libo zhang †, % baruchel · 30/12/2015 · when exposed to hypoxia overnight, a two to 65-fold...

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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 *,†, Sushil Kumar *, †, Paul Thorner †, % , Sylvain

Baruchel *,‡,§

*New Agent and Innovative Therapy Program, †Department of Paediatric Laboratory Medicine, ‡

Division of Hematology and Oncology, Department of Paediatrics, The Hospital for Sick

Children, Toronto, Canada; %Department of Laboratory Medicine and Pathobiology, University

of Toronto; § Institute of Medical Sciences, University of Toronto, Toronto, Canada

Running title: Preclinical study of evofosfamide and topotecan

Key words: neuroblastoma, rhabdomyosarcoma, tumor models, preclinical study, hypoxia

Address all correspondence to: Sylvain Baruchel, New Agent and Innovative Therapy Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada

M5G 1X8. Phone: 416-813-5977; Fax: 416-813-5327; E-mail: [email protected]

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed by the authors

on April 3, 2021. © 2015 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 30, 2015; DOI: 10.1158/1078-0432.CCR-15-1853

http://clincancerres.aacrjournals.org/

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Translational Relevance

Tumor hypoxia is known to correlate with increased malignancy, metastatic potential, drug

resistance and poor patient prognosis in a number of tumor types. Targeting tumor hypoxia with

hypoxia-activated prodrugs (HAP), such as evofosfamide, may improve cancer therapy. So far,

the effect of evofosfamide has been studied in combination with cytotoxic agents in adult

cancers, whereas the impact of evofosfamide on pediatric cancers has not been investigated. This

study focuses on the preclinical evaluation of efficacy of evofosfamide and its combination with

topotecan, a topoisomerase I inhibitor widely used in recurrent neuroblastoma and

rhabdomyosarcoma. We observed that targeting tumor hypoxia with HAPs improves antitumor

effects of topotecan in our tumor models, which provides a new therapeutic approach for the

treatment of pediatric solid tumors. All data gathered from this study will help us build the

rationale for future phase 1 clinical trials in treating patients with relapsed/ refractory

neuroblastoma and rhabdomyosarcoma.




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Abstract:

Purpose: Tumor cells residing in tumor hypoxic zones are a major cause of drug resistance and

tumor relapse. In this study, we investigated the efficacy of evofosfamide, a hypoxia-activated

prodrug, and its combination with topotecan in neuroblastoma (NBL) and rhabdomyosarcoma

(RMS) preclinical models.

Experimental Design: NBL and RMS cells were tested in vitro to assess the effect of

evofosfamide on cell proliferation, both as a single agent and in combination with topotecan. In

vivo antitumor activity was evaluated in different xenograft models. Animal survival was studied

with the NBL metastatic tumor model.

Results: All tested cell lines showed response to evofosfamide under normoxic condition, but

when exposed to hypoxia overnight, a two to 65-fold decrease of IC50 was observed. Adding

topotecan to the evofosfamide treatment significantly increased cytotoxicity in vitro. In NBL

xenograft models, single-agent evofosfamide treatment delayed tumor growth. Complete tumor

regression was observed in the combined topotecan / evofosfamide treatment group after two-

week treatment. Combined treatment also improved survival in a NBL metastatic model,

compared to single-agent treatments. In RMS xenograft models, combined treatment was more

effective than single agents. We also showed that evofosfamide mostly targeted tumor cells

within hypoxic regions while topotecan was more effective to tumor cells in normoxic regions.

Combined treatment induced tumor cell apoptosis in both normoxic and hypoxic regions.

Conclusions: Evofosfamide shows antitumor effects in NBL and RMS xenografts. Compared to

single-agent, evofosfamide / topotecan, combined therapy improves tumor response, delays

tumor relapse and enhances animal survival in preclinical tumor models.




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Introduction:

Neuroblastoma (NBL) is the most common extra-cranial childhood malignancy, responsible for

15% of all childhood cancer deaths (1). Despite intensive treatment protocols including

megatherapy with hematopoietic stem cell transplantation and immunotherapy, 3-year disease-

free survival is only about 50% for metastatic disease compared to 95% for localized tumors (2).

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and the third most

common extracranial solid tumor in children, with an annual incidence of 4-7 cases per million

children under the age of 16 years (3). Prognosis of metastatic RMS remains poor despite

aggressive therapies.

