Target Identification in Small Cell Lung Cancer via Integrated Phenotypic Screening and Activity-Based Protein Profiling

Abstract

To overcome hurdles in identifying key kinases in small cell lung cancer (SCLC), we integrated a target-agnostic phenotypic screen of kinase inhibitors with target identification using activity-based protein profiling (ABPP) in which a desthiobiotin-ATP probe was used. We screened 21 SCLC cell lines with known c-MYC amplification status for alterations in viability using a chemical library of 235 small-molecule kinase inhibitors. One screen hit compound was interrogated with ABPP, and, through this approach, we reidentified Aurora kinase B as a critical kinase in MYC-amplified SCLC cells. We next extended the platform to a second compound that had activity in SCLC cell lines lacking c-MYC amplification and identified TANK-binding kinase 1, a kinase that affects cell viability, polo-like kinase-1 signaling, G2-M arrest, and apoptosis in SCLC cells lacking MYC amplification. These results demonstrate that phenotypic screening combined with ABPP can identify key disease drivers, suggesting that this approach, which combines new chemical probes and disease cell screens, has the potential to identify other important targets in other cancer types. Mol Cancer Ther; 15(2); 334-42. ©2016 AACR.

Figure 1. Workflow for therapeutic target identification in SCLC via integrated phenotypic screen with ABPP ATP probe and LC-MS/MS

A compound library (A) was used for cell viability screen in SCLC cell lines (B), with paired active and inactive compounds based on screen results chosen for further drug profiling using desthiobiotin-ATP probe (C) tandem liquid chromatography-mass spectrometry. The identified peptides were quantified by MaxQuant, and the candidate hits (orange nodes) of active drug were determined based on those that inhibited ATP binding by >60% compared with DMSO treatment and filtered by potential hits (blue nodes) of inactive compound (D). Potential targets were validated in SCLC cells using relevant small RNA interference and drugs (E).

Figure 2. Compound screen across SCLC cell lines

We investigated 235 compounds for the cell viability screen in the 21 SCLC cell lines; 1,000 cells/well were seeded in 384-well plate and treated with individual compounds at 1 µM concentration for 3 days. Cell viability was performed by CellTiter Glo. A heat map of the average percent cell survival from duplicates compared with DMSO control was plotted by the “imagesc” function in MatLab. Blue indicates less cell survival; red indicates more cell survival. Bottom right (color key legend) shows range of average values. Left panel shows the chemical structures of the active and inactive compound pairs (E08 versus N09 and K14 versus B13).

Figure 3. Aurora kinase B (AURKB) identified by combining drug screen with ABPP ATP probe in H82 cells and drive cell survival in MYC-amplified SCLC cells

A: Binding proteins of E08 identified by ABPP ATP probe in H82 cells. Drug profiling of compounds E08 and N09 was performed in H82 cells. The compound’s candidate binding proteins (shown on the human kinome tree) were defined as those that inhibited binding of the ATP peptide by >60% compared with DMSO treatment. The orange nodes represent the final candidate targets of active compound E08, and the blue nodes represent the targets of both active and inactive compounds. B: AURKB knockdown by siRNA dramatically inhibited cell viability in H82 cells. H82 cells were 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. C: MYC-amplified SCLC are more sensitive to depletion of AURKB by siRNA. SCLC cell lines with MYC amplification (n=6) and no MYC amplification (n=14) were transfected with pooled siRNA targeting AURKB. Cell viability was performed by CellTiter Glo 5 days after transfection. The box plot between MYC amplified (MYC+_SCLC) and lacking MYC amplification (MYC−_SCLC) SCLC cell lines based on cell viability changes was constructed by GraphPad Prism 6 (P value based on the Wilcoxon rank sum test). D: MYC amplified SCLC are more sensitive to AURKB inhibitors. SCLC cell lines with MYC amplification (n=6) and lacking MYC amplification (n=11) were treated with 0.5 µM of indicated AURKB inhibitors. Cell viability was performed by CellTiter Glo 3 days after treatment. A heat map of average percent cell survival from duplicates compared with DMSO control was plotted by the “imagesc” function in MatLab. Bottom right (color key legend) shows range of average values. P value shown at left bottom was based on the Wilcoxon rank sum test between MYC amplification (MYC+) and MYC lacking amplification (MYC−) groups.

Figure 4. TBK1 identified by ABPP ATP probe in SW210.5 cells plays an important role in cell survival in a subset of SCLC cells lacking MYC amplification

A: Depletion of TBK1 by siRNA affected cell growth in SCLC cells. Ten SCLC cell lines with no MYC amplification were transfected with pooled siRNA targeting TANK-binding kinase 1 (TBK1) and lung cancer cell line A549 as the positive control. Cell viability was performed by CellTiter Glo 5 days after transfection. Cell viability changes were constructed by GraphPad Prism 6 after normalizing with ON-TARGET plus Non-Targeting pool control siRNA. B: TBK1 inhibitors affected cell viability in SW210.5 and H1607 cells. Four SCLC cell lines with no MYC amplification were treated with TBK1 inhibitors Compound II and BX-795 at the titration concentration. Cell viability was performed by CellTiter Glo 4 days after treatment. The growth curve was constructed by GraphPad Prism 6. C. TBK1 inhibitors induced cell apoptosis in SW210.5 and H1607 cells. SW210.5 and H1607 cells were treated with indicated concentrations of TBK1 inhibitors Compound II and BX-795 for 48 hours, and PARP cleavage was evaluated by Western blotting. Equal protein loading was confirmed by β-actin evaluation. D: TBK1 mediated activation of the mitotic kinase PLK1. SW210.5 cells were treated with indicated TBK1 inhibitors for 1 hour and then exposed to 50 ng/mL of nocodazole for 18 hours. Western blot detected the signaling change using indicated antibodies, and β-actin evaluation was confirmed the equal protein loading.