Olaratumab Exerts Antitumor Activity in Preclinical Models ... malignant rhabdoid tumor, and this activity

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  • Cancer Therapy: Preclinical

    Olaratumab Exerts Antitumor Activity in Preclinical Models of Pediatric Bone and Soft Tissue Tumors through Inhibition of Platelet-Derived Growth Factor Receptor a Caitlin D. Lowery1,Wayne Blosser1, Michele Dowless1, Shelby Knoche1, Jennifer Stephens1, Huiling Li1, David Surguladze1, Nick Loizos1, Debra Luffer-Atlas1, Gerard J. Oakley III1, Qianxu Guo1, Seema Iyer1, Brian P. Rubin2,3, and Louis Stancato1

    Abstract

    Purpose: Platelet-derived growth factor receptor a (PDGFRa) is implicated in several adult and pediatric malignancies, where activated signaling in tumor cells and/or cells within the micro- environment drive tumorigenesis and disease progression. Olar- atumab (LY3012207/IMC-3G3) is a humanmAb that exclusively binds to PDGFRa and recently received accelerated FDA approval and conditional EMA approval for treatment of advanced adult sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in preclinical models of pediatric bone and soft tissue tumors.

    Experimental Design: PDGFRa expression was evaluated by qPCR and Western blot analysis. Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric osteosarcoma and rhabdoid tumor cell lines. In vivo activity of

    olaratumabwas assessed in preclinical mousemodels of pediatric osteosarcoma and malignant rhabdoid tumor.

    Results: In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced proliferation and inhibited invasion driven by individual platelet-derived growth factors (PDGFs) or serum. Furthermore, olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin.

    Conclusions: Overall, these data indicate that olaratumab, alone and in combination with standard of care, blocks the growth of some preclinical PDGFRa-expressing pediatric bone and soft tissue tumor models. Clin Cancer Res; 24(4); 847–57. �2017 AACR.

    Introduction The platelet-derived growth factor (PDGF) pathway is com-

    posed of two receptors (PDGFRa and PDGFRb), four homodi- meric ligands (PDGF-AA, -BB, -CC, and -DD), and one hetero- dimeric ligand (PDGF-AB). After ligand binding, PDGFRa and/or PDGFRb form complexes as homo- or heterodimers, resulting in receptor transphosphorylation and activation of downstream signaling pathways, which in turn regulate normal cellular pro- cesses, such as proliferation, migration, and survival (1). Aberrant activation of the PDGF pathway through overexpression of key

    nodes or receptor mutation often facilitates tumorigenesis and disease progression across several cancer subtypes (2, 3). In addition, PDGF signaling in tumor-associated stromal cells pro- motes fibroblast activation and angiogenesis (4–7).

    The PDGF pathway has been implicated in bone and soft tissue sarcoma, a collection of mesenchymal malignancies comprised of nearly 80 distinct histologies. Olaratumab (LY3012207/ IMC-3G3) is a fully human mAb that specifically binds to and inhibits PDGFRa (8). In a phase II clinical study, olaratumab in combination with doxorubicin improved advanced adult sarco- mapatient outcomewith amedian overall survival benefit of 11.8 monthswhen comparedwith doxorubicin alone (9).On the basis of these data, olaratumab received accelerated approval from the FDA and conditional approval from the European Medicines Agency for treatment of advanced soft tissue sarcoma in combi- nation with doxorubicin in adult patients.

    Sarcoma subtypes occurring primarily in the pediatric popu- lation, including osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma, account for approximately 15% of childhood cancers (10). Despite intensive multimodal therapy, which typically includes a combination of chemotherapies, surgery, and/or radio- therapy, the current 5-year overall survival rate for pediatric sarcoma patients is approximately 60%; for those who experience a relapse or havemetastatic disease, survival drops to only 20% to 30%(11).Malignant rhabdoid tumor (MRT) is ahighly aggressive pediatric cancer typically occurring in the kidney and soft tissues or the central nervous system [where it is referred to as atypical teratoid/rhabdoid tumor (AT/RT)] and is characterized by a loss

    1Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana. 2Depart- ment of Pathology, Robert J Tomsich Pathology and Laboratory Medicine Institute and Cleveland Clinic, Cleveland, Ohio. 3Department of Cancer Biology, Robert J Tomsich Pathology and Laboratory Medicine Institute and Cleveland Clinic, Cleveland, Ohio.

    Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

    Current address for S. Knoche: 1) Fred & Pamela Buffett Cancer Center and 2) Eppley Institute for Research in Cancer &Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska.

