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Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, CanadaDepartment of Medical Biophysics, University of Toronto, Toronto, Ontario, CanadaDepartment of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
Address for correspondence: Ming-Sound Tsao, MD, Princess Margaret Cancer Centre, University Health Network, Room 14-301, 101 College Street, Toronto, Ontario, M5G 1L7, Canada
Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, CanadaDepartment of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
The tumor suppressor p53 is frequently inactivated in non–small cell lung cancer (NSCLC). Activation of the p53 pathway by inhibition of its negative regulator MDM2 may offer an attractive approach for NSCLC therapy. We evaluated the antitumor activity of the small-molecule MDM2 inhibitor RG7388 in patient-derived xenograft (PDX) models of NSCLC.
Methods:
We investigated the effect of RG7388 treatment on cell proliferation, cell cycle arrest, and apoptosis using a panel of human NSCLC cell lines (A549, H157, H1650, H1395, and H358) and PDX cell lines (human lung cell lines 12, 137, 277, and 196). PDX-bearing mice were used to test the therapeutic efficacy and pharmacodynamic effects of RG7388 treatment.
Results:
We demonstrated that RG7388 promotes low nanomolar antiproliferative activity selectively in cell lines with wild-type p53 and p53 pathway activation, resulting in cell cycle arrest and apoptosis. In PDX models, oral administration of RG7388 led to potent dose-dependent and time-dependent activation of p53 and had a significant impact on p53 downstream targets. Daily treatment of RG7388 in mice at 50 and 80 mg/kg/day inhibited tumor growth in three wild-type p53 PDX models. Activation of the p53 pathway inhibited cell proliferation as observed by reduced Ki-67-positive cells in xenograft tumors. However, induction of apoptotic caspase activity was not observed in these tumors. Notably, RG7388 treatment remains effective in tumors lacking MDM2 amplification but expressing wild-type p53.
Conclusions:
MDM2 small-molecule inhibitor is effective in treating NSCLC tumors with wild-type p53, supporting further clinical investigation as a potential NSCLC therapy.
In the last decade, molecularly targeted drugs such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors have emerged as viable therapeutic options for lung adenocarcinoma patients.
Spanish Lung Cancer Group in Collaboration with Groupe Français de Pneumo-Cancérologie and Associazione Italiana Oncologia Toracica. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.
However, these two proteins represent possible targets in only a fraction of NSCLC patients. Therefore, there is a need to investigate the efficacy of new agents directed against other potential driver oncogenes in NSCLC.
The tumor suppressor p53 plays an important role in protecting against cancer development. The p53 protein is a transcription factor that prevents accumulation of genetic damage in response to stress by regulating DNA repair, the cell cycle, and apoptosis through transactivation of key downstream genes.
Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis.
Indeed, known tobacco carcinogens, such as benzo[a]pyrene, have been shown to form diol epoxide adducts at lung cancer mutation hotspots within the human TP53 gene.
Furthermore, even in the presence of functional p53, tumor cells can overexpress MDM2 to reduce p53 activity by means of an autoregulatory feedback loop.
Inhibition of p53-MDM2 interaction in tumor cells could be a potential strategy to restore p53 function and to counteract the oncogenic consequences of MDM2 overproduction in NSCLC patients.
Members of the nutlin class of imidazole compounds interact specifically with the p53-binding pocket of MDM2 to free p53 from MDM2-mediated negative control.
Restoration of p53 function was observed after oral administration of RG7112 to mice with tumors bearing human osteosarcoma cells with MDM2 gene amplification.
A neoadjuvant clinical study found partial response or stable disease in 15 of 20 liposarcoma patients treated preoperatively with RG7112 for up to three 28-day cycles.
Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study.
Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study.
However, cells transplanted after prolonged artificial in vitro culture typically lack the molecular and histological characteristics and heterogeneity of the original patient tumors.