There is strong evidence that tumor cells residing in the tumor hypoxic regions cause drug

resistance and tumor relapse (4). Tumor hypoxic zones, which occur in tumors due to non-

uniform and inefficient vasculature, hinder the accessibility of chemotherapeutics to tumor cells.

Hypoxia can be a direct cause of therapeutic resistance due to limited blood supply and drug

distribution (5). Hypoxia also inhibits tumor cell proliferation and induces cell cycle arrest,

further enhancing chemo-resistance through regulating drug transporters, anti-apoptotic proteins

and pro-angiogenic factors (6). Our previous studies have shown that tumor hypoxic

microenvironment may serve as a niche for the highly tumorigenic fraction of tumor cells which

exhibit the cancer stem cell features in a diverse group of solid tumor cell lines, including

neuroblastoma, rhabdomyosarcoma, and small-cell lung carcinoma (7). We also found that

hypoxia induces drug resistance to etoposide, melphalan, doxorubicin, and cyclophosphamide in

neuroblastoma cells, which is mediated through an expanded autocrine loop between VEGF/Flt1

and HIF-1 alpha expression (8). There is evidence that some cells in hypoxic regions remain

alive in a dormant state for prolonged duration and become drug resistant since anticancer drugs




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preferentially target rapidly proliferating cells (6). Hypoxia-inducible factors also induce stem

cell phenotype and suppress differentiation of NBL cells (9). In RMS, HIF-1 alpha, induced by

hypoxia, upregulates anti-apoptotic proteins and glycolytic enzymes (10). HIF-1 alpha

inactivation by the inhibition of PI3K/Akt pathway is reported to sensitize tumor cells to

apoptosis in RMS (11). Moreover, HIF-1 alpha stabilization as the result of hypoxia increases

VEGF expression, increases glycolysis and resistance to chemotherapy (12).

Evofosfamide (previously known as TH-302), a hypoxia activated prodrug (HAP) has been

designed to penetrate to hypoxic regions of tumors. Evofosfamide is reduced at the

nitroimadazole site of the prodrug by intracellular reductases and when exposed to hypoxic

conditions, leads to the release of the alkylating agent bromo-isophosphoramide mustard (Br-

IPM). Br-IPM can then act as a DNA crosslinking agent at the tumor hypoxic region and may

diffuse to adjacent normoxic regions via a bystander effect (13). Consequently, evofosfamide can

target the malignant cells residing in hypoxic zones, while having little or no cytotoxic effect on

the normal cells (14, 15). The anti-tumor efficacy of evofosfamide correlated with hypoxic

fractions in tumor xenografts of lung cancer, melanoma, pancreatic cancer, renal cell carcinoma

and hepatocellular carcinoma. In adult soft tissue sarcoma Phase-II trial, evofosfamide in

combination with doxorubicin demonstrated 2% complete response (CR) and 34% partial

response (PR) (16). As a single maintenance therapy, it had limited hematologic toxicity and

cardiotoxicity, whereas with doxorubicin, it caused manageable hematological toxicity without

aggravating cardiotoxicity. A phase-III study in advanced and metastatic adult soft tissue

sarcoma is ongoing (NCT01440088). To date, no studies investigating the efficacy of

evofosfamide in pediatric cancers have been conducted.




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Topotecan, a topoisomerase-I inhibitor has shown activity against both NBL and RMS as a

single agent (17). The combination of cyclophosphamide plus topotecan has been active in

rhabdomyosarcoma, neuroblastoma, and Ewing sarcoma patients with recurrent or refractory

disease who have not received topotecan previously (18-20). The combination of topotecan,

cyclophosphamide, and etoposide is tolerable and effective in relapsed and newly diagnosed

neuroblastoma in phase II clinical trials and upfront phase III clinical trials (18, 21). Single agent

topotecan has also been found to be antiangiogenic in preclinical models of pediatric cancers (22,

23). Despite initial response, tumors eventually acquire resistance to topotecan. Mechanisms of

resistance include mutations in topoisomerase-I and involvement of transporters like BCRP, P-

glycoprotein and Multidrug Resistance Associated Protein-Type IV (24-26).

