Open Full Page

Published OnlineFirst December 17, 2014; DOI: 10.1158/1535-7163.MCT-14-0337

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Interactions of Multitargeted Kinase Inhibitors and Nucleoside Drugs: Achilles Heel of Combination Therapy? Vijaya L. Damaraju1, Michelle Kuzma1, Delores Mowles1, Carol E. Cass1, and Michael B. Sawyer1,2

Abstract

Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazo- leading to reduced intracellular gemcitabine and FLT accumu- panib, and sunitinib are used to treat many solid tumors. lation. Pretreatment or cotreatment of Caki-1 cells with TKIs Combination trials of TKIs with gemcitabine, a nucleoside reduced cellular accumulation of [3H]nucleosides, suggesting anticancer drug, in pancreas, renal, lung, ovarian, and other that TKI scheduling with nucleoside drugs would influence malignancies resulted in little benefit to patients. TKI interac- cytotoxicity. In combination cytotoxicity experiments that com- tions with human nucleoside transporters (hNT) were studied pared sequential versus simultaneous addition of drugs in by assessing inhibition of [3H]uridine uptake in yeast produc- Caki-1 cells, cytotoxicity was greatest when gemcitabine was ing recombinant hNTs individually and in cultured human added before TKIs. In clinical settings, TKI inhibitor concentra- cancer cell lines. Axitinib, pazopanib, and sunitinib inhibited tions in tumor tissues are sufficient to inhibit hENT1 activity, hENT1 at low micromolar concentrations. In A549, AsPC-1, thereby reducing nucleoside chemotherapy drug levels in can- and Caki-1 cells, [3H]uridine, [3H]thymidine, [3H]gemcitabine, cer cells and reducing efficacy in combination schedules. An and [3H]fluorothymidine (FLT) accumulation was blocked by additional unwanted interaction may be reduced FLT uptake in all three TKIs. Pazopanib > axitinib sunitinib inhibited tumor tissues that could lead to aberrant conclusions regarding hENT1 with IC50 values of 2, 7, and 29 mmol/L, respectively, tumor response. Mol Cancer Ther; 14(1); 236–45. 2014 AACR.

Introduction (8). Similarly axitinib and pazopanib are used to treat advanced RCC, soft tissue sarcomas, pancreatic, and lung cancers (6, 9–16). Oral multitargeted tyrosine kinase inhibitors (TKI) have Combinations of conventional cytotoxic drugs such as activity towards VEGF receptors (VEGFR), platelet-derived gemcitabine, a nucleoside analog that targets cells in S-phase of growth factor receptors (PDGFR), stem cell factor receptor the cell cycle, with novel agents that target key signaling pathways (KIT), and other tyrosine kinases. VEGF is a mediator of that control cancer cell survival, proliferation, and/or invasion is a angiogenesis and contributes to tumor growth and metastasis promising approach, and has been attempted in several clinical (1, 2), whereas PDGF activates growth and survival of vascular trials (17–21). Clinical trials of combinations of TKIs with smooth muscle cells and recruitment and differentiation of gemcitabine have been attempted in pancreatic, bladder, pericytes (3, 4). TKI inhibition of tumor angiogenesis and NSCLC, ovarian, and other malignancies with little benefitto other signaling pathways associated with tumor development patients (9, 22–24). These disappointing results suggest that there resulted in promising antitumor activity against many solid may be unfavorable interactions between TKIs and nucleoside tumor types including renal cell cancer (RCC), pancreatic chemotherapy drugs. cancer, gastrointestinal stromal tumors (GIST), and non–small In humans, nucleoside transport is mediated by two cell lung cancer (NSCLC; refs. 5–7). unrelated protein families, the SLC28 family of concentrative Among oral TKIs approved for clinical use, axitinib (AG- nucleoside transporters (CNT) and the SLC29 family 013736), pazopanib (GQ786034), and sunitinib (SU11248) of equilibrative nucleoside transporters (ENT; ref. 25). SLC28 are used to treat several solid tumor types. Sunitinib is used to and SLC29 families have three human concentrative (hCNT1/ treat advanced RCC, pancreatic neuroendocrine tumors, and GIST 2/3) and four human equilibrative members (hENT1/2/3/ 4), respectively. Nucleosides and nucleoside analog drugs are transported into cells by hENT1 and hENT2) and 1Department of Oncology, University of Alberta, Edmonton, Alberta, hCNT1, hCNT2, and hCNT3. Roles of hNTs in transport of Canada. 2Department of Medical Oncology, Cross Cancer Institute, nucleoside and nucleoside drugs are summarized in recent Edmonton, Alberta, Canada. reviews (25–27). Corresponding Author: Michael B. Sawyer, Department of Medical Oncology, Earlier studies indicated that TKIs may interfere with uptake of Cross Cancer Institute, 11560 University Avenue NW, Edmonton, Alberta T6G nucleoside chemotherapy drugs (28–31) and more recently 1Z2, Canada. Phone: 780-432-8248; Fax: 780-432-8888; E-mail: Damaraju and colleagues (32) showed that erlotinib, gefitinib, [email protected] and vandetanib compete with nucleoside drugs for cellular uptake doi: 10.1158/1535-7163.MCT-14-0337 and hence lead to reduced efficacy of combination treatments in 2014 American Association for Cancer Research. cytotoxicity studies. As TKIs are widely used, in the current study

