenesis, Netherlands Cancer Institute, Amsterdam, The Netherlands. Collaorative Innovation Centre for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China. AstraZeneca, Personalised Healthcare & Biomarkers, Molecular Diagnostics, Darwin, Building 3, Camridge Science Park, Milton Road, Camridge CB4 WG, UK. # These authors have contriuted equally to the work. Corresponding authors: o.pardo@imperial.ac.uk, huiru_tang@fudan.edu.cn, m.seckl@imperial.ac.uk

2 SUPPLEMEARY TABLES Supplementary Tale : NMR data for metaolites identified and assigned. Keys refer to the peaks laelled on Figure A. Both the chemical shifts for proton (δ H) and caron (δ 3 C) NMR are taulated. a Multiplicity: singlet(s), doulet(d), triplet(t), quartet(q), doulet of doulets(dd), doule of triplets (dt), multiplet(m). The signals or the multiplicities were not determined. Supplementary Tale 2: Metaolites showed statistically significant differences in oth cell line pairs etween erlotini-resistant and erlotini-sensitive cells. "decrease" or "increase" meant statistically significant such differences were detected in the erlotini-resistant cells as compared to sensitive ones. Keys refer to the peaks laelled on Figure B and C. The chemical shifts for the identified metaolites and the OPLS-DA coefficient for oth cell line pairs are shown. SUPPLEMEARY FIGURES Supplementary Figure : (A and C) ER and H975 cells are resistant to the EGFR TKIs., ER, and H975 cells were treated with increasing concentrations of erlotini (A) or (C) for 48 h efore crystal violet staining. (B) and ER cells do not display differential sensitivity to classical chemotherapeutic agents. and ER cells were treated with increasing concentrations of cisplatin, taxol and etoposide for 48 hours prior to crystal violet staining. Results shown are representative of at least three independent experiments. Data are average ± SEM of quadruplicates, P <.. Supplementary Figure 2: (A-C) Schematic representation of additional metaolic pathways differentially modulated in erlotini-resistant and sensitive cells. Red; metaolites with increased and Blue; decreased levels in resistant cells as compared to their sensitive counterparts. (D) Representative H-NMR spectra from the cell culture media of, ER, and H975 cells. Cells were grown for 4 days in complete medium. The media were then collected and analysed y NMR for their GSH content. The spectra are zoomed onto the chemical shifts area corresponding to GSH and GSSG. The top line corresponds to a purchased GSH/GSSG mixture internal control. The spectra shown are representative of replicates per cell lines. Supplementary Figure 3: qpcr controls for -mediated silencing of GSH metaolic enzymes and accompanying changes in GSH levels. (A) and ER cells were transfected with s targeting the indicated enzymes. 48 h later, transfected cells were sujected to RT-qPCR to assess the down-regulation of the target mrnas. Data are shown are average of quadruplicates ± SEM. (B) GSH levels were measured y colorimetric assay in or ER cells downstream of the silencing of selected targets. Data are shown are representative of at least three experiments. Results are average of quadruplicates ± SEM. (B) Statistical analysis, Student t-test with taken as reference. P <.5, P <.. (C) H975 cells were transfected with s for the indicated enzymes or a non-targeting sequence () prior to treatment with the IC 5 concentration of erlotini for cells. Cell survival was determined y crystal violet staining and normalised to that of the condition. (D) Intracellular GSH levels were measured in H975 cells treated with or without mercaptosuccinate (MS) using a colorimetric assay. (E) H975 cells were incuated in the presence or asence of MS for 2 h prior to treatment with or without erlotini for 48 h. Cell