Because early-passage xenografts derived from fresh surgical tumor specimens are not cultured on plastic but are propagated in immune-deficient mice, these models may predict clinical response better than conventional cell line–derived tumors.
reported that response to cisplatin in ovarian patient-derived xenograft (PDX) models was consistent with clinical response in patients. In the present study, we demonstrate that the MDM2 inhibitor RG7388 can reactivate p53 signaling and inhibit tumor growth in clinically relevant NSCLC PDX models expressing wild-type p53 in vitro and in vivo, supporting further clinical evaluation of MDM2 inhibitors in NSCLC.
MATERIALS AND METHODS
Cell Lines and Reagents
Human NSCLC cell lines, NCI-A549, NCI-H1395, NCI-H157, NCI-H1650, and NCI-H358, were obtained from the American Type Culture Collection (Manassas, VA). All cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (Hyclone Europe, Ltd., Cramlington, United Kingdom) and antibiotics at 37°C and 5% carbon dioxide. Authentication of human cell lines was performed by short tandem repeat DNA profiling analysis.
NSCLC PDX models were established using protocols approved by the University Health Network Research Ethics Board and Animal Care Committee as previously described.
and were processed into a single-cell suspension to establish long-term cell culture growth on plastic dishes. Tumor fragments were minced, digested in collagenase for 1 hour at 37°C, and treated with DNase for 10 minutes at 37°C. Magnetic columns (MACS Technology) coated with H2Kb/H2Dd anti-mouse antibodies were used to deplete stromal mouse fibroblast cells in the tumor cell population. RG7388 and solvents were provided by Hoffmann-La Roche (Nutley, NJ).
Fluorescence In Situ Hybridization
Dual-color interphase fluorescence in situ hybridization was performed on 4-μm paraffin sections of tissue microarrays as previously described.
Two bacterial artificial chromosome clones RP11-154P16 (starts 69,128,534 ends 69,309,251) and RP11-450G15 (starts 69,080,491 ends 69,256,798) that hybridize to the 12q15 region containing MDM2 (starts 69,201,971 ends 69,239,320) were selected from the University of California Santa Cruz (UCSC) Human Genome Browser Assembly (February 2009 CRch37/hg19) and were obtained from the Centre for Applied Genomics (TCAG, Toronto, ON, Canada). The bacterial artificial chromosome clones were directly labeled with Spectrum Orange (SpO) fluorochrome using commercially available nick translation kit according to the manufacturer’s protocol and were used together with CEP12 control probe labeled with SpG (Abbott Molecular, Abbott Park, IL). Probes were verified on normal blood metaphases to confirm correct localization and on polymerase chain reaction (PCR). Probe numbers were counted in 50 nonoverlapping tumor cells per core, and the scores were analyzed to determine the mean MDM2:CEP12 ratio per cell.
Western Blot Analysis and Immunohistochemistry
Whole-cell extracts were lysed with lysis buffer (10 mM Tris [pH 8.0], 1% NP-40, 2 mM ethylenediaminetetraacetic acid, 150 mM, 0.1 mM Na3VO4, and protease inhibitors [Roche]), resolved by sodium dodecyl sulphate- polyacrylamide gel electrophoresis and transferred to polyvinyliden fluoride membranes. Primary antibodies included anti-p53 (SC126; Santa Cruz, CA), anti-MDM2 (SMP14; Santa Cruz), p21 (C24420; BD Biosciences, Mississauga, ON), and beta-actin (Clone AC-74; Sigma, St. Louis, MI). After blocking, membranes were incubated with relevant antibodies and probed with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, Beverly, MA). All films were developed with ECL-Plus reagents (GE Healthcare, Piscataway, NJ).