236 Mol Cancer Ther; 14(1) January 2015

Human Nucleoside Transporter Inhibition by Kinase Inhibitors

we examined interactions of human nucleoside transporters replicates per concentration and experiments were repeated three (hNTs) with multitargeted TKIs axitinib, pazopanib, and times. sunitinib and resulting effects on combination cytotoxicity in cultured human cancer cell lines. To study interactions of TKIs Nucleoside transport inhibition in A549, AsPC-1, and Caki-1 with hNTs, we examined inhibition of [3H]uridine transport in cells yeast cells producing each of five recombinant hNTs individually Cells (100,000/well) were seeded in 12-well plates and on the as well as inhibition of uridine and thymidine uptake and third day, uptake of [3H]nucleosides was measured at room gemcitabine accumulation in three human cancer cell lines, temperature in transport buffer (pH 7.4) containing 20 mmol/ pancreatic adenocarcinoma AsPC-1, NSCLC A549, and RCC L Tris, 3 mmol/L K2HPO4, 1 mmol/L MgCl2, 1.4 mmol/L CaCl2, Caki-1. We also examined sequential versus simultaneous and 5 mmol/L glucose with 144 mmol/L NaCl hereafter termed combination cytotoxicity with gemcitabine and TKIs in Caki-1 transport buffer. For uridine uptake assays, cell growth medium cells. was aspirated, cells were washed with sodium or sodium-free buffer, [3H]uridine was added and uptake was measured over fi Materials and Methods xed time points in the presence or absence of established NT inhibitors (NBMPR, dilazep) or TKIs. Sunitinib's effects on Materials kinetics of uridine uptake were determined in A549 cells at Nitrobenzylmercaptopurineribonucleoside(NBMPR),dilazep, graded concentrations of [3H]uridine (0–1,000 mmol/L) at 0, dipyridamole, unlabeled nucleosides, and other chemicals were 25, 50, or 100 mmol/L of sunitinib using 30-second incubations obtained from Sigma Chemical Company. Tritiated nucleosides from a period during which initial time courses of [3H]uridine were purchased from Moravek Biochemicals. Tissue culture (96- uptake were shown to be linear. At the end of uptake intervals, and 12-well) plates and flasks were from VWR International. Cell permeant-containing solutions were removed by aspiration, culture media and FBS were from Gibco BRL. Ecolite was from ICN and cells were quickly rinsed twice with sodium buffer and Pharmaceuticals. Dojindo Cell Counting Kit-8 (CCK-8) was from solubilized with 5% TritonX-100. Radioactivity in solubilized Dojindo Molecular Technologies, Inc. Axitinib, pazopanib, and extracts was measured by liquid scintillation counting. Uptake sunitinib were from LC Laboratories. values were expressed as pmol/106 cells and graphs were generated using the Prism software. Each experiment was Cell culture conducted two or three times with triplicate measurements. Human cancer cell lines, A549 (NSCLC), AsPC-1 (pancreatic For [3H]uridine and [3H]thymidine accumulation experiments, cancer), and Caki-1 (RCC) were obtained from ATCC in 2005, A549, AsPC-1, and Caki-1 cells were exposed to either 10 mmol/L 2001, and 2002, respectively. Cell lines were sent to DDC [3H]uridine or 1 