Combination of MDM2 and Targeted Kinase Inhibitors Results in Prolonged Tumor Control in Lung Adenocarcinomas With Oncogenic Tyrosine Kinase Drivers and MDM2 Amplification

Abstract

PURPOSE

MDM2, a negative regulator of the TP53 tumor suppressor, is oncogenic when amplified. MDM2 amplification (MDM2amp) is mutually exclusive with TP53 mutation and is seen in 6% of patients with lung adenocarcinoma (LUAD), with significant enrichment in subsets with receptor tyrosine kinase (RTK) driver alterations. Recent studies have shown synergistic activity of MDM2 and MEK inhibition in patient-derived LUAD models with MDM2amp and RTK driver alterations. However, the combination of MDM2 and RTK inhibitors in LUAD has not been studied.

METHODS

We evaluated the combination of MDM2 and RTK inhibition in patient-derived models of LUAD.

RESULTS

In a RET-fusion LUAD patient-derived model with MDM2amp, MDM2 inhibition with either milademetan or AMG232 combined with selpercatinib resulted in long-term in vivo tumor control markedly superior to either agent alone. Similarly, in an EGFR-mutated model with MDM2amp, combining either milademetan or AMG232 with osimertinib resulted in long-term in vivo tumor control, which was strikingly superior to either agent alone.

CONCLUSION

These preclinical in vivo data provide a rationale for further clinical development of this combinatorial targeted therapy approach.

Preclinical in vivo data support personalized genomically informed combination targeted therapy in lung cancer.

INTRODUCTION

Since the first reports of sensitizing EGFR mutations in 2004, advances in molecular diagnostics have uncovered a growing list of clinically actionable drivers in non–small cell lung cancer (NSCLC) including recurrent gene rearrangements involving ALK, ROS1, NTRK, RET, and NRG1, activating single-nucleotide and small insertion variants in BRAF and ERBB2, and MET exon 14 skipping alterations (METex14). For these oncogenic drivers, most often detected by large-panel next-generation sequencing (NGS), the use of targeted therapies results in response rates that exceed those achieved with systemic chemotherapy.^1-4 On the basis of these findings, there is a growing number of tyrosine kinase inhibitors (TKIs) approved by various regulatory agencies for the treatment of patients with specific oncogene-driven NSCLCs.^5-8 Despite these advances, the development of resistance and hence the eventual need for cytotoxic chemotherapies are often inevitable across the spectrum of oncogenic drivers.^4,9-12

CONTEXT

• Key Objective

• What is the antitumor activity of MDM2 and receptor tyrosine kinase (RTK) inhibition in preclinical models of lung adenocarcinoma (LUAD)?

• Knowledge Generated

• Combination of MDM2 inhibition and either EGFR or RET inhibition provided long-term tumor control in preclinical, patient-derived models of EGFR or RET-altered LUAD with concurrent MDM2 amplification (MDM2amp).

• Relevance

• Our findings provide preclinical support to justify clinical strategies testing MDM2 inhibition in combination with RTK inhibition in LUAD with MDM2amp and either EGFR or RET alterations.

In addition to identifying candidate targetable alterations, the adoption of broad genomic profiling in NSCLC has permitted the routine identification of multiple potentially targetable alterations at diagnosis.¹³ Indeed, there is growing interest in genomically informed, combinatorial therapeutic strategies^14-16 inspired by the successes of combination therapies in infectious diseases.^17,18 Recent studies aimed to match patients with a combinatorial approach according to molecular profiling suggested that rationally selected, molecularly guided targeted therapy combinations could be associated with improved disease control.¹⁹ The overarching hope is that both primary and acquired resistance to therapies may become less frequent if combination regimens are used earlier in the course of disease, at the critical point where tumor heterogeneity is lower.²⁰

