Peptidomimetic Src/Pretubulin Inhibitor KX-01 Alone and in Combination with Paclitaxel Suppresses Growth, Metastasis in Human ER/PR/HER2-Negative Tumor Xenografts

Published OnlineFirst July 10, 2012; DOI: 10.1158/1535-7163.MCT-12-0146

Molecular Cancer Preclinical Development Therapeutics

Muralidharan Anbalagan1, Alaa Ali1, Ryan K. Jones1, Carolyn G. Marsden1, Mei Sheng1, Latonya Carrier1, Yahao Bu2, David Hangauer2, and Brian G. Rowan1

Abstract Src kinase is elevated in breast tumors that are ER/PR negative and do not overexpress HER2, but clinical trials with Src inhibitors have shown little activity. The present study evaluated preclinical efficacy of a novel peptidomimetic compound, KX-01 (KX2-391), that exhibits dual action as an Src and pretubulin inhibitor. KX- 01 was evaluated as a single-agent and in combination with paclitaxel in MDA-MB-231, MDA-MB-157, and MDA-MB-468 human ER/PR/HER2-negative breast cancer cells. Treatments were evaluated by growth/ apoptosis, isobologram analysis, migration/invasion assays, tumor xenograft volume, metastasis, and measurement of Src, focal adhesion kinase (FAK), microtubules, Ki67, and microvessel density. KX-01 inhibited cell growth in vitro and in combination with paclitaxel resulted in synergistic growth inhibition. KX-01 resulted in a dose-dependent inhibition of MDA-MB-231 and MDA-MB-157 tumor xenografts (1 and 5 mg/kg, twice daily). KX-01 inhibited activity of Src and downstream mediator FAK in tumors that was coincident with reduced proliferation and angiogenesis and increased apoptosis. KX01 also resulted in microtubule disruption in tumors. Combination of KX-01 with paclitaxel resulted in significant regression of MDA-MB-231 tumors and reduced metastasis to mouse lung and liver. KX-01 is a potently active Src/pretubulin inhibitor that inhibits breast tumor growth and metastasis. As ER/PR/HER2-negative patients are candidates for paclitaxel therapy, combination with KX-01 may potentiate antitumor efficacy in management of this aggressive breast cancer subtype. Mol Cancer Ther; 11(9); 1936–47. 2012 AACR.

Introduction c-Src is an oncogenic non–receptor tyrosine kinase that Patients with ER/PR/HER2-negative breast cancer is upregulated in approximately half of all breast cancers subtype have been difficult to treat due to tumor hetero- (5). Src kinase has been associated with breast cancer geneity and lack of definitive targets for targeted thera- proliferation, angiogenesis, cell motility, migration/inva- peutics (1). Chemotherapy remains the standard of care sion, and metastasis (6, 7). The role of Src in proliferation, for patients, as hormonal therapies and HER2 targeting migration, and invasion coupled with the elevated Src agents are not indicated (2). Paclitaxel is a microtubule- expression in breast cancer make Src a promising target stabilizing agent widely used in breast cancer (3). for development of therapeutics. Moreover, Src inhibition Although ER/PR/HER2-negative breast cancer responds has been identified as a therapeutic target for ER/PR/ to these and other conventional agents, patients become HER2-negative breast cancer (8). However as a single resistant to treatment and relapse more frequently than agent, the broad specificity Src/tyrosine kinase inhibitor patients with other breast cancer subtypes (4). dasatinib resulted in only a modest response rate of 5% in a phase II trial of 43 patients with advanced ER/PR/ HER2-negative breast cancer (4, 9). Another Src inhibitor, sarcatinib failed as monotherapy in patients with hor- Authors' Affiliations: 1Department of Structural and Cellular Biology, mone receptor–negative breast cancer (10). It is unknown Tulane University School of Medicine, New Orleans, Louisiana; and 2Kinex why these compounds failed in these early trials or Pharmaceuticals LLC, New York State Center of Excellence in Bioinfor- matics and Life Sciences, Buffalo, New York whether combinations with other drugs may have resulted in better response. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Peptidomimetics represent a novel class of drugs that interact with the peptide substrate sites of proteins. KX-01 Corresponding Author: Brian G. Rowan, Department of Structural & Cellular Biology SL49, Tulane University School of Medicine, 1430 Tulane (clinical-reference, KX2-391) was developed as a "first-in- Avenue, New Orleans, LA 70112. Phone: 504-988-1365; Fax: 504-988- class" peptidomimetic Src kinase inhibitor that binds to 1687; E-mail: [email protected] the peptide substrate site and inhibits Src kinase activity doi: 10.1158/1535-7163.MCT-12-0146 and downstream targets (11, 12). Orally bioavailable KX- 2012 American Association for Cancer Research. 01 completed phase I clinical testing (13) and is currently

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in phase II trials for prostate cancer (14) as well as phase Ib The number of cells invaded was counted and percentage trial for acute myeloid leukemia (15). In addition to Src invasion was calculated by setting vehicle as 100%. The inhibition, KX-01 has a second mechanism of action invasion was normalized to cell number as follows: (MOA) through binding to novel sites on a-b tubulin Percent invaded cells heterodimer that results in inhibition of microtubule Relative invasion % ¼ 100 polymerization (16). The dual activity of KX-01 to both Percent viable cells inhibit Src and disrupt microtubules may provide KX-01 with additional antitumor activity for ER/PR/HER2-neg- Three-dimensional on-top assay ative breast cancer in comparison to compounds that This assay was conducted as described (20) and is exhibit Src inhibition alone. described in Supplementary Methods. KX-01 was added A previous study from this laboratory has shown that to the medium (10, 25, 50 nmol/L) at the time of cell KX-01 combined with tamoxifen resulted in synergistic plating. The Matrigel medium mixture and KX-01 was growth inhibition in ER-positive xenograft tumors, in replaced every 2 days for the duration of the 4 days and part, through reduced phosphorylation and transcrip- photomicrographed. tional activity of estrogen receptor-a (ERa; ref. 11). Given the elevated expression of Src in ER/PR/HER2-negative Scratch assay breast cancer (17), the present study evaluated the pre- Scratch assay was conducted as described (19). clinical activity of peptidomimetic Src inhibitor KX-01 Cells were incubated with vehicle or KX-01 (5, 10, and alone and in combination with chemotherapeutic drug 25 nmol/L) for 24 hours after scratch and wound closure paclitaxel. Because Src has been implicated in invasion was photographed at 40 magnification. Wound closure and metastasis, this study also examined the anti-invasive was evaluated by measuring wound width by ImageJ. and antimetastatic potential of KX-01 both in vitro and in Percentage of gap closure was calculated as: vivo. % gap closure ¼ Gap area at time 0 Gap area at time 24 hours Materials and Methods 100% Gap area at time 0 Cell lines, culture conditions, and reagents The human breast cancer cell lines MDA-MB-231, MDA-MB-157, and MDA-MB-468 were obtained from the Animals American Type Culture Collection. These cell lines lack Female athymic nude mice (BALB/c) aged between 4 expression of ERa and progesterone receptor (PR) and do and 5 weeks obtained from Charles River were housed in not exhibit amplification/overexpression of HER2 (18). sterile cages and maintained in pathogen-free aseptic The cells were cultured in Dulbecco’s Modified Eagle’s rooms with 12-hours light/dark schedule. Mice were fed Medium (DMEM; Invitrogen, Gibco) supplemented with with autoclaved food pellets and water ad libitum.All 10% FBS, 1% penicillin/streptomycin in a humidified experiments were carried out in accordance with approved incubator at 37 C containing 5% CO . No further authen- 2 Institutional Animal Care and Use Committee protocol tication was conducted for cell lines. KX-01was provided #2941R2 from Tulane University, New Orleans, LA. by Kinex Pharmaceuticals in powder form [KX-01:MSA (methanesulfonic acid) salt] that was water-soluble. Pac- Tumor xenograft studies litaxel was purchased from Hospira Inc. and doxorubicin Xenograft procedures and KX-01 oral dosing were as from Bedford laboratories. described (11). Briefly, mammary fat pad tumors were established by injecting 5 106 MDA-MB-231 cells in MTT and apoptosis assay 150 mL of PBS–Matrigel mixture (1:2) orthotopically and MTT and apoptosis assays (ELISA, Roche) were con- bilaterally into the mammary fat pads of female nude mice ducted as described (11). Cell growth was measured in (2 tumors/mouse). Treatments were started when tumors MDA-MB-231, MDA-MB-157, MDA-MB-468 cells after reached approximately 80 to 100 mm3. The first study incubation with vehicle and varying concentrations of used MDA-MB-231 xenografts and was conducted using KX-01, paclitaxel, doxorubicin, or dasatinib (5, 10, 25, vehicle (ultra-pure water) and 2 doses of KX-01 (1 and 50, 100, 250 nmol/L) for 48 hours. Cell growth was 5 mg/kg) administered twice daily by oral gavage (using expressed as percent of vehicle. Apoptosis was evaluated metal 22-G feeding needle) for 28 days. A similar exper- 24 hours after drug incubation. iment was carried out with MDA-MB-157 xenografts (another ER/PR/HER2-negative model) to assess KX- Invasion assay 01 response. A second study was conducted to test Invasion assays were conducted as described (19). Cells combination of KX-01 with paclitaxel on tumor growth. were incubated with KX-01 (10, 25, 50 nmol/L) for MDA-MD-231 tumor xenograft–bearing mice were trea- 24 hours. Invaded cells were counted (ImageJ software) ted with vehicle or KX-01 (5 mg/kg) twice daily, paclitaxel and photomicrographed. The correction for growth inhi- by intraperitoneal (i.p.) injection once a week, or combi- bition by KX-01 is described in Supplementary Methods. nation of KX-0 þ paclitaxel. Treatments were for 40 days

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for all groups. A third study used MDA-MB-157 xeno- organ were generated by serial additions of MCF-7 human grafts with the same combination treatment. A fourth breast cancer cells (106,105,104,103,102, 0 cells) to mouse study tested the effect of KX-01 or combination with whole lung, whole liver, or bone marrow (from one femur) paclitaxel for 24 days on larger MDA-MB-231 tumors just before homogenization of the tissues and isolation of (300 mm3). Tumors were allowed to reach approximate- DNA. Mouse organs were derived from untreated female ly 300 mm3 before beginning treatments. In this experi- nude mice at the same age as mice from the treatment ment, mice were treated with KX-01 at a higher dose of 15 groups. Human/mouse GAPDH was used as an internal mg/kg, and mice were treated once a day instead of twice control. Real-time RT-PCR for human/mouse GAPDH a day. Paclitaxel was used at a dose of 20 mg/kg i.p. once a was carried out as described (22). Real-time PCR assays week. In all experiments, tumor caliper measurements were conducted in triplicate. The standard curves for were taken twice a week and tumor volume was calcu- lung, liver, and femur were used to calculate the absolute lated by the formula: 0.523 LM2 (where L is large number of human cells present in the lung, liver, and diameter and M is small diameter). At the end the experi- bone. Nonlinear regression analysis was used to interpo- ments, animals were sacrificed and tumors and mouse late unknowns from the standard curves (GraphPad organs removed. Tissues were either stored in 10% neu- Prism5). The detection limit for real-time PCR in this tral-buffered formalin for paraffin embedding, or snap- assay was 100 human cells per mouse lung, liver, or femur. frozen for measurement of chromosome-17 by real-time PCR, and embedded for frozen sectioning for CD-31 Statistical analysis staining. Immunohistochemistry (IHC) was conducted as Data were expressed as mean SD. P < 0.05 was described (11) on paraffin-embedded tumor tissues. The considered significant. The mean and SD were calculated detailed immunohistochemical procedure is described in using Microsoft Excel or GraphPad Prism 5 software. Supplementary Methods. Statistical significance was determined by 2-sample Stu- dent t tests (P < 0.05; 2-tailed) and one-way ANOVA a Immunofluorescence staining of -tubulin in MDA- followed by Newman–Keuls multiple comparison test. MB-231 cells and tumors Combination indices (CI) for combination drug treat- For in vitro experiments, MDA-MB-231 cells were incu- ments were conducted using CalcuSyn software (Biosoft). bated with 200 nmol/L KX-01 for 24 hours, fixed and The extent of cooperation between KX-01 with paclitaxel a immunostained with -tubulin antibody conjugated with was determined from the CI as follows: CI < 1.0 indicated AlexaFluor-488 (Invitrogen #32-2588). Fluorescence synergism; CI ¼ 1.0 indicated additivity; CI > 1.0 indicated images were captured with a BD Pathway 855 Confocal antagonism (23). fluorescence microscope at 60 using Attovision soft- a ware. Immunofluorescence for -tubulin was conducted Results in MDA-MB-231 tumors as described in Supplementary Methods. All fluorescent images were captured using an Chemical structures of KX-01, dasatinib, and paclitaxel identical exposure time to permit comparison of staining are shown in Fig. 1A. intensity between treatment groups. Effect of KX-01 and dasatinib on ERa-negative breast Quantification of micrometastases cancer cells Human DNA within the mouse organs was measured The ATP analogue Src inhibitor dasatinib was shown to using quantitative real-time reverse transcriptase (RT)- inhibit growth of MDA-MB-231 breast cancer cells but not a PCR to detect human chromosome 17. The detection is ER /PR/HER2-negative MDA-MB-468 cells (ref. 18; Fig. based on a human-specific a-satellite DNA sequence of 1B). KX-01 at varying concentrations (10–250 nmol/L) the centromere region of human chromosome 17 (21). inhibited in vitro growth of both MDA-MB-231 and a After necropsy (n ¼ 5 animals per group), mouse organs MDA-MB-468 cells as well as another ER /PR/HER2- were removed, homogenized, and DNA extracted and negative cell line, MDA-MB-157 (Fig. 1C). For dasatinib- quantified using a NanoDrop Spectrophotometer (Ther- resistant MDA-MB-468 cells, KX-01 inhibited growth by moScientific). Quantitative real-time PCR procedure is approximately 75.0% at the same concentration range described in Supplementary Methods. The CT value where dasatinib inhibited growth by only approximately obtained for human chromosome 17 was normalized 20.0% (Fig. 1C). Moreover, KX-01 induced significant to housekeeping gene glyceraldehyde-3-phosphate dehydro- apoptosis in MDA-MB-468 cells at 25 nmol/L, whereas genase (GAPDH; CT value) that detected both mouse dasatinib did not induce apoptosis at a 10 higher con- and human GAPDH as a measure of total DNA for the centration (250 nmol/L; Supplementary Fig. S1). samples (GAPDH served as a loading control). Because the extracted DNA could have both mouse and human Combination of KX-01 with paclitaxel resulted in DNA, we used primers that detected both mouse and synergistic growth inhibition of breast cancer cells in D ¼ vitro human GAPDH. CT CT value of human chromosome 17 gene minus CT value of mouse/human GAPDH. Stan- A previous study from this laboratory showed that KX- dard curves for number of human cells within a mouse 01 inhibited growth of a panel of breast cancer cell lines

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Figure 1. KX-01 induced growth inhibition and apoptosis in ER/PR/HER2-negative breast cancer cells and synergized with paclitaxel in vitro. A, chemical structures of KX-01, dasatinib, and paclitaxel. B and C, growth-inhibitory effects of the broad speciﬁcity Src inhibitor dasatinib (B) and KX-01 (C) at varying concentrations (10, 25, 50, 100, 250 nmol/L) on ER/PR/HER2-negative MDA-MB-231, MDA-MB-157, and MDA-MB-468 cells. Cells were incubated with drugs for 48 hours and growth inhibition was assessed by MTT assay. Results presented as % vehicle SD (n ¼ 3). D, KX-01 induced synergistic growth inhibition in MDA-MB-231 cells when combined with paclitaxel (PAX). Cell growth was measured by MTT assay. Cells were incubated with KX-01 (25 nmol/L), PAX (5 nmol/L), or combination for 24, 48, and 72 hours. Results presented as % vehicle SD (n ¼ 3). E, KX-01 in combination with PAX resulted in enhanced apoptosis in MDA-MB-231 cells. Apoptosis assay was conducted following 24-hour incubation with vehicle KX-01 (25 nmol/L), PAX (5 nmol/L), or KX-01þPAX. , P < 0.05 (Student t test), signiﬁcantly different compared with vehicle (VC), #, P < 0.05 (Student t test), statistical evidence of enhanced apoptosis in combination treatment compared with either drug alone. Data are representative of 3 independent experiments.

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with different ERa status (ER-positive MCF-7 cells and phosphorylation (Fig. 2C) with no significant effect on ER-negative MDA-MB-231, MDA-MB-157, MDA-MB- expression of total Src or FAK. 468, and BT-549 cells) but did not affect growth of nontumorigenic breast epithelial MCF-10A cells (11). KX-01 reduced proliferation and angiogenesis and After 24-hour incubation, KX-01 (25 nmol/L) alone or increased apoptosis in MDA-MB-231 tumor paclitaxel (5 nmol/L) alone at suboptimal growth inhib- xenografts itory concentrations produced modest growth inhibition Terminal deoxynucleotidyl transferase–mediated of 11.2% and 14.1%, respectively (Fig. 1D). Co-incubation dUTP nick end labeling (TUNEL) was used to measure of KX-01 with paclitaxel resulted in synergistic 43.2% apoptotic cells. KX-01 significantly enhanced the number growth inhibition of MDA-MB-231 cells compared with of apoptotic cells 3- to 4-fold compared with vehicle- vehicle-treated cells (Fig. 1D). A dose response for growth treated tumors (P < 0.001; Fig. 2D and G). KX-01 reduced inhibition was determined over concentration ranges of the percentage of tumor cells positive for proliferation 5 to 100 nmol/L for KX-01 alone and 1 to 10 nmol/L for marker Ki67-positive tumor cells to 37.3% compared with paclitaxel (data not shown). From these data, CI values vehicle (58.7%; P < 0.001; Fig. 2E and H). To evaluate the were calculated (as described in ref. 23) for KX-01 þ effect of KX-01 on angiogenesis, microvessel density paclitaxel over a concentration range (Supplementary (MVD) was assessed by counting CD31-positive micro- Table S1A). Similar patterns of synergistic growth inhibi- vessels. KX-01 significantly reduced MVD (P < 0.05) tion of KX-01 with paclitaxel at various concentrations compared with vehicle (Fig. 2F and I). In summary, were observed in MDA-MB-157 and MDA-MB-468 cells these data show that KX-01 significantly reduced (Supplementary Table S1A). growth of MDA-MB-231 tumors that was attributed, in part, to reduced tumor cell proliferation, increased Combination of KX-01 with paclitaxel induced apoptosis, and decreased blood vessel density (modest- apoptosis in MDA-MB-231 breast cancer cells ly) that was coincident with reduced Src kinase activity To determine whether synergistic growth inhibition in the tumors. by combination of paclitaxel þ KX-01 resulted from increased apoptosis, an ELISA-based DNA fragmentation Combination of KX-01 with paclitaxel resulted in assay was used. KX-01 or paclitaxel at the concentrations regression of ER/PR/HER2-negative tumor used in Fig. 1D resulted in modest apoptosis in MDA-MB- xenografts 231 cells (Fig. 1E). Combination of KX-01 with paclitaxel The in vitro observations of synergistic growth inhibi- enhanced apoptosis 2-fold compared with single drug tion of breast cancer cell lines co-incubated with KX-01 treatments (Fig. 1E). and paclitaxel were evaluated in vivo using MDA-MB-231 and MDA-MB-157 tumor xenografts. KX-01 administered Oral administration of KX-01 reduced growth of ER/ alone (5 mg/kg, twice daily) decreased MDA-MB-231 PR/HER2-negative breast tumor xenografts in mice tumor volume by 61.1%, and treatment with paclitaxel MDA-MB-231 and MDA-MB-157 xenograft tumors alone (3 mg/kg, once a week) decreased tumor volume (80–100 mm3) in nude mice were established to determine by 33.4% compared with vehicle (P < 0.01, Fig. 3A). the effect of KX-01 on tumor growth in vivo. Mice were Combination of KX-01 with paclitaxel reduced tumor treated with vehicle (ultra-pure water), KX-01 at 1 mg/kg volume by 93.0% (Fig. 3A). Most significantly, combi- and KX-01 at 5 mg/kg for 28 days, twice daily. KX-01 nation of KX-01 with paclitaxel resulted in complete resulted in marked tumor growth inhibition in both tumor regression of tumors in 2 of 5 mice. In MDA-MB-157 models (Fig. 2A and Supplementary Fig. S2A). Mice xenografts, the same single-agent doses of KX-01 and receiving KX-01 at 1 and 5 mg/kg showed reduced paclitaxel reduced tumor volumes by 42.0% and 16.3%, MDA-MB-231 tumor volume compared with vehicle from respectively (Fig. 3B). Combination of KX-01þ paclitaxel days 18 and 15, respectively (P < 0.05; Fig. 2A). KX-01 at 1 reduced MDA-MB-157 tumor volumes by 71.1% com- and 5 mg/kg reduced MDA-MB-231 tumor volume 35.3% pared with vehicle (P < 0.001; Fig. 3B). No noticeable and 79.0%, respectively (Fig. 2A), and reduced MDA-MB- toxicity or significant body weights reduction was ob- 157 tumor volume 23.5% and 42.0%, respectively (Sup- served in either drug alone or combinational treatment plementary Fig. S2A). groups (Supplementary Fig. S3A). Hematoxylin and eosin (H&E) staining of MDA-MB-231 tumor sections revealed KX-01 decreased activity of Src and downstream more acellular, necrotic regions in tumors from the com- target focal adhesion kinase in MDA-MB-231 tumor bination treatment groups than form single drug treat- xenografts ments (Supplementary Fig. S3B, arrows). To determine whether KX-01 effectively inhibited activation of Src in primary tumors, tumors were prepared Combination of KX-01 with paclitaxel resulted in for IHC using antibodies to the active forms of Src kinase regression of larger, established MDA-MB-231 (p-Y416-Src) and its downstream target focal adhesion tumors kinase (FAK; p-Y861-FAK). KX-01 significantly inhibited Xenograft experiments described in Figs. 2 and 3 and Src phosphorylation (Supplementry Fig. S2B) and FAK Supplementary Fig. S2 began drug treatments when