In this study, we selected the metronomic dose of topotecan for in vivo studies. Low Metronomic

Dose (LDM) chemotherapy usually refers to administration of low dose of cytototoxic agent at

close intervals without drug free breaks. LDM chemotherapy has lower acute toxicity due to

lower exposure of the cytotoxic agents. It has been shown active in diverse tumor types,

including metastatic disease, especially when combined with antiangiogenic drugs (27-30) . The

availability of the oral topotecan, along with our previous studies combining metronomic

topotecan with pazopanib in NBL preclinical models (23), suggest that oral metronomic

topotecan may be an ideal candidate for pediatric solid tumors. Considering the limited effects

on hypoxic tumors with conventional chemotherapy, hypoxia-targeting cytotoxic agents along

with topotecan therapy may achieve greater antitumor activities. Therefore, in this study, we

investigated the effect of combined therapy with topotecan and evofosfamide in NBL and RMS

preclinical tumor models.




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Materials and Methods

Drugs and reagents

Evofosfamide was provided by Threshold Pharmaceuticals Inc. Topotecan HCl was obtained

from Selleck (S1231). Stock solutions were prepared in DMSO and aliquots were stored at

−80°C. They were further diluted with culture media or saline for in vitro and in vivo studies,

respectively.

Cells and Cell Culture

RH4, RH30, and RD rhabdomyosarcoma cells (31) were gifts from Dr. David Malkin (The

Hospital for Sick Children, Toronto). LAN-5 (32), SK-N-BE(2) (33), BE(2)C (34), and SH-

SY5Y (34) neuroblastoma cells were kindly provided by Dr. Herman Yeger (The Hospital for

Sick Children, Toronto). CHLA-15, CHLA-20 and CHLA-90 (35) were obtained from the

Children's Oncology Group (COG) Cell Culture and Xenograft Repository under a signed and

approved Material Transfer Agreement. Cell line authentication was performed using short

tandem repeats (STR) DNA profiling (Promega’s GenePrint® 10 System)(36) conducted by the

Genetic Analysis Facility at the Centre for Applied Genomics of The Hospital for Sick Children

(Toronto, Canada). The DNA (STR) profile for all cell lines match to the profile listed in the

Children's Oncology Group (COG) STR Database (http://strdb.cogcell.org). RH4, RH30 and RD

rhabdomyosarcoma cells were cultured in DMEM supplemented with 10% FBS. CHLA-15,

CHLA-20 and CHLA-90 neuroblastoma cells were cultured in Iscove’s modified Dulbecco’s

medium supplemented with 3 mM l-glutamine, insulin, and transferin 5 μg/ml each and 5 ng/ml

selenous acid (ITS Culture Supplement; Collaborative Biomedical Products, Bedford, MA) and




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20% fetal bovine serum (FBS, complete medium). SK-N-BE(2) and SH-SY5Y neuroblastoma

cells were cultured in AMEM with 10% FBS.

Test Animals

Non-obese diabetic-severe combined immunodeficiency (NOD/SCID) mice (Jackson laboratory,

Maine) were used for xenograft mouse models at 4 weeks of age. The mice were housed in an

isolated sterile containment facility. All animal studies were approved by Animal Care Committee

at the Hospital for Sick Children (Animal Use Protocol#1000019356). During the study the mice

were observed daily for possible adverse effects due to treatments. Morbidity signs of ill health,

such as ruffled/thinning fur, abnormal behaviors or local erosion from the tumor, were noted and

experiments terminated as indicated.

Cell Viability Assay

Cell viability was assessed by Alamar Blue assay as previously described (37). Alamar Blue cell

proliferation assay is based on a reducing environment that indicates metabolically active 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, cells were exposed

to increasing concentrations of evofosfamide and topotecan either as single agents or combined

treatment for 72 hours. Alamar Blue was added to a final concentration of 5%, incubated for 4

hours at 37oC and quantitated on a plate reader at 530/590 nm. IC50 was determined by

GraphPad Prism software.

We also evaluated drug efficacy under the condition of hypoxia. To simulate tumor hypoxia in

vitro, after drug addition, the plates were incubated for 2 hours at 37 °C in an anaerobic chamber.

The anaerobic chamber was evacuated and gassed with the hypoxic gas mixture (94% N2/5%




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CO2/1% O2) to create a hypoxic environment. After 2 hours or overnight exposure to hypoxic

condition, cells were removed from the hypoxia chamber and further incubated for 3 days in

standard tissue-culture incubator. Cell viability was assessed by Alamar Blue assay as described

above.