MDM2 is a known oncogene when amplified and functions as a key negative regulator of the p53 tumor suppressor pathway, and single-agent MDM2 inhibition to restore p53 function has been studied with variable success.^21-23 A decade ago, Saiki et al²⁴ first observed synergy of MDM2 inhibition with compounds targeting the MAPK or PI3K pathways. However, these results predated routine broad NGS of cancer patient samples and were therefore not guided by the presence of relevant co-occurring alterations. Enabled by routine broad genomic profiling using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) large panel assay, we recently reported that MDM2 amplification (MDM2amp) occurred in over 6% of lung adenocarcinoma (LUAD) with further enrichment of MDM2amp in a subset of LUAD tumors with a receptor tyrosine kinase (RTK) driver alteration, specifically METex14 (36%), EGFR (8%), RET (12%), and ALK (10%).²⁵ This observation prompted us to evaluate the combination of MDM2 and MEK inhibition, as a model for an approach that could be applied broadly in the second line, in the context of acquired resistance to TKI therapy.²⁵ Indeed, we found that combined MDM2 and MEK inhibition resulted in effective tumor control and enhanced apoptosis across several patient-derived models harboring MDM2amp and different RTK alterations. However, the efficacy of combined MDM2 and RTK inhibition in the absence of an acquired resistance alteration (hence modeling the first-line setting) has not yet been explored. In this brief report, we evaluate the combination of inhibitors of MDM2 and a second concurrent RTK driver alteration in the context of representative MDM2amp patient-derived LUAD models.

METHODS

Materials

All investigations on human samples were performed with the approval Memorial Sloan Kettering Cancer Center Institutional Review Board (IRB) and in accordance with an assurance filed with and approved by the Department of Health and Human Services, where appropriate. In addition, the data were anonymized to protect the identities of individuals involved in the research. All individuals provided informed consent under MSK protocols 06-107, 12-245, and 14-091.

Cell culture growth media (DME-F12) and phosphate-buffered saline without calcium or magnesium were prepared by the MSK Media Preparation Core Facility. Fetal bovine serum (FBS) was procured from Atlanta Biologicals (Flowery Branch, GA). Antibiotics (Gibco, Cat. 15240062), tissue culture plastics (100-mm dishes, six-well plates, and clear 96-well plates), dimethyl sulfoxide, alamarBlue viability dye, and pClick-IT (Alexa Fluor 647, Cat. C10640) and Annexin-V (Cat. 556454) assays were procured from Thermo Fisher (Waltham, MA). Black polystyrene and 96-well flat bottom clear plates along with protease and phosphatase inhibitor cocktail, sodium orthovanadate, and radioimmunoprecipitation assay (RIPA) lysis buffer (10X) were procured from EMD-Millipore Sigma (St Louis, MO). Milademetan was provided by Rain Oncology (Newark, CA). AMG232 was obtained from Medchem Express (Monmouth Junction, NJ). All other small molecule inhibitors were obtained from Selleckchem (Houston, TX). Details of antibodies raised against total or phosphorylated proteins used for western blotting and immunohistochemistry are provided in Appendix Table A1. Precision plus protein kaleidoscope molecular weight markers used for western blotting were procured from Bio-Rad (Hercules, CA). Films for western blot film developing were procured from Ewen Parker X-Ray (East Orange, NJ). RNA and DNA isolation kits were procured from Qiagen (Germantown, MD).

Generation of Patient-Derived Xenograft Models and Cell Lines and Efficacy Studies

Tumor tissue was collected under IRB-approved protocols (MSK protocol 14-091) for LX-285 (passage number 9 used for study). Tumor tissue was immediately minced, mixed (50:50) with Matrigel (Corning, New York, NY), and implanted subcutaneously in 6- to 8-weeks-old female NOD scid gamma (NSG) mice (Jackson Laboratory, Bar Harbor, ME) to generate patient-derived xenografts (PDX) as previously described.²⁶ Mice were monitored daily and models were transplanted in mice three times before being deemed established. PDX tumor histology was confirmed by pathology review of hematoxylin and eosin slides and direct comparison with the corresponding patient biospecimens.

