1078-0432.CCR-15-1853.Full-Text.Pdf

Published OnlineFirst December 30, 2015; DOI: 10.1158/1078-0432.CCR-15-1853

Cancer Therapy: Preclinical Clinical Cancer Research Combined Antitumor Therapy with Metronomic Topotecan and Hypoxia-Activated Prodrug, Evofosfamide, in Neuroblastoma and Rhabdomyosarcoma Preclinical Models Libo Zhang1,2, Paula Marrano2, Bing Wu1,2, Sushil Kumar1,2, Paul Thorner2,4, and Sylvain Baruchel1,3,5

Abstract

Purpose: Tumor cells residing in tumor hypoxic zones are a growth. Complete tumor regression was observed in the major cause of drug resistance and tumor relapse. In this study, we combined topotecan/evofosfamide treatment group after 2- investigated the efﬁcacy of evofosfamide, a hypoxia-activated week treatment. Combined treatment also improved survival prodrug, and its combination with topotecan in neuroblastoma in a neuroblastoma metastatic model, compared to single- and rhabdomyosarcoma preclinical models. agent treatments. In rhabdomyosarcoma xenograft models, Experimental Design: Neuroblastoma and rhabdomyosarco- combined treatment was more effective than single agents. ma cells were tested in vitro to assess the effect of evofosfamide on We also showed that evofosfamide mostly targeted tumor cells cell proliferation, both as a single agent and in combination with within hypoxic regions while topotecan was more effective to topotecan. In vivo antitumor activity was evaluated in different tumor cells in normoxic regions. Combined treatment xenograft models. Animal survival was studied with the neuro- induced tumor cell apoptosis in both normoxic and hypoxic blastoma metastatic tumor model. regions. Results: All tested cell lines showed response to evofosfa- Conclusions: Evofosfamide shows antitumor effects in neu- mide under normoxic conditions, but when exposed to hyp- roblastoma and rhabdomyosarcoma xenografts. Compared oxia overnight, a 2- to 65-fold decrease of IC50 was observed. with single-agent, evofosfamide/topotecan, combined therapy Adding topotecan to the evofosfamide treatment signiﬁcantly improves tumor response, delays tumor relapse, and enhances increased cytotoxicity in vitro. In neuroblastoma xenograft animal survival in preclinical tumor models. Clin Cancer Res; 1–12. models, single-agent evofosfamide treatment delayed tumor 2016 AACR.

Introduction tissue sarcoma and the third most common extracranial solid tumor in children, with an annual incidence of 4 to 7 cases per Neuroblastoma is the most common extracranial childhood million children under the age of 16 years (3). Prognosis of malignancy, responsible for 15% of all childhood cancer-related metastatic rhabdomyosarcoma remains poor despite aggressive deaths (1). Despite intensive treatment protocols including mega- therapies. therapy with hematopoietic stem cell transplantation and immu- There is strong evidence that tumor cells residing in the tumor notherapy, 3-year disease-free survival is only about 50% for hypoxic regions cause drug resistance and tumor relapse (4). metastatic disease compared with 95% for localized tumors Tumor hypoxic zones, which occur in tumors due to nonuniform (2). Rhabdomyosarcoma is the most common pediatric soft and inefﬁcient vasculature, hinder the accessibility of chemotherapeutics to tumor cells. Hypoxia can be a direct cause of thera- 1New Agent and Innovative Therapy Program, The Hospital for Sick peutic resistance due to limited blood supply and drug distribu- Children, Toronto, Ontario, Canada. 2Department of Paediatric Labo- tion (5). Hypoxia also inhibits tumor cell proliferation and ratory Medicine, The Hospital for Sick Children, Toronto, Ontario, induces cell-cycle arrest, further enhancing chemoresistance Canada. 3Division of Hematology and Oncology, Department of Pae- diatrics, The Hospital for Sick Children, Toronto, Ontario, Canada. through regulating drug transporters, antiapoptotic proteins, and 4Department of Laboratory Medicine and Pathobiology, University of proangiogenic factors (6). Our previous studies have shown that Toronto, Toronto, Ontario, Canada. 5Institute of Medical Sciences, tumor hypoxic microenvironment may serve as a niche for the University of Toronto, Toronto, Ontario, Canada. highly tumorigenic fraction of tumor cells which exhibit the Note: Supplementary data for this article are available at Clinical Cancer cancer stem cell features in a diverse group of solid tumor cell Research Online (http://clincancerres.aacrjournals.org/). lines, including neuroblastoma, rhabdomyosarcoma, and small- Corresponding Author: Sylvain Baruchel, New Agent and Innovative Therapy cell lung carcinoma (7). We also found that hypoxia induces drug Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario resistance to etoposide, melphalan, doxorubicin, and cyclophos- M5G 1X8, Canada. Phone: 416-813-5977; Fax: 416-813-5327; E-mail: phamide in neuroblastoma cells, which is mediated through an [email protected] expanded autocrine loop between VEGF/Flt1 and HIF-1a expres- doi: 10.1158/1078-0432.CCR-15-1853 sion (8). There is evidence that some cells in hypoxic regions 2016 American Association for Cancer Research. remain alive in a dormant state for prolonged duration and