    Corresponding Author: Louis F. Stancato, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285. Phone: 1-317-655-6910; Fax: 1-317-276-1414; E-mail: stancato_louis@lilly.com

    doi: 10.1158/1078-0432.CCR-17-1258

    �2017 American Association for Cancer Research.

    Clinical Cancer Research

    www.aacrjournals.org 847

    on October 7, 2020. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

    Published OnlineFirst November 30, 2017; DOI: 10.1158/1078-0432.CCR-17-1258

    http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-1258&domain=pdf&date_stamp=2018-1-29 http://clincancerres.aacrjournals.org/ http://clincancerres.aacrjournals.org/

  • of SMARCB1 (12, 13). Currently, no standard of care exists for this patient population, and overall survival remains poor (14–16). For pediatric cancer survivors, the possibility of debilitating long- term side effects, chronic health conditions, and even secondary cancers resulting from demanding therapeutic regimens remains (17, 18). Therefore, it is of the utmost importance to identify and evaluate targeted agents in the pediatric setting to improve patient outcome.

    Although the prevalence of PDGFRA genetic aberrations in pediatric cancer is reported to be only about 2% (19), members of the PDGF pathway are highly expressed in several subtypes of pediatric bone and soft tissue tumors, including rhabdomyosar- coma (20, 21), synovial sarcoma (22), osteogenic sarcoma (23), Ewing sarcoma (24), andMRT (25). In addition, PDGFRa expres- sion is linked to adverse outcomes in pediatric patients with rhabdomyosarcoma (26). In this study, we investigated olaratu- mab alone and in combination with standard-of-care (SOC) chemotherapy in preclinical models of pediatric bone and soft tissue tumors.

    Materials and Methods Test compounds

    Olaratumab (LY3012207/IMC-3G3, Eli Lilly and Company) was prepared in PBS for both in vitro and in vivo use. The mouse anti-PDGFRa antibody, 1E10, was also prepared in PBS for in vivo experiments.

    Cell culture The HuO9 osteosarcoma cell line was purchased from the

    Japanese Collection of Research Bioresources (JCRB) Cell Bank. Rh18, Rh28, Rh36, and Rh41 rhabdomyosarcoma cell lines were obtained from St. Jude Children's Research Hospital (Memphis, TN). Other pediatric cancer cell lines and WS-1 normal human fibroblasts were ordered from ATCC. Cell lines were Mycoplasma negative prior to freezing working stocks. The frozen working stocks of each cell line were authenticated by STR-based DNA profiling and multiplex PCR. The genetic profiles for the samples were identical to the genetic profiles reported for these cell lines. BT-12 and BT-16 AT/RT cell lines were a gift from Dr. Peter Houghton (Greehey Children's Cancer Research Institute, San

    Antonio, TX). Cell line details are listed in Supplementary Table S1. All cells were maintained at 37�C and 5% CO2 in tissue culture–treated flasks. Cells were used for experiments within 10 to 20 passages and then discarded.

    Reagents and antibodies Specific details regarding recombinant human PDGFs (AA, BB,

    CC, DD, and AB) are displayed in Supplementary Table S2. Antibodies against PDGFRa, PDGFRa Y754, PDGFRb, PDGFRb 751, ERK1/2, ERK1/2 T202/Y204, AKT, and AKT S473 were purchased from Cell Signaling Technology or Santa Cruz Bio- technology; details are included in Supplementary Table S2.

    Western blot analysis To determine receptor status and baseline pathway signaling in

    a panel of pediatric bone and soft tissue tumor lines, cells were serum starved overnight and harvested into 1% SDS lysis buffer containing 1� HALT protease and phosphatase inhibitor (Thermo Fisher Scientific, cat #78441). To determine effects of olaratumab treatment, cell lines were treated with either an IgG control antibody or olaratumab for 15 minutes and then stim- ulated with individual PDGFs (final concentration, 5 nmol/L) for an additional 15 minutes. Cells were immediately washed with PBS and harvested into 1% SDS lysis buffer containing 1� HALT protease and phosphatase inhibitor.

    Protein expression and phosphorylation status was assessed by SDS-PAGE and immunoblotting as described previously (27). Briefly, whole-cell lysates (30–50 mg of protein/sample) were separated on gradient Tris-Glycine protein gels (Novex, Thermo Fisher Scientific) and transferred to nitrocellulose via TransBlot Turbo (Bio-Rad, cat #170–4159). After blocking with 5%milk in TBST, membranes were probed with primary antibody overnight at 4�C, washed 3 times with TBST, and incubated with secondary antibodies for 1 hour at room temperature. Following 3 washes with TBST, membranes were developed