Mouse Xenograft Models

Two NBL cell lines (CHLA-20 and SK-N-BE(2)) and two RMS cell lines (RH4 and RD) were

used to establish murine models (38). Briefly, tumor cells were washed three times with HBSS

before injection. Cell suspensions were mixed 1:1 with Matrigel (BD Bioscience). Subcutaneous

xenografts were developed by injecting 1 x 106 tumor cells into the subcutaneous fat pad of

NOD/SCID mice. Treatments commenced when the tumor sizes reached about 0.25cm3.

Animals were randomized into four groups with 10 animals in each group: control;

evofosfamide, topotecan as a single agent, and the combination of evofosfamide and topotecan.

The doses of evofosfamide and topotecan were 50 mg/kg (qd × 5/wk, IP) and 1 mg/kg (qd ×

5/wk by oral gavage), respectively. The injection or gavage volume was 200 µL. Control mice

received the same volume of the vehicle (saline). Tumor growth was measured three times a

week in two dimensions, using a digital caliper. Tumor volume was calculated as width2 × length

×0.5. When tumor volume reached 1.5cm3, mice were sacrificed, and tumors were dissected and

weighed. Tumor growth curves were plotted with the relative tumor volumes at different time

points. The relative tumor volume of each tumor is defined as the tumor volume divided by its

initial volume. Potential drug toxicity was assessed in tumor-bearing mice by monitoring animal

body weight, appetite, diarrhea, signs of animal distress, or any marked neurologic symptoms.

Animal Survival Study




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SK-N-BE(2) cells were prepared and injected i.v. into the lateral tail vein (26-gauge needle, 1 x

106 cells in 100µl total volume). Mice were randomized into four groups: control, evofosfamide,

topotecan and the combination of evofosfamide and topotecan, with 8 mice in each group. 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 stress. Tumor-

bearing mice were euthanized at the endpoint when there were signs of distress, including 20%

of body weight loss, fur ruffling, rapid respiratory rate, hunched posture, reduced activity, and

progressive ascites formation.

Immunohistochemistry

Double immunofluorescence staining was performed on 5 μm thick frozen sections. Tissue

sections were fixed for 10 minutes in acetone and allow to air dry for 5 minutes. Endogenous

biotin, biotin receptors and avidin sites were block with the Avidin/Biotin Blocking Kit (SP-

2001, Vector Laboratories, Burlingame, CA). The tissue sections were incubated with rabbit

anti-cleaved caspase-3 antibody (1:50; #9661, Cell Signalling, Beverly, MA) or rabbit anti-Ki67

antibody (1:200; ab16667, Abcam, Toronto, Canada) for 1 hour at room temperature. Detection

of the rabbit antibodies was performed by incubation with Texas Red goat anti-rabbit IgG

antibody (1:200; TI-1000, Vector Laboratories,) for 30 minutes. Following the washing of the

tissue sections the Mouse IgG1 anti-pimonidazole monoclonal antibody clone 4.3.11.3 (1:50;

Hypoxyprobe, Inc., Burlington, MA) was added overnight at 4°C. This antibody was detected

with Fluorescein horse anti-mouse IgG Antibody (1:200; #FI-2000, Vector Laboratories) for 30

minutes. The tissue sections were washed and then mounted with Vectashield mounting medium

with DAPI (H-1200, Vector Laboratories).

Statistical Analysis




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Data from different experiments were presented as mean ± SD. For statistical analysis, Student's

t test for independent means was used. A P value of

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All these cell lines were exposed to increased concentrations of evofosfamide in vitro for 72

hours. Under normoxic condition, all the tested lines responded to evofosfamide treatment in a

dose-dependent manner, with IC50 values ranging from 4.6 to 151µM. When exposing NBL and

RMS cells under hypoxic conditions (1% O2) for 2 hours, no significant IC50 change was

documented. However, when tumor cells were exposed to hypoxia (1% O2) overnight, there was

a two to 65-fold decrease of evofosfamide IC50 (Fig. 1A, 1B, Tab 1) with the IC50 values

ranging from 0.07 to 21.9µM.

Improved anti-proliferation effects of evofosfamide when combined with topotecan in vitro

In our previous studies, we showed that maximal plasma concentration (Cmax) of topotecan was

19.8 ng/ml with the metronomic oral dose of 1 mg/kg drug administration in NOD/SCID mouse

model (23). In patients, Cmax of topotecan is 10.6 ± 4.4 ng/ ml with the oral dose of 2.3 mg /m2

/day. Since the terminal half-life of oral topotecan (3.9 ± 1.0 h) (40) is significantly longer than

evofosfamide (0.6 ± 0.1 h) (41), a constant concentration of topotecan (20 nM= 8.4 ng/ml) was

selected for in vitro combined treatment.

All selected tumor cell lines were treated in vitro with increasing concentrations of evofosfamide

with or without the presence of topotecan (20 nM). The dose response curves were generated by

Graphpad software and IC50 values were compared between IC50 concentrations of

evofosfamide when tested alone and in combination with 20nM topotecan in neuroblastoma and

rhabdomyosarcoma cell lines.As shown in Tables 1 and 2, with the presence of topotecan,

evofosfamide induced cytotoxicity was significantly enhanced under overnight hypoxia as

indicated by decreased IC50s in most tested tumor cell lines.




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To understand whether the combination of the drugs was additive or synergistic, a Bliss analysis

was performed with MacSynergy II software with 95% confidence limits. The effect of the

combination was additive in all cell lines.

Topotecan and evofosfamide delay tumor growth and enhance animal survival in NBL model

After verifying in vitro activity of evofosfamide and topotecan, the in vivo effectiveness of

topotecan / evofosfamide as single-agents or combine therapy was evaluated in CHLA-20 and

SK-N-BE(2) xenograft models. Since both CHLA-20 and SK-N-BE(2) cell lines are derived

from samples of patients who relapsed after chemotherapy, we use them to develop aggressive

tumor models for rigorous screening of anti-cancer agents. All treatments commenced when the

tumor sizes reached 0.25cm3. In both NBL models, after 2 weeks of treatment, tumor growth

delay was observed with evofosfamide or topotecan as monotherapies (Fig. 2A). In SK-N-BE(2)

xenograft model, combine regimen induced complete tumor regression, while topotecan induced

partial tumor regression. When we compared the tumor sizes between these two arms, the

difference is statistically significant (p

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Treatment was well tolerated, with weight loss >10% observed in single-agent and combined

treatment groups within the first 8 days but returned to baseline thereafter. No animal death was

observed in any of the groups during the experimental period (Fig. 2C).

In order to determine the antitumor activities of evofosfamide and topotecan on late-stage

metastatic disease, we further assess animal survival in the SK-N-BE(2) intravenous metastatic

model. Fourteen days after tumor cell inoculation, we initiated drug treatment with evofosfamide

and topotecan as single agents or combined treatment. As shown in Fig. 2D, evofosfamide or

topotecan treatment showed a substantial increase in animal survival compared to the control

mice with a median survival of 22.5 days for control group, 25 days for evofosfamide group and

36 days for topotecan group. However, treatment with a combination of evofosfamide and

topotecan had the greatest impact on animal survival (p < 0.01) with a median survival of 46

days.

Topotecan and evofosfamide delays tumor growth in RMS models

To test the antitumor activity of evofosfamide and topotecan in rhabdomyosarcoma (RMS), we

chose an alveolar RMS cell line (RH4) and an embryonal RMS cell line (RD) to generate

xenograft models. Tumor bearing NOD/SCID mice were randomized to therapy with

evofosfamide, topotecan, or combined therapy. In both RH4 and RD xenograft models, both

evofosfamide and topotecan monotherapy significantly delayed tumor growth with 2 weeks of

treatment (Fig. 3). Compared to NBL models, single-agent evofosfamide was more effective in

RMS models with partial tumor regression in all treated xenografts. The combination of

topotecan and evofosfamide were superior to the respective drugs given alone at different time

points from day 26 to day 30 (P < 0.01; Fig. 3). There was no significant drug-related toxicity in

any of the treatment arms.




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Colocalization of cell apoptosis and tumor hypoxia with evofosfamide and topotecan treatment

As combine treatment with evofosfamide and topotecan significantly improved anti-tumor

activity both in vitro and in vivo, we have particular interest to examine its potential mechanism

of action in vivo. To this end, tumor cell apoptosis was assessed by cleaved caspase-3

immunoreactivity analysis in our xenograft models. On day 5 of drug treatment, RH4 xenografts

from different regimens were harvested for immunohistochemical analysis (Fig. 4A). Hypoxic

regions were identified as the area of pimonidazole positive staining. Apoptotic cells were

detected by anti-cleaved caspase-3 antibody. Apoptosis was quantified as the percentage of

positive cleaved caspase-3 staining area using ImageJ software (Fig. 4B). As shown in Fig. 4A

and 4B, evofosfamide induced tumor cell apoptosis in hypoxic regions, with a remarkably higher

percentage of cleaved caspase-3 positive cells in hypoxic regions compared to normoxic regions

(Student's t test, 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 was observed throughout the tumor, in both normoxic and hypoxic areas.

