Olaratumab Exerts Anti-Tumor Activity in Preclinical Models of Pediatric

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Author Manuscript Published OnlineFirst on November 30, 2017; DOI: 10.1158/1078-0432.CCR-17-1258 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Olaratumab exerts anti-tumor activity in preclinical models of pediatric bone and soft 2 tissue tumors through inhibition of platelet-derived growth factor receptor α

3 Running Title: Preclinical activity of olaratumab in pediatric tumor types

4 Caitlin D. Lowery1, Wayne Blosser1, Michele Dowless1, Shelby Knoche1*, Jennifer Stephens1, 5 Huiling Li1, David Surguladze1, Nick Loizos1, Debra Luffer-Atlas1, Gerard J. Oakley III1, Qianxu 6 Guo1, Seema Iyer1, Brian P. Rubin2, Louis Stancato1

7 1Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana.

8 2Departments of Pathology and Cancer Biology, Robert J Tomsich Pathology and Laboratory 9 Medicine Institute and Cleveland Clinic

10 *Former employee of Eli Lilly and Company

11 Keywords: Olaratumab, PDGFR, platelet derived growth factor receptor alpha, pediatrics

12 Financial support: This study was funded by Eli Lilly and Company, Lilly Corporate Center, 13 Indianapolis, Indiana, USA.

14 Corresponding Author:

15 Louis F. Stancato 16 Oncology TME & Translational Research 17 Lilly Corporate Center, Indianapolis, Indiana 46285 18 Phone: 317-655-6910 19 Fax: 317-276-1414 20 Email: [email protected] 21

22 Conflict of Interest Statement: All authors except S Knoche and BP Rubin are full-time 23 employees of Eli Lilly and Company.

24 Manuscript Information:

25 Word counts: 26 • Statement of translational relevance: 115 27 • Abstract: 209 28 • Manuscript text: 4,997 29 30 References: 47 31 32 Display items: 33 • Figures: 6 34 • Supplementary Material: 4 tables, 3 figures 35 36

Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 30, 2017; DOI: 10.1158/1078-0432.CCR-17-1258 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

37 Statement of Translational Relevance

38 Olaratumab (LY3012207/IMC-3G3) is a human monoclonal antibody against the platelet-derived 39 growth factor receptor alpha (PDGFRα) which recently received accelerated approval from the 40 FDA and conditional EMA approval for treatment of adult advanced soft tissue sarcoma patients 41 in combination with doxorubicin. Pediatric patients with bone and soft tissue tumors are treated 42 with intensive, multimodal therapeutic regimens which often results in debilitating long term side 43 effects. PDGFRα has been implicated in these pediatric malignancies. In this study, we 44 demonstrate the anti-tumor activity of olaratumab in combination with standard chemotherapy in 45 preclinical models of pediatric bone and soft tissue tumors. These data support clinical 46 evaluation of olaratumab in combination with chemotherapy in pediatric patients with solid 47 tumors (NCT02677116).

48 Abstract

49 Purpose: Platelet-derived growth factor receptor alpha (PDGFRα) is implicated in several adult 50 and pediatric malignancies, where activated signaling in tumor cells and/or cells within the 51 microenvironment drive tumorigenesis and disease progression. Olaratumab (LY3012207/IMC- 52 3G3) is a human monoclonal antibody that exclusively binds to PDGFRα and recently received 53 accelerated FDA approval and conditional EMA approval for treatment of advanced adult 54 sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in 55 preclinical models of pediatric bone and soft tissue tumors.

56 Experimental Design: PDGFRα expression was evaluated by qPCR and western blot analysis. 57 Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric 58 osteosarcoma and rhabdoid tumor cell lines. In vivo activity of olaratumab was assessed in 59 preclinical mouse models of pediatric osteosarcoma and malignant rhabdoid tumor.

60 Results: In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced 61 proliferation and inhibited invasion driven by individual PDGFs or serum. Furthermore, 62 olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and 63 malignant rhabdoid tumor and this activity was enhanced by combination with either doxorubicin 64 or cisplatin.

65 Conclusion: Overall, these data indicate that olaratumab, alone and in combination with 66 standard of care, blocks the growth of some preclinical PDGFRα-expressing pediatric bone and 67 soft tissue tumor models.

68 Introduction

69 The platelet-derived growth factor (PDGF) pathway is composed of two receptors (PDGFRα and 70 PDGFRβ), four homodimeric ligands (PDGF-AA, -BB, -CC, and -DD), and one heterodimeric 71 ligand (PDGF-AB). After ligand binding, PDGFRα and/or PDGFRβ form complexes as homo- or 72 heterodimers, resulting in receptor transphosphorylation and activation of downstream signaling 73 pathways which in turn regulate normal cellular processes such as proliferation, migration, and 74 survival (1). Aberrant activation of the PDGF pathway through overexpression of key nodes or 75 receptor mutation often facilitates tumorigenesis and disease progression across several cancer

76 subtypes (2,3). In addition, PDGF signaling in tumor-associated stromal cells promotes 77 fibroblast activation and angiogenesis (4-7).

78 The PDGF pathway has been implicated in bone and soft tissue sarcoma, a collection of 79 mesenchymal malignancies comprised of nearly 80 distinct histologies. Olaratumab 80 (LY3012207/IMC-3G3) is a fully human monoclonal antibody that specifically binds to and 81 inhibits PDGFRα (8). In a phase II clinical study, olaratumab in combination with doxorubicin 82 improved advanced adult sarcoma patient outcome with a median overall survival benefit of 83 11.8 months when compared to doxorubicin alone (9). Based on these data, olaratumab 84 received accelerated approval from the Food and Drug Administration and conditional approval 85 from the European Medicines Agency for treatment of advanced soft tissue sarcoma in 86 combination with doxorubicin in adult patients.