3 viaility was assessed y crystal violet staining. Results shown are representative of experiments performed at least three times. Data are average of quadruplicates ± SEM. Statistical analysis: (C- D) t-test, (E) ANOVA. P <., P <., P <.5. Supplementary Figure 4: ER cells treated with or without Ethacrynic Acid (EA) were exposed to varying doses of Gefitini for 48 hours and cell survival monitored y crystal violet staining (A). Fold change in cell survival at the IC5 dose was determined and data normalised to the cell viaility with Gefitini alone (B). Fold change in GSH was measured in cells treated with or without Gefitini, EA or comination (C). P <., P <.. Supplementary Figure 5: Three replicates for the Western lots shown in Fig 4A and B were quantified using the optical densitometry function in ImageJ and the results otained for (A) NRF2, (B) KEAP, (C) SQSTM and (D) PALB2 normalised to the corresponding control cell line and plotted. (E-F) mrna level of SQSTM was quantified for 4 cell lines and results were normalised to the corresponding control cell line. Results shown are representative of experiments performed at least three times. Data are average of triplicates ± SEM. For ER, cells were used as control while for H975, were used for normalisation. Statistical analysis: t-test. P <., P <.,P <.5. Supplementary Figure 6: (A, C and E) qpcr controls for silencing of NRF2, SQSTM and KEAP in the indicated cell lines. (B, D and F) Accompanying changes in the mrna levels for GSH synthesising enzymes as determined y qpcr. Data shown are representative of at least three independent experiments. Results are average of quadruplicates ± SEM. Statistical analysis: t-test, P <.5, P <.. Supplementary Figure 7: Silencing of PALB2 in cells does not modulate their sensitivity to erlotini. (A and B) Cells were transfected with targeting PALB2 or with a non-targeting control for 48 h prior to exposure to a dose range of erlotini for 2 days (A) or GSH levels measurements using a colorimetric assay (B). Cell survival was assessed using crystal violet staining. Results shown are representative of experiments performed in triplicate. Data are average of quadruplicates ± SEM. Silencing of T79M-EGFR using selective does only modify EGFR expression in T79M-EGFR containing cells as assessed y qpcr. and ER cells were transfected with non-targeting () or two separate T79M-targeting s (C and D) and sujected to qpcr for EGFR using primers detecting equally T79M and non-t79m EGFRs. Results shown are normalised to the corresponding condition. Statistical analysis: Student t-test, P <., P <.. (E) Response to erlotini during systemic EA administration in mouse xenografts. Nude mice (n=/condition) were injected sucutaneously with cells and treatment started when tumours reached mm 3. Tumour volume was monitored for 2 weeks.

4 Supplementary Tale : NMR data for metaolites identified and assigned. Keys refer to the peaks laelled on Figure A. Both the chemical shifts for proton (δ H) and caron (δ 3 C) NMR are taulated. a Multiplicity: singlet(s), doulet(d), triplet(t), quartet(q), doulet of doulets(dd), doule of triplets (dt), multiplet(m). The signals or the multiplicities were not determined. Key Metaolites Assignment δ H(multiplicity a ) δ 3 C.92(s) 26. Acetate CH 3 COO Adenosine CH-ring CH-ring CH-riose C2H-riose C4H-riose C3H-riose C5H-riose 8.34(s) 8.78(s) 6.8(d) / Alanine (Ala) α-ch β-ch 3 4 Aspartate (Asp) α-ch β-ch β -CH γ 3.78(q).48(d) 3.9(m) 2.69(dd) 2.8(dd) Choline α-ch 2 β-ch 2 N-CH (s) Creatine CH 3 CH 2 N=C 3.4(s) 3.93(s) Formate H COO (s) 6.52(s) Fumarate C2,3H COO - 9 Glutamate (Glu) α-ch β-ch 2 γ-ch 2 δco 3.76(t) 2.8(m) 2.35(m) Glycerophosphocho line(gpc) -CH 2 2-CH 3-CH 2 α-ch 2 β-ch 2 N-CH 3 3.6(dd) 3.89(m) 3.72(dd) 4.32(t) 3.68(t) 3.23(s) 56.7 Glycine (Gly) α-ch (s) Glycogen 5.4(road peak)