mmol/L [3H]thymidine for 60 minutes in the Medical to verify their authenticity by STR profiling in July absence or presence of 5 or 25 mmol/L sunitinib or 100 mmol/L 2013 for A549 and AsPC-1 and February 2014 for Caki-1 cells dilazep (an inhibitor of hENT1 and hENT2; ref. 36) and processed and mycoplasma status. Results showed that A549 and AsPC-1 as described above for uptake assays. Effects of TKIs on 1 mmol/L were 100%, whereas Caki-1 was >80% matched to the ATCC [3H]gemcitabine or 10 mmol/L [3H]uridine uptake (15 minutes) panel of markers and all three were free of mycoplasma. Cells into cells were studied in the absence or presence of 10 mmol/L were maintained in RPMI1640 medium supplemented with inhibitor. 10% FBS, 2 mmol/L L-glutamine. All cultures were kept at 37 C For sequencing of exposure to nucleosides and TKIs, uptake in 5% CO2/95% air and subcultured at 2- to 3-day intervals to experiments were conducted as follows. In the first set of maintain exponential growth. Transport and cytotoxicity experiments, control cells were incubated in buffer without experiments were conducted with cells in the exponential drug for 15 minutes after which 10 mmol/L [3H]uridine, growth phase. 1 mmol/L [3H]gemcitabine, or 1 mmol/L [3H]fluorothymidine (FLT) was added and uptake was measured for 15 minutes. In Uridine transport in Saccharomyces cerevisiae the second set of experiments, cells were incubated with 10 mmol/ Saccharomyces cerevisiae yeast were separately transformed L axitinib, pazopanib, or sunitinib for 15 minutes after which the with plasmids (pYPhENT1, pYPhENT2, pYPhCNT1, pYPhCNT2, drug was removed and uptake of 10 mmol/L [3H]uridine, 1 mmol/ or pYPhCNT3) encoding hNTs (hENT1, hENT2, hCNT1, hCNT2, L[3H]gemcitabine, or 1 mmol/L [3H]FLT was measured for or hCNT3, respectively) as described elsewhere (33, 34). Uptake 15 minutes. In the third set of experiments, 10 mmol/L of 1 mmol/L [3H]uridine (Moravek Biochemicals) into yeast [3H]uridine, 1 mmol/L [3H]gemcitabine, or 1 mmol/L [3H]FLT was measured as previously described (34, 35) using the was added for 15 minutes after which media was removed and 10 semiautomated cell harvester (Micro96 HARVESTER; Skatron mmol/L axitinib, pazopanib, or sunitinib was added. In the last set Instruments). Yeast were incubated at room temperature with 1 of experiments, cells were incubated in buffer without any drug for mmol/L [3H]uridine in yeast growth media (pH 7.4) in the presence 15 minutes after which the buffer was removed and either 10 or absence (uninhibited controls) of graded concentrations of test mmol/L [3H]uridine, 1 mmol/L [3H]gemcitabine, or 1 mmol/L compounds. Uridine self-inhibition was used to determine [3H]FLT was added together with 10 mmol/L axitinib, 10 mmol/ maximum inhibition of mediated transport. L pazopanib, or 10 mmol/L sunitinib and incubated for 15 Concentration–effect curves were subjected to nonlinear minutes. At the end of these time points, media was removed regression analysis using Prism software (version 4.03; and cells were processed for radioactivity as described above. GraphPad Software Inc.