Inhibiting MDM2-p53 Interaction Suppresses Tumor Growth in Patient-Derived Non– Small Cell Lung Cancer Xenograft Models  Josephine Hai, PhD, Shingo Sakashita, MD, Ghassan Allo, MD, Olga Ludkovski, MSc, Christine Ng, MPhil, Frances A. Shepherd, MD, Ming-Sound Tsao, MD  Journal of Thoracic Oncology  Volume 10, Issue 8, Pages 1172-1180 (August 2015) DOI: 10.1097/JTO.0000000000000584 Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 RG7388 activates p53 pathway and inhibits cell proliferation in p53 wild-type NSCLC and HLC cells. A, RG7388 dose dependently accumulates p53 protein and its transcriptional targets MDM2 and p21 in A549, H1395, and HLC 12 cells. Western blot analysis was performed on whole-cell lysates 24 hours after RG7388 treatment. B, Three wild-type p53 and (C) six mutant p53 exponentially growing cell lines were incubated with RG7388 for 5 days, and cell viability was measured by MTS assay. Results represent means ± standard error of the mean from three independent experiments in quadruplicates. D, RG7388 cytotoxicity depends on the p53 status of lung cancer cells. E, Basal MDM2 protein levels were compared in cell lines by Western blot analysis. HLC, Human lung cell lines; NSCLC, non–small-cell lung cancer; MW, molecular weight. Journal of Thoracic Oncology 2015 10, 1172-1180DOI: (10.1097/JTO.0000000000000584) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 RG7388 induces cell cycle arrest and apoptosis. A–C, Cell cycle analysis was analyzed by PI staining after treatment of A549 (A), HLC 12 (B), and H1395 (C) cells with indicated doses of RG7388 or DMSO for 24 hours. D–F, Apoptotic activity was measured by Annexin V and PI staining after incubation of A549 (D), HLC 12 (E), and H1395 (F) cells with indicated doses of RG7388 for 48 hours. Results represent mean ± standard error of the mean from three independent experiments (*p < 0.05; **p < 0.01; ****p < 0.0001). ns, not significant; HLC, human lung cell lines; PI, propidium iodide. Journal of Thoracic Oncology 2015 10, 1172-1180DOI: (10.1097/JTO.0000000000000584) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Pharmacodynamics of RG7388 in PDX-bearing mice. A, Representative immunohistochemical sections of MDM2 staining intensities from zero to three. B, Immunohistochemical detection of MDM2 and p53 in tumors formed from PHLC 12 and PHLC 189 (×10 magnification). MDM2 staining was measured using H-score criteria. (C, D) Western blot analysis of changes in p53, MDM2, and p21 expression in PHLC 189 (C) and PHLC 12 (D) xenografts. Lysates were prepared from tumors treated with vehicle or 50 or 80 mg/kg RG7388 (n = 3/group). Monoclonal p53 antibody (DO-1) recognizes full length (FL) and β isoforms. IHC, immunohistochemistry; H&C, hematoxylin and eosin; PDX, patient-derived xenograft. Journal of Thoracic Oncology 2015 10, 1172-1180DOI: (10.1097/JTO.0000000000000584) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 In vivo antitumor activity of RG7388 on PDX models. A, Effect of RG7388 treatment on tumor growth of PHLC 189 in NOD-SCID mice at 80 mg/kg. Final PHLC 189-treated tumor weights at time of killing compared with control tumors. The data points represent the averages of eight mice per treatment group. B–D, Effect of RG7388 treatment on tumor growth of PHLC 12 (B), 229 (C), and 193 (D) in NOD-SCID mice at 50 or 80 mg/kg. C, Final PHLC 229-treated tumor weights at the time of killing compared with control tumors. The p values were calculated using analysis of variance (ANOVA)-mixed model testing and Student t test for tumor growth and final tumor weights, respectively (**p < 0.01; ***p < 0.001; ****p < 0.0001). PDX, patient-derived xenograft. Journal of Thoracic Oncology 2015 10, 1172-1180DOI: (10.1097/JTO.0000000000000584) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5 RG7388 inhibits cell proliferation but not apoptotic caspase activity in vivo. A, Representative histologic sections of xenografts from PHLC 189 tumors were immunostained with Ki-67 and CC3. B and C, The percentage of positive Ki-67 cells were quantified in PHLC 189 tumor sections and the number of CC3-positive cells were scored at 10 high-power fields (n = 8 per group; ×10 magnification; *p < 0.05). H&C, hematoxylin and eosin; CC3, cleaved caspase-3; ns, not significant. Journal of Thoracic Oncology 2015 10, 1172-1180DOI: (10.1097/JTO.0000000000000584) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 6 RG7388 activates p53 pathway and its downstream targets in vivo. A, Quantitative real-time PCR was performed on total tumor RNA extracted at 4 hours after treatment on PHLC12 mice to measure transcript levels of indicated genes relative to levels in untreated cells (n = 5 mice/group). B and C, Representative tumor sections of PHLC 12 xenografts were immunostained with p53 and scored using H-score criteria (*p < 0.05). H&C, hematoxylin and eosin; PCR, polymerase chain reaction. Journal of Thoracic Oncology 2015 10, 1172-1180DOI: (10.1097/JTO.0000000000000584) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions