Darinaparsin Inhibits Prostate Tumor–Initiating Cells and Du145

Published OnlineFirst November 7, 2014; DOI: 10.1158/1535-7163.MCT-13-1040

Small Molecule Therapeutics Molecular Cancer Therapeutics Darinaparsin Inhibits Prostate Tumor–Initiating Cells and Du145 Xenografts and Is an Inhibitor of Hedgehog Signaling Nitu Bansal1, Nadine Johnson Farley1, Lisa Wu1, Jonathan Lewis2, Hagop Youssouﬁan2, and Joseph R. Bertino1

Abstract

Prostate cancer is the leading cause of cancer-related death in also inhibits growth of the castrate-resistant Du145 prostate men in the United States. A major cause of drug resistance in tumor propagated as xenograft in mice and inhibits the tumor- prostate and other epithelial tumors may be due to the presence of initiating potential of prostate cancer cells. Although the mech- a fraction of tumor cells that retain the ability to initiate tumors anism by which darinaparsin acts is not completely known, we and hence are termed tumor-initiating cells (TIC) or cancer stem show that it kills prostate cancer cells by blocking cells in the cells. Here, we report that darinaparsin, an organic derivative of G2–M phase of the cell cycle and inhibits Hedgehog signaling by arsenic trioxide, is cytotoxic to prostate cancer cell lines as well as downregulating Gli-2 transcriptional activity. These data provide fresh prostate cancer cells from patients at low micromolar con- a rationale for evaluating darinaparsin in patients with castrate- centrations, and importantly inhibits the TIC subpopulations. It resistant prostate cancer. Mol Cancer Ther; 14(1); 23–30. 2014 AACR.

Introduction weeks was well tolerated (6). Phase II studies in both hematologic malignancies and solid cancers are currently under way Arsenic derivatives have been therapeutically used for more (7, 8). In vitro studies showed that darinaparsin more potently than 2,000 years. In the early 20th century, use of arsenic induces growth arrest, apoptosis, and oxidative stress than ATO trioxide(ATO)intreatingleukemiawasﬁrst reported, and by in several hematologic malignancies (8). Also, unlike ATO, it the mid-20th century, its effectiveness in patients with relapsed does not increase bcl-2 protein levels. Importantly, darinapar- acute promyelocytic leukemia (APL) was demonstrated (1). A sin is effective in ATO-resistant leukemic cell lines that over- randomized clinical trial in United States led to FDA approval express multidrug-resistant protein 1/ATP-binding cassette, of ATO for relapsed or refractory APL (1). Recently, the com- subfamily C, member1 (MRP1/ABCC1; refs. 3, 9). Darinaparsin bination of ATO and all trans-retinoic acid has been recom- showed increased antiangiogenic activity in both in vitro human mended as ﬁrst-line treatment of APL (2). ATO has been umbilical vascular endothelial cell microtubule formation and investigated in the treatment of other non-APL cancers; how- in vivo Matrigel plug models (10). Recent investigations have ever, it was less effective at clinically relevant doses and was also shown that darinaparsin is a multivalent molecule that can highly toxic at higher concentrations (3). Therefore, other induce an incomplete stress response by disrupting microtu- arsenicals with antitumor activity and with less toxicity and bules and sonic hedgehog (Shh) signaling (11). oral availability have been sought. Darinaparsin is an organic In this study, we examined the cytotoxic effects of darinaparsin arsenic (S-dimethylarsino-glutathione; Z-101) made by conju- in both established prostate cancer cell lines and in fresh prostate gating dimethylarsenic to glutathione (4, 5). Screening of the cancer cells from patients. Here, we show that darinaparsin is a NCI-60 panel of cells indicated that Ic-50 concentrations with potent cytotoxic against various human prostate cancer cell lines darinaparsin ranged from 0.02 to 7.3 mmol/L. Mouse toxicity as well as primary prostate cancer cells and is effective in inhibiting studies showed that the LD of darinaparsin was approximate- 50 prostate tumor–initiating cells (TIC). Studies with the Du145 cell ly 50-fold higher than that of ATO. Phase I studies with line also showed synergistic cell kill with taxotere. Du145 tumors darinaparsin in patients with advanced refractory solid tumors propagated in nude mice were also sensitive to darinaparsin. showed that 300 mg/m2 i.v. for 5 consecutive days every 4 Prompted by studies showing that ATO inhibited Hedgehog signaling (12), we show that darinaparsin also inhibits Hedgehog 1Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey. signaling in prostate cancer by downregulating Gli-2 transcrip- 2Ziopharm Oncology Inc., New York, New York. tional activity. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Materials and Methods Corresponding Author: Joseph R. Bertino, Rutgers Cancer Institute of New Reagents Jersey, Room 3033, 185 Little Albany Street, New Brunswick, NJ 08903. Phone: Collagen I (rat tail collagen) was purchased from BD Bios- 732-235-8510; Fax: 732-235-8181; E-mail: [email protected] ciences, darinaparsin/Z101 was obtained from Ziopharm doi: 10.1158/1535-7163.MCT-13-1040 Oncology Inc., NOD/SCIDg mice were purchased from The 2014 American Association for Cancer Research. Jackson Laboratory, Gli-2, GAPDH, and b-tubulin antibodies