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toma, and Ewing sarcoma patients with recurrent or refractory Translational Relevance disease who have not received topotecan previously (18–20). Tumor hypoxia is known to correlate with increased malig- The combination of topotecan, cyclophosphamide, and etopo- nancy, metastatic potential, drug resistance, and poor patient side is tolerable and effective in relapsed and newly diagnosed prognosis in a number of tumor types. Targeting tumor neuroblastoma in phase II clinical trials and upfront phase III hypoxia with hypoxia-activated prodrugs (HAP), such as evo- clinical trials (18, 21). Single-agent topotecan has also been fosfamide, may improve cancer therapy. So far, the effect of found to be antiangiogenic in preclinical models of pediatric evofosfamide has been studied in combination with cytotoxic cancers (22, 23). Despite initial response, tumors eventually agents in adult cancers, whereas the impact of evofosfamide on acquire resistance to topotecan. Mechanisms of resistance pediatric cancers has not been investigated. This study focuses include mutations in topoisomerase-I and involvement of on the preclinical evaluation of efficacy of evofosfamide and transporters like BCRP, P-glycoprotein, and multidrug resis- its combination with topotecan, a topoisomerase I inhibitor tance associated protein-type IV (24–26). widely used in recurrent neuroblastoma and rhabdomyosar- In this study, we selected the metronomic dose of topotecan for coma. We observed that targeting tumor hypoxia with HAPs in vivo studies. Low metronomic dose (LDM) chemotherapy improves antitumor effects of topotecan in our tumor models, usually refers to administration of low dose of cytototoxic agent which provides a new therapeutic approach for the treatment at close intervals without drug free breaks. LDM chemotherapy of pediatric solid tumors. All data gathered from this study will has lower acute toxicity due to lower exposure of the cytotoxic help us build the rationale for future phase I clinical trials in agents. It has been shown active in diverse tumor types, including treating patients with relapsed/refractory neuroblastoma and metastatic disease, especially when combined with antiangio- rhabdomyosarcoma. genic drugs (27–30). The availability of the oral topotecan, along with our previous studies combining metronomic topotecan with pazopanib in neuroblastoma preclinical models (23), suggest that oral metronomic topotecan may be an ideal candidate for become drug resistant as anticancer drugs preferentially target pediatric solid tumors. Considering the limited effects on hypoxic rapidly proliferating cells (6). Hypoxia-inducible factors also tumors with conventional chemotherapy, hypoxia-targeting cyto- induce stem cell phenotype and suppress differentiation of neu- toxic agents along with topotecan therapy may achieve greater roblastoma cells (9). In rhabdomyosarcoma, HIF-1a, induced by antitumor activities. Therefore, in this study, we investigated the hypoxia, upregulates antiapoptotic proteins and glycolytic effect of combined therapy with topotecan and evofosfamide in enzymes (10). HIF-1a inactivation by the inhibition of PI3K/Akt neuroblastoma and rhabdomyosarcoma preclinical tumor pathway is reported to sensitize tumor cells to apoptosis in models. rhabdomyosarcoma (11). Moreover, HIF-1a stabilization as the result of hypoxia increases VEGF expression, increases glycolysis Materials and Methods and resistance to chemotherapy (12). Drugs and reagents Evofosfamide (previously known as TH-302), a hypoxia- Evofosfamide was provided by Threshold Pharmaceuticals Inc. activated prodrug (HAP) has been designed to penetrate to Topotecan HCl was obtained from Selleck (S1231). Stock solu- hypoxic regions of tumors. Evofosfamide is reduced at the tions were prepared in DMSO and aliquots were stored at 80C. nitroimadazole site of the prodrug by intracellular reductases They were further diluted with culture media or saline for in vitro and when exposed to hypoxic conditions, leads to the release of and in vivo studies, respectively. the alkylating agent bromoisophosphoramide mustard (Br- IPM). Br-IPM can then act as a DNA crosslinking agent at the tumor hypoxic region and may diffuse to adjacent normoxic Cells and cell culture regions via a bystander effect (13). Consequently, evofosfamide RH4, RH30, and RD rhabdomyosarcoma cells (31) were gifts can target the malignant cells residing in hypoxic zones, while from Dr. David Malkin (The Hospital for Sick Children, Toronto, having little or no cytotoxic effect on the normal cells (14, 15). Ontario, Canada). LAN-5 (32), SK-N-BE(2) (33), BE(2)C (34), The antitumor efficacy of evofosfamide correlated with hypoxic and SH-SY5Y (34) neuroblastoma cells were kindly provided by fractions in tumor xenografts of lung cancer, melanoma, pan- Dr. Herman Yeger (The Hospital for Sick Children, Toronto, creatic cancer, renal cell carcinoma, and hepatocellular carci- Ontario, Canada). CHLA-15, CHLA-20, and CHLA-90 (35) were noma. In adult soft tissue sarcoma phase II trial, evofosfamide obtained from the Children's Oncology Group (COG) Cell Cul- in combination with doxorubicin demonstrated 2% complete ture and Xenograft Repository under a signed and approved response (CR) and 34% partial response (PR; ref. 16). As a Material Transfer Agreement. Cell line authentication was per- single maintenance therapy, it had limited hematologic toxicity formed using short tandem repeats (STR) DNA profiling (Pro- and cardiotoxicity, whereas with doxorubicin, it caused man- mega's GenePrint 10 System; ref. 36) conducted by the Genetic ageable hematologic toxicity without aggravating cardiotoxi- Analysis Facility at the Centre for Applied Genomics of The city. A phase III study in advanced and metastatic adult soft Hospital for Sick Children (Toronto, Ontario, Canada). The DNA tissue sarcoma is ongoing (NCT01440088). To date, no studies (STR) profile for all cell lines match to the profile listed in the investigating the efficacy of evofosfamide in pediatric cancers Children's Oncology Group (COG) STR Database (http://strdb. have been conducted. cogcell.org). RH4, RH30, and RD rhabdomyosarcoma cells were Topotecan, a topoisomerase-I inhibitor has shown activity cultured in DMEM supplemented with 10% FBS. CHLA-15, against both neuroblastoma and rhabdomyosarcoma as a sin- CHLA-20, and CHLA-90 neuroblastoma cells were cultured in gle agent (17). The combination of cyclophosphamide plus Iscove's modified Dulbecco's medium supplemented with 3 topotecan has been active in rhabdomyosarcoma, neuroblas- mmol/L L-glutamine, insulin, and transferrin 5 mg/mL each and

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5 ng/mL selenous acid (ITS Culture Supplement; Collaborative were sacrificed, and tumors were dissected and weighed. Tumor Biomedical Products) and 20% FBS (complete medium). SK-N- growth curves were plotted with the relative tumor volumes at BE(2) and SH-SY5Y neuroblastoma cells were cultured in AMEM different time points. The relative tumor volume of each tumor is with 10% FBS. defined as the tumor volume divided by its initial volume. Potential drug toxicity was assessed in tumor-bearing mice by Test animals monitoring animal body weight, appetite, diarrhea, signs of NOD/SCID mice (Jackson Laboratory) were used for xenograft animal distress, or any marked neurologic symptoms. mouse models at 4 weeks of age. The mice were housed in an isolated sterile containment facility. All animal studies were Animal survival study approved by Animal Care Committee at the Hospital for Sick SK-N-BE(2) cells were prepared and injected intravenously 6 Children (Animal Use Protocol#1000019356). During the study, into the lateral tail vein (26-gauge needle, 1 10 cells in 100 the mice were observed daily for possible adverse effects due to mL total volume). Mice were randomized into four groups: treatments. Morbidity signs of ill health, such as ruffled/thinning control, evofosfamide, topotecan, and the combination of fur, abnormal behaviors, or local erosion from the tumor were evofosfamide and topotecan, with eight mice in each group. noted and experiments terminated as indicated. All the treatments were initiated 14 days after tumor cell inoculation at the the same schedule and dosage as above. Animals were monitored for body weight and any signs of Cell viability assay stress. Tumor-bearing mice were euthanized at the endpoint Cell viability was assessed by Alamar Blue assay as previously when there were signs of distress, including 20% of body described (37). Alamar Blue cell proliferation assay is based on weight loss, fur ruffling, rapid respiratory rate, hunched pos- a reducing environment that indicates metabolically active ture, reduced activity, and progressive ascites formation. cells. Briefly, exponentially growing tumor cells was seeded into 48-well plates at 1 105 cells per well and grown overnight prior to initiating treatment. On the day of the test, IHC cells were exposed to increasing concentrations of evofosfa- Double immunofluorescence staining was performed on mide and topotecan either as single agents or combined treat- 5-mm-thick frozen sections. Tissue sections were fixed for 10 ment for 72 hours. Alamar Blue was added to a final concen- minutes in acetone and allow to air dry for 5 minutes. Endog- tration of 5%, incubated for 4 hours at 37C and quantitated on enous biotin, biotin receptors, and avidin sites were blocked a plate reader at 530/590 nm. IC50 was determined by Graph- with the Avidin/Biotin Blocking Kit (SP-2001, Vector Labora- Pad Prism software. tories). The tissue sections were incubated with rabbit anti- We also evaluated drug efficacy under the condition of cleaved caspase-3 antibody (1:50; #9661, Cell Signaling Tech- hypoxia. To simulate tumor hypoxia in vitro, after drug addi- nology) or rabbit anti-Ki67 antibody (1:200; ab16667, Abcam) tion, the plates were incubated for 2 hours at 37Cinan for 1 hour at room temperature. Detection of the rabbit anti- anaerobic chamber. The anaerobic chamber was evacuated and bodies was performed by incubation with Texas Red goat anti- gassed with the hypoxic gas mixture (94% N2/5% CO2/1% O2) rabbit IgG antibody (1:200; TI-1000, Vector Laboratories) for to create a hypoxic environment. After 2 hours or overnight 30 minutes. After the washing of the tissue sections, the mouse exposure to hypoxic condition, cells were removed from the IgG1 anti-pimonidazole mAb clone 4.3.11.3 (1:50; Hypoxyp- hypoxia chamber and further incubated for 3 days in standard robe, Inc.) was added overnight at 4 C. This antibody tissue culture incubator. Cell viability was assessed by Alamar was detected with Fluorescein horse anti-mouse IgG Antibody Blue assay as described above. (1:200; #FI-2000, Vector Laboratories) for 30 minutes. The tissue sections were washed and then mounted with Mouse xenograft models Vectashield mounting medium with DAPI (H-1200, Vector Two neuroblastoma cell lines [CHLA-20 and SK-N-BE(2) and Laboratories). two rhabdomyosarcoma cell lines (RH4 and RD) were used to establish murine models (38). Briefly, tumor cells were washed Statistical analysis three times with Hank balanced salt solution before injection. Data from different experiments were presented as mean SD. Cell suspensions were mixed 1:1 with Matrigel (BD Bios- For statistical analysis, Student t test for independent means was ciences). Subcutaneous xenografts were developed by injecting used. A P value of <0.05 was considered significant. To compare 1 106 tumor cells into the subcutaneous fat pad of NOD/ the effects of different treatments on tumor growth in vivo, one- SCID mice. Treatments commenced when the tumor sizes way ANOVA with Dunnett multiple comparison test was used. reached about 0.25 cm3. Survival curve comparisons were performed using GraphPad Animals were randomized into four groups with 10 animals in Prism software for Kaplan–Meier survival analysis. Statistical each group: control; evofosfamide, topotecan as a single agent, differences between the various groups were compared pair-wise and the combination of evofosfamide and topotecan. The doses using log-rank (Mantel–Cox) analysis. of evofosfamide and topotecan were 50 mg/kg (every day 5 Combination synergism was analyzed with MacSynergy II days/week, i.p.) and 1 mg/kg (every day 5 days/week by oral software (kindly provided by M.N. Prichard) with 95% confi- gavage), respectively. The injection or gavage volume was 200 mL. dence limits according to the Bliss independence model (39). The Control mice received the same volume of the vehicle (saline). effect attributed to combination is determined by subtracting the Tumor growth was measured three times a week in two dimen- experimental values from theoretical additive values defined by sions, using a digital caliper. Tumor volume was calculated as the equation: Exy ¼ Ex þ Ey ExEy, where Exy is the additive effect 2 3 width length 0.5. When tumor volume reached 1.5 cm , mice of drugs x and y as predicted by their individual effects Ex and Ey.

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Results All these cell lines were exposed to increased concentrations of evofosfamide in vitro for 72 hours. Under normoxic condi- Improved antiproliferation effects of evofosfamide under tion, all the tested lines responded to evofosfamide treatment prolonged hypoxic conditions with neuroblastoma and in a dose-dependent manner, with IC values ranging from rhabdomyosarcoma cell lines 50 4.6 to 151 mmol/L. When exposing neuroblastoma and rhab- We used a panel of neuroblastoma and rhabdomyosarcoma domyosarcoma cells under hypoxic conditions (1% O )for cell lines that represented heterogeneity and different stages of 2 2hours,nosignificant IC change was documented. However, therapy, including five neuroblastoma [CHLA-15, CHLA-20, 50 when tumor cells were exposed to hypoxia (1% O )overnight, CHLA-90, SK-N-BE(2), and SH-SY5Y] and three rhabdomyosar- 2 there was a 2- to 65-fold decrease of evofosfamide IC (Fig. 1A coma cell lines (RH4, RH30, and RD), to assess the effects of 50 and B; Table 1) with the IC values ranging from 0.07 to evofosfamide on cell proliferation in vitro. SH-SY5Y is MYCN 50 21.9 mmol/L. nonamplified. CHLA-15 and CHLA-20 cell lines are MYCN nonamplified cell lines obtained from tumors of treatment-na€ve and chemotherapy-treated neuroblastoma patients, respectively. Both Improved antiproliferation effects of evofosfamide when CHLA-90 (MYCN nonamplified) and SK-N-BE(2) (MYCN-ampli- combined with topotecan in vitro fied) cell lines are obtained from bone marrow metastases of In our previous studies, we showed that maximal plasma patients who relapsed after chemotherapy with mutant p53 (35). concentration (Cmax) of topotecan was 19.8 ng/mL with the RH4 and RH30 cell lines are alveolar rhabdomyosarcoma cell metronomic oral dose of 1 mg/kg drug administration in lines, whereas RD is an embryonal rhabdomyosarcoma cell line. NOD/SCID mouse model (23). In patients, Cmax of topotecan

Figure 1. Increased cytotoxicity of evofosfamide under prolonged hypoxic conditions with neuroblastoma and rhabdomyosarcoma cells. A panel of 5 different neuroblastoma cell lines [CHLA-15, CHLA-20, CHLA-90, SK-N-BE(2), and SH-SY5Y; A] and 3 rhabdomyosarcoma cell lines (RH4, RH30, and RD; B) were selected to assess the effects of evofosfamide on cell proliferation in vitro. Cell viability was measured by Alamar Blue assays after exposing tumor cells to increasing concentrations of evofosfamide in vitro for 72 hours under both normoxic and two hypoxic conditions, 1% O2 for 2 hours or 1% O2 overnight. Dose–response curves were ﬁt to the data and IC50 values were calculated by GraphPad Prism software.

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Table 1. Comparison of IC concentrations of evofosfamide when tested alone 50 overnight hypoxia as indicated by decreased IC50sinmost and in combination with 20 nmol/L topotecan in neuroblastoma and tested tumor cell lines. rhabdomyosarcoma cell lines To understand whether the combination of the drugs was IC50 (mmol/L) þ additive or synergistic, a Bliss analysis was performed with Mac- Evofosfamide ﬁ Evofosfamide Topotecan P Synergy II software with 95% con dence limits. The effect of the Neuroblastoma cell lines combination was additive in all cell lines. CHLA-15-normoxia 4.6 5.8 3 3.5 0.39 CHLA-15-1% O2; 2 hours 9.5 6.3 7.4 4.1 0.57 Topotecan and evofosfamide delay tumor growth and enhance < CHLA-15-1% O2; O/N 0.07 0.03 0.04 0.01 0.01 animal survival in neuroblastoma model CHLA-20-normoxia 8.9 2.7 1.2 0.6 <0.01 After verifying in vitro activity of evofosfamide and topotecan, CHLA-20-1% O ; 2 hours 5.6 1.1 0.83 0.12 <0.01 2 the in vivo effectiveness of topotecan/evofosfamide as single CHLA-20-1% O2; O/N 0.16 0.01 0.1 0.01 <0.01 CHLA-90-normoxia 13.9 4.3 4.9 2.5 <0.05 agents or combination therapy was evaluated in CHLA-20 and

CHLA-90-1% O2; 2 hours 10.7 2.7 3.5 1.3 <0.05 SK-N-BE(2) xenograft models. As both CHLA-20 and SK-N-BE < CHLA-90-1% O2; O/N 0.47 0.10 0.24 0.03 0.01 (2) cell lines are derived from samples of patients who relapsed SK-N-BE(2)-normoxia 51.9 9.3 46.2 16.9 0.55 after chemotherapy, we use them to develop aggressive tumor SK-N-BE(2)-1% O2; 2 hours 54.9 5.8 49 15.6 0.47 models for rigorous screening of anticancer agents. All treat- SK-N-BE(2)-1% O2; O/N 2.4 0.59 1.7 0.47 0.22 3 SH-SY5Y-normoxia 66.1 9.2 57.5 13.8 <0.05 ments commenced when the tumor sizes reached 0.25 cm .In

SH-SY5Y- 1% O2; 2 hours 66.1 5.9 39.8 5.3 <0.01 both neuroblastoma models, after 2 weeks of treatment, tumor SH-SY5Y- 1% O2; O/N 1.3 0.23 0.5 0.07 <0.01 growth delay was observed with evofosfamide or topotecan as Rhabdomyosarcoma cell lines monotherapies (Fig. 2A). In SK-N-BE(2) xenograft model, com- RH4-normoxia 20.9 1.4 16.2 1.1 <0.01 bined regimen induced complete tumor regression, while topo- RH4-1% O ; 2 hours 24 1.1 13.8 1.7 <0.01 2 tecan induced partial tumor regression. When we compared RH4-1% O2; O/N 3.3 0.27 1.7 0.22 <0.01 RH30-normoxia 151 34.7 120 6.5 <0.01 the tumor sizes between these two arms, the difference is fi P < RH30-1% O2; 2 hours 115 3.8 95.5 2.7 <0.01 statistically signi cant ( 0.05) at different time points (from RH30-1% O2; O/N 21.9 8.2 15.1 1.5 <0.01 day 14 to day 33). In CHLA-20 xenograft model, both single- RD-normoxia 19.1 4.9 11 2.0 <0.01 agent topotecan and combined treatment induced complete < RD-1% O2; 2 hours 18.2 4.7 11 1.9 0.01 tumor regression, but all those tumors relapsed after drug RD-1% O ; O/N 9.55 0.33 4.07 0.15 <0.01 2 treatment was stopped. Therefore, we compared tumor recur- fi NOTE: P values represent statistically signi cant differences using the extra rence between single-agent topotecan and combination groups sum-of-squares F test. after we stopped drug treatment. As shown in Figure 2B, the combination group had significant longer recurrent-free interval compared with single-agent topotecan group (P < 0.05). For is 10.6 4.4 ng/mL with the oral dose of 2.3 mg/m2/day. As the topotecan-treated mice, median time of recurrence was 30 days, terminal half-life of oral topotecan (3.9 1.0 hours; ref. 40) is while median time of recurrence for combined therapy was significantly longer than evofosfamide (0.6 0.1 hours; ref. 41), a postponedto57days.Inaddition, when we rechallenged those constant concentration of topotecan (20 nmol/L ¼ 8.4 ng/mL) relapsed tumors with the combined evofosfamide/topotecan was selected for in vitro combined treatment. therapy, those tumors remained responsive with the original All selected tumor cell lines were treated in vitro with treatment schedule (Fig. 2A). increasing concentrations of evofosfamide with or without the Treatment was well tolerated, with weight loss >10% presence of topotecan (20 nmol/L). The dose–response curves observed in single-agent and combined treatment groups with- were generated by GraphPad software and IC50 values were in the first 8 days but returned to baseline thereafter. No animal compared between IC50 concentrations of evofosfamide when death was observed in any of the groups during the experi- tested alone and in combination with 20 nmol/L topotecan in mental period (Fig. 2C). neuroblastoma and rhabdomyosarcoma cell lines. As shown To determine the antitumor activities of evofosfamide and in Tables 1 and 2, with the presence of topotecan, evofosfa- topotecan on late-stage metastatic disease, we further assess mide-induced cytotoxicity was significantly enhanced under animal survival in the SK-N-BE(2) intravenous metastatic model.

Table 2. IC50 of topotecan in neuroblastoma and rhabdomyosarcoma cell lines

IC50 (nmol/L) a Normoxia 1% O2, 2 hours 1% O2,O/N P Neuroblastoma cell lines CHLA-15 17.6 3.9 15.6 0.9 13.7 1.4 NSb CHLA-20 12.9 1.2 12.4 1.0 14.1 0.6 NS CHLA-90 4,170 1,030 4,550 1,590 5,260 724 NS SK-N-BE(2) 96.2 11.8 110 13.8 95.0 14.3 NS SH-SY5Y 19.2 7.0 12.4 1.1 14.1 0.6 NS Rhabdomyosarcoma cell lines RH4 132 10.0 110 21.8 119 3.7 NS RH30 2,251 685 1,963 416 1,621 174 NS RD 387 55.8 250 87.5 317 22.9 NS aP value from one-way ANOVA. bNS, not signiﬁcant, P > 0.05

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Figure 2. Antitumor effects of evofosfamide and topotecan in neuroblastoma xenograft models. A, a total of 1 106 cells were implanted subcutaneously into SCID/SCID mice. Once tumors were palpable (about 0.25 cm3), mice were randomized into vehicle control (n ¼ 10), or three treatment groups (n ¼ 10 for each group). Mice bearing human neuroblastoma [SK-N-BE(2) and CHLA-20] xenografts were treated for 2 weeks with evofosfamide (50 mg/kg; every day 5 days/week, i.p.) or topotecan (1 mg/kg; every day 5 days/week, orally) as single agents or in combination. The shaded area indicates the period of drug treatment. Tumor growth curves were plotted with the tumor volumes at different time points. One-way ANOVA was used to compare tumor volumes between experimental groups at different time points during the experimental period (, P < 0.05; , P < 0.01). Data are expressed as mean SEM. After 1 cycle of therapy, the combination treatment with evofosfamide and topotecan led to complete tumor regression in both SK-N-BE(2) and CHLA-20 xenograft models. In the CHLA-20 model, tumor recurrence was observed in the combined treatment group. On day 55, we rechallenged the tumor bearing animals with the original treatment dosage and schedule on day 55 (identified by shaded area between day 55 and 69).All the relapsed tumors remained responsive to combined treatment. B, recurrence-free interval curves were estimated in CHLA-20 xenograft model by the Kaplan–Meier method and compared between topotecan and combination of evofosfamide and topotecan treatment using the log-rank (Mantel–Cox) analysis. The tumor-free interval was defined from the end of treatment to the first appearance of a palpable mammary tumor at least 100 mm3 in size. , P < 0.05. C, mouse body weight was measured in CHLA-20 tumor-bearing mice in different treatment groups and control animals. Percentage of body weight loss was calculated and plotted to monitor the potential drug toxicity. D, Kaplan–Meier survival curves of the SK-N-BE(2) metastatic tumor model. NOD/SCID mice (n ¼ 8/group) were injected with 1 106 SK-N-BE(2) cells intravenously. Fourteen days after tumor cell inoculation, mice were treated for 2 weeks with control vehicle, evofosfamide, topotecan, or the combination of evofosfamide and topotecan with the same dose as indicated above. Statistical differences between the various groups were compared pair-wise using log-rank (Mantel–Cox) analysis. , P < 0.05.

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Fourteen days after tumor cell inoculation, we initiated drug different regimens were harvested for immunohistochemical treatment with evofosfamide and topotecan as single agents or analysis (Fig. 4A). Hypoxic regions were identified as the area of combined treatment. As shown in Fig. 2D, evofosfamide or pimonidazole-positive staining. Apoptotic cells were detected by topotecan treatment showed a substantial increase in animal anti-cleaved caspase-3 antibody. Apoptosis was quantified as the survival compared with the control mice with a median survival percentage of positive cleaved caspase-3 staining area using Ima- of 22.5 days for the control group, 25 days for the evofosfamide geJ software (Fig. 4B). As shown in Fig. 4A and B, evofosfamide group, and 36 days for the topotecan group. However, treatment induced tumor cell apoptosis in hypoxic regions, with a remark- with a combination of evofosfamide and topotecan had the ably higher percentage of cleaved caspase-3–positive cells in greatest impact on animal survival (P < 0.01) with a median hypoxic regions compared with normoxic regions (Student t test, survival of 46 days. P < 0.01). On the contrary, topotecan induced more extensive cell apoptosis in normoxic regions than hypoxic regions (P < 0.05). With the combination treatment, significant tumor cell apoptosis Topotecan and evofosfamide delays tumor growth in was observed throughout the tumor, in both normoxic and rhabdomyosarcoma models hypoxic areas. To test the antitumor activity of evofosfamide and topotecan in rhabdomyosarcoma, we chose an alveolar rhabdomyosarcoma cell line (RH4) and an embryonal rhabdomyosarcoma cell line Decreased cell proliferation with evofosfamide and topotecan (RD) to generate xenograft models. Tumor bearing NOD/SCID combined treatment in vivo mice were randomized to therapy with evofosfamide, topotecan, We further evaluated tumor cell proliferation in different or combined therapy. In both RH4 and RD xenograft models, treatment groups by immunostaining of the proliferation marker, þ both evofosfamide and topotecan monotherapy significantly Ki67. As shown in Fig. 4C and D, the Ki67 proliferating tumor delayed tumor growth with 2 weeks of treatment (Fig. 3). Com- cell population was mainly located in normoxic regions. After the pared with neuroblastoma models, single-agent evofosfamide 4-day in vivo treatment, single-agent topotecan showed a limited was more effective in rhabdomyosarcoma models with partial effect on cell proliferation as measured by Ki67 positivity. Sur- tumor regression in all treated xenografts. The combination of prisingly, decreased cell proliferation was observed in evofosfa- topotecan and evofosfamide were superior to the respective drugs mide-treated tumors in normoxic regions with significantly lower given alone at different time points from day 26 to day 30 (P < percentage of Ki67 positive cells than control tumors (P < 0.05), 0.01; Fig. 3). There was no significant drug-related toxicity in any suggesting a hypoxia-independent bystander effect. Combined of the treatment arms. evofosfamide and topotecan treatment led to a further decrease in þ the Ki67 -proliferating tumor cell population compared with single-agent evofosfamide group (combined group vs. single- Colocalization of cell apoptosis and tumor hypoxia with agent topotecan, P < 0.01; combined group vs. single-agent evofosfamide and topotecan treatment evofosfamide, P < 0.05). As combined treatment with evofosfamide and topotecan significantly improved antitumor activity both in vitro and in vivo, we have particular interest to examine its potential mechanism of Discussion action in vivo. To this end, tumor cell apoptosis was assessed by Topotecan has demonstrated efficacy in preclinical and clinical cleaved caspase-3 immunoreactivity analysis in our xenograft studies in neuroblastoma and rhabdomyosarcoma. However, models. On day 5 of drug treatment, RH4 xenografts from total eradication of highly tumorigenic populations of tumor

Figure 3. Effects of evofosfamide and topotecan on the growth of rhabdomyosarcoma xenografts. A total of 1 106 cells were implanted subcutaneously into SCID/SCID mice. Once tumors were palpable (about 0.25 cm3), mice were randomized into vehicle control group (n ¼ 10) or three treatment arms (n ¼ 10 for each group). RH4 and RD xenografts were treated with evofosfamide (50 mg/kg; every day 5 days/week, i.p.) or topotecan (1 mg/kg; every day 5 days/week, orally) as single agents or in combination. The shaded area indicates the period of drug treatment. Tumor growth was monitored as described above. One-way ANOVA was used to compare tumor volumes between experimental groups at different time points during the experimental period (, P < 0.05; , P < 0.01). Data are expressed as mean SEM.

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Figure 4. Localization of tumor cell apoptosis, proliferation, and tumor hypoxia after evofosfamide and topotecan treatment. RH4 xenografts from different regimens were harvested 4 days after drug treatment for immunohistochemical analysis. Hypoxic regions were identified as the area of pimonidazole-positive staining (circled area). A, apoptotic cells were detected by anti-cleaved caspase-3 antibody. B, Ten high-power fields per treatment arm were quantified for cell apoptosis in both tumor hypoxic and normoxic regions expressed by percentage of positive cleaved caspase-3 staining area using ImageJ software. Statistical analysis was done by t test (, P < 0.05; , P < 0.01). Error bars, SD. (Continued on the following page.)

cells is required to achieve relapse-free survival in patients with HAPs, which deliver the cytotoxic payload in hypoxic zones, cancer. Incomplete distribution of chemotherapeutics to poorly have been developed to solve this issue. PR-104, an earlier HAP, perfused areas of primary tumors and metastatic sites is a major was found to have a steep dose–response relationship in a hindrance in complete eradication of these tumor cells. These panel of pediatric tumor xenograft models, which raises safety sparsely vascularized areas are characterized by hypoxia which concerns in pediatric patients (42). Evofosfamide, however, has confers drug and radiation resistance, and a proangiogenic and demonstrated survival beneﬁt in various adult cancers without invasive phenotype to tumor cells. Despite the advent of novel causing prohibitively additive toxicity with conventional che- chemotherapies, complete eradication of tumor cells cannot be motherapy in clinical trials (16). Evofosfamide shares the accomplished until hypoxic tumor cells are targeted. Therefore, similar active metabolites as ifofosfamide, but it is preferen- targeting hypoxic zone of solid tumor may reverse drug resis- tially activated in hypoxic tissues and does not have the same tance and make cytotoxic agent more effective. In this study, we toxic metabolites as ifosfamide. It is anticipated that evofosfa- are proposing new strategies to improve the efﬁcacy of chemo- mide should have less hematologic, renal, and CNS toxicity therapy by incorporating a novel agent targeting tumor hypoxic than ifosfamide. There is no direct clinical studies comparing regions. evofosfamide with ifosfamide, but evofosfamide shows

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Figure 4. (Continued. ) C, immunohistochemical staining of Ki67 was performed to identify proliferating cells in different groups of xenografts. D, cell proliferation in normoxic regions was quantiﬁed by percentage of positive Ki67 staining area using ImageJ software. Statistical analysis was done by t test (, P < 0.05; , P < 0.01). Data are means SEM.

superior efficacy and less toxicity than ifosfamide in preclinical Our observation that single-agent topotecan is more effective in lung carcinoma models (43). In our xenograft models, we delaying tumor growth in neuroblastoma than in rhabdomyo- observed tumor growth inhibition with single-agent evofosfa- sarcoma is in agreement with our previous finding (23). From our mide, but no significant antitumor effect was achieved in the observations in vitro, evofosfamide showed more activity in neu- CHLA-20 model with ifosfamide (50 mg/kg; every day 5 roblastoma cell lines than in rhabdomyosarcoma cells. However, days/week, i.p.; Supplementary Fig. S1). In clinical trials in vivo, rhabdomyosarcoma xenografts were more sensitive to (NCT02047500), evofosfamide is administered at a dose rang- evofosfamide than neuroblastoma xenografts. In this study, we ing from 170 to 340 mg/m2. This could be converted to 32–64 cannot simply translate in vitro observation to in vivo activity. In mg/kg in the tumor bearing mice, calculated by the pharma- vitro, we simulated tumor hypoxia by incubating tumor cells cokinetic data from the clinical studies and preclinical mouse inside the hypoxia chamber. Therefore, 100% of cell population studies (44). In this study, we tested evofosfamide at the dose was under hypoxia and exposed to activated evofosfamide. In vivo, of 50 mg/kg, as single agent and in combination with topotecan the percentage of hypoxic tumor cells varies among different in two of the most common pediatric extracranial solid tumors, tumor types (45, 46). In vivo tumor microenvironment is also neuroblastoma and rhabdomyosarcoma. To our knowledge, more complex in which HAP activation would be affected by this is the first study evaluating the effectiveness of evofosfa- many other factors, including the duration and the intensity of mide in pediatric tumors. oxidative stress, tumor angiogenesis, drug penetration in different

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solid tumor types, and acquired drug resistance under prolonged An ideal HAP requires selective activation in hypoxic regions as hypoxic conditions. Although it was beyond the scope of this well as good penetration of multiple cell layers to exert a local study to examine the tumor microenvironment among different cytotoxic bystander effect. Using a multicellular tumor spheroid tumor types, it remains an important area for our future and multicellular layer (MCL) coculture model systems, Meng investigation. and colleagues (49) showed that evofosfamide has penetrability From our study, evofosfamide showed enhanced cytotoxicity and bystander effect. In our study, with evofosfamide single-agent under overnight and 3-day (Supplementary Fig. S2) prolonged treatment, we observed decreased cell proliferation even at nor- hypoxia compared with short-term (2-hour) hypoxia exposure. moxic regions. This phenomenon confirms that once activated in When we test the effect of evofosfamide and its combination with hypoxic tissues, evofosfamide releases Br-IPM which can diffuse topotecan on five neuroblastoma and three rhabdomyosarcoma into surrounding oxygenated regions of the tumor and kill cells in cell lines under normoxia, short-term hypoxia and prolonged those regions via a "bystander effect." hypoxia, it is also under overnight hypoxia (1% O2) that adding In summary, topotecan and evofosfamide demonstrated anti- topotecan significantly lowered IC50 of evofosfamide in most tumor activities in our preclinical models of various subtypes of tumor cell line, except SK-N-BE(2). However, in the SK-N-BE(2) neuroblastoma and rhabdomyosarcoma. Compared with single xenograft model, combined therapy achieved significant antitu- agents, combination treatment induces more tumor regression, mor effects in vivo. Combined treatment induced complete tumor delays tumor relapse, and enhances animal survival in our pre- regression in all treated animals, which was superior to evofosfa- clinical tumor models. In this study, we explored the mechanism mide or topotecan single-agent treatment. This is likely because of action with combined evofosfamide/topotecan therapy. We SK-N-BE(2) xenografts have more severe and more chronic hyp- observed increased reactive oxygen species (ROS) under hypoxic oxic environment than in vitro cell culture (1% O2, overnight), condition (1% O2, overnight; Supplementary Fig. S3). We also which could more effectively activate evofosfamide to exert its found that adding topotecan enhanced the oxidative stress in cytotoxic activity in vivo. rhabdomyosarcoma cells under hypoxic condition (Supplemen- In all the neuroblastoma and rhabdomyosarcoma xenograft tary Fig. S4). In vivo, evofosfamide showed significant cytotoxicity models tested, combined treatment showed superior antitumor against hypoxic tumor cells, while topotecan plays complemen- effects compared with single-agent treatments, and induced tary roles by targeting tumor cells residing at normoxic regions. tumor regression after a 2-week treatment period. The advantage The ability of this combination to target cells in both normoxic of the combination regimen was also approved in the metastatic and hypoxic tumor zones, as demonstrated by immunofluores- neuroblastoma model. Our experimental metastatic model has cence, provides a proof-of-principle for its superior efficacy. These been developed to simulate residual disease in neuroblastoma. preclinical data support the development of a clinical trial in high- Residual disease refers to disseminated tumor cells which survive risk neuroblastoma and rhabdomyosarcoma. after therapy (47). More than 50% of children with high-risk neuroblastoma develop recurrence due to the presence of minimal residual disease. In our metastatic model derived from a Disclosure of Potential Conflicts of Interest disseminated MYCN-amplified cell line, mice treated with com- S. Baruchel reports receiving commercial research grants from Merck TH302. No potential conflicts of interest were disclosed by the other authors. bined topotecan and evofosfamide had extended survival compared with single-agent topotecan treatment. This result is significant in promoting new therapies for neuroblastoma minimal Authors' Contributions residual disease or refractory neuroblastoma by combining HAPs Conception and design: L. Zhang, S. Baruchel with existing chemotherapeutic drugs. We believe that incorpo- Development of methodology: L. Zhang, P.S. Thorner, S. Baruchel rating HAPs, such as evofosfamide, could potentially eradicate the Acquisition of data (provided animals, acquired and managed patients, tumor cells hiding in hypoxic zones which are largely responsible provided facilities, etc.): L. Zhang, P. Marrano, P.S. Thorner, S. Baruchel for eventual relapse and metastasis. Eventually, this new regimen Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Zhang, P. Marrano, B. Wu, S. Kumar, P.S. Thorner, might prolong progression-free survival in high-risk neuroblas- S. Baruchel toma patients. Writing, review, and/or revision of the manuscript: L. Zhang, P. Marrano, In this study, we were able to assess the correlation between S. Kumar, P.S. Thorner, S. Baruchel tumor hypoxia and tumor cell apoptosis with immunostaining of Administrative, technical, or material support (i.e., reporting or organizing hypoxic marker pimonidazole and apoptoic marker cleaved cas- data, constructing databases): L. Zhang, P. Marrano, B. Wu, S. Baruchel pase-3. As expected, in evofosfamide-treated tumors, apoptotic Study supervision: L. Zhang, S. Baruchel The costs of publication of this article were defrayed in part by the payment of cells were mostly confined to hypoxic zones while those in page charges. This article must therefore be hereby marked advertisement in topotecan-treated tumors were located in normoxic areas. Homo- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. geneous population of tumor cells undergoing apoptosis was observed in both normoxic and hypoxic regions, which supported the mechanism of synergistic efficacy of the combination therapy Grant Support in vivo. In a previous study, the accessibility of topotecan, doxo- This work was supported by Threshold Pharmaceuticals, Merck-Serono, rubicin, and mitoxantrone was found to decrease with increasing James Birrell Neuroblastoma Research Fund and CJ Memorial Fund/Sick Kids distance from functional blood vessels in breast cancer xenografts Foundation. The costs of publication of this article were defrayed in part by the paymentof (48). Clearly, poor drug penetration into solid tumors limits drug fi page charges. This article must therefore be hereby marked advertisement in ef cacy. In our study, we achieved tumor cytotoxicity in both accordance with 18 U.S.C. Section 1734 solely to indicate this fact. normoxic and hypoxic regions by combining evofosfamide with topotecan, which explains the superior efficacy of our combina- Received July 31, 2015; revised December 4, 2015; accepted December 17, tion regimen in all tumor models. 2015; published OnlineFirst December 30, 2015.

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Combined Antitumor Therapy with Metronomic Topotecan and Hypoxia-Activated Prodrug, Evofosfamide, in Neuroblastoma and Rhabdomyosarcoma Preclinical Models

Libo Zhang, Paula Marrano, Bing Wu, et al.

Clin Cancer Res Published OnlineFirst December 30, 2015.

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