Figure S1. Compounds Screen Across SCLC Cell Lines

SUPPLEMENTARY FIGURE AND TABLE LEGENDS

Figure S1. Compounds screen across SCLC cell lines.

A. The categories of Roche published kinase inhibitor set. 235 compounds were classified based on their primary target and number of compounds for each category was shown in Pie plot.

B. c-MYC amplification in SCLC cells. MYC amplification was assessed in 21 of SCLC cell lines as described in Materials and Methods. c-MYC amplification in each cell line was defined by copy number more than 10.

Figure S2. Aurora kinase B identified by drug profiling in H82 cells and drive cell survival in MYC amplified SCLC.

A. The effect of E08 on cell viability in SCLC cells. 21 SCLC cell lines were seeded in 384-well plate with 1000 cells per well and treated with the active compound E08 and inactive compound N09 in duplicates at 1mM concentration. Cell viability assay was performed by CellTiter Glo 3 days after treatment. The average percentage of cell viability for each cell line was calculated compared with DMSO control and shown in Radar chart made by Excel.

B. The effect of E08 on cell viability in H82 cells. H82 cells were treated with compounds E08 and N09 in triplicates at the indicated titration concentration and cell viability assay was performed by CellTiter Glo 3 days after treatment. The average percentage of cell viability was calculated compared with DMSO control and shown in line chart made by Excel.

C. Hits of E08 identified by ATP probe-based drug profiling in H82 cells. Compound E08 and N09 drug profile were performed in H82 cells as described in Materials and Methods. Protein kinases were annotated via MetaCore (http://portal.genego.com ) (MetaCore, CA) based on protein ID generated by MaxQuant. Hits of each compound were chosen based on the inhibition on each peptide more than 60% compared with DMSO control. Candidate hits of the active compound E08 were obtained after removing the hits of inactive compound N09. The inhibition for each peptide corresponds to protein kinase was calculated compared with DMSO treatment. “#” represents the average of inhibition from the two individual experiments; otherwise, the hit was detected by only one experiment.

D. 15 kinases RNAi screen in SCLC cells. Small cell lung cancer cell lines were transfected with transfected with pooled siRNA targeting 15 indicated genes as described in Materials and Methods. Cell viability was performed by CellTiter Glo 5 days after transfection. Cell Viability changes were determined for each target gene after normalization on ON-TARGET plus Non-Targeting pool control siRNA and plotted by GraphPad Prism 6.

E. Depletion of AURKB by siRNA in SCLC cells. Small cell lung cancer cell lines were transfected with pooled siRNA targeting AURKB. Cell viability was performed by CellTiter Glo 5 days after transfection. Cell viability changes were constructed by GraphPad Prism 6.

Figure S3. TBK1 identified by drug profiling in SW210.5 cells

A. The effect of K14 on cell viability in SCLC cells. The indicated SCLC cell lines with no MYC amplification were seeded in 384-well plate with 1000 cells per well and treated with the active compound K14 and inactive compound B13 in duplicates at 1mM concentration. Cell viability assay was performed by CellTiter Glo 3 days after treatment. The average percentage of cell viability for each cell line was calculated compared with DMSO control and shown in Radar chart made by Excel.

B. Hits of K14 identified by ATP probe-based drug profiling in SW210.5 cells. Compound K14 and B13 drug profile were performed in SW210.5 cells as described in Materials and Methods. Hits of each compound were chosen based on the inhibition on each peptide more than 60%. Candidate hits of the active compound K14 were obtained after removing the hits of inactive compound B13. “#” represents the average of inhibition from the two individual experiments; otherwise, the hit was detected by only one experiment.

C. Depletion of TBK1 not affected the cell proliferation in MYC-amplified SCLC cells. Cells were transfected with pooled siRNA targeting TBK1, and A549 and SW210.5 as the positive control. Cell viability was performed by CellTiter Glo 5 days after transfection and cell viability changes were constructed by GraphPad Prism 6.

D. TBK1 expression in SCLC cell lines and human tissues. The expression of the phosphorylation of TBK1 and total TBK1 were evaluated by Western blotting in small cell lung cancer cell lines (upper panel) and small cell lung cancer human tissues (bottom panel). Equal protein loading was confirmed by β-actin evaluation.

E. TBK1 activation in mitosis in the relative TBK1 sensitive SCLC cell lines. The relative TBK1 sensitive cell lines SW210.5, H1607, H841 and DMS114 and TBK1 resistant cell lines H146, DMS79, H526 and H209 were exposed to 50 ng/ml of nocodazole for 18 hours, and Western blot detected the signaling change using indicated antibodies and b-actin evaluation was confirmed the equal protein loading.

F. TBK1 inhibitors affected cell viability in SCLC cells. 10 of small cell lung cancer cell lines with no MYC amplification were treated with TBK1 inhibitors CompII and BX-795 at the titration concentration. Cell viability was performed by CellTiter Glo 4 days after treatment and half maximal inhibitory concentration IC50 was calculated by CalcuSyn software version 2.11(http://www.biosoft.com/w/calcusyn.htm).

G. Depletion of TBK1 by siRNA in SCLC cells. 4 of small cell lung cancer cell lines were transfected with pooled siRNA targeting TBK1. Cell viability was performed by CellTiter Glo 5 days after transfection and cell viability changes were constructed by GraphPad Prism 6 (upper panel) and cells were exposed to 20nM of TBK1 siRNA for 72 hours and western analysis used to determine target modulation (bottom panel).

H. TBK1 inhibitors not affected aurora kinase in SW210.5 cells. SW210.5 cells were exposed to 50 ng/ml of nocodazole for 18 hours, and then treated with indicated TBK1 inhibitors for 10 minutes. Western blot detected the signaling change using indicated antibodies and b-actin evaluation was confirmed the equal protein loading.

I. TBK1 inhibitors induced cell apoptosis in SW210.5 and H1607 cells. SW210.5 and H1607 cells were treated with 0.5mM concentrations of TBK1 inhibitors Compound II and BX-795 for 48 hours, and caspase 3 activity was measured by flow cytometry as described in Materials and Methods.

J. TBK1 inhibitors affected cell division in SW210.5. SW210.5 cells were treated with TBK1 inhibitors CompII and BX-795 at 1mM for 48 hours and cell cycle assay was analyzed by flow cytometry as described in Materials and Methods.

SUPPLEMENTARY TABLE LEGENDS

Supplementary Table S1. Cell viability screen in SCLC cells against Roche published kinase inhibitor set. 235 compounds were applied for cell viability screen in the 21 SCLC cell lines. 1000 cells per well were seeded in 384-well plate and total 235 compounds were respectively delivered in duplicates to each well by a PrecisionTM microplate liquid handler and DMSO as the control. Cells were treated with final concentration of 1mM of individual compound for 3 days and cell viability assay was performed by CellTiter Glo. The average percentage of cell survival from duplicates compared with DMSO control was shown in the table.

Supplementary Table S2. Hits of E08 and N09 identified by ATP probe-based drug profiling in H82 cells. The drug profiling for E08 and N09 were performed in H82 cells twice and candidate hits of each compound were defined as described in Materials and Methods. The percentage of inhibition for each peptide represents the average data points from two individual MS runs.

Supplementary Table S3. Hits of K14 and B13 identified by ATP probe-based drug profiling in SW210.5 cells. The drug profiling for K14 and B13 were performed in SW210.5 cells twice and candidate hits of each compound were defined as described in Materials and Methods. The percentage of inhibition for each peptide represents the average data points from two individual MS runs.