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Bansal et al.

were purchased from Cell Signaling Technology, a2-FITC, CD44- tatectomy on an Institutional Review Board (IRB)–approved APC (BD Biosciences), SAG (Smoothened agonist), and ATO were protocol. To obtain single cells from fresh prostate tumors, the purchased from Sigma-Aldrich. Gli-2 luciferase reporter plasmid specimen was minced in small pieces and incubated in 200 mg/mL was obtained as a kind gift from P.A. Beachy (Stanford University of collagenase I in RPMI for 2 to 4 hours, the longer time for larger School of Medicine, Stanford, CA). size specimens. After 4 hours, the media containing the minced tissue was strained and the supernatant was collected and washed Cell culture twice in 1 PBS at 250 g for 30 seconds (this step eliminates the Du145, LnCap, PC3, and CWR22 cells were purchased from fibroblasts from the epithelial cells). The pellet was suspended in the ATCC in 2010. The ATCC uses isoenzymology method PROSTAlife medium (Life Science Technologies) and plated in a for species determination and short-tandem repeats methods flask. The cells were incubated at 37C for 3 to 4 days to allow the for identity verification of the cell line. The cells were also epithelial organoids to attach. The media was replaced and cells tested for Mycoplasma (http://www.atcc.org/~/media/PDFs/ passaged upon 70% confluency. The cells were stained with anti- Technical%20Bulletins/tb08.ashx). Upon receipt, the cells were EPCAM and anti-androgen receptor antibodies and analyzed with maintained at low passage numbers in RPMI (Gibco BRL) flow cytometry to confirm that the isolated cells were prostate supplemented with 10% fetal bovine serum and 1% penicil- epithelial cells. lin–streptomycin. The cells were routinely tested for Mycoplas- ma contamination in the laboratory using the Lonza Myco- Collagen attachment assay plasmaTestingKit.Noothermethodwasusedtoauthenticate For enrichment of prostate TICs from early-passage Du145 the cells. cells, a collagen I attachment assay was used to harvest 5-minute attached cells (13). These cells, enriched for TICs, were main- Isolation of primary prostate cells from prostate tumor tissues tained in keratinocyte serum-free medium (KSFM) supplemented Human primary prostate tumors were obtained from the with epidermal growth factor (EGF) and bovine pituitary extract Robert Wood Johnson Hospital (New Brunswick, NJ) after pros- (13). Briefly, tissue culture plates were coated with 70 mg collagen I

Figure 1. Prostate cancer cells are sensitive to Z101 (darinaparsin) and ATO in micromolar concentrations. A, prostate cancer cell lines LnCap, Du145, and PC3 were plated in 96-well plates. Twenty-four hours later, cells were treated with darinaparsin and ATO at different concentrations. Seventy-two hours after treatment, MTS reagent was added and color change was monitored at 490 nm. Data were analyzed using prism software. B, primary prostate tumor cells isolated from the prostate of 5 different patients with Gleason score 8–9 were also plated in 96-well plates and treated with darinaparsin (see Materials and Methods). After 72 hours, MTS reagent was added and color change was monitored at 490 nm. Data were analyzed using prism software.

24 Mol Cancer Ther; 14(1) January 2015 Molecular Cancer Therapeutics

Darinaparsin Kills Prostate Cancer Cells by Inhibiting Shh Signaling

A Du145 cells PC3 cells

*P = 0.03 140 *P = 0.004 120 *P = 0.002 *P = 0.06 *P = 0.007 120 100 *P = 0.28 100 80 80 60 60 40 40 20 20 0 0 Untreated ATO (5 µmol/L) DAR (2.5 µmol/L) DAR (5 µmol/L) Untreated ATO (5 µmol/L) DAR (2.5 µmol/L) DAR (5 µmol/L)

% Cells attached on collagen I in 5 min Drugs % Cells attached on collagen I in 5 min Drugs

BC*P = 0.01 *P = 0.013 80 *P = 0.012 *P = 0.14 50 *P = 0.013 45 70 40 60

cells 35 + 50 30 40

/CD44 25 hi 30

20 % a 2 b 1/CD44 15 20 10 10 % of a 2 b 1 5 0 0 Untreated Taxotere ATO Darinaparsin Untreated Darinaparsin Taxotere Drugs Drugs

Figure 2. Darinaparsin decreases the number of cells that attach on collagen I. A, Du145 and PC3 cells treated with darinaparsin and ATO at IC50 concentrations or higher for 72 hours were collected and counted. Equal numbers of untreated and treated cells were plated on collagen I–coated dishes for 5 minutes. Attached cells were counted and calculated as a percentage of total cells. The graph represents the average values of three independent experiments. Error bars, SEM; P 0.05 is considered statistically significant. B, darinaparsin decreases the percentage of a2b1hi/CD44hi cells in parental Du145 cells. C, a2b1hi/CD44hi cells grown as spheroids, treated with darinaparsin or taxotere for 72 hours were collected, washed and stained with a2-FITC and CD44-APC. The cells were acquired with flow cytometry for staining of a2-FITC and CD44-APC. The bar graph shows the cells positive for a2hi/CD44hi cells (the staining of cells is expressed in percentage). The experiments were repeated three independent times. Error bars, SEM. P 0.05 is considered statistically significant. and blocked with 1% BSA. Early-passage cells were allowed to Cytotoxicity assay attach on collagen I–coated dishes for 5 minutes. The 5-minute Du145, PC3, and LnCap prostate cancer cells and primary attached cells were collected after trypsinization (0.5% trypsin). prostate cells were plated at a density of 3,500 cells/200 mLin The unattached cells after 20 minutes were also collected and used 96-well plates. Twenty-four hours after plating, darinaparsin, as a control (non-TICs). taxotere and other drugs were added at various concentrations. For three-dimensional spheroid formation, 5-minute Seventy-two hours after treatment, the MTS reagent was added attached tumor cells enriched for TIC's and 20-minute nonat- (Promega) to the cells. Color change was monitored at 490 nm tached cells were suspended and plated in 1% agarose-coated 6- and data were acquired by SOFT max pro. Cell viability and IC50 well plates. Every 3 days, half of the media was supplemented concentrations were determined using PRISM software. For synergy with fresh KSFM. For drug treatment assays, drugs at specified experiment, CalcuSyn software was used to determine the combi- IC50 concentrations were added to the spheroids. After 48 or 72 nation index (CI) values between taxotere and darinaparsin (14). hours, spheroids were collected and dissociated and used for colony-forming assays. For colony formation assay from the Flow cytometry spheroids, 48 or 72 hours after drug treatment, floating cell Flow cytometry using FACS was used to determine cell-surface spheroids were collected, washed, and dissociated with 0.1% markers: a2-FITC and CD44-APC. Data were analyzed using the trypsin to form single cells. Equal numbers of single cells (25– CellQuest software. For cell-cycle analysis, the cells were collected 300 cells) were then plated in 6-well tissue culture plates. After at 72 hours, washed, and fixed in ethanol overnight. The fixed cells 14 days, colonies were stained with 0.25% crystal violet in 95% were then washed and stained with PI-RNase (BD Pharmigen) ethanol. Colonies consisting of more than 50 cells were and cells were acquired using flow cytometry. The data were counted. analyzed using MODFIT software.