To generate cell lines from fresh PDX tumors, tumors were cut into 5-mm pieces and then digested in a cocktail of tumor dissociation enzymes obtained from Miltenyi Biotech (130-095-929) in 5-mL serum-free DMEM:F12 media for 1 hour at 37°C, with vortexing every 10 minutes for 1 hour, according to manufacturer's instructions. Digested samples were resuspended in 45-mL complete growth media to inactivate the dissociation enzymes, and then cells were pelleted by centrifugation. Finally, the cells were plated in growth media supplemented with 2% FBS and antibiotics, and allowed to propagate for multiple generations, passaging with trypsin when approaching confluence. The PDX and cell line models were generated and propagated in the absence of small molecule inhibitors.

For efficacy studies, fresh PDX tumor samples were minced, mixed with Matrigel, and implanted into a subcutaneous flank of female NSG mice to generate xenografts. For ECLC5-GLx, cell line xenografts were generated by implanting 10 million cells into the flanks of the animals. After randomizing into groups of five to eight, tumor-bearing animals were treated with vehicle, MDM2 inhibitor (milademetan 100 mg/kg once daily on a 5 day on, 2 day off schedule, AMG232 15 mg/kg once daily), RTK inhibitor: selpercatinib 2.5 or 10 mg/kg twice daily on a 5 day on, 2 day off schedule, osimertinib 2.5 or 5 mg/kg twice daily on a 5 day on, 2 day off schedule, capmatinib 5 mg twice daily on a 5 day on, 2 day off schedule, or combination of respective MDM2 inhibitors and RTK inhibitors when tumors reached approximately 150 mm³ volume. Drugs were prepared as follows: selpercatinib and osimertinib were resuspended in 0.5% hydroxypropyl methylcellulose, 0.2% Tween 80, pH 8.0, and milademetan was resuspended in 0.5% methylcellulose. Stock solutions were made fresh weekly and stored at 4°C. Tumor size and body weight were measured twice weekly, and tumor volume was calculated using the modified ellipsoid formula as done previously.²⁷

Genomic Characterization of Preclinical Models

Cell lines (ECLC5-GLx, ECLC5-B) and PDXs (LX-285) were profiled by the MSK-IMPACT platform, a large panel NGS assay designed to detect mutations, copy-number alterations, and select fusions involving up to 505 cancer-associated genes.²⁸ Paired analysis of PDX tissue or cell line and matched-normal sample was performed to unambiguously identify somatic mutations. MDM2 total copy number was computed using the FACETS algorithm as previously described.²⁹

Growth and Propagation of Cell Lines

Cell lines were maintained in a humidified incubator infused with 5% carbon dioxide at 37°C and subcultured when stock flasks reached approximately 75% confluence at a 1:4 dilution. SW1573 cell line was maintained in DMEM:F12 growth medium supplemented by 5% FBS and 1% antibiotics. All other cell lines were maintained in DMEM:F12 growth medium supplemented by 10% FBS and 1% antibiotics. The molecular alteration of interest was confirmed by polymerase chain reaction each time a cell line vial was thawed. Cells were tested for mycoplasma contamination every 6 months using MycoAlert Plus kit procured from Lonza (Morristown, NJ).

Viability Drug Assays

For viability assays, the cells were plated in 96-well flat bottom, black polystyrene plates at a density of 7,500 cells per well for ECLC5-GLx and incubated with compounds for 96 hours. For A549, 5,000 cells per well were plated and treated with compound for 72 hours. The relative number of viable cells was determined using alamarBlue viability dye, and fluorescence was measured using a Molecular Devices SpectraMax M2 multimodal plate reader (Ex: 555 nm, Em: 585 nm). Data were analyzed by nonlinear regression and curves fitted using Prism 9 to generate concentration that inhibits 50% values (IC50). For synergy drug studies, data were analyzed using the Combenefit software presenting the highest single agent synergy method.^30,31 All data are expressed relative to control values and an average of two to five independent experiments in which each condition was assayed in at least triplicate.