87 Sarcoma subtypes occurring primarily in the pediatric population, including osteosarcoma (OS), 88 rhabdomyosarcoma (RMS), and Ewing’s sarcoma, account for approximately 15% of childhood 89 cancers (10). Despite intensive multimodal therapy which typically includes a combination of 90 chemotherapies, surgery, and/or radiation therapy, the current 5-year overall survival rate for 91 pediatric sarcoma patients is ~60%; for those who experience a relapse or have metastatic 92 disease, survival drops to only 20-30% (11). Malignant rhabdoid tumor (MRT) is a highly 93 aggressive pediatric cancer typically occurring in the kidney and soft tissues or the central 94 nervous system (where it is referred to as atypical teratoid/rhabdoid tumor [AT/RT]) and is 95 characterized by a loss of SMARCB1 (12,13). Currently, no standard of care exists for this 96 patient population and overall survival remains poor (14-16). For pediatric cancer survivors, the 97 possibility of debilitating long-term side effects, chronic health conditions, and even secondary 98 cancers resulting from demanding therapeutic regimens remains (17,18). Therefore, it is of the 99 utmost importance to identify and evaluate targeted agents in the pediatric setting to improve 100 patient outcome.

101 While the prevalence of PDGFRA genetic aberrations in pediatric cancer is reported to be only 102 about 2% (19), members of the PDGF pathway are highly expressed in several subtypes of 103 pediatric bone and soft tissue tumors, including RMS (20,21), synovial sarcoma (22), osteogenic 104 sarcoma (23), Ewing’s sarcoma (24), and MRT (25). In addition, PDGFRα expression is linked 105 to adverse outcomes in pediatric patients with RMS (26). In this study, we investigated 106 olaratumab alone and in combination with standard-of-care (SOC) chemotherapy in preclinical 107 models of pediatric bone and soft tissue tumors.

108 Materials and Methods

109 Test compounds

110 Olaratumab (LY3012207/IMC-3G3, Eli Lilly and Company) was prepared in phosphate buffered 111 saline (PBS) for both in vitro and in vivo use. The mouse anti-PDGFRα antibody, 1E10, was 112 also prepared in PBS for in vivo experiments.

113 Cell culture

114 The HuO9 osteosarcoma cell line was purchased from the Japanese Collection of Research 115 Bioresources (JCRB) Cell Bank. Rh18, Rh28, Rh36, and Rh41 rhabdomyosarcoma cell lines 116 were obtained from St. Jude Children’s Research Hospital. Other pediatric cancer cell lines and 117 WS-1 normal human fibroblasts were ordered from American Type Culture Collection (ATCC). 118 Cell lines were Mycoplasma negative prior to freezing working stocks. The frozen working 119 stocks of each cell line were authenticated by STR-based DNA profiling and multiplex PCR. The 120 genetic profiles for the samples were identical to the genetic profiles reported for these cell 121 lines. BT-12 and BT-16 atypical teratoid/rhabdoid tumor (AT/RT) cell lines were a gift from Dr. 122 Peter Houghton (Greehey Children's Cancer Research Institute). Cell line details are listed in

123 Supplemental Table 1. All cells were maintained at 37°C and 5% CO2 in tissue-culture treated 124 flasks. Cells were used for experiments within 10-20 passages and then discarded.

125 Reagents and antibodies

126 Specific details regarding recombinant human PDGFs (AA, BB, CC, DD, and AB) are displayed 127 in Supplemental Table 2. Antibodies against PDGFRα, PDGFRα Y754, PDGFRβ, PDGFRβ 128 Υ751, ERK1/2, ERK1/2 T202/Y204, AKT, and AKT S473 were purchased from Cell Signaling 129 (Danvers, MA) or Santa Cruz Biotechnology (Dallas, TX); details are included in Supplemental 130 Table 2.

131 Western blot analysis

132 To determine receptor status and baseline pathway signaling in a panel of pediatric bone and 133 soft tissue tumor lines, cells were serum starved overnight and harvested into 1% SDS lysis 134 buffer containing 1x HALT protease and phosphatase inhibitor (ThermoFisher, cat#78441). To 135 determine effects of olaratumab treatment, cell lines were treated with either an IgG control 136 antibody or olaratumab for 15 minutes and then stimulated with individual PDGFs (final 137 concentration: 5 nM) for an additional 15 minutes. Cells were immediately washed with PBS and 138 harvested into 1% SDS lysis buffer containing 1x HALT protease and phosphatase inhibitor.

139 Protein expression and phosphorylation status was assessed by SDS-PAGE and 140 immunoblotting as described previously (27). Briefly, whole cell lysates (30-50 μg of protein per 141 sample) were separated on gradient Tris-Glycine protein gels (Novex, ThermoFisher) and 142 transferred to nitrocellulose via TransBlot Turbo (Bio-Rad, cat#170-4159). After blocking with 143 5% milk in TBST, membranes were probed with primary antibody overnight at 4°C, washed 3 144 times with TBST, and incubated with secondary antibodies for 1 h at room temperature. 145 Following 3 washes with TBST, membranes were developed with SuperSignal West Femto 146 Chemiluminescent Substrate (ThermoFisher, cat#34095) and imaged with a Bio-Rad ChemiDoc 147 XRS.

148 Cell proliferation assay

149 In order to determine anti-proliferation EC50 values for olaratumab, pediatric cancer cells were 150 seeded in 96-well plates. The next day, cells were incubated with increasing concentrations of 151 olaratumab or the IgG antibody control in serum free media for 2 h at 37°C and then stimulated 152 with individual PDGFs. Cell proliferation was assayed by CellTiter Glo™ Luminescent Cell

153 Viability Assay (Promega, cat# G7571) 72 h after stimulation. Data were normalized to the

154 unstimulated IgG-treated control. Relative EC50 values were calculated using GraphPad Prism 155 software. Experiments were repeated in triplicate and data from a representative experiment are 156 displayed in Table 1.