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Bansal et al.

A Colonies formed in 14 days (n)

100 *P = 0.09 *P = 0.03 *P = 0.04 80 Figure 3. 60 Darinaparsin inhibits TICs. hi hi 40 A, a2b1 /CD44 cells selected from Du145 cells were grown as spheroids, 20 treated with darinaparsin for 72

Colonies (cells >40) hours; the spheroids were dissociated 0 and plated in 6-well dishes to form Untreated 1.5 mmol/L 3 mmol/L 6 mmol/L colonies. After 14 days, colonies were stained with crystal violet and Darinaparsin counted. Error bars, SEM. B, primary prostate cancer cells had decreased C Untreated hi B 8 levels of CD44 and a2b1 cells. Dar-treated Prostate cells isolated from four 7 70 Untreated different patient samples were 6 treated with darinaparsin for 72 hours 60 Dar-treated 5 (respective IC50 concentrations) and 50 hi

hi analyzed for the expression of a2b1 4 hi 40 cells and (C) CD44 and using ﬂow 30 % CD44 3 cytometry. Data were represented as

% a 2 b 1 20 2 percentage bar graph, normalized to IgG controls. 10 1 0 0 T38 T84 T64 T92 T38 T84 T64 T92 Tumor samples Tumor samples

Luciferase reporter assay lated using the formula width2 (length/2). For the study Du145 cells were plated at 100,000 cells/mL in 24-well plates. involving PC3 cells, 2 106 cells in 100 mL of PBS were injected Sixteen hours after plating, cells were cotransfected with 0.5 mg subcutaneously into the right flank of NOD/SCIDg mice. Once pcDNA-Gli2 and 0.5 mg 8xGli-luciferase reporter. After 8 hours of tumors were palpable, the mice were randomized into two groups transfection, cells were treated with a Shh agonist (10 nmol/L SAG (n ¼ 4), control and darinaparsin. Mice were treated with the same from Sigma-Aldrich) with or without darinaparsin for 24 hours. dosage and schedule as described above for Du145 cells. Results Luciferase assays were performed with the Promega Luciferase are presented as mean SEM. System. Assays were done in triplicates and the results were normalized to total protein levels. Statistical analysis All experiments were performed three times, and each exper- Western blot analysis iment was done in triplicate. Statistical analysis was performed Du145 cells treated with darinaparsin for 72 hours were har- using Prism software (GraphPad). In all cases, ANOVA followed vested and total protein was extracted with RIPA lysis buffer. Fifty by two-tailed, unpaired Student t tests were performed to analyze mg of total protein was electrophoresed on a SDS-PAGE gel and statistical differences between groups. P values of <0.05 were then transferred to nitrocellulose membrane (Millipore). Mem- considered statistically significant. branes were then blocked in 5% milk and probed with anti-Gli-2, b-tubulin, GAPDH, and Gli-1 antibodies overnight. Results Animal studies Darinaparsin is toxic to prostate cancer cells For the study involving Du145 cells, 106 cells in 100 mL of PBS Prostate cancer cell lines LnCap, Du145, and PC3 cells were were injected subcutaneously into the right flank of 4-week-old treated with darinaparsin or ATO at various concentrations for 72 nude female mice. Once tumors were palpable, the mice were hours. After 72 hours, cell viability was measured using the MTS, randomized to different groups, each group had 8 mice: control, and as shown in Fig. 1A, all three cell lines were sensitive to both taxotere, darinaparsin, and combinations of taxotere and darina- darinaparsin and ATO; IC50 concentrations of Du145, PC3, and parsin. Mice were treated with taxotere (6 mg/kg) i.p. once a week LnCap cells ranged from approximately 5 to 10 mmol/L. Du145 for 3 weeks and with darinaparsin (100 mg/kg) i.p. every other day and LnCap cells were more sensitive to darinaparsin than ATO, for 10 days. Treatment with the combination followed the same while PC3 cells were equally sensitive to both darinaparsin and schedule and doses. For the study involving TICs, 20,000 a2b1hi/ ATO. Similar to the prostate cancer cell lines, primary prostate CD44hi cells (5-minute collagen-attached cells) or 20,000 cancer cells isolated from five different patients (Gleason score 8) low low a2b1 /CD44 cells (20-minute unattached cells) were were equally sensitive to darinaparsin with IC50 concentrations injected subcutaneously in nude mice and the next day treatment ranging from 2.5 to 20 mmol/L. (Fig. 1B). Primary prostate cancer was initiated with injections of darinaparsin or taxotere (doses cells isolated from primary prostate tumors were confirmed for and schedules as above). Tumor size and body weights were the prostate epithelial cells by testing for EpCam and AR markers measured three times a week and the tumor volume was calcu- (Supplementary Fig. S1A and S1B).