Formalin-fixed paraffin-embedded tissues were cut at 4-µm thickness and dried in a 60°C oven overnight. Immunohistochemistry was performed using the peroxidase antiperoxidase technique following a microwave antigen retrieval procedure. Ki-67 and cleaved caspase-3 antibodies were used at 1:500 dilution, and MDM2 and p53 were used at 1:100 after pepsin digestion. Whole slides were scanned using the Aperio Scanscope XT (Vista, Canada). Tumor cells stained positive for Ki-67 were scored in 500 tumor cells. Similarly, cleaved caspase-3–positive cells were scored in 10 fields at high-power fields (×400 magnification). For MDM2- and p53-stained slides, pathologists (GA and SS) scored the slides using the hybrid (H) scoring method: H-score = % cells of 0 intensity + (% cells of 1+ intensity × 1) + (% cells of 2+ intensity × 2) + (% cells of 3+ intensity × 3). The intensity score was defined as 0 if no appreciable staining in the tumor cells; 1+ barely detectable staining; 2+ readily appreciable brown staining distinctly marking the tumor cell cytoplasm and/or nucleus; and 3+ very strong dark staining in tumor cells.
Quantitative Real-Time PCR Analysis and DNA Sequencing
Total cellular RNA was extracted from xenograft tumors and cell lines using TRIzol reagent (Invitrogen, Burlington, ON) and RNeasy Mini kit (Qiagen, Mississauga, ON), respectively. One microgram of total RNA was reverse transcribed using Superscript II RNase H reverse transcription kit (Invitrogen). Real-time quantitative PCR was performed with the human-specific primers listed in Supplementary Table S1 (Supplemental Digital Content, http://links.lww.com/JTO/A845), and 10 ng of the first-strand cDNA synthesis as a template using Stratagene MX3000P and SYBR Green (Stratagene, La Jolla, CA). Messenger RNA (mRNA) copy number was adjusted by the geometric mean of the two house-keeping genes ACTB and BAT1 as previously described.
Genomic DNA was extracted from xenograft tumors and cell lines using TRIzol reagent (Invitrogen). Using TP53 primers listed in Supplementary Table S1 (Supplemental Digital Content, http://links.lww.com/JTO/A845), PCR was used to prepare gene products for DNA sequencing.
Cell Viability, Cell Cycle, and Apoptosis Assays
Cell proliferation/viability was evaluated by the tetrazolium dye (MTS) assay (Promega, Madison, WI). Each cell line was plated at a seeding density to give logarithmic growth over the course of the assay in a 96-well tissue culture plate. The concentration of final product was measured by absorbance at 490 nm, which is proportional to the viable cell number in each well. Apoptosis was determined using the Annexin V assay (Biolegend, San Diego, CA), and cell cycle was analyzed using the propidium iodide assay (Invitrogen, Waltham, MA). Cells were seeded in culture plates 24 hours before incubation with RG7388 for the indicated doses and times. Cells were collected and stained with Annexin V-FITC and/or propidium iodide solution as per the manufacturer’s protocol and analyzed by flow cytometry. Cell death was recorded in a FACSCanto II (BD Biosciences) in the total population (10,000 cells), and data were analyzed using FACSCanto II software and using ModFit Software (Verity Software House, Topsham, ME).
PDX Models
Non-obese (NOD)-SCID mice were bred on site at the Princess Margaret Cancer Centre animal facility. Mice were fed autoclaved food and water ad libitum. All manipulations were performed under sterile conditions in a laminar flow hood, in accordance with the procedures approved by the Institutional Animal Care Committee. PHLC 12, 189, 229, and 193 were revived by implanting cryopreserved early-passage tumor fragments (<6 passages) into the subcutaneous tissue of the NOD-SCID mice. The histological subtype and mutational status for PDX models are listed in the Supplementary Table S3 (Supplemental Digital Content, http://links.lww.com/JTO/A845).
For treatment, mice were randomized into groups of six to 10 mice with similar mean tumor volumes of 50 to 150 mm3. RG7388 was administered by oral gavage at nontoxic doses from 50 to 80 mg/kg daily (Supplementary Figure S1, Supplemental Digital Content, http://links.lww.com/JTO/A845). Tumor size was measured in two dimensions using a caliper every 3 to 4 days. Tumor volume was calculated using the following formula (length × width2)π/6. Mice were killed once the humane end point (approximately 1.5 cm diameter) was reached. At the time of killing, tumors were weighed and tumor portions were snap frozen and stored in liquid nitrogen or fixed in 10% buffered formalin for routine histopathologic processing.