Decreased cell proliferation with evofosfamide and topotecan combined treatment in vivo

We further evaluated tumor cell proliferation in different treatment groups by immunostaining of

the proliferation marker, Ki67. As shown in Fig. 4C and 4D, the Ki67+ proliferating tumor cell

population was mainly located in normoxic regions. After the 4-day in vivo treatment, single-

agent topotecan showed a limited effect on cell proliferation as measured by Ki67 positivity.

Surprisingly, decreased cell proliferation was observed in evofosfamide-treated tumors in

normoxic regions with significantly lower percentage of Ki67 positive cells than control tumors

(p < 0.05), suggesting a hypoxia-independent bystander effect. Combined evofosfamide and

topotecan treatment led to a further decrease in the Ki67+proliferating tumor cell population




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compared to single-agent evofosfamide group (combined group vs. single-agent topotecan, p <

0.01; combined group vs. single-agent evofosfamide, p < 0.05).




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Discussion

Topotecan has demonstrated efficacy in preclinical and clinical studies in neuroblastoma and

rhabdomyosarcoma. However, total eradication of highly tumorigenic populations of tumor cells

is required to achieve relapse-free survival in cancer patients. Incomplete distribution of

chemotherapeutics to poorly perfused areas of primary tumors and metastatic sites is a major

hindrance in complete eradication of these tumor cells. These sparsely vascularized areas are

characterized by hypoxia which confers drug and radiation resistance, and a pro-angiogenic and

invasive phenotype to tumor cells. Despite the advent of novel chemotherapies, complete

eradication of tumor cells cannot be accomplished until hypoxic tumor cells are targeted.

Therefore, targeting hypoxic zone of solid tumor may reverse drug resistance and make cytotoxic

agent more effective. In this study, we are proposing new strategies to improve the efficacy of

chemotherapy by incorporating a novel agent targeting tumor hypoxic regions.

Hypoxia activated prodrugs (HAP), which deliver the cytotoxic payload in hypoxic zones, have

been developed to solve this issue. PR-104, an earlier HAP, was found to have a steep dose-

response relationship in a panel of pediatric tumor xenograft models, which raises safety

concerns in pediatric patients (42). Evofosfamide, however, has demonstrated survival benefit in

various adult cancers without causing prohibitively additive toxicity with conventional

chemotherapy in clinical trials (16). Evofosfamide shares the similar active metabolites as

ifofosfamide, but it is preferentially activated in hypoxic tissues and does not have the same toxic

metabolites as ifosfamide. It is anticipated that evofosfamide should have less hematologic, renal

and CNS toxicity than ifosfamide. There is no direct clinical studies comparing evofosfamide

with ifosfamide, but evofosfamide shows superior efficacy and less toxicity than ifosfamide in

preclinical lung carcinoma models (43). In our xenograft models, we observed tumor growth




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inhibition with single agent evofosfamide, but no significant antitumor effect was achieved in the

CHLA-20 model with ifosfamide (50 mg/kg; qd × 5/wk, IP) (Supplementary Figure 1). In

clinical trials (NCT02047500), evofosfamide is administered at a dose ranging from 170-

340 mg/m2. This could be converted to 32–64 mg/kg in the tumor bearing mice, calculated by the

pharmacokinetic data from the clinical studies and preclinical mouse studies (44). In this study,

we tested evofosfamide at the dose of 50mg/kg, as single agent and in combination with

topotecan in two of the most common pediatric extracranial solid tumors, NBL and RMS. To our

knowledge, this is the first study evaluating the effectiveness of evofosfamide in pediatric

tumors.

Our observation that single agent topotecan is more effective in delaying tumor growth in NBL

than in RMS is in agreement with our previous finding (23). From our observations in vitro,

evofosfamide showed more activity in neuroblastoma cell lines than in rhabdomyosarcoma cells.