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Darinaparsin Kills Prostate Cancer Cells by Inhibiting Shh Signaling

A i. ii.

Figure 4. A, Gli-2 is upregulated in prostate cancer stem cells. Du145 cells were plated on collagen I–coated dishes for 20' unattached cells 5' attached cells Total Du145 Total 5' attached cells 5 minutes. Unattached cells were 20' unattached cells Gli-2 replated in other collagen I–coated dishes for another 20 minutes. Both Gli-2 5-minute attached and 20-minute GAPDH unattached cells were collected. GAPDH i, 50 mg of total protein was loaded on a SDS gel and analyzed by Western blot analysis for Gli-2 proteins. ii, RT-PCR was done for the expression of Gli2 in 5-minute attached and B 20-minute unattached cells using Gli2 primers. B, darinaparsin decreases Gli-2 levels in Du145 cells. Du145 cells were treated with darinaparsin for 72 hours. Cells were harvested and 50 g of total protein was subjected

m Untreated 1.5 m mol/L DAR 3 m mol/L DAR to SDS-PAGE followed by Western blotting with anti-Gli-2 and anti-GAPDH. Gli-2

GAPDH

Darinaparsin reduces the number of the tumor-initiating Gli-2 levels as compared with the parental or 20 minutes subpopulation of cells unattached b1low/CD44low cells, at both the protein and mRNA Relapse in most solid tumors after conventional chemother- level (Fig. 4A, i and ii). Furthermore, treatment of cells with apy has been attributed to the existence of drug-resistant TICs. darinaparsin decreased the protein levels of Gli-2 (Fig. 4B). We previously developed a novel assay for TIC prostate tumor Interestingly, Gli-2 levels were decreased at 1.5 mmol/L enrichment based on rapid adherence of TICs to collagen I (13). concentration but not at 3 mmol/L. This experiment was repeat- In brief, cells that attach on collagen I have high expression of ed three times and the same pattern was observed. The mech- CD44 and a2b1(a2b1hi/CD44hi) as compared with 20-min- anism by which Gli2 levels come back may be due to some ute unattached cells (a2b1low/CD44low). We also showed that feedback mechanism. We also investigated whether darinapar- a2b1hi/CD44hi cells have increased colony forming, migration, sin inhibits Gli-2 activity by measuring its effect on the Gli and adhesion ability and can form tumors in nude mice and are responsive luciferase promoter pGL38xGli, which contains the TICs. As shown in Fig. 2A, darinaparsin treatment at an IC50 8 GLI DNA-binding sites attached to the chicken lens crystalline concentration or 2-fold higher reduced the number of Du145 promoter followed by the luciferase gene (12). Du145 cells cells and PC3 cells that attached on collagen I after 5 minutes. were transfected with the luciferase plasmid and cotransfected Darinaparsin-treated cells as compared with taxotere or ATO- with pcDNA-Gli-2 plasmids. After 8 