157 To evaluate the effects of combination treatment on pediatric cancer cell proliferation, cells were 158 plated in 96-well microtiter plates in normal media containing 10% FBS. The next morning, 159 media was changed to media containing 0.5% FBS. After 4 h, cells were co-stimulated with 5 160 nM PDGF-AA and 5 nM PDGF-CC and treated with 1 μM olaratumab, 0.5 μM doxorubicin, or 2 161 μM cisplatin or combination of olaratumab and chemotherapy. Cell proliferation was assayed 162 after 72 h by CellTiter Glo. Luminescence was normalized to the average of the untreated 163 control for each cell line. Results are presented as the mean of duplicate or triplicate 164 experiments (indicated in figure legend) ± standard error of the mean (SEM). Statistical 165 significance was determined by the Student’s t-test.

166 Evaluation of cell invasion

167 Invasion assays were performed using the CultreCoat® Low BME Cell Invasion Assay 168 (Trevigen, cat#3481-096-K) per the manufacturer’s instructions. Briefly, 2.5 x 104 pediatric 169 cancer cells were seeded in the upper chamber of the well with 1 μM IgG control antibody or 170 olaratumab. Serum-free media was added to the lower chamber and individual PDGFs were 171 used as a chemoattractant. Media with 10% FBS as a chemoattractant served as a positive 172 control. Final concentration of ligand was 5 nM for each, with the exception of PDGF-DD, which 173 was 20 nM. After 48 h, cells able to invade through the membrane to the lower chamber were 174 dissociated and plates were read at 485 nm excitation, 520 nm emission. Results were 175 normalized to the serum free media - IgG control for each cell line. Results are presented as the 176 average of duplicate or triplicate experiments (indicated in figure legend) ± SEM. Significance 177 was determined by the Student’s t-test.

178 Expression profiling of patient-derived xenograft mouse models

179 Samples from pediatric sarcoma patient-derived xenografts were obtained from Champions 180 Oncology (Hackensack, NJ), START Discovery (San Antonio, TX), and Oncotest (Charles River 181 Labs, Freiberg, Germany). Frozen tumors were manually dissociated using a TissueLyser 182 (Qiagen). RNA extraction was performed using the MagMAX-96 total RNA Isolation Kit 183 (ThermoFisher, cat#AM1830) per the manufacturer’s instructions. The High Capacity cDNA 184 Reverse Transcription Kit (ThermoFisher, cat#4374967) was used to generate cDNA. TaqMan 185 Gene Expression Master Mix (cat#4369016) and TaqMan probes for human PDGFRA, 186 PDGFRB, PDGFA, PDGFC, and 18S were purchased from ThermoFisher (cat#4331182). 187 Probe IDs are as follows: PDGFRA – HS00998018_m1; PDGFRB – HS01019589_m1; PDGFA 188 – HS00964426_m1; PDGFC – HS01044219_m1; and 18S – HS03928985_g1. A standard 189 curve was generated for each gene using a plasmid DNA template and data were extracted 190 using this curve. Values are expressed as copies of mRNA/ng of cDNA.

191 In vivo evaluation of olaratumab

192 A colorimetric enzyme linked immunosorbent assay (ELISA) was used to quantify the 193 concentration of olaratumab in mouse serum and mouse plasma. Briefly, the PDGFRα 194 extracellular domain was immobilized in coating buffer in 96-well microtitre plate. After washing 195 the plates, wells were blocked with block/diluent buffer between 1 and 2 h. Plates were then 196 washed and analytes (diluted 1:500 in block/diluent buffer) were added to each well, incubated

197 for approximately 1 h, and washed. Goat α-human IgG F(ab)2 was added to each well and 198 incubated for approximately 1 h. The plates were washed, followed by the addition of 199 tetramethylbenzidine (TMB) to each well and incubation for 15 minutes. The reaction was 200 terminated with the addition of a Stop Solution. Plates were read at 450 nm for detection of 201 olaratumab and at 620 nm for background signal. The concentration of olaratumab was 202 determined on a standard curve. 203 204 In vivo studies were performed in accordance with American Association for Laboratory Animal 205 Care institutional guidelines. A-204 and HuO9 in vivo experiments were approved by the Eli Lilly 206 and Company Animal Care and Use Committee. To evaluate the effects of olaratumab in HuO9 207 and A-204 cell line-derived xenografts, cells were harvested during log phase growth and 208 resuspended in Hank’s Balanced Salt Solution (HBSS). Cell suspensions containing 10 x 106 209 (HuO9) or 5 x 106 (A-204) cells (0.2 mL total volume) were subcutaneously injected into the right 210 flank of female athymic nude mice and tumor growth monitoring started 1 week post-injection. 211 When tumor volumes averaged 200 mm3, mice were randomized into treatment groups 212 (n=6/group) based on tumor volume and body weight. In vivo experiments utilizing the 213 osteosarcoma PDX model CTG-1095 were conducted at Champions Oncology (Hackensack, 214 NJ). The CCSARC005 osteosarcoma patient derived-xenograft model was generated and 215 evaluated at Covance, Inc (Indianapolis, IN).

216 Mice with A-204 xenografts were treated with 40 mg/kg IgG or olaratumab three times a week 217 for 4 weeks. Animals bearing HuO9 or CTG-1095 tumors were treated with control (80 mg/kg 218 IgG, ip + vehicle), cisplatin (5 mg/kg once weekly, ip), doxorubicin (5 mg/kg once weekly, iv), 219 olaratumab + 1E10 (60 mg/kg and 20 mg/kg, respectively, twice weekly, ip), or combination of 220 olaratumab + 1E10 and a SOC for 5 weeks. Mice with CCSARC005 xenograft tumors were 221 treated with control (60 mg/kg IgG, ip + vehicle), olaratumab (60 mg/kg, twice weekly, ip), 222 cisplatin (4 mg/kg once weekly, ip) or a combination of the two. Tumor volume was monitored 223 twice weekly. For all studies, experiments were terminated within 2 weeks following the end of 224 the treatment period.