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Figure 2. KX-01 inhibited growth of MDA-MB-231 xenografts. A, athymic nude mice bearing MDA- MB-231 tumors (100 mm3) were separated into 3 treatment groups randomly (n ¼ 10 tumors per group). The vehicle group received ultra- pure water and the other groups were treated with 2 different doses of KX- 01 (1 and 5 mg/kg) for 28 days and tumor volumes were recorded as mm3 SD. , P < 0.05 (one-way ANOVA followed by Newman–Keuls multiple comparison test). b. wt., body weight. B and C, KX-01 inhibited phosphorylation of Src and downstream mediator FAK in MDA- MB-231 xenografts. IHC was conducted as described in Supplementary Methods for total Src, phospho-Src-Y416 (p-Y416- Src), FAK, and phospho-FAK-Y861 (p-Y861-FAK). Representative photomicrographs are presented (200). D–F, KX-01 induced apoptosis and reduced proliferation and angiogenesis in MDA-MB-231 xenografts. Paraffin-embedded tumor sections from vehicle and KX- 01 (5 mg/kg) groups were stained for TUNEL (D) and Ki67 (E). Fluorescent (TUNEL) and bright-field (Ki67) photomicrographs were taken and representative images are presented (200). F, frozen tumor sections were stained for mouse CD31. Bright-field microscope images (40) were taken, and representative photomicrographs are presented. G- I, quantitative representation of TUNEL, Ki67, and CD-31 staining. VC, vehicle control.

tumors reached a palpable size of approximately 80 to was needed for efficacy against larger, established MDA- 100 mm3. To determine whether combination of KX-01 MB-231 tumors as compared with smaller tumors (24, 25). with paclitaxel would cause regression of larger and more For this reason, paclitaxel was used at a dose of 20 mg/kg, established tumors, MDA-MB-231 xenografts were once a week. KX-01 alone resulted in significant tumor allowed to reach approximately 300 mm3 before treatments growth inhibition but without tumor regression; tumor began. Animals were treated with KX-01 at a higher dose of volume was decreased by 48.2% (P < 0.001 vs. 15 mg/kg only once a day, which when compared with 5 vehicle; Fig. 4A). Paclitaxel alone resulted in significant mg/kg KX-01 twice a day showed similar tumor growth tumor growth inhibition but without tumor regression; inhibition in MDA-MB-231 xenografts (data not shown). tumor volume was decreased by 35.4% (P < 0.001; Previous studies showed that a higher dose of paclitaxel Fig. 4A) with no significant reduction in mouse body

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KX-01 disrupted microtubules in MDA-MB-231 cells and tumors In addition to Src inhibition, KX-01 was shown to inhibit microtubule polymerization in vitro indicating a second MOA (16, 26). KX-01 (200 nmol/L, 24 hours) markedly inhibited microtubule formation in MDA- MB-231 cells in vitro (Supplementary Fig. S4A). To assess the effect of KX-01 on microtubule organization in tumor tissues, paraffin-embedded MDA-MB-231 tumor sections were incubated with an anti-tubulin fluorescent antibody. In vehicle-treated animals, tumor cells exhibited staining of distinct microtubule fibers demonstrative of an intact microtubule network (Fig. 4B). In KX-01–treated mice, tumor cells exhibited a more diffuse staining pattern with markedly reduced fluorescence intensity indicative of a disrupted and reduced microtubule network in the cells. In contrast, paclitaxel resulted in more intensely stained microtubule arrays, consistent with the microtubule- stabilizing activity of paclitaxel (Fig. 4B). Interestingly, the tumors from mice treated with KX-01 þ paclitaxel showed a microtubule-staining pattern that was markedly different from either single treatment. KX-01 þ paclitaxel resulted in a diffuse tubulin-staining pattern indicating loss of the microtubule network, as well as evidence of fragmented microtubules (arrows). Taken together, these data provide direct evidence that KX01 alone, and in combination with paclitaxel, resulted in disruption of the microtubule network in tumors.