hours of transfection, cells treated cells had fewer a2b1hi/CD44hi cells when grown as were treated with a SAG and cotreated with darinaparsin monolayer (Fig. 2B) or as spheroids (Fig. 2C; IC50 of darina- or ATO or left untreated. Twenty-four hours later, cells were parsin in spheroids from Du145 was determined by the MTS harvested and luciferase activity was assayed. The luciferase assay; Supplementary Fig. S2). Dot plots are in Supplementary levels were normalized with total protein. As shown in Fig. 5A, Figs. S3 and S4. Darinaparsin treatment also reduced secondary such as ATO (15), darinaparsin also inhibited Gli transcrip- colonies formed from dissociating a2b1hi/CD44hi spheroids tional activity compared with vehicle control. The effect of (Fig. 3A), indicating an effect on the TICs. Darinaparsin also darinaparsin on the cell cycle was also examined. Darinaparsin hi hi decreased levels of CD44 and a2b1 cells (Fig. 3B and C) blocks the cells in G2–M phase of cell cycle and but did not from primary prostate cells, further validating the inhibitory induce apoptosis (Fig. 5B). Therefore, the cytotoxic effect of effect of darinaparsin on TICs. darinaparsin may be attributed to the block in the G2–Mphase as well as inhibition of Gli2. Effect of darinaparsin on Shh signaling Darinaparsin induces an incomplete stress response with Darinaparsin inhibits growth of the Du145 and PC3 prostate disruption of microtubules and Shh signaling (11). As darina- tumors parsin is a potent inhibitor of growth of Du145 cells, we tested To show the antitumor effect of darinaparsin and to deter- the effect of darinaparsin on Shh signaling. Prostate cancer– mine whether the combination of darinaparsin with taxotere initiating cells from Du145 cultures (early-passage cells) iso- produced additive or synergistic antitumor effects in vivo,nude lated by the collagen attachment assay showed an increase in mice bearing the Du145 tumor were injected with darinaparsin

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Bansal et al.

A 60

40 Figure 5. 30 Darinaparsin inhibits Gli2 transcriptional activity. Du145 cells

protein) were transfected with pcDNA-Gli-2 20 and a Gli luciferase reporter plasmid. After 8 hours of transfection, cells 10 were treated with ATO or darinaparsin

Luciferase (normalized to total for 24 hours. Cells were then collected 0 and harvested and analyzed for m m Untreated 5 mol/L As2O3 5 mol/L DAR luciferase activity using Promega luciferase kits. B, darinaparsin blocks B cells in G2–M phase of the cell cycle. Du145 cells were treated with 1.5 or 3 G1: 38.32% G1: 8.06% G1: 5.04% mmol/L darinaparsin, 72 hours after G2: 37.62% G2: 66.72% G2: 58.41% treatment cells were collected and stained with PI/RNAse (BD Biosciences) and analyzed for the cell cycle with ﬂow cytometer and MODFIT software.