225 Results

226 Expression of PDGF pathway components is detected in several pediatric sarcoma cell lines

227 Gene expression of PDGFRA and PDGFRB and of PDGFRα-activating ligands PDGFA and 228 PDGFC was evaluated in 7 pediatric bone and soft tissue tumor cell lines (Fig. 1). The 229 osteosarcoma (OS) cell line HuO9 and the malignant rhabdoid tumor (MRT) cell line A-204 230 exclusively expressed PDGFRA while the transcripts of both receptors were detected in MG-63, 231 KHOS/NP, and MNNG/Hos OS cell lines (Fig. 1A). A-204, HuO9, and MG-63 also expressed 232 PDGFA and/or PDGFC, indicating the potential for autocrine activation of PDGFRα (Fig. 1B). 233 Gene expression correlated with PDGFRα and PDGFRβ protein expression (Supplemental Fig.

234 1). Furthermore, PDGFRα expression was detected in G-402 MRT cells but not in G-401 MRT 235 cells nor in BT-12 or BT-16 atypical teratoid/rhabdoid tumor (AT/RT) cells (Supplemental Fig. 236 1A). Expression of PDGFRα and β was also not observed in the embryonal rhabdomyosarcoma 237 (RMS) cell line RD or the Ewing’s sarcoma cell line RD-ES (Supplemental Fig. 1B). Surprisingly, 238 although previous studies reported robust PDGFRα expression in human and murine RMS 239 (20,28), low levels of PDGFRA and PDGFRB transcript were detected in the alveolar RMS 240 (aRMS) cell line SJCRH30 (Fig. 1A). Similarly, PDGFRα was not detected at the protein level in 241 3 out of 4 RMS cell lines (Supplemental Fig. 1C). As olaratumab is a selective anti-PDGFRα 242 antibody, this study focused on receptor-expressing pediatric models (Table 1, Fig. 1, and 243 Supplemental Fig. 1). Receptor status for all evaluated cell lines is summarized in Table 1.

244 PDGFRα phosphorylation is blocked by olaratumab in pediatric osteosarcoma cell lines

245 Olaratumab specifically binds PDGFRα and prevents ligand binding to the receptor and 246 stimulating transphosphorylation (8). The influence of PDGFRβ expression and potential 247 PDGFRα:PDGFRβ heterodimers on olaratumab activity is currently not well understood; 248 therefore, in vitro evaluation of olaratumab was conducted using cell lines with varying 249 expression levels of PDGFRα and/or PDGFRβ. Furthermore, BT-16, an AT/RT cell line which 250 does not express either receptor, was investigated alongside PDGFR+ cell lines as a negative 251 control. The WS-1 normal fibroblast cell line was used as a nontransformed cell control.

252 Phosphorylation status of the PDGF receptors and their downstream effectors was assessed 253 following stimulation by individual PDGFs (Fig. 2, Supplemental Fig. 2 and 3A). Phosphorylation 254 of PDGFRα at tyrosine 754 (Y754) was detected in PDGFRα-positive cell lines, albeit to varying 255 degrees, and indicated receptor activation. Stimulation with PDGF-BB or –DD elicited 256 phosphorylation of PDGFRβ at tyrosine 771 (Y771) in PDGFRβ-expressing cell lines (Fig. 2B 257 and 2C and Supplemental Fig. 2); however, phospho-PDGFRβ was not detected in BT-12 258 AT/RT cells (Supplemental Fig. 3A). Stimulation of HuO9 and MG-63 OS cells with PDGFs 259 resulted in activation of downstream PI3K and MAPK pathway signaling (as measured by 260 phosphorylation of AKT at serine 473 [S473] and ERK1/2 threonine 202/tyrosine 204 261 [T202/Y204]) (Fig. 2A and 2B). Baseline AKT and ERK1/2 phosphorylation was observed in all 262 MRT and AT/RT cell lines evaluated regardless of PDGF receptor status (Fig. 2, Supplemental 263 Fig. 3A). Stimulation with any PDGF (in the case of A-204 and G-402) (Fig. 2A and 2B) or 264 PDGFRβ-activating ligands (in G-401 and BT-12 cells) (Fig. 2C, Supplemental Fig. 3A) resulted 265 in a detectable increase in pAKT and pERK1/2. Surprisingly, PDGF-CC stimulation elicited a 266 marked increase in AKT and ERK1/2 phosphorylation in BT-16 cells, suggesting that PDGFRα 267 may be expressed below the level which can be detected by western blot (Supplemental Fig. 268 3A).

269 As expected, incubation of cells with olaratumab prior to ligand stimulation blocked PDGFRα 270 Y754 phosphorylation in PDGFRα-positive cell lines (Fig. 2, Supplemental Fig. 2). Interestingly, 271 olaratumab blocked activation of AKT and ERK1/2 resulting from stimulation with any ligand in 272 HuO9 cells (Fig. 2A). Similarly, phosphorylation of AKT was strongly inhibited with olaratumab 273 treatment in A-204 cells, while ERK1/2 phosphorylation was reduced to baseline signal with 274 treatment (Fig. 2A). PDGFRβ phosphorylation at Y771 was sustained following olaratumab

275 treatment in PDGFRβ-expressing MG-63 and G-401 as well as WS-1 fibroblasts (Fig. 2B and 276 2C, Supplemental Fig. 2). However, PDGF-BB and –AB-stimulated PDGFRβ phosphorylation 277 was noticeably reduced with olaratumab treatment in G-402 MRT cells (Fig. 2B). Reduced 278 phosphorylation of AKT and ERK1/2 in PDGFRα/PDGFRβ co-expressing cells (Fig. 2B, 279 Supplemental Fig. 2) was observed only when olaratumab prevented PDGFRα stimulation by 280 PDGF-AA, –AB, or (with the exception of MG-63) PDGF-CC; furthermore, AKT and ERK1/2 281 phosphorylation in G-401 cells did not change with olaratumab treatment (Supplemental Fig. 282 2A), indicating that PDGFRβ homodimeric signaling is not affected by olaratumab. Interestingly, 283 olaratumab treatment slightly reduced pAKT and pERK1/2 in BT-16 cells stimulated with PDGF- 284 AA or –CC (Supplemental Fig. 3A).