KX-01 inhibited breast cancer cell outgrowth, invasion, and migration in vitro Cell invasiveness was assessed using a 3D invasion assay in which MDA-MB-231 cells were cultured in recon- stituted extracellular matrix to mimic an in vivo microen- vironment (20). Treatment of MDA-MB-231 cells with 25 Figure 3. KX-01 combined with paclitaxel (PAX) resulted in regression of and 50 nmol/L KX-01 reduced invasive stellate structures MDA-MB-231 and MDA-MB-157 xenografts. Mice bearing MDA-MB- that are a hallmark of invasive cells (Fig. 5A). There was 231 or MDA-MB-157 xenografts (100 mm3) were separated into 4 almost complete inhibition of stellate structures at treatment groups randomly with 5 mice per group (2 tumors per mouse). 50 nmol/L KX-01. The effect of KX-01 on MDA-MB-231 MDA-MB-231 (A) and MDA-MB-157 (B) xenograft–bearing mice were treated with ultra-pure water, KX-01 alone (5 mg/kg) twice daily, paclitaxel invasion in vitro was assessed using the Boyden chamber (PAX; 3 mg/kg) once a week i.p. as indicated by the arrows, and KX-01 þ Matrigel invasion assay. A significant decrease in MDA- PAX. Tumors volumes were recorded as mm3 SD. , P < 0.05 (one-way MB-231 cell invasion by 52.9% occurred with 50 nmol/L ANOVA followed by Newman–Keuls multiple comparison test). b. wt., KX-01 (Fig. 5B; P < 0.05; Supplementary Fig. S4B). The body weight. anti-invasive effect was independent of the growth-inhibitory effect of KX-01, as invasion data were normalized to weight (Supplementary Fig. S3C). Combination of KX-01 cell number (see Materials and Methods). Cancer cell (15 mg/kg) with paclitaxel (20 mg/kg) resulted in 90.6% migration was assessed by a wound-healing/scratch tumor regression to a volume that was markedly below the assay. Concentrations of 5, 10, and 25 nmol/L KX-01 starting tumor volume (P < 0.001; Fig. 4A) with approxi- significantly decreased MDA-MB-231 cell migration by mately 10% mouse body weight reduction but without 11.7%, 15.6% and 39.9%, respectively, compared with noticeable toxicity (initial mean body weight ¼ 19.4 g; final vehicle (Fig. 5C; P < 0.05; Supplementary Fig. S4C). mean body weight ¼ 17.6 g; Supplementary Fig. S3C). By day 12, mice receiving KX-01 þ paclitaxel showed signif- Effect of KX-01 on MDA-MB-231 micrometastasis in icant tumor regression compared with single treatments vivo (P < 0.001, Fig. 4A). H&E staining of MDA-MB-231 tumor Although the relatively short treatment time (40 days) sections showed more acellular, necrotic regions in tumors for the MDA-MB-231 xenograft tumors was not suffi- from combination treatments than from single treatments cient for tumors to exhibit visual macrometastases to (Supplementary Fig. S3D, arrows). mouse organs, 40 days were sufficient to measure

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KX-01 Plus Paclitaxel Blocks Tumor Growth and Metastasis

extrapolated from the standard curve, Supplementary Fig. S5A). KX-01 alone (5 mg/kg) or KX-01 þ paclitaxel (3 mg/kg) significantly reduced metastasis to the lungs (P < 0.05, Fig. 6A). No human cells were detected in the lungs of mice treated with either KX-01 alone or KX-01 þ paclitaxel. The incidence of metastasis to the bone marrowwas3of5mice(60%),andthemeannumberof MDA-MB-231 cells in the bone marrow for the vehicle group was 7.1 105 cells. Although there were no detectable human cells in the bone marrow from mice treated with KX-01 alone or paclitaxel þ KX-01, the differences between these treatment groups and vehicle were not statistically significant (Fig. 6B). In addition, therewasnostatisticaldifferencebetweenvehicleand paclitaxel alone for metastatic cells in the bone marrow (Fig. 6B). The incidence of micrometastasis to liver in the vehiclegroupwas4of5mice(80%)andthemean number of MDA-MB-231 cells in the liver for the vehicle group was 3.0 106 cells. As with the lungs and bone marrow, no detectable human cells were found in the livers of mice treated with KX-01 alone or KX-01 þ paclitaxel (Fig. 6C). There was a statistically significant reduction in liver metastasis in the KX-01 alone group and the KX-01 þ paclitaxel group compared with the vehicle group (P < 0.05; Fig. 6C). Paclitaxel alone did not significantly alter liver metastasis compared with vehicle. Taken together, these data show that KX-01 alone and in combination with paclitaxel significantly reduced metastasis of MDA-MB-231 cells to the lung and liver.

Figure 4. Combination of high-dose KX-01 with paclitaxel induced regression of established, large MDA-MB-231 tumors. A, MDA-MB-231 3 Discussion tumors were established in nude mice (tumor volume 300 mm ) before treatments began. Mice (n ¼ 5 per group) were treated with vehicle, KX-01 Activation of Src kinase has been shown to decrease (15 mg/kg) by oral gavage once daily, paclitaxel (PAX; 20 mg/kg) once a apoptosis of cancer cells and to promote cell mitosis, cell week i.p. (arrows), or KX-01þPAX. Tumor volumes were recorded as mm3 SD. , P < 0.05 (one-way ANOVA followed by Newman–Keuls multiple motility/invasion, and neo-angiogenesis to tumors (6, 7). comparison test). B, paraffin tumor sections from treatment groups were In the present study, the clinical Src/pretubulin inhibitor analyzed by immunofluorescence with anti-tubulin antibody conjugated KX-01 as a single-agent inhibited growth, migration, and with Alexafluor-488 (green) and imaged with a confocal microscope invasive potential and increased apoptosis of ER/PR/ þ ( 60). White arrows indicate fragmented microtubules in the KX-01 HER2-negative breast cancer cells in vitro. Treatment of PAX group. mice bearing ER/PR/HER2-negative tumors with KX-01 resulted in a dose-dependent decrease in tumor volume micrometastasis by quantitating human DNA in mouse with concomitant reduction in proliferation, MVD, and tissues. At the termination of experiments described increased apoptosis. When KX-01 was combined with in Fig. 3A, lung, liver, and the bone marrow from paclitaxel, there was significantly reduced tumor volume femurs were removed (n ¼ 5 mice), and the amount of and tumor regression compared with either drug alone human DNA in these mouse tissues was measured by without noticeable toxicities. KX-01 also exhibited an quantitative real-time RT-PCR directed toward an antimetastatic effect against MDA-MB-231 tumors. The a-satellite sequence specific for human chromosome antitumor and antimetastatic effects of KX-01 were coin- 17. Standard curves for the number of human cells cident with marked inhibition of Src activity and disrup- within control mouse organs were generated by serial tion of the microtubule network in the primary tumors. additions of human breast cancer cells to whole lung, ER/PR/HER2-negative breast cancers are intrinsically whole liver, and bone marrow (one femur) just before more chemoresistant than other breast cancer subtypes homogenization of the tissues and DNA isolation (Sup- (27). Given the lack of targeted agents and the rapid onset plementary Fig. S5A). The incidence of metastasis to the of chemoresistance in metastatic ER/PR/HER2-negative lung in the vehicle group was 4 of 5 mice (80%). The breast cancers, there is urgent need for novel therapeutic mean number of MDA-MB-231 cells in the mouse lung approaches to target these tumors. Combinations of tar- for the vehicle group was 6.0 106 cells (number geted agents with conventional chemotherapeutic agents

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Anbalagan et al.