Untreated 1.5 mmol/L 3 mmol/L

every other day for 5 times, taxotere every once a week for three Discussion times, or with both taxotere and darinaparsin in the same doses In this study, we report that darinaparsin is effective in killing and schedule to determine toxicity. Interestingly, darinaparsin both prostate cancer cells from cell lines as well as cells from fresh inhibited tumor growth as effectively as taxotere. However, prostate tumors at low micromolar concentrations. The IC unlike the in vitro study (Supplementary Fig. S5), the combi- 50 concentration of darinaparsin was lower than ATO in Du145 nation of taxotere and darinaparsin did not have more of an and LnCap cells, whereas in PC3 cells, IC s of both darinaparsin effect than either of the drugs alone (Fig. 6A). Neither drug, nor 50 and ATO were similar. Importantly, darinaparsin was efficient the combination caused toxicity, was measured by weight loss is reducing the number of a2b1hi/CD44hi cells and reducing (Fig. 6b). We also tested the efficacy of darinaparsin in human the number of primary and secondary colonies formed from PC3 xenografts, also castrate resistant, to determine whether a2b1hi/CD44hi spheroids, indicating that darinaparsin and ATO other prostate cancers also were inhibited by darinaparsin. As are cytotoxic to TICs, and darinaparsin is more potent. We shown in Fig. 6C, darinaparsin treatment inhibited tumor previously showed that clinically used chemotherapeutic drugs growth without evidence of toxicity, as measured by a decrease did not affect TICs from Du145 TICs (12). in body weight (Fig. 6D). ATO antagonizes Shh signaling by reducing Gli2, a transcriptional effector (12). ATO treatment inhibits the ciliary accumu- Effect of darinaparsin on tumor growth initiated by TICs lation of Gli2 and hence blocks trafficking required for activation. Mice were injected with 20,000 a2b1hi/CD44hi cells (enriched In addition, ATO caused a reduction of overall levels of Gli2. for TICs) or with 20,000 a2b1low/CD44low cells (depleted of Moreover, in SMO inhibitor–resistant cells, ATO efficiently inhib- TICs) and treated with darinaparsin or with taxotere before ited tumor growth either alone or in combination with itracona- tumors were palpable (day 1 after implantation). As shown zole, a compound that also inhibited Shh signaling (16). In this in Table 1, only 3 of 8 darinaparsin-treated mice developed study, we show that darinaparsin also inhibited Shh signaling. We tumors, whereas 5 of 8 taxotere-treated mice developed tumors. also show that darinaparsin blocks cell in G –M. Reports showing Six of 8 untreated mice formed tumors (Table 1). 2 that darinaparsin inhibits microtubule polymerization and induces stress granule formation (11) may be the mechanism by which it blocks cells in G2–M (Fig. 3D). Table 1. Darinaparsin inhibits tumor growth initiated by TICs The mechanism by which darinaparsin kills cells appears to be Cells (10,000) Tumors remaining Drugs injected complex. In hematopoietic cell lines, an increase in oxidative stress hi hi b1 /CD44 6/8 Untreated was shown to play an important role in darinaparsin action. In solid 1hi/CD44hi 5/8 Taxotere-6 mg/kg b cancers, gene expression studies comparing darinaparsin to ATO b1hi/CD44hi 3/8 DAR-100 mg/kg (17) showed that darinaparsin decreased the expression of genes NOTE: 20,000 a2b1hi cells (TICs fraction) mixed with Matrigel were injected s.c. into the abdominal flanks of nude mice. Staring on day 2, the drugs were injected that were upregulated by Myc. In this study, we show that darina- as described; taxotere was given once a week for 3 weeks and darinaparsin was parsin inhibited growth of two human prostate cancer xenografts in administered every other day for six times. The tumor growth was monitored nude mice and reduced the number of mice with tumors that were every day for 45 days. (n ¼ 8; see in Materials and Methods). initiated with TICs. However, in contrast to the in vitro results that

28 Mol Cancer Ther; 14(1) January 2015 Molecular Cancer Therapeutics

Darinaparsin Kills Prostate Cancer Cells by Inhibiting Shh Signaling

AB2,500 Control 30 Taxotere Darinaparsin 2,000 Taxotere + darinaparsin 25 )

3 20 1,500 Control Darinaparsin alone 15 Taxotere alone 1,000

Weight (g) Weight Taxotere + darinaparsin 10

500 Tumor volume (mm volume Tumor 5

0 0 D7 D10 D13 D16 D19 D21 D25 D28 D31 D35 D40 0 5 10 15 20 25 30 Days Days

CD35 Ctrl 700 Ctrl 30 Dar 600 Dar ) 25 3 500 20 400 15 Weight (g) Weight 300 10 200 5 Tumor volume (mm volume Tumor 100 0 D1 D4 D7 D10 D12 0 D1 D4 D7 D10 D12 Days Days