285 PDGF-stimulated cell proliferation is inhibited by olaratumab alone and in combination with 286 chemotherapeutic agents

287 As the PDGF pathway supports tumor cell growth (1), the potential antiproliferative effect of 288 olaratumab was assessed in a panel of pediatric bone and soft tissue tumor cell lines 289 (summarized in Table 1). In addition, olaratumab was evaluated in combination with either 290 doxorubicin or cisplatin in OS and rhabdoid tumor cell lines (Supplemental Fig. 3B, 3C, and 4 291 and Supplemental Table 3). Stimulation with PDGF-AA and PDGF-CC increased cell 292 proliferation in PDGFRα-expressing cell lines when compared to unstimulated control cells 293 (Supplemental Fig. 4A-4D; p values reported in Supplemental Table 3). The increase in A-204 294 and HuO9 proliferation in response to ligand stimulation was modest when compared to the 295 unstimulated control; however, these cell lines also express PDGFRα-activating ligands and 296 likely activate the receptor in an autocrine manner (Supplemental Fig. 4A and 4B). As expected, 297 PDGFRα-null rhabdoid cell lines G-401, BT-12, and BT-16 did not respond to PDGF-AA/-CC 298 treatment with increased proliferation (Supplemental Fig. 3B and 3C and Supplemental Fig. 4E).

299 Doxorubicin significantly reduced tumor cell proliferation regardless of receptor status or 300 stimulation with exogenous ligand (Supplemental Fig. 3B and 3C and Supplemental Fig. 4). 301 Combination of doxorubicin with olaratumab in A-204 and HuO9 modestly enhanced this 302 reduction by an additional 10% (Supplemental Fig. 4A and 4B). Cisplatin plus olaratumab 303 resulted in a 20-30% further reduction in proliferation in PDGFRα-positive cell lines OS cell lines 304 when compared to single agent treatment (Supplemental Fig. 4A-4D). As expected, olaratumab 305 treatment did not affect proliferation in cells which do not express the receptor (Supplemental 306 Fig. 3B and 3C, Supplemental Fig. 4E).

307 Olaratumab has varying effects on PDGF-driven tumor cell invasion

308 The ability of olaratumab to modulate cell invasion through PDGFRα inhibition was also 309 evaluated in OS and rhabdoid tumor cell lines (Fig. 3 and Supplemental Fig. 3D and 3E). Serum 310 elicited tumor cell invasion when used as a chemoattractant regardless of PDGF receptor 311 status. Interestingly, serum-stimulated A-204, HuO9, and G-402 cell invasion was reduced with 312 olaratumab treatment by approximately 50% when compared to the IgG control treatment group 313 (Fig. 3A-3C). Unexpectedly, A-204 and HuO9 cell invasion did not increase substantially when 314 any of the individual PDGFs were used as the chemoattractant which again suggests that 315 PDGFRα is already activated via an autocrine loop (Fig. 3A and 3B). In contrast, all PDGFs

316 (with the exception of PDGF-AB in the case of MG-63) promoted invasion of cells which co- 317 expressed PDGFRα and PDGFRβ (Fig. 3C and 3D). MG-63 cells were particularly responsive 318 to the ligands when compared to the 10% FBS control chemoattractant (Fig. 3D). Olaratumab 319 treatment significantly blocked HuO9, G-402, and MG-63 cell invasion stimulated by the 320 different ligands; however, while olaratumab treatment reduced HuO9 invasion, these changes 321 were less than 20% of the untreated control. Furthermore, olaratumab did not inhibit the 322 invasive potential of tumor cells which lacked PDGFRα expression (G-401, BT-12, and B-16; 323 Fig. 3E and Supplemental Fig. 3D and 3E).

324 Olaratumab, alone or in combination with chemotherapy, delays xenograft growth

325 Single agent olaratumab was first investigated in mice bearing A-204 MRT xenografts, as the A- 326 204 cell line expresses both PDGFRα and its activating ligand PDGFC. Tumor-bearing animals 327 were given 40 mg/kg olaratumab or control antibody three times weekly for four weeks. Using 328 this dosing schedule, olaratumab treatment significantly delayed A-204 xenograft growth in all 329 treated animals, with stable disease observed in 55% of the treated arm (Fig. 4A and B, 330 Supplemental Table 4).

331 A dose-dependent increase in mean trough serum concentration was measured following 332 olaratumab dose-escalation in two cell-line derived xenograft mouse models (Supplemental Fig. 333 5A). Based on these results, 60 mg/kg olaratumab twice weekly was selected as the dosing 334 schedule used in future in vivo experiments. Trough mean serum concentrations of olaratumab 335 were determined in several cell line-derived and patient-derived xenograft mouse models and 336 was found to be relatively consistent across the evaluated models (Supplemental Fig. 5B).

337 Olaratumab was further investigated in mice bearing HuO9 OS xenografts (Fig. 4C and D, 338 Supplemental Table 4). To interrogate and potentially disrupt the relationship between the 339 human tumor and mouse stroma, the mouse anti-PDGFRα antibody 1E10 was given in 340 conjunction with olaratumab. In addition to single agent olaratumab/1E10 treatment, 341 combination with doxorubicin or cisplatin (two SOC agents used in the treatment of 342 osteosarcoma patients) was also evaluated. Administration of olaratumab/1E10, cisplatin, or 343 doxorubicin alone delayed tumor growth when compared to the control arm (Fig. 4C). 344 Furthermore, stable disease was achieved in 1 or 2 animals in each group (Fig. 4D). The 345 combination of olaratumab/1E10 plus cisplatin (Fig. 4C, left) resulted in stable disease in 4/6 346 mice (Fig. 4D), while combination with doxorubicin (Fig. 4C, right) elicited a partial regression in 347 one animal in addition to 3/6 animals with stable disease.