Statistics
All cell line and animal data are presented as mean ± standard error of the mean. Statistical significance was determined using two-tailed Student’s t test. Differences in tumor growth rates of xenografts were tested using mixed-model analysis of variance (ANOVA). Tests that produced p value 0.05 or less were considered to be significant. All statistical analyses were performed with GraphPad Prism 6.0 (La Jolla, CA).
RESULTS
RG7388 Activates p53 Pathway and Inhibits Cell Proliferation in p53 Wild-Type NSCLC Cells
RG7388 dose dependently induced p53 accumulation and up-regulation of the p53 downstream transcriptional targets p21 and MDM2 in multiple cell lines, including A549, H1395, and HLC 12 cells (Fig. 1A).
FIGURE 1RG7388 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.
We next evaluated the ability of RG7388 to inhibit cell proliferation in five NSCLC cell lines (H1395, A549, H157, H1650, and H358) and four PDX-derived cell lines (HLC 12, 277, 137, and 196). DNA sequencing of TP53 revealed that HLC 12 cells were wild type for TP53, whereas HLC 137, 196, and 277 cells harbored an S183stop and I251N and R337P mutation in the TP53 gene, respectively (Supplementary Table S2, Supplemental Digital Content, http://links.lww.com/JTO/A845). Previous studies have reported that A549 and H1395 are wild type for TP53 and H157, H1650, and H358 contain c.892G>T, c.673-2A>G, and deletion in the TP53 gene, respectively.
RG7388 potently inhibited cell growth in all three p53 wild-type lines (HLC 12, A549 and H1395), giving rise to the half maximal inhibitory concentration (IC50) values ranging from 0.05 to 0.75 μM (Fig. 1B). In contrast, in the p53 mutant lines (H157, H1650, H358, HLC 137, HLC 196, and HLC 277), treatment induced a 20- to 1000-fold lower cytotoxicity, with IC50 values ranging from 16 to 50 μM (Fig. 1C). RG7388 induced the greatest response in the H1395 cell line, which harbors an MDM2 gene amplification (IC50: 0.05 μM).
RG7388 Induces Cell Cycle Arrest and Apoptosis in NSCLC Cells
One of the primary cellular functions of activated p53 is inhibition of cell cycle progression in G1 and G2 phases. Treatment of exponentially proliferating A549 cells with RG7388 for 24 hours led to a dose-dependent cell cycle block in G1 and G2/M phases and a depletion of the S-phase compartment. At 1.5 μM concentration, the cell population in S-phase decreased from approximately 37% to 2%, whereas accumulation of cells at G1 and G2 phases increased (approximately 49% to 74% and 14% to 24%, respectively; Fig. 2A). The same effect was observed in HLC 12 and H1395 cell line models, which showed a reduction of the S-phase population to 2% and 6%, respectively (Fig. 2B, C).
FIGURE 2RG7388 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.
We then examined the effect of p53 activation on apoptosis in A549, HLC 12, and H1395 cells. In A549 cells, apoptosis was significantly induced after 48 hours treatment of 1 to 10 μM of RG7388 (Fig. 2D). The concentration of RG7388 required to achieve 50% of the maximum cell cycle arrest (IC50: 0.75 μM) was approximately seven-fold lower than the concentration required to induce apoptosis (IC50: >5 μM). In HLC 12 and H1395 cells, RG7388 induced a minor 1.5-fold increase in cell death at 10 and 5 μM, respectively (Fig. 2E, F).
RG7388 Activates the p53 Pathway in PDX Tumors In Vivo
Pharmocodynamic effects of RG7388 were assessed in mice bearing PHLC 12 and PHLC 189 tumors. Because previous reports have shown that osteosarcoma tumors with high levels of MDM2 protein or amplification are highly sensitive to RG7112, we examined the MDM2 amplification status in our PDX models.