Preparation of Whole-Cell Extracts and Western Blotting

Protein levels and phosphorylation state were detected by western blotting. Samples from cell lines were lysed in RIPA assay lysis buffer diluted to 1× in double distilled water containing phosphatase and protease inhibitors and 1 µg/mL sodium orthovanadate. For samples derived from PDX models, tumor pieces were dissociated using gentleMACS Dissociator (Auburn, CA). Protein concentration was quantified using Bradford assay (Bio-Rad, Cat. 5000006). Lysates were denatured in 2× Laemmli sample buffer at 55°C for 10 minutes, resolved on 4%-12% Bolt gels (Thermo Fisher, Waltham, MA), and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 3% bovine serum albumin (Sigma, Marlborough, MA) in tris-buffered saline supplemented with 0.1% Tween 20 (Thermo Fisher; vol/vol) for 1 hour at room temperature and probed with primary antibodies with specificity as outlined in Appendix Table A1. Bound antibodies were detected with peroxidase-labeled goat antibody raised to mouse or rabbit IgG (R&D Systems, Minneapolis, MN) and imaged with enhanced chemiluminescence western blotting detection reagent (GE Healthcare, Chicago, IL). Images were captured on radiograph films. Experiments were repeated at least two times from independently prepared samples.

RESULTS

Combining MDM2 and RTK Inhibition Is Synergistic In Vitro and Induces Expression of Proteins Involved in Cell Cycle Arrest and Apoptosis

We first assessed in vitro the combination of MDM2 and RTK inhibition in the patient-derived cell line ECLC5-GLx (TRIM33::RET, MDM2amp, TP53 wildtype). All patient-derived cell lines have been described previously.²⁵ In ECLC5-GLx, combination of the RET inhibitor selpercatinib and the MDM2 inhibitor milademetan was synergistic and induced a dose-response shift (Figs 1A-1C). We next evaluated the effects of the combination of MDM2 and RTK inhibition on expression of cell cycle and apoptosis regulators in ECLC5-GLx by western blot. In these cells, the combination of inhibitors caused an increase in the p27 (Kip1) above that seen with individual agents, while milademetan alone caused increased p21 (Cip1) expression and this was not further elevated by combination with selpercatinib (Fig 1D). As we and others have previously shown,²⁵ milademetan caused increased expression of cyclin D1 (CCND1)—previously described as a resistance mechanism to single-agent MDM2 inhibition³²—but this induction was inhibited in combination with selpercatinib (Fig 1D). As we showed previously,²⁵ E2F1, a p53-independent target of MDM2 and a cell cycle progression marker, was decreased by milademetan and selpercatinib individually, which translated to a more profound decrease with the combination compared with either agent alone (Fig 1D). Increased expression of phosphorylated p53 (p-p53) and the proapoptotic protein PUMA was observed with single-agent milademetan and selpercatinib, and this level of expression was maintained in the combination (Fig 1D).

FIG 1.

Combination activity in vitro of MDM2 inhibition and TKI in ECLC5-GLx model of lung adenocarcinoma. (A) Synergy matrix for ECLC5-GLx. The synergy matrices are synergy scores, calculated according to the HSA synergy model (which measures whether the expected combination effect equals to the higher effect of individual drugs). The larger numeral in each box is the synergy score; negative values indicate antagonism. The number below the synergy score is the standard deviation. Boxes are colored blue if the synergy score significant by t-test. (B) Milademetan dose-response shift observed in the presence of increasing concentrations of RET inhibition with selpercatinib. (C) D-r shift observed for selpercatinib in the presence of increasing concentrations of milademetan. At least two independent synergy experiments were conducted. (D) Western blot of whole-cell extracts prepared from ECLC5-GLx cells treated with milademetan (100 nM), selpercatinib (100 nM), or combination of milademetan and selpercatinib (100 nM each) at 6, 24, and 48 hours. GAPDH was used as a loading control. D-r, dose response; HSA, highest single agent; TKI, tyrosine kinase inhibitor. *P < .05, **P < .01, ***P < .001.