However, in vivo, rhabdomyosarcoma xenografts were more sensitive to evofosfamide than

neuroblastoma xenografts. In this study, we can’t simply translate in vitro observation to in vivo

activity. In vitro, we simulated tumor hypoxia by incubating tumor cells inside the hypoxia

chamber. Therefore, 100% of cell population was under hypoxia and exposed to activated

evofosfamide. In vivo, the percentage of hypoxic tumor cells varies among different tumor types

(45, 46). In vivo tumor microenvironment is also more complex in which HAP activation would

be affected by many other factors, including the duration and the intensity of oxidative stress,

tumor angiogenesis, drug penetration in different solid tumor types, and acquired drug resistance

under prolonged hypoxic conditions. Although it was beyond the scope of this study to examine

the tumor microenvironment among different tumor types, it remains an important area for our

future investigation.




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From our study, evofosfamide showed enhanced cytotoxicity under overnight and 3-day

(Supplementary Figure 2) prolonged hypoxia compared to short-term (2-hour) hypoxia

exposure. When we test the effect of evofosfamide and its combination with topotecan on five

neuroblastoma and three rhabdomyosarcoma cell lines under normoxia, short-term hypoxia and

prolonged hypoxia, it is also under overnight hypoxia (1% O2) that adding topotecan

significantly lowered IC50 of evofosfamide in most tumor cell line, except SK-N-BE(2).

However, in the SK-N-BE(2) xenograft model, combined therapy achieved significant antitumor

effects in vivo. Combined treatment induced complete tumor regression in all treated animals,

which was superior to evofosfamide or topotecan single-agent treatment. This is likely because

SK-N-BE(2) xenografts have more severe and more chronic hypoxic environment than in vitro

cell culture (1% O2, overnight), which could more effectively activate evofosfamide to exert its

cytotoxic activity in vivo.

In all the NBL and RMS xenograft models tested, combined treatment showed superior

antitumor effects compared to single-agent treatments, and induced tumor regression after a 2-

week treatment period. The advantage of the combination regimen was also approved in the

metastatic NBL model. Our experimental metastatic model has been developed to simulate

residual disease in neuroblastoma. Residual Disease refers to disseminated tumor cells which

survive after therapy (47). More than 50% of children with high-risk NBL develop recurrence

due to the presence of minimal residual disease. In our metastatic model derived from a

disseminated MYCN-amplified cell line, mice treated with combined topotecan and

evofosfamide had extended survival compared to single-agent topotecan treatment. This result is

significant in promoting new therapies for NBL minimal residual disease or refractory NBL by




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combining HAPs with existing chemotherapeutic drugs. We believe that incorporating HAPs,

such as evofosfamide, could potentially eradicate the tumor cells hiding in hypoxic zones which

are largely responsible for eventual relapse and metastasis. Eventually, this new regimen might

prolong Progression Free Survival in high-risk NBL patients.

In this study, we were able to assess the correlation between tumor hypoxia and tumor cell

apoptosis with immunostaining of hypoxic marker pimonidazole and apoptoic marker cleaved

caspase-3. As expected, in evofosfamide-treated tumors, apoptotic cells were mostly confined to

hypoxic zones while those in topotecan-treated tumors were located in normoxic areas.

Homogeneous 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 in vivo. In a previous study, the accessibility of topotecan, doxorubicin and mitoxantrone

was found to decrease with increasing distance from functional blood vessels in breast cancer

xenografts (48). Clearly, poor drug penetration into solid tumors limits drug efficacy. In our

study, we achieved tumor cytotoxicity in both normoxic and hypoxic regions by combining

evofosfamide with topotecan, which explains the superior efficacy of our combination regimen

in all tumor models.

An ideal HAP requires selective activation in hypoxic regions as well as good penetration of

multiple cell layers to exert a local cytotoxic bystander effect. Using a multicellular tumor

spheroid and multicellular layer (MCL) co-culture model systems, Meng (49) et al. showed that

evofosfamide has penetrability and bystander effect. In our study, with evofosfamide single-

agent treatment, we observed decreased cell proliferation even at normoxic regions. This

phenomenon confirms that once activated in hypoxic tissues, evofosfamide releases Br-IPM




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which can diffuse into surrounding oxygenated regions of the tumor and kill cells in those

regions via a “bystander effect”.