Figure 6. Xenograft studies with darinaparsin. NOD/SCIDg were injected with Du145 cells, once the tumor was palpable; mice were randomized into four groups (n ¼ 8 per group). Control (group 1), darinaparsin (group 2), taxotere (group 3), and darinaparsin and taxotere (group 4). Darinaparsin group was injected every other day 7 days with darinaparsin at 100 mg/kg; taxotere group was injected with taxotere at dose of 6 mg/kg once a week for 2 weeks and the combination group got darinaparsin and taxotere as mentioned above. A, darinaparsin suppresses growth of a Du145 tumor xenograft; tumor volume was measured three times a week, and the tumor volume calculated using the formula width2 (length/2). Results are presented as mean SEM. B, the mice were weighed every day for 28 days; the graph represents average weight of 8 mice in each group. C, NOD/SCIDg were injected with PC3 cells, once the tumor was palpable; mice were randomized into two groups, Control and Darinaparsin. Darinaparsin group was injected with similar dosage and schedule as mentioned for Du145 cells. Darinaparsin also suppresses growth of a PC3 tumor xenograft; tumor volume was measured and calculated as mentioned for Du145 cells. D, the mice were weighed every other day for 15 days; the graph represents average of weight of 8 mice in each group. Results are presented as mean the SEM. Error bars, SEM. , P 0.05. showed synergistic cell kill between taxotere and darinaparsin and is a consultant/advisory board member for ScientificReviewCom- fl (Supplementary Fig. S3), the in vivo study with a combination of mittee of Ziopharm Oncology, Inc. No potential con icts of interest were taxotere and darinaparsin was not even additive. A possible expla- disclosed by the other authors. nation for lack of additivity or synergy is that darinaparsin and – taxotere both block cells in G2 M. It is also possible that different Authors' Contributions doses and schedules would cause additive or synergistic effects. Conception and design: N. Bansal, L. Wu, J.R. Bertino Itraconazole, also inhibits Shh signaling by inhibiting downstream Development of methodology: N. Bansal, H. Youssoufian gli-2 transcription, such as ATO and darinaparsin, (16). In a phase II Acquisition of data (provided animals, acquired and managed patients, study, itraconazole showed only modest antitumor activity, and provided facilities, etc.): N. Bansal, N.J. Farley, L. Wu dose escalation was limited by toxicity (15). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Wu, J. Lewis, H. Youssoufian, J.R. Bertino The development of an oral form of darinaparsin and Writing, review, and/or revision of the manuscript: N. Bansal, J. Lewis, evidence of antitumor activity in phase I and II trials (6, 18), H. Youssoufian, J.R. Bertino together with the results of this study, provide a rationale for Administrative, technical, or material support (i.e., reporting or organizing evaluating darinaparsin in patients with castrate-resistant prostate data, constructing databases): N. Bansal, J. Lewis, H. Youssoufian cancer. Study supervision: N. Bansal, J.R. Bertino

Disclosure of Potential Conﬂict of Interest Acknowledgments J. Lewis has ownership interest (including patents) in Ziopharm Oncol- The authors thank Dr. P.A. Beachy for providing them with 8xGli-luciferase ogy Inc. J.R. Bertino reports receiving a commercial research grant from reporter plasmid construct.

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Bansal et al.

Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate This work was supported by a grant from Ziopharm Oncology Inc. to J.R. this fact. Bertino. The costs of publication of this article were defrayed in part by the Received December 17, 2013; revised October 21, 2014; accepted October 25, payment of page charges. This article must therefore be hereby marked 2014; published OnlineFirst November 7, 2014.

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30 Mol Cancer Ther; 14(1) January 2015 Molecular Cancer Therapeutics

Darinaparsin Inhibits Prostate Tumor−Initiating Cells and Du145 Xenografts and Is an Inhibitor of Hedgehog Signaling

Nitu Bansal, Nadine Johnson Farley, Lisa Wu, et al.

Mol Cancer Ther 2015;14:23-30. Published OnlineFirst November 7, 2014.

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