Combination of MDM2 and RTK Inhibition Improves Tumor Control Compared With RTK Inhibition Alone In Vivo

We next assessed the combinatorial activity of MDM2 inhibition and RTK inhibition in vivo. In ECLC5-GLx, the combination of selpercatinib and milademetan resulted in improved tumor control compared with either agent alone (Fig 2A), without significant effect on animal weight (Appendix Fig A1A). Similarly, in LX-285 (EGFRex19, EGFR T790M, MDM2amp, TP53 WT), the combination of osimertinib and milademetan resulted in improved tumor control compared with either agent alone (Fig 2B), without significant effect on animal weight (Appendix Fig A1B).

FIG 2.

Combination of milademetan and TKI is effective in patient-derived models of MDM2amp with concurrent driver alterations. Lung adenocarcinoma models were treated with the indicated dose of either vehicle, milademetan, respective RTK TKI, or combination daily on a 5-day schedule (five mice/group). The tumor volume over time is shown. (A) ECLC5-GLx patient-derived cell line xenograft model (TRIM33::RET fusion, MDM2amp, TP53 wildtype status). For selpercatinib and combination milademetan and selpercatinib arms, dosing was stopped after 62 days of implantation as indicated by the orange arrowhead. (B) LX-285 patient-derived xenograft model (EGFRex19 deletion, T790M mutation, MDM2amp, TP53 wildtype), zoomed in figure in dashed box. For osimertinib and combination milademetan and osimertinib arms, dosing was stopped after 62 days of implantation as indicated by the orange arrowhead. Note that the vehicle and single-agent MDM2 inhibitor milademetan arms in this model were previously published.²⁵ MDM2amp, MDM2 amplification; RTK, receptor tyrosine kinase; TKI, tyrosine kinase inhibitor.

To confirm generalizability across MDM2 inhibitors, we also assessed the combination of RTK inhibition with a different MDM2 inhibitor, AMG232, in the ECLC5B (also generated from the same patient as ECLC5-GLx tumor model). No effect was observed with AMG232 monotherapy, and only modest deceleration of tumor growth compared with the vehicle-treatment group was observed for selpercatinib (Fig 3A) as expected with the doses used. By contrast, the mice treated with AMG232 and selpercatinib combination achieved significant tumor regression that lasted for over 75 days. None of the treatment regimens caused significant reduction in animal weight (Appendix Fig A2A). Similar findings were observed with osimertinib plus AMG232 in LX-285 (Fig 3B). Specifically, although single-agent AMG232 caused a significant deceleration in growth of LX-285 tumors, the effect was not sustained (Fig 3B). Osimertinib monotherapy and osimertinib + AMG232 combined therapy, however, produced profound and durable tumor control, with shrinkage of the tumor volume observed in all animals in this group. Eventually, however, the tumor growth resumed in the osimertinib monotherapy group 89 days after treatment was initiated (Fig 3B), peaking on day 193, when the experiment was concluded because of large tumor volume. Combined osimertinib + AMG232 treatment resulted in significantly superior tumor control, and although small tumor regrowth was eventually observed in this group as well, the curves between osimertinib monotherapy and the combined therapy group separated on day 64, with continuous significant difference in tumor volumes until the end of the experiment on day 196. None of the treatment regimens caused significant reduction in animal weight (Appendix Fig A2B). Taken together, these results suggest that MDM2 and RTK inhibition may be an effective strategy to prolong responses to RTK inhibition in the first-line setting for EGFR- and RET-altered lung cancers.

FIG 3.

Combination of AMG232 and TKI is effective in patient-derived models of MDM2amp with concurrent driver alterations. Lung adenocarcinoma models were treated with the indicated dose of either vehicle, AMG232, respective RTK TKI, or combination daily on a 5-day schedule (five mice/group). The tumor volume over time is shown. (A) ECLC5-B patient-derived cell line xenograft model (TRIM33::RET fusion, MDM2amp, TP53 wildtype status). (B) LX-285 patient-derived xenograft model (EGFRex19 deletion, T790M mutation, MDM2amp, TP53 wildtype), zoomed in figure in dashed box. Results shown represent mean ± SEM. MDM2amp, MDM2 amplification; RTK, receptor tyrosine kinase; SEM, standard error of mean; TKI, tyrosine kinase inhibitor.