Figure 5. KX-01 inhibited invasive stellate formation, invasion, and migration of MDA-MB-231 cells in vitro. A, invasive stellate structures of MDA-MB-231 cells in 3D culture following vehicle or KX-01 treatment were photomicrographed on day 4. B, MDA-MB-231 cells were incubated with vehicle (VC) or KX-01 (10, 25, 50 nmol/L) for 24 hours, and the number of cells invaded was quantified and normalized to cell number and presented as % relative invasion SD. , P < 0.05, statistically significant compared with VC. C, monolayer cultures of MDA-MB-231 cells were gently scratched with a pipette tip to produce a wound. Photographs of cultures were taken immediately after the scratch (0 hour) and after 24-hour incubation with vehicle or KX-01 (5, 10, 25 nmol/L). Migration is presented as % gap closure SD, , P < 0.05, significantly different from VC. Data are representative of 3 independent experiments.

are currently being investigated and have proven clini- with fragmentation of many of the remaining micro- cally successful in several cancers including breast cancer tubules. It was recently shown that microtubule-inhibit- (28, 29). The present study presents KX-01 as a first-in- ing drugs can cause microtubule minus-end detach- class clinical Src/pretubulin inhibitor that results in sig- ment from the microtubule-organizing centers (ref. 31). nificant tumor regression when combined with standard- It is possible that KX-01 through inhibition of tubulin of-care chemotherapy agent paclitaxel showing that this polymerization has a similar effect in causing microtu- combination could be considered in the clinical setting. bule detachment from microtubule-organizing centers. KX-01 efficacy in preclinical tumor models was corre- However in the KX-01 þ paclitaxel group, the resulting lated with strong inhibition of Src and FAK activity, detached microtubules are protected by paclitaxel from suggesting that Src inhibition contributed to the antitumor further KX-01–induced depolymerization, thus giving effect. In addition to Src inhibition, KX-01 also inhibits rise to fragmented microtubules. It is likely that these 2 microtubule polymerization as a second MOA (16, 26). different mechanisms for microtubule disruption con- This second MOA likely contributes to the robust apo- tribute to the antitumor synergy observed when KX-01 ptosis and antitumor activity in comparison with drugs and paclitaxel were combined. that exhibit only Src inhibition. In this regard, KX-01 as a Invading cells produce various membrane protrusions single compound may exert similar effects as combination that promote migration and proteolysis of extracellular of clinical Src inhibitors with microtubule-targeting che- matrix (ECM; 32). Src has been shown to regulate micro- motherapy (30) and may also be effective in Src inhibitor– tentacles and invadopodia (32). Src is both necessary and resistant tumors. sufficient for the formation of invadopodia at sites of Paclitaxel stabilizes microtubules resulting in mitotic integrin-mediated ECM adhesion (33). Cells coordinate blockade and subsequent cell-cycle arrest or apoptosis. focal adhesions and invadopodia to provide traction (34). Interestingly, KX-01 and paclitaxel disrupt microtu- MDA-MB-231 cells are highly invasive and migratory and bules by different means; inhibition of microtubule are shown to produce microtentacle protrusions and assembly and stabilization of microtubules, respective- invadopodia (32). Src inhibition by KX-01 significantly ly, as was observed by the markedly different microtu- reduced formation of invasive stellate structures in MDA- bule-staining patterns in tumors exposed to these 2 MB-231 cells and inhibited cell migration and invasion. agents (Fig. 4B). The combination of KX-01 with pacli- These findings correlated with KX-01 inhibition of Src and taxel resulted in a reduced microtubule network along FAK in tumors.

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KX-01 Plus Paclitaxel Blocks Tumor Growth and Metastasis

Figure 6. Quantiﬁcation of micrometastasis of MDA-MB-231 tumors in mouse organs. After necropsy, lung, bone marrow (femur), and liver were collected from treatment groups described in Fig. 3 [vehicle control (VC), KX-01, PAX, KX-01 þ PAX (n ¼ 5 mice per group)] and genomic DNA was isolated. Micrometastases originating from human primary MDA-MB-231 xenografts were detected by quantitative real-time RT-PCR for an a-satellite DNA sequence in the centromere region of human chromosome 17. Test samples (the number of human cells in each mouse tissue sample) were interpolated from a standard curve as described in Materials and Methods. Graphs represent the number of human cells in intact mouse lung (A), bone marrow from one femur (B), and intact mouse liver (C). , P < 0.05, signiﬁcantly different compared with VC.

Metastasis is the ﬁnal step in the progression of many 17 microsatellite regions by PCR using an established solid tumors with the lungs, liver, and bone as frequent procedure (21, 37) that we have previously used (38). sites for breast cancer metastasis (35, 36). The brief treat- Although precaution was taken to rinse mouse organs ment period (40 days) of MDA-MB-231 xenografts per- thoroughly with PBS to remove blood and blood clots mitted an evaluation of micrometastasis to mouse organs before DNA isolation, a caveat to this procedure is the from the primary tumor. KX-01 alone or in combination presence of any circulating tumor cells in the vasculature with paclitaxel resulted in complete absence of detectable of the organs that could contribute to the DNA quantita- micrometastasis to the lung and liver. It should be noted tion. Future studies will evaluate KX-01 in impacting that the reduction in micrometastasis could be attributed, multiple steps of the metastatic process using appropriate in part, to the reduced primary tumor burden in the metastatic models and histopathologic methods to quan- treated animals. Because the tumor xenografts had to be titate metastatic burden. terminated within 30 to 40 days due to the large size of To determine whether KX-01 could synergize with the primary tumors, micrometastases to mouse organs cytotoxic drugs other than paclitaxel, doxorubicin (a were limited, and sectioning/H&E staining of mouse topoisomerase II inhibitor widely used for the treatment organs to quantitate micrometastases was not possible. of breast cancer) was used in combinational treatments. As an alternate approach to measure very early micro- In ER/PR/HER2/neu-negative cell lines, lower concen- metastases, we quantitated levels of human chromosome trations of KX-01 (10 and 25 nmol/L) synergized with

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doxorubicin for cell growth inhibition (Supplementary showed that targeting 3 MOAs (Src inhibition, tubulin Table S1B). However, at higher KX-01 concentrations disruption, and tubulin stabilization) can achieve greater (50 nmol/L), combination with doxorubicin resulted in efficacy in tumor growth inhibition than treatment with antagonism with CI values above 1.0 (Supplementary compounds that would target only a single MOA. Fur- Table S1B). In MDA-MB-231 and MDA-MB-157 tumor thermore, paclitaxel dose is limited in the clinic due to xenograft experiments, combination of KX-01 þ doxoru- neurotoxicity. The inclusion of KX-01 with paclitaxel bicin resulted in significant tumor growth inhibition that therapy may permit lower doses of paclitaxel to be used was greater than either drug alone (Supplementary while maintaining or improving efficacy. Fig. S2B and S2C). However unlike combination with paclitaxel, the antimetastatic effect of KX-01 in vivo was Disclosure of Potential Conflicts of Interest antagonized by combination with doxorubicin (Supple- D. Hangauer is Chief Scientific Officer for Kinex Pharmaceuticals LLC mentary Fig. S5B). Whereas KX-01 alone reduced lung that supplied KX-01 and dasatinib for this study. B.G. Rowan received a $25,000 research grant from Kinex Pharmaceuticals to support the research metastasis, when combined with doxorubicin, there was presented in this study. No potential conflicts of interest were disclosed by no significant difference in lung metastasis compared the other authors. with vehicle. It is unknown why combination of KX-01 with doxorubicin did not result in complete synergy for Authors' Contributions cell growth inhibition and reduced metastasis across a Conception and design: M. Anbalagan, D. Hangauer, B.G. Rowan broad concentration range of KX-01 as was shown for Development of methodology: M. Anbalagan, C.G. Marsden, B.G. Rowan Acquisition of data (provided animals, acquired and managed patients, combination with paclitaxel. The 2 cytotoxic agents have provided facilities, etc.): M. Anbalagan, A. Ali, R.K. Jones, L. Carrier, B.G. different MOAs that likely underlie the efficacy when Rowan combined with KX-01. As noted above, the synergy Analysis and interpretation of data (e.g., statistical analysis, biostatis- tics, computational analysis): M. Anbalagan, A. Ali, R.K. Jones, Y. Bu, D. between KX-01 and paclitaxel may be due to targeting Hangauer, B.G. Rowan 2 different mechanisms for microtubule inhibition. Doxo- Writing, review, and/or revision of the manuscript: M. Anbalagan, D. rubicin as a DNA-damaging agent may not complement Hangauer, B.G. Rowan Administrative, technical, or material support (i.e., reporting or orga- KX-01 as effectively. It will be important to identify con- nizing data, constructing databases): M. Sheng, B.G. Rowan centrations of doxorubicin þ KX-01 that will exhibit Study supervision: B.G. Rowan desired clinical outcomes. Additional studies are needed Provided KX-01 used in study: D. Hangauer to identify the mechanisms underlying the mixed efficacy of doxorubicin combined with KX-01. Acknowledgments Louisiana Cancer Research Consortium FACS core facility performed ER/PR/HER2-negative breast cancer represents 15% of the confocal microscopy. breast cancers, but this tumor subtype expresses elevated Src kinase compared with ER-positive breast cancer (39, 40). ER/PR/HER2-negative breast cancer presents more Grant Support The study was supported by R01DK068432, Breast Cancer Relief Foun- frequently in younger women and in African-American dation, New Orleans, LA; Kinex Pharmaceuticals LLC, Buffalo, NY; women (41). In urban hospitals that treat a larger propor- Clinical and Translational Research, Education and Commercialization Project, Tulane, to B.G. Rowan. tion of African-American women, the incidence of ER/ The costs of publication of this article were defrayed in part by the PR/HER2-negative breast cancer is higher than 15%. payment of page charges. This article must therefore be hereby marked Patients who present with this tumor subtype represent advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. an unmet medical need for clinical care of breast cancer. Although it is unlikely that inhibition of Src alone would Received February 13, 2012; revised July 5, 2012; accepted July 5, 2012; significantly impact patient care, the present study published OnlineFirst July 10, 2012.

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Peptidomimetic Src/Pretubulin Inhibitor KX-01 Alone and in Combination with Paclitaxel Suppresses Growth, Metastasis in Human ER/PR/HER2-Negative Tumor Xenografts

Muralidharan Anbalagan, Alaa Ali, Ryan K. Jones, et al.

Mol Cancer Ther 2012;11:1936-1947. Published OnlineFirst July 10, 2012.

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