In summary, topotecan and evofosfamide demonstrated antitumor activities in our preclinical

models of various sub-types of neuroblastoma and rhabdomyosarcoma. Compared to single

agents, combination treatment induces more tumor regression, delays tumor relapse, and

enhances animal survival in our preclinical tumor models. In this study, we explored the

mechanism of action with combined evofosfamide/topotecan therapy. We observed increased

reactive oxygen species (ROS) under hypoxic condition (1%O2, overnight) (Supplementary

Figure 3). We also found that adding topotecan enhanced the oxidative stress in

rhabdomyosarcoma cells under hypoxic condition (Supplementary Figure 4). In vivo,

evofosfamide showed significant cytotoxicity against hypoxic tumor cells, while topotecan plays

complementary roles by targeting tumor cells residing at normoxic regions. The ability of this

combination to target cells in both normoxic and hypoxic tumor zones, as demonstrated by

immunofluorescence, provides a proof of principle for its superior efficacy. These preclinical

data support the development of a clinical trial in high-risk neuroblastoma and

rhabdomyosarcoma.

Acknowledgments This work was supported by Threshold Pharmaceuticals, Merck-Serono, James Birrell

Neuroblastoma Research Fund and CJ Memorial Fund / SickKids Foundation.




22

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25

Figure Legends:

Figure 1. Increased cytotoxicity of evofosfamide under prolonged hypoxic conditions with NBL

and RMS cells. A panel of 5 different NBL cell lines (CHLA-15, CHLA-20, CHLA-90, SK-N-

BE(2) and SH-SY5Y) and 3 rhabdomyosarcoma cell line (RH4, RH30 and RD) 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.

Figure 2. Anti-tumor effects of evofosfamide and topotecan in NBL xenograft models. A, A

total of 1 × 106 cells were implanted subcutaneously into SCID/SCID mice. Once tumors were

palpable (about 0.25cm3), mice were randomized into vehicle control (n=10) or three treatment

groups (n = 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; qd × 5/wk, IP) or

topotecan (1 mg/kg; qd × 5/wk, 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 ± s.e.m. 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 re-challenged the tumor bearing animals with

the original treatment dosage and schedule on day 55 (identified by shaded area between day 55




26

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 = 8/group) were injected with 1 × 106 SK-N-BE(2)

cells intravenously. 14 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.

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 0.25cm3), mice were randomized into vehicle control group (n=10)

or three treatment arms (n = 10 for each group). RH4 and RD xenografts were treated with

evofosfamide (50 mg/kg; qd × 5/wk, IP) or topotecan (1 mg/kg; qd × 5/wk, 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 ± s.e.m.




27

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. 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.




28

Table 1: Comparison of IC50 concentrations of evofosfamide when tested alone and in

combination with 20nM topotecan in neuroblastoma and rhabdomyosarcoma cell lines.

IC50 (µM) P Value Evofosfamide Evofosfamide+Topotecan

NBL cell lines: CHLA-15-normoxia 4.6 ± 5.8 3 ± 3.5 0.39 CHLA-15-1% O2; 2hr 9.5 ± 6.3 7.4 ± 4.1 0.57 CHLA-15-1% O2; O/N 0.07 ± 0.03 0.04 ± 0.01

29

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

IC50 (nM) p Value1 Normoxia 1% O2, 2hr 1% O2, O/N

NBL cell lines: CHLA-15 17.6 ± 3.9 15.6 ± 0.9 13.7 ± 1.4 NS2

CHLA-20 12.9 ± 1.2 12.4 ± 1.0 14.1 ± 0.6 NS CHLA-90 4170 ± 1030 4550 ± 1590 5260 ± 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

RMS cell lines: RH4 132 ± 10.0 110 ± 21.8 119 ± 3.7 NS

RH30 2251 ± 685 1963 ± 416 1621 ± 174 NS RD 387 ± 55.8 250 ± 87.5 317 ± 22.9 NS

1 p value from one-way ANOVA.

2 NS: not significant, p>0.05




Fig. 1

B

A




Fig. 2

B C

A

D

P

Fig. 3

** **




Fig. 4

Control

Evofosfamide

Topotecan

Evofosfamide + Topotecan

DAPI Anti-cleaved

caspase-3 Anti-

pimonidazole Superimpose

B

A

**

*




Control

Evofosfamide

Topotecan

Evofosfamide + Topotecan

Anti-Ki67 Anti-pimonidazole Superimpose

Fig. 4

D

C

**

**

*

*




Published OnlineFirst December 30, 2015.Clin Cancer Res Libo Zhang, Paula Marrano, Bing Wu, et al. rhabdomyosarcoma preclinical models

andhypoxia-activated prodrug, evofosfamide, in neuroblastoma Combined antitumor therapy with metronomic topotecan and

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