DISCUSSION

Despite the improvement in outcomes because of targeted therapies in subsets of NSCLCs driven by a kinase alteration, the development of resistance to these targeted therapies represents an important challenge.³³ The adoption of NGS of NSCLCs has opened the possibility of identifying multiple potentially targetable alterations at the time of cancer diagnosis. Our earlier work demonstrated MDM2 and MEK inhibition was effective in preclinical models of LUADs with MDM2amp and a mitogenic driver alteration activating MAPK signaling.²⁵

MDM2 is an E3 ubiquitin ligase that functions as the primary cellular antagonist of the tumor suppressor TP53.³⁴ TP53 and MDM2 have a cyclical coexistence: when TP53 is activated, it regulates transcription of a host of dependent genes including regulators of the cell cycle (eg, p21and p27, etc), apoptosis (PUMA, NOXA, etc), and MDM2. Once MDM2 matures, it binds and ubiquitinates p53 leading to proteasomal degradation of p53 and subsequent downregulation of the p53 pathway.³⁵ MDM2amp, leading to high expression of MDM2 protein, keeps p53 levels consistently low, thereby removing a major hurdle to uncontrolled cell proliferation. Therefore, MDM2 is a rational therapeutic target in cancers with wildtype TP53, and this notion has led to the development of multiple small molecule inhibitors of MDM2.³⁶ Various stage clinical trials are ongoing to evaluate different MDM2 inhibitors alone or in combination with chemotherapy, predominantly in cancers with wildtype TP53.³⁷

In this study, we focused on exploiting MDM2 as a target for therapy in lung cancers that harbor MDM2amp and a known kinase alteration. In the ECLC5-GLx and ECLC5B cell lines with a TRIM33RET fusion and MDM2amp, RET inhibitors are poor activators of apoptosis.³⁸ However, we have shown in our previous work that MDM2 inhibition is very effective at activating apoptosis and inhibiting growth of these cells.²⁵

The two MDM2 inhibitors used in our study function by preventing interaction between MDM2 and p53, freeing p53 to resume its normal proapoptotic activity through upregulation of proapoptotic proteins such PUMA and loss of cell cycle inhibitors such as p21 and p27, among others. The RET inhibitor selpercatinib, however, caused relatively little proapoptotic proteins in ECLC5-GLx cells. These contrary results suggested to us that MDM2 inhibitor, given their predilection for inducing massive cell death, can prolong the growth inhibitory effect of RET and other kinase inhibitors in cells with an RTK driver and MDM2amp. To this end, we found that combination of selpercatinib and milademetan was synergistic in blocking growth of the RET fusion + MDM2-amplified cell line in vitro and induced potent tumor control in vivo, more prolonged than either agent alone. We expanded our in vivo analysis to look at combination of MDM2 inhibitor and osimertinib in vivo in a PDX model with EGFRex19 deletion and MDM2amp (LX-285). The combination of AMG232 or milademetan and osimertinib was more effective than monotherapy at blocking growth of LX-285 PDX tumors. Although LX-285 PDX tumors treated with MDM2 inhibitors started to grow 100 days past initiation of therapy, the combination of AMG232 and osimertinib controlled tumor growth for almost 200 days after treatment initiation. These results suggest that cotargeting MDM2 and the respective RTK is effective at prolonging response to therapy and delaying the onset of primary resistance to TKI, and in the case of the EGFR TKI with milademetan, raising the prospect of eventually preventing it altogether.