348 PDX models of pediatric bone and soft tissue sarcoma were profiled for expression of PDGFRA, 349 PDGFRB, PDGFA, and PDGFC (Supplemental Table 5). Expression of each gene was variable, 350 both within and between sarcoma subtypes. PDGFRA transcript was detectable in 50% (11/22) 351 of models assayed with a cutoff of at least 500 copies mRNA/ng cDNA. OS PDXs made up 352 more than half (6/11) of the PDGFRA-expressing models. Activity of olaratumab was evaluated 353 in the CTG-1095 model, an OS PDX which expressed moderate levels of PDGFRA, PDGFRB, 354 and PDGFC (Supplemental Table 5). Olaratumab/1E10 treatment impaired tumor growth, as 355 evidenced by a reduction in tumor volume when compared to the control arm (Fig. 5A and B, 356 Supplemental Table 4). While doxorubicin did not demonstrate any activity in the xenograft

357 tumors, single agent cisplatin significantly delayed tumor growth. In addition, stable disease was 358 achieved in two animals receiving olaratumab/1E10 in combination with cisplatin (Fig. 5B).

359 Olaratumab treatment, alone or in combination with cisplatin, was also investigated in the 360 CCSARC005 OS PDX model. Immunohistochemical analysis of xenograft tumors revealed that 361 a small number of tumor cells express PDGFRα; however, PDGFRβ was readily detectable by 362 western blot (Supplemental Fig. 6). Treatment with olaratumab or cisplatin alone had no effect 363 on tumor growth when compared to tumors from vehicle-treated animals (Fig. 5C). The 364 combination of olaratumab and cisplatin significantly inhibited xenograft tumor growth with 365 tumors in 2/5 mice meeting the criteria for stable disease (Fig. 5C and D) and the remaining 366 mice within the cohort responding with strong tumor growth delay. Tumors from the combination 367 arm were significantly smaller when compared to vehicle- or single agent-treated tumors at 368 study termination (Fig. 5C, Supplemental Table 4).

369 Discussion

370 Aberrant expression and/or activation of PDGFRα is implicated in several subtypes of adult and 371 pediatric sarcoma and supports protumorigenic processes in both the tumor cells and 372 associated stroma (1). Therefore, anti-PDGFRα therapies are of high interest; several 373 molecules targeting PDGFRα have been developed and evaluated both preclinically and 374 clinically in adult and pediatric malignancies (29-31). Olaratumab (LY3012207/IMC-3G3) is a 375 fully human IgG1 monoclonal antibody which specifically binds to PDGFRα thus inhibiting ligand 376 binding and receptor activation (8). Following a pivotal randomized phase II trial, olaratumab 377 received accelerated FDA approval and conditional EMA approval for treatment of advanced 378 soft tissue sarcoma in adult patients in combination with doxorubicin (9). In this study, we 379 demonstrate olaratumab activity in preclinical pediatric bone and soft tissue tumor models. 380 Olaratumab specifically blocked phosphorylation of PDGFRα and attenuated associated 381 downstream MAPK and PI3K pathway signaling in PDGFRα-positive tumor cell lines. 382 Furthermore, as a single agent, olaratumab significantly delayed osteosarcoma and rhabdoid 383 xenograft growth; combination with doxorubicin or cisplatin resulted in stable disease in three 384 mouse models of human osteosarcoma.

385 Therapeutic targeting of PDGFRα with small molecule inhibitors such as sunitinib and imatinib 386 has been investigated preclinically and clinically in multiple pediatric cancer types, including 387 osteosarcoma and malignant rhabdoid tumor (25,32-35). As many of these drugs are designed 388 to bind the ATP site within the kinase domain, target specificity is often lacking, resulting in 389 inhibition of multiple receptor tyrosine kinases and associated toxicity (34,36). This target 390 promiscuity makes it difficult to assign the direct contribution of PDGFRα to tumor growth and 391 cancer cell survival in preclinical studies using small molecule inhibitors. In contrast, monoclonal 392 antibodies are engineered towards a specific target and cross-reactivity has not been reported 393 (36). Olaratumab blocks ligand binding and receptor dimerization by specifically binding to 394 PDGFRα and as such is able to suppress potential kinase domain-independent signaling (37). 395 Furthermore, olaratumab-bound PDGFRα is internalized and degraded which attenuates 396 downstream pathway activation and likely further contributes to drug activity (8,37).

397 While PDGFRA is not frequently altered in childhood cancers (19,38,39), receptor expression is 398 associated with progressive disease and poor prognosis in some pediatric malignancies of bone 399 and soft tissue (11,26,40). Expression of the receptor was readily detected in several 400 osteosarcoma cell lines and PDX models, which was expected, as 50% of osteosarcoma 401 primary samples were previously found to be PDGFRα positive (23). In addition, PDGFRα- 402 activating ligands (namely, PDGFA and/or PDGFC) were also expressed in these cells lines. 403 Conversely, PDGFRα was not expressed at the gene or protein level in the majority of RMS cell 404 lines and PDX models in our study nor in a previous study which examined PDGFRα in 405 SJCRH30 and other RMS cell lines (41). These findings were particularly surprising, as 406 PDGFRα is readily detectable in RMS clinical specimens by qPCR or immunohistochemistry 407 and PDGFRA is a transcriptional target of the fusion protein driving alveolar RMS (PAX3-FOXO) 408 (20,22,26). Similarly, the Ewing’s sarcoma PDX models evaluated in this study were negative 409 for PDGFC expression, despite previous work identifying PDGFC as a target gene of the EWS- 410 FLI1 fusion transcription factor (42). These data are suggestive of potential alterations in the 411 tumor transcriptome when cultured or propagated outside of its native environment and are 412 reminiscent of a previous report describing changes in the epigenome and transcriptome of 413 mammalian cells isolated from primary tissues upon exposure to cell culture (43). Furthermore, 414 a recent study demonstrated that an anti-PDGFRα polyclonal antibody frequently used in 415 previous immunohistochemical analyses cross-reacts with PDGFRβ, further confounding 416 previous reports regarding the prevalence of PDGFRα expression in pediatric bone and soft 417 tissue tumor patient samples (44). In light of this discordance in receptor expression between 418 available preclinical bioresources and clinical specimens, it is difficult to investigate the full 419 range of olaratumab activity in pediatric sarcoma in a preclinical setting and to identify potential 420 predictive biomarkers. Furthermore, while PDGFRα expression in cell lines and xenograft 421 models was necessary for olaratumab to demonstrate some anti-tumor activity in preclinical 422 models, expression alone was insufficient to predict sensitivity, echoing the findings of the 423 pivotal phase II study in adult STS patients (9). Further development of preclinical models that 424 better recapitulate human disease is necessary to predict the clinical activity of novel agents 425 and to understand additional factors that may influence sensitivity.