Using fluorescence in situ hybridization, MDM2 copy number (MDM2/CEP12 ratio per cell) for PHLC 12 and PHLC 189 tumor was determined to be 1.04 and 1.06, respectively (Supplementary Figure S2, Supplemental Digital Content, http://links.lww.com/JTO/A845). Similarly, silver-enhanced in situ hybridization assays determined that MDM2 copy number for PHLC 12 and PHLC 189 tumor to be 1.10 and 1.57, respectively (Supplementary Figure S3, Supplemental Digital Content, http://links.lww.com/JTO/A845). These results were corroborated by the nuclear MDM2 protein levels in PHLC 12 (H-score 80) and 189 xenograft tumor sections (H-score of 120; Fig. 3A, B).
FIGURE 3Pharmacodynamics 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.
We further verified that both xenograft models harbored wild-type p53 by sequencing the TP53 gene and immunohistochemistry analysis (Fig. 3B and Supplementary Table S3, Supplemental Digital Content, http://links.lww.com/JTO/A845). To examine the ability of RG7388 to activate the p53 pathway in vivo, mice bearing these primary xenograft tumors were treated with a single oral dose of 50 or 80 mg/kg of RG7388 for 4, 8, or 24 hours. Western blot analysis showed a dose-dependent increase in p53 protein and its transcriptional targets, p21 and MDM2 (Fig. 3C, D). For PHLC 189 tumors treated with 50 mg/kg RG7388, p53 protein levels increased 4 hours after treatment, peaked at 8 hours, and returned to basal levels by 24 hours. However, at the higher 80 mg/kg dose, p53 levels increased after 4 hours and remained elevated after 24 hours. Elevated levels of p21 and MDM2 accompanied these changes in p53 levels. Similarly, p53 and MDM2 protein levels in PHLC 12 tumors peaked at 8 hours and persisted up to 24 hours after dosing at both 50 and 80 mg/kg concentrations (Fig. 3D).
RG7388 Suppresses PDX Tumor Growth in Mice
We next tested the antitumor activity of RG7388 on mice bearing PHLC 189, 12, and 229 PDX models. Daily oral administration of RG7388 80 mg/kg significantly inhibited PHLC 189 xenograft tumor growth rate (Fig. 4A). At the time of killing, tumors treated with RG7388 weighed approximately 50% less than the vehicle-treated group (p = 0.0002; Fig. 4A). Treatment with RG7388 for 28 days suppressed tumor growth (baseline 107 ± 9; post-treatment 109 ± 13 mm3), whereas the vehicle-treated tumors grew from to 105 ± 11 to 217 ± 43 mm3. We also observed significant growth suppression upon chronic administration of RG7388 on mice bearing PHLC 12 and PHLC 229 (Fig. 4B and C; p < 0.0001). In PHLC 12-bearing mice, equivalent antitumor response was achieved at both 80 and 50 mg/kg doses, suggesting that a lower dose regimen may be sufficient to achieve the same efficacy despite more transient pharmacodynamics effects (Fig. 4B).
FIGURE 4In 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.
To evaluate the ability of RG7388 to induce cellular arrest or apoptosis in vivo, levels of the cell proliferation marker Ki-67 and the apoptotic marker cleaved caspase-3 were measured in PHLC 189 tumors 24 hours after dosing with a single 80 mg/kg dose compared with vehicle alone (n = 8 per group; Fig. 5A). We detected significantly reduced (on average 30%) numbers of Ki-67-positive tumor cells in the RG7388-treated tumors compared with its control (39%; p = 0.0149; Fig. 5B). However, we did not detect a significant difference in the number of cleaved caspase-3–stained cells between groups (Fig. 5C).
FIGURE 5RG7388 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.