5 3 Guanosine CH-ring CH-riose C3H-riose C4H-riose C5H-riose 4 Histidine (His) C4H,ring C2H,ring 5 Inosine CH-ring CH-ring CH-riose C2H-riose C3H-riose C4H-riose C5H-riose 6 Inosine-5 - monophosphate(im P) CH-ring CH-ring CH-riose C2H-riose C3H-riose C4H-riose C5H-riose 7 Isoleucine (Ile) α-ch β-ch γ-ch γ -CH δ-ch 3 3- CH 3 8 Lactate α-ch β-ch 3 COO - 9 Leucine(Leu) α-ch β-ch 2 γ-ch δ-ch 3 δ -CH 3 2 Myo-inositol C,3H C2H C5H C4,6H 2 N-acetyl Aspartate α-ch β-ch β -CH CH 3 22 NAD N5ring N4ring N2ring (s) 5.92(d) 4.4(dd) 4.24(dt) (s) 7.85(s) 8.35(s) 8.24(s) 6.(d) (s) 8.24(s) 6.4(d) (m).98(m).27(m).47(m).94(t).(d) 4.(q).33(d) 3.73(t).72(m).69(m).97(d).96(d) 3.54(dd) 4.6(t) 3.26(t) 3.62(t) 4.39(dd) 2.68(dd) 2.49(dd) 2.2(s) 8.2(m) 8.83(d) 9.34(s)

6 23 Oxidative Glutathione (GSSG) N6ring A2Hring A8H ring A4ring A H N H S-CH2 CH CH 2 CH 2 CH 2 C=O COOH 24 Phenylalanine (Phe) α-ch β-ch β -CH C,ring C2,6,ring C3,5,ring C4,ring 25 Phosphocholine (PC) 26 Reduced Glutathione (GSH) α-ch 2 β-ch 2 N-CH 3 Glu α Glu β Glu γ Cys α Cys β Gly α C=O 9.4(d) 8.(s) 8.43(s) 6.9(d) (dd)/2.98(dd) (m) 2.6(m) 2.5(m) 3.99(dd) 3.3(dd) 3.27(dd) 7.33(m) 7.42(m) 7.38(m) 3.6(m) 4.7(m) 3.22(s) 3.78(t) 2.6(m) 2.56(m) 4.57(dd) 2.94(m) , (s) Succinate CH 2 COO Taurine N-CH 2 S-CH (t) 3.42(t) Tyrosine (Tyr) C3,5H,ring C2,6H,ring C,ring α-ch β-ch 2 6.9(d) 7.9(d) 3.94(dd) Uridine 5 - diphosphate (UDP) 3 UDP-glucose (UDPG) CH-ring CH-ring CH-riose C3H-riose C2H-riose C4H-riose C5H-riose G-H C6,ring C5,ring 7.99(d) 5.97(d) 5.96(road,s) 4.43(t) 4.39(t) 4.27(m) 4.23(c) (d) 5.98(d)

7 C H,riose C2 3 H,riose C4,riose C5 H,riose G2-H G6-H G3-H G4-H G5-H 5.99(d) 4.38(m) 4.29(m) 4.26/4.2(m) / / UDP-N-acetyl glucosamine C6,ring C H,riose C5,ring C2 3 H,riose C5 H,riose C4 H,riose C2,ring G-H G2-H G3-H G4-H G5-H NA-H NA-C=O 7.96(d) 5.98(d) 5.97(d) 4.37(m) 4.23/4.7(m) 4.29(m) 5.52(dd) / UDP-N-Acetyl Galactosamine G-H G2-H G3-H G4-H G5-H G6-H C2 3 H,riose C4,riose C5 H,riose NA-H C H,riose C5,ring C6,ring 5.55(dd) 4.5(m) 3.97(dd) 3.76(m) (m) 4.29(m) 4.25/4.9(m) 2.89(s) 5.99(d) 5.97(d) 7.96(d) Uracil C5H C6H 5.8(d) 7.54(d) 35 Uridine C6,ring C H,riose C5,ring C3 H,riose C5 H,riose C4 H,riose C2,ring 7.87(d) 5.92(d) 5.9(d) 4.34(t) 4.2(q) 4.23(t) Valine (Val) α-ch β-ch γ-ch 3 γ -CH 3 3.6(d) 2.28(m).99(d).4(d)

