Volume 160, Issue 5, Pages 977-989 (February 2015) Drug-Induced Death Signaling Strategy Rapidly Predicts Cancer Response to Chemotherapy  Joan Montero, Kristopher A. Sarosiek, Joseph D. DeAngelo, Ophélia Maertens, Jeremy Ryan, Dalia Ercan, Huiying Piao, Neil S. Horowitz, Ross S. Berkowitz, Ursula Matulonis, Pasi A. Jänne, Philip C. Amrein, Karen Cichowski, Ronny Drapkin, Anthony Letai  Cell  Volume 160, Issue 5, Pages 977-989 (February 2015) DOI: 10.1016/j.cell.2015.01.042 Copyright © 2015 Elsevier Inc. Terms and Conditions

Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 DBP Predicts Chemotherapy Sensitivity in PC9 Cell Lines (A) To perform DBP we obtain a single cell suspension from a cell line or a primary sample, and we expose the cells to the different drug treatments to be tested. After this incubation, we permeabilize, stain with the fluorescent dye JC-1 and expose the cells to different BH3 peptides that will promote mitochondrial depolarization and MOMP, the ultimate event that triggers apoptosis. By comparing the non-treated cells with the treated ones, DBP will determine the Δ% priming for each agent and identify which are most effective to induce apoptosis in that particular sample. All this analysis is performed in less than 24 hr, minimizing ex vivo culture. (B) DBP was performed on three different PC9 cell lines: parental PC9, PC9GR (gefitinib resistant, T790M mutation present), and PC9WZR (gefitinib and WZ4002 resistant, T790M mutation present), using a 16 hr incubation of: gefitinib 1 μM, WZ4002 100 nM, CI-1040 3 μM (MEK inhibitor), and WZ4002+CI-1040. Results expressed as Δ% priming (increase in priming compared to non-treated cells). Values indicate mean values ± SEM, at least three independent experiments were performed (n ≥ 3). (C) Cell death measurements at 72 hr for the same cell lines under the same treatments by FACS using Annexin V/PI staining. Results are expressed as increase on cell death or Δ% cell death, compared to non-treated cells. Values indicate mean values ± SEM, at least three independent experiments were performed (n ≥ 3). (D) Plot showing correlation between Δ% priming at 16 hr and Δ% cell death at 72 hr. ROC curve analysis at right. (E) Western blot analysis, showing changes in the BCL-2 family of proteins. See also Figures S1, S2 and S3. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 DBP Predicts Chemotherapy Sensitivity in Breast Cancer Cell Lines (A) DBP was performed in five breast cancer cell lines: BT20, HCC1954, SKBR3, T47D, and HCC2218 showing different pattern of response to the treatments tested (16 hr incubation): (1) gefitinib 1 μM, (2) lapatinib 1 μM, (3) MK-2206 1 μM, (4) AZD6244 1 μM, (5) BEZ235 1 μM, (6) dinaciclib 10 nM (SCH 727965), (7) ABT-888 5 μM and the combination (8) AZD6244 + BEZ235. Results expressed as Δ% priming (increase in priming compared to non-treated cells). Values indicate mean values ± SEM, at least three independent experiments were performed (n ≥ 3). Cell death measurements at 96 hr for the same cell lines under the same treatments by FACS using Annexin V/PI staining. Results are expressed as increase on cell death or Δ% cell death, compared to non-treated cells. Values indicate mean values ± SEM, at least three independent experiments were performed (n ≥ 3). (B) Plot showing the significant correlation between Δ% priming at 16 hr and Δ% cell death at 96 hr. (C) ROC curve analysis. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Identifying the Optimal Treatment in Hematological Malignancies Using DBP We selected several drugs targeting either key membrane receptors: (1) gefitinib 1 μM, (2) imatinib 1 μM, (3) lapatinib 1 μM, (4) PD173074 1 μM and (5) TAE684 1 μM; or important intracellular kinases: (6) MK-2206 1 μM, (7) PLX4032 10 μM, (8) AZD6244 1 μM and (9) BEZ235 1 μM, and we tested them with several human hematological cancer cell lines: K562, DHL6, LP1, DHL4, and AML3. (A) DBP (16 hr incubation) results expressed as Δ% priming and cell death measurements at 72 hr using Annexin V/PI staining expressed as Δ% cell death. Values indicate mean values ± SEM, at least three independent experiments were performed (n ≥ 3). (B) Plot showing the significant correlation between Δ% priming at 16 hr and Δ% cell death at 72 hr. (C) ROC curve analysis shows AUC = 0.83. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Identifying the Optimal Treatment in Solid Tumors Using DBP We tested the same panel of kinase inhibitors on several human solid tumor cell lines: MCF7, PC9, SK-MEL-5, HCT116 and MDA-MB-231. (A) DBP (16 hr incubation) results expressed as Δ% priming and cell death measurements at 72 or 96 hr (as indicated) using Annexin V/PI staining expressed as Δ% cell death. Values indicate mean values ± SEM, at least three independent experiments were performed (n ≥ 3). (B) Plot showing the significant correlation between Δ% priming at 16 hr and Δ% cell death at 72/96 hr. (C) The ROC curve analysis has an AUC = 0.96, indicating that DBP is an excellent binary predictor for chemotherapy response in solid tumor cell lines. See also Figure S4. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 DBP Is a Good Binary Predictor for Cell Lines (A) Compilation of Figures 1, 2, 3, 4 and S3 results, showing a significant correlation between Δ% priming and Δ% cell death for all cell lines analyzed. (B) The total area under the ROC curve is 0.89, indicating that is a good binary predictor for chemotherapy response in all the cell lines and treatments tested. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 DBP Can Identify the Best In Vivo Treatment among Several Options Braf/Nf1 mutant melanoma cells were treated ex vivo with PLX4720 1 μM, PD0325901 (referred to as PD-901) 0.25 μM, GDC-0941 1 μM, rapamycin 0.1 μM, PD-901 + rapamycin, and PLX4720 + rapamycin. (A) DBP (16 hr incubation) results expressed as Δ% priming. Values indicate mean values ± SEM, at least three independent experiments were performed (n ≥ 3). (B) In vivo response for this Braf/Nf1 mutant allograft melanoma model (adapted from Figure 4C Maertens et al., 2013) expressed as change in tumor volume (log2) after 7 days of treatment. (C) Correlation between Δ% priming and change in tumor volume. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 DBP Can Stratify In Vivo Drug Response to Imatinib in a Cohort of CML Patients and to Carboplatin in Ovarian Adenocarcinoma Patients (A) 24 frozen Ficoll purified bone marrow primary CML samples were treated for 16 hr with imatinib 1 and 5 μM, and DBP was then performed. Results are expressed as Δ% priming. Values indicate mean ± SEM. Unpaired t-test, two-tailed, ∗ p < 0.05. (B) A ROC curve analysis for this set of samples. The AUC is 0.89. (C) 16 ovarian adenocarcinoma patient samples were analyzed by DBP with carboplatin. We treated the samples for 16 hr with carboplatin 100 μg/ml, and DBP was then performed. Shown is a Kaplan -Meier plot of the patients’ progression-free survival in response to carboplatin and taxol. A significant difference was observed between those patients whose samples showed a Δ% priming >20% from those that were <20%, as assessed by Mantel-Cox statistical analysis. See also Figures S5 and S6, and Table S1. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S1 Dynamic Range and Peptide Concentration Optimization, Related to Figure 1 We observe that as we increase the peptide concentration (as shown with BIM) the mitochondria depolarizes due to mitochondrial outer membrane permeabilization (MOMP). When the cancer cells are effectively treated, the permeabilizing effect of the peptide at a given concentration increases. Thus we identify the “dynamic range” as the peptide concentrations where we see an increase in this mitochondrial depolarization, and we define the maximal response to the peptide [Δ% depolarization (optimal)] as Δ% priming. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S2 Gefitinib and WZ4002 Response Over Time, Related to Figure 1 Cell death was monitored over time on parental PC9, PC9GR and PC9WZR cell lines in response to gefitinib 1 μM and WZ4002 100 nM. Measurements at 24, 48 and 72 hr were performed by FACS using Annexin V/PI staining. Values indicate mean ± SEM, at least three independent experiments were performed (n ≥ 3). Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S3 DBP Predicts Chemotherapy Sensitivity in Non-Small Cell Lung Cancer Cell Lines, Related to Figure 1 (A) DBP was performed in six non-small cell lung cancer cell lines: H460, H1650, A549, Hop62, HCC827, H1975 showing different pattern of response to the treatments tested (16 hr incubation): gefitinib 1 μM, WZ4002 100 nM, AZD6244 1 μM (MEK inhibitor), BEZ235 1 μM (PI3K/mTOR inhibitor) and AZD6244 + BEZ235. Results are expressed as Δ% priming (increase in priming compared to non-treated cells). Cell death measurements at 72 hr for the same cell lines under the same treatments by FACS using Annexin V/PI staining. Results are expressed as increase in cell death (Δ% cell death), compared to non-treated cells. Values indicate mean ± SEM, at least three independent experiments were performed (n ≥ 3). (B) Plot showing the significant correlation between Δ% priming at 16 hr and Δ% cell death at 72 hr. (C) Receiver operating characteristic curve analysis. The area under the ROC curve is 0.895, indicating that is a good binary predictor for chemotherapy response in the NSCLC cell lines tested. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S4 Comparison between Dynamic BH3 Profile Using JC-1 Based Whole-Cell Analysis and Cytochrome c Release, Related to Figure 4 (A and B) We compared the results obtained with JC-1 whole cell DBP analysis with the cytochrome c release detected by FACS in PC9 (A) and SK-MEL-5 (B) when the cells were exposed to BIM 1 μM and 0.3 μM respectively. Values indicate mean ± SEM, at least three independent experiments were performed (n ≥ 3) in the top two graphs, representative experiments in the bottom two graphs. As shown, similar results were obtained by both methods demonstrating that MOMP measurements by transmembrane potential loss (top) and cytochrome c release (bottom) are comparable. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S5 Imatinib Inhibition of Phospho-ABL Induces Priming Increase and Later Cell Death, Related to Figure 7 (A) DBP was performed in two BCR-ABL positive human CML cell lines, K562 and Ku812 exposed to increasing concentration of imatinib for 16 hr: 0.01, 0.1, 1, and 10 μM. Results are expressed as Δ% priming (increase in priming compared to non-treated cells). Cell death measurements at 72 hr for the same cell lines under the same treatments by FACS using Annexin V/PI staining. Results are expressed as increase in cell death (Δ% cell death), compared to non-treated cells. Values indicate mean ± SEM, at least three independent experiments were performed (n ≥ 3). (B) Representative western blots against Phospho-c-ABL (Tyr 412) and Phospho-CRKL (Tyr 207), using Actin as a loading control, for the same two cell lines exposed for 16 hr to different imatinib concentrations. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S6 DBP Analysis in CML Primary Samples, Related to Figure 7 We repeated the calculations performed in Figure 6 applying the criteria of only accepting tracings for which there was at least a difference of 100 relative fluorescent units between our positive control (FCCP) and negative control. (A) 17 frozen Ficoll purified bone marrow primary CML samples were treated for 16 hr with imatinib 1 and 5 μM, and DBP was then performed. Results are expressed as Δ% priming. Those samples obtained from patients that had a complete hematologic response to imatinib treatment in clinic showed significantly higher Δ% priming in our DBP analysis, as opposed to those samples obtained from patients that relapsed. Values indicate mean ± SEM. Unpaired t-test, two-tailed, ∗ p < 0.05. (B) We performed a receiver operating characteristic curve analysis for this set of samples. The area under the ROC curve is 0.88. Cell 2015 160, 977-989DOI: (10.1016/j.cell.2015.01.042) Copyright © 2015 Elsevier Inc. Terms and Conditions