To confirm that p53 accumulation was due to decreased degradation of the protein rather than elevated transcription of the p53 gene, quantitative real-time PCR was conducted on RNA extracted from PHLC 12 and PHLC 189 after final RG7388 dosing. RG7388 significantly increased mRNA levels of genes: p21, GADD45, MDM2, and PUMA, which are known transcriptional p53 targets
(Fig. 6A and Supplementary Figure S4, Supplemental Digital Content, http://links.lww.com/JTO/A845). In contrast, the level of the p53 transcript itself was unaffected by RG7388, suggesting that RG7388 up-regulates p53 by means of a post- translational mechanism (Fig. 6A). Immunohistochemical detection of p53 protein levels in PHLC 12 and 229 demonstrated the increased p53 nuclear accumulation in the RG7388-treated tumors compared with controls (p = 0.0120; Fig. 6B, C; Supplementary Figure S5, Supplemental Digital Content, http://links.lww.com/JTO/A845).
FIGURE 6RG7388 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.
To confirm that the tumor inhibition was not due to p53-independent mechanisms induced by RG7388 exposure, we treated PHLC 193 mice harboring a R283H mutation in TP53 and MDM2 amplification (MDM2/CEP12: 8.88) with 50 and 80 mg/kg of RG7388 for 1 week and found no tumor growth changes (Fig. 4D).
DISCUSSION
In this article, we demonstrate that blocking the p53-MDM2 interaction using the small-molecule MDM2 inhibitor RG7388 can reactivate p53 in NSCLC cells, resulting in cell cycle arrest and apoptotic effects. Treatment of mice bearing PDX tumors with RG7388 led to the accumulation of p53 and significant inhibition of tumor growth, supporting the notion that established NSCLC tumors remain persistently vulnerable to the p53 tumor suppressor function.
These results provide compelling evidence that a single oral dose of RG7388 is highly effective in activating p53 and its major function in cell cycle arrest.
The nutlin class of small-molecule MDM2 inhibitors have been reported to inhibit the proliferation of a variety of standard cancer cell lines with wild-type p53.
This is the first study to report RG7388 efficacy in a panel of NSCLC cells including cells derived from early-passage PDX models (HLC). Given that mutant p53 protein is unable to bind to DNA to exert its normal function, we expected that the growth inhibition and cytotoxic effects of RG7388 would be dependent on the p53 status of NSCLC cells. Indeed, our data confirm that RG7388 potently and selectively inhibits cell growth in a panel of NSCLC and HLC cells harboring wild-type p53 and not in p53 mutant cells. RG7388 induced the greatest response in the H1395 cell line, which harbors an MDM2 gene amplification. This result is consistent with previous studies showing that RG7112, the first generation of this class of drug, was most effective in selectively killing osteosarcoma cell lines with MDM2 gene amplifications.
One of the major functions of p53 is to suppress cell cycle progression, which typically precedes the induction of apoptosis. Our data show that A549, HLC 12, and H1395 cells undergo cell cycle block at G1 phase after treatment with RG7388 in a dose-dependent manner. However, the onset of cell death was relatively slow compared with growth suppression. A much lower RG7388 concentration was required to reach maximum cell cycle inhibition in A549 compared with maximum apoptotic activity. This was expected given that cell cycle arrest is one of the primary functions of p53 to protect cells from propagation of damaged DNA, whereas apoptosis is the ultimate step for elimination of cells with unrepairable damage.
The lower drug threshold for cell cycle arrest in some cell lines may allow for delineation of the two effects of activated p53 in the clinical setting.
An important factor when considering drug combinations with RG7388 is cell cycle–mediated drug resistance. The effects of antimetabolite drugs, such as gemcitabine, are cell cycle dependent, in that they are most effective during a specific part of the cell cycle.
Previous studies have shown that mitogen-activated protein kinase kinase (MEK) 1 and 2 inhibitors inhibitors suppress DNA synthesis and antagonize the effect of gemcitabine.
Therefore, appropriate sequencing of the drug combinations with RG7388 should be considered when MDM2 inhibitor is being evaluated for clinical use.