8 Supplementary Tale 2: Metaolites showed statistically significant differences in oth cell line pairs etween erlotini-resistant and erlotini-sensitive cells. "decrease" or "increase" meant statistically significant such differences were detected in the erlotini-resistant cells as compared to sensitive ones. Keys refer to the peaks laelled on Figure B and C. The chemical shifts for the identified metaolites and the OPLS-DA coefficient for oth cell line pairs are shown. Key Metaolite Variation tendency Chemical shift Coefficient vs ER Coefficient 975 vs Adenosine Decrease Aspartate Decrease Choline Increase Inosine Decrease Isoleucine Increase Leucine Increase N-acetyl aspartate Decrease GSSG Decrease Phosphocholine Increase GSH Decrease UDP Increase UDPG Increase Uridine Increase Valine Increase

9 Supplementary Figure A. B Cell numer (fold) Cell numer (fold) ER Erlotini (nm) ER Cisplatin (µm) Cell numer (fold) H975 ER Taxol ( M) Erlotini (nm) ER Etoposide ( M) C. Cell numer (fold) ER Cell numer (fold) H EGFRI (µm) EGFRI (µm)

10 A. -ketoisocaproate Leu Val Ile 4-methyl-2- oxopentanoate 3-methyl-2- oxoutanoate Isoutyryl-CoA 3-methyl-2- oxopentanoate 3-methylutanoyl -CoA Branched chain fatty acid 2-methylutanoyl-CoA B. Acetylcholine Phosphocholine Choline Betaine aldehyde Betaine Dimethylglycine CDP-choline Gly Sarcosine Phosphatidyl choline Glycerophospho choline Guanidinoacetate Creatine C. N-acetylaspartate Uridine Ala Asp N-caramoyl- Asp UMP UDP UDPG Asn PRPP IMP Inosine Adenosine AMP D GSH GSSG ER ER H chemical shift (ppm)

11 Supplementary Figure 3 A. Relative mrna levels GPX GPX GSTpi GSPpi GSS GSS GCLC GCLC GSR GSR ER ER ER ER ER B. Relative GSH levels (fold) ER GSTpi Relative GSH levels (fold) ER GPX Relative GSH levels (fold) ER GGT Relative GSH levels (fold) GSS Relative GSH levels (fold) GSR Relative GSH levels (fold) GCLC C. Relative cell survival GGT H975 GPX GSTpi D. E. H975 GSH levels (fold) MS - + Relative cell survival Erlo MS H

12 Supplementary Figure 4 A ER B. IC 5 R e l a t i v e s u r v i v a l ( % ) F o ld c h a n g e Log [Gefitini]. G e f it in i G e f it in i + E A C G S H le v e l F o ld c h a n g e N o t r e a t m e n t G e f it in i G e f it in i + E A Gefitini Gefitini + EA

13 Supplementary Figure 5 A. B. NRF2 levels (fold OD) Cyto n.s. ER Nuclear n.s. H975 ER H975 KEAP levels (fold OD) ER H975 C. D. SQSTM levels (fold) ER H975 PALB2 levels (fold) ER H975 E. F. mrna levels (fold) SQSTM..5 ER mrna levels (fold) SQSTM H975

14 Supplementary Figure 6 A. NRF2 mrna levels (fold).5 NRF2 B. mrna levels (fold).5.5 sinrf2 GCLC GSR GSS C. SQSTM mrna (fold).5 SQSTM D. mrna levels (fold).5 GCLC sisqstm GSR GSS E. mrna levels (fold) ER F. mrna (Fold) sikeap KEAP GCLC GSR GSS

15 Supplementary Figure 7 A. Relative Survival (%) sipalb B. GSH levels (fold) Erlotini (nm).2 PALB2 C. D. EGFR mrna levels (fold) T79M # ER EGFR mrna levels (fold) T79M #2 E. 8 Tumour Volume ( mm 3 ) Con EA ER ER+EA Days