426 PDGFC has been reported to be highly expressed in A-204 cells, suggesting the potential for an 427 autocrine manner of PDGFRα activation (41); indeed, shRNA-mediated silencing of PDGFC 428 significantly reduced A-204 proliferation, comparable to the knockdown of PDGFRA. We 429 confirmed that PDGFC was expressed in A-204 cells and found that HuO9 expressed both 430 PDGF-AA and -CC, indicating another potential autocrine loop. Furthermore, we observed that 431 exogenous PDGFs were not chemoattractive and could not promote invasion of serum-starved 432 HuO9 or A-204 cells. Insensitivity of HuO9 and A-204 to exogenously supplied PDGFs may be 433 explained by saturation of PDGFRα via endogenously produced ligands. Interestingly, the 434 addition of serum elicited an increase in A-204 and HuO9 osteosarcoma cell invasion well 435 above that observed when PDGFs were used as chemoattractants. Olaratumab subsequently 436 blocked this serum-induced invasion, suggesting that PDGFRα may accommodate signaling 437 initiated by non-PDGF ligands and/or through non-canonical receptor dimerization partners.

438 As expected, olaratumab blocked PDGF-stimulated PDGFRα phosphorylation in alpha-positive 439 normal and pediatric tumor cell lines. PDGFRβ phosphorylation and PDGFRβ-activated

440 downstream pathways were not inhibited by treatment, further illustrating the specificity of 441 olaratumab for the alpha receptor. Interestingly, varied effects on downstream MAPK and PI3K 442 pathway signaling were observed following ligand stimulation and olaratumab treatment in MG- 443 63 and WS-1 cells, potentially influenced by co-expression of PDGFRα and PDGFRβ. 444 Dimerization and subsequent activation of PDGFRα is primarily driven by binding PDGF-AA and 445 PDGF-CC, while PDGFRβ has a higher affinity for PDGF-BB and PDGF-DD (1,45). 446 Heterodimerization can occur after binding any ligand except for PDGF-AA. The expression of 447 PDGFRα alone in HuO9 OS cells and A-204 MRT cells seemingly heightens the affinity of the 448 receptor for individual PDGFs and olaratumab is therefore highly effective at blocking MAPK 449 and PI3K pathway activation caused by PDGFRα signaling. In contrast, MG-63 and WS-1 cells 450 express both PDGFRα and PDGFRβ and downstream signaling persisted after PDGF-BB, -DD, 451 and -AB stimulation, even in the presence of olaratumab. Furthermore, PDGFRβ 452 phosphorylation and downstream pathway signaling was unaffected by olaratumab in G-401 453 MRT cells which only express the beta receptor; however, olaratumab reduced PDGF-BB or – 454 AB-stimulated PDGFRβ phosphorylation in G-402 MRT cells. The differences in signal 455 propagation initiation and maintenance are most likely influenced by the patterns of homo- and 456 heterodimerization. Furthermore, although it is generally accepted that specific PDGFs activate 457 PDGFRα or PDGFRβ, our data suggest that receptor affinity for ligand may be flexible and 458 dependent on relative expression levels of PDGFRα and PDGFRβ protein.

459 To our knowledge, this study is the first to demonstrate that the anti-tumor activity of olaratumab 460 observed in adult sarcomas can extend to preclinical models of pediatric bone and soft tissue 461 malignancies. In the phase II clinical trial combining olaratumab with doxorubicin, progression- 462 free survival was improved by 2.5 months and overall survival was extended by a median gain 463 of 11.8 months with combination treatment when compared to single agent doxorubicin (9). In 464 this study, single agent olaratumab delayed tumor growth in preclinical mouse models of OS 465 and MRT, indicating that PDGFRα signaling is necessary to drive some aspect of xenograft 466 growth; however, a recent report demonstrated that inhibition of PDGFRα alone is insufficient 467 for durable control of MRT growth (25) and combination with other targeted agents or 468 chemotherapy is most likely necessary. Indeed, we observed enhanced anti-tumor activity with 469 co-administration of olaratumab and either doxorubicin or cisplatin in OS mouse models. These 470 data indicate that the improvement in activity observed upon combining SOC with olaratumab is 471 not limited to doxorubicin. Therefore, other chemotherapies commonly used in pediatric tumors, 472 such as ifosfamide, cyclophosphamide, or vincristine (46,47), could potentially combine with 473 olaratumab to enhance anti-tumor activity in preclinical models of pediatric bone and soft tissue 474 tumors.

475 Acknowledgments

476 The authors would like to thank Dr. Peter Houghton for his gift of the atypical teratoid/rhabdoid 477 tumor cell lines BT-12 and BT-16.

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623 Table

624 Table 1. Receptor status and relative EC50 values for proliferation inhibition in a panel of 625 pediatric bone and soft tissue tumor cell lines.