In lung cancer therapy, combinations therapies have been used for patients who have progressed on a particular drug and when a new molecular mechanism of resistance is detected. For example, osimertinib and savolitinib demonstrated clinical utility in patients with EGFR mutations who progressed on EGFR TKI and had an acquired MET alteration.³⁹ However, such TKI combinations remain susceptible to activation of yet another bypass pathway reactivating MAPK signaling, as acquired resistance mechanisms to TKIs are myriad.^10,33,40-42 Acquired resistance to MDM2 inhibitors is mainly because of new TP53 mutations.^43,44 Thus, resistance to combination TKI and MDM2 inhibition would require either a single acquired alteration that could reactivate both the MAPK and TP53 pathways, or the simultaneous acquisition of a TP53 mutation and a TKI resistance alteration in a given cancer cell, neither of which is likely. Our present study provides preclinical data supporting the efficacy of combined inhibition of targetable driver alterations in independent pathways, as detected by clinical genomic profiling at the time of initial diagnosis, an approach potentially relevant to the substantial number of patients whose tumors harbor a targetable kinase driver and MDM2amp.²⁵ The present data could inform a phase I trial design evaluating the safety and tolerability of MDM2 inhibition in combination with TKI inhibition in patients with LUAD, and, subsequently, provided no adverse safety signals are observed, would provide the rationale for a phase II trial evaluating the activity of this combination in patients with LUAD containing these concurrent targetable alterations. The overarching goal of this approach would be to delay or ideally prevent the development of resistance to TKI therapy, which unfortunately is inevitable for most or all patients treated with single-agent TKIs.

APPENDIX

FIG A1.

Effect of combination milademetan and TKI on tumor-bearing animal weight. (A) Tumor-bearing animal weights in grams during course of treatment for ECLC5-GLx. (B) Tumor-bearing animal weights in grams during course of treatment for LX-285. Results shown represent mean ± SEM. BID, twice a day; MDM2amp, MDM2 amplification; SEM, standard error of mean; TKI, tyrosine kinase inhibitor.

FIG A2.

Effect of combination AMG232 and TKI on tumor-bearing animal weight. (A) Tumor-bearing animal weights in grams during course of treatment for ECLC5B. (B) Tumor-bearing animal weights in grams during course of treatment for LX-285. Results shown represent mean ± SEM. MDM2amp, MDM2 amplification; QD, once daily; SEM, standard error of mean; TKI, tyrosine kinase inhibitor.

TABLE A1.

Antibodies Used for Western Blot Experiments

Antibody Clone	Catalog No.	Company
GAPDH (D16H11) XP Rabbit mAb	5174S	Cell Signalling Technologies
p27 Kip1 (D69C12) XP Rabbit mAb	3686S	Cell Signalling Technologies
p21 Waf1/Cip1 (12D1) Rabbit mAb	2947S	Cell Signalling Technologies
Cyclin D1 (E3P5S) XP Rabbit mAb	55506S	Cell Signalling Technologies
Phospho-p53 (Ser15) antibody	9284	Cell Signalling Technologies
Puma (E2P7G) Rabbit mAb	98672	Cell Signalling Technologies
E2F-1 antibody	3742	Cell Signalling Technologies
Vinculin (E1E9V) XP Rabbit mAb	13901	Cell Signalling Technologies
β-actin	4967	Cell Signalling Technologies
β-actin HRP	5125	Cell Signalling Technologies

AUTHOR CONTRIBUTIONS

Conception and design: Arielle Elkrief, Igor Odintsov, Roger S. Smith, Zebing Liu, Michael D. Offin, Romel Somwar, Marc Ladanyi

Financial support: Romel Somwar, Marc Ladanyi

Administrative support: Igor Odintsov, Marc Ladanyi

Provision of study materials or patients: Igor Odintsov, Inna Khodos, Charles M. Rudin, Elisa de Stanchina, Romel Somwar

Collection and assembly of data: Arielle Elkrief, Igor Odintsov, Roger S. Smith, Morana Vojnic, Takuo Hayashi, Inna Khodos, Vladimir Markov, Zebing Liu, Jamie L. Bloom, Michael D. Offin, Charles M. Rudin, Elisa de Stanchina, Gregory J. Riely, Romel Somwar, Marc Ladanyi

Data analysis and interpretation: Arielle Elkrief, Igor Odintsov, Roger S. Smith, Morana Vojnic, Zebing Liu, Allan J.W. Lui, Michael D. Offin, Elisa de Stanchina, Romel Somwar, Marc Ladanyi

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.

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