In this article, we demonstrate the first in vivo efficacy of RG7388 in a panel of patient-derived NSCLC xenograft models, which closely mirror the histology and molecular profiles of the original patient tumors and may, therefore, be stronger predictors of clinical response.
We show that daily oral administration of RG7388 on PHLC 12-, 189-, and 229-bearing mice resulted in significant tumor growth inhibition. As predicted by the mechanistic model of p53 regulation, antitumor effects resulted from decreased cell proliferation as indicated by up-regulation of cell cycle genes p21 and GADD45 and reduced Ki-67-positive proliferative cells in treated tumor sections compared with controls. Cleaved caspase-3 detection is often used as a marker for apoptotic induction.
However, in vivo caspase activity was not observed in either PHLC 12 or 189 xenografts. The presence of activated caspase 3 is viewed as an “early” marker of apoptosis, as it is often detected before plasma membrane blebbing and DNA fragmentation. Therefore, it is possible that most of the apoptotic cells were eliminated before detection. Our in vivo data show that RG7388 treatment can elevate transcript levels of the downstream proapoptotic gene PUMA in both PHLC 12 and 189 tumors. Therefore, it is conceivable that RG7388 may also induce caspase-independent mechanisms of cell death.
Previous studies have demonstrated that MDM2-amplified osteosarcoma cells are highly sensitive to RG7112, suggesting that MDM2 gene amplification could be a useful predictor of RG7112 response.
In this article, we found potent antitumor response to RG7388 in both MDM2-amplified (H1395) and nonamplified (A549 and HLC12) NSCLC cells with wild-type p53. Functional p53 activation and significant tumor growth inhibition was observed in all three nonamplified MDM2 PDX models upon RG7388 treatment. Consistent with this, a phase-1 RG7112 dose escalation study in sarcoma patients recently observed disease control and p53 biomarker activity in both MDM2-amplified and nonamplified patients.
Although it may be ideal to select patients with MDM2-amplified lung adenocarcinoma with wild-type p53 for RG7388 treatment, our data suggest that the use of p53 wild-type status will identify a larger cohort of NSCLC patients who may respond and benefit from RG7388 treatment.
Clinical antitumor activity has been demonstrated recently with the first pharmacological MDM2 inhibitor RG7112 in liposarcoma patients.
Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study.
Here, using RG7388, a second-generation MDM2 antagonist with superior selectivity and potency, we demonstrated that RG7388 stimulates rapid p53 accumulation in clinically relevant NSCLC PDX tumor models, resulting in inhibition of cell proliferation and tumor growth. This is the first study to provide strong support for the clinical evaluation of MDM2 small-molecule inhibitors in NSCLC patients and suggests that p53 wild-type status may be a useful predictor of response to p53-activating therapy by this class of agents.
ACKNOWLEDGEMENTS
The authors thank Ming Li and Emin Ibrahimov for technical animal assistance and Hoffmann-La Roche and Dr. Steven Middleton for making RG7388 available for this study. The authors also thank Drs. Leigh A. Henricksen and Larry Morrison at Ventana Medical System (Tucson, Arizona) for performing the MDM2 silver in situ hybridization study.
This study was supported by the Canadian Cancer Society Research Institute grant #020527, the Ontario Ministry of Health and Long Term Care, and the Princess Margaret Cancer Foundation.
Spanish Lung Cancer Group in Collaboration with Groupe Français de Pneumo-Cancérologie and Associazione Italiana Oncologia Toracica. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.
Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis.
Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study.
Disclosure: Drs. Sakashita and Allo are supported by the Terry Fox Foundation Training Program in Molecular Pathology of Cancer at Canadian Institutes of Health Research (CIHR) (STP 53912). Dr. Tsao holds the M. Qasim Choksi Chair in Lung Cancer Translational Research. Dr. Shepherd holds the Scott Taylor Chair in Lung Cancer Research. All other authors declare no conflict of interest.
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