Relative EC50 (nM) - antiproliferation Cell Line Disease PDGFRα† PDGFRβ† +AA +BB +CC +DD +AB A-204 MRT* + - 45.74 170.7 71.42 ND†† 123 BT-12 AT/RT^ - + Not evaluated BT-16 AT/RT - - Not evaluated G-401 MRT - + Not evaluated G-402 MRT + + 167.3 498.2 70.95 ND ND RD Embryonal RMS^^ - - Not evaluated RD-ES Ewing's sarcoma - - Not evaluated Rh18 Alveolar RMS - + Not evaluated Rh28 Alveolar RMS - - Not evaluated Rh36 Embryonal RMS - - Not evaluated Rh41 Alveolar RMS + - Not evaluated SJCRH30 Alveolar RMS - - Not evaluated HuO9 Osteosarcoma + - 204.7 311.3 35.11 20.1 164 KHOS/NP Osteosarcoma + + ND MG-63 Osteosarcoma + + 52.37 186 38.03 ND 101.1 MNNG/Hos Osteosarcoma + + ND Saos-2 Osteosarcoma + - Not evaluated SJSA-1 Osteosarcoma + + ND WS-1 Normal fibroblast + + Not evaluated *MRT: malignant rhabdoid tumor †: expression ^AT/RT: determined by western ††ND: Not determined due to ambiguous or atypical teratoid/rhabdoid tumor blot; boldface indicates non-converged curves ^^RMS: rhabdomyosarcoma strong signal observed 626 627

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636 Figure Legends

637 Figure 1. Pediatric bone and soft tissue tumor cell lines express platelet-derived growth 638 factors and their receptors. A panel of pediatric bone and soft tissue tumor cell lines were 639 evaluated for receptor and ligand expression by qPCR. (A) Results are plotted as expression of 640 PDGFRA by PDGFRB on a log10 scale. Note: expression of PDGFRB did not exceed 1000 641 copies/ng cDNA. (B) Expression of PDGFA and PDGFC are presented on a linear scale.

642 Figure 2. Olaratumab prevents ligand-induced phosphorylation of PDGFRα. (A) A-204 and 643 HuO9 cells, which only express PDGFRα, (B) G-402 and MG-63 cells, which express both 644 PDGF receptors, or (C) G-401 cells, which are PDGFRβ-positive, were treated with 1 μM IgG or 645 olaratumab for 15 minutes, then stimulated with individual PDGFs (5 nM). Whole cell lysates 646 were probed for phosphorylation of PDGF receptors and downstream effectors AKT and 647 ERK1/2.

648 Figure 3. Olaratumab reduces osteosarcoma and malignant rhabdoid tumor cell invasion 649 in vitro. (A) A-204, (B) HuO9, (C) G-402, (D) MG-63, or (E) G-401 cells were evaluated for cell 650 invasion using a simplified Boyden chamber assay. Cells were incubated with IgG or 651 olaratumab; individual PDGFs (5 nM each, with the exception of 25 nM PDGF-DD) or FBS 652 served as chemoattractants. After 48 h, the number of invasive cells was quantified using a 653 fluorescence-based method. Results are presented as the summary of duplicate (G-402) or 654 triplicate experiments ± standard error of the mean (SEM). p values were calculated using the 655 Student’s t-test. Significance indicators: *: p < 0.05, ***: p < 0.001. 656 657 Figure 4. Olaratumab inhibits tumor growth in A-204 and HuO9 cell line-derived 658 xenograft mouse models. (A) and (B) Mice with A-204 subcutaneous xenografts were treated 659 with either 40 mg/kg IgG control or 40 mg/kg olaratumab three times weekly for four weeks. (A) 660 Average tumor volume ± standard error of the mean (SEM) (n = 11/group) during the treatment 661 period is shown. (B) Waterfall plot of %ΔTumor/Control or %Regression for individual animals 662 at Day 30. One animal in the treatment arm was found dead on day 17 and was not included in 663 downstream analysis. (C) and (D) Mice bearing HuO9 subcutaneous xenografts were treated 664 with olaratumab (60 mg/kg)/1E10 (20 mg/kg) twice weekly and/or cisplatin or doxorubicin (5 665 mg/kg) once weekly. Treatment began at day 44 (dotted line) and continued for 5 weeks. (C) A 666 single HuO9 in vivo experiment is displayed in as two separate graphs with the same control 667 and olaratumab/1E10 arms but different chemotherapy-containing arms containing 668 chemotherapy (left: cisplatin and right: doxorubicin). The graph displays average tumor volume 669 ± SEM (n = 6/group). (D) Waterfall plot of individual %ΔTumor/Control or %Regression at Day 670 60. p values for tumor growth curves are displayed in Supplemental Table 4. PD: progressive 671 disease (>10% growth); SD: stable disease (-50% to 10% growth); PR: partial regression (≤- 672 50% and >14mm3). 673 674 Figure 5. Olaratumab, alone or in combination with cisplatin, reduces tumor volume in 675 two osteosarcoma PDX mouse models. (A) and (B) Mice bearing CTG-1095 osteosarcoma 676 PDX tumors were treated with olaratumab/1E10 (60 mg/kg olaratumab/20 mg/kg 1E10) twice 677 weekly and/or SOC (cisplatin or doxorubicin [5 mg/kg]) once weekly (n = 5/group). One animal

678 in the olaratumab/1E10 group was sacrificed prior to experiment termination due to 679 circumstances unrelated to treatment. (A) Dosing began on Day 0 and average tumor volume ± 680 SEM over the five week dosing period is shown. (B) Waterfall plot of %ΔTumor/Control or 681 %Regression for individual animals at Day 21. (C) and (D) Animals with CCSARC005 682 osteosarcoma PDX tumors were treated with olaratumab (60 mg/kg) twice weekly and/or 4 683 mg/kg cisplatin once weekly (n = 5/group). (C) Dosing period is indicated by the dotted vertical 684 lines. Average tumor volume ± SEM is shown. (D) Waterfall plot of %ΔTumor/Control or 685 %Regression for individual mice at Day 62. p values for tumor growth curves are displayed in 686 Supplemental Table 4. PD: progressive disease (>10% growth); SD: stable disease (-50% to 687 10% growth); PR: partial regression (≤-50% and >14mm3). 688

Olaratumab exerts anti-tumor activity in preclinical models of pediatric bone and soft tissue tumors through inhibition of platelet-derived growth factor receptor α

Caitlin D Lowery, Wayne Blosser, Michele Dowless, et al.

Clin Cancer Res Published OnlineFirst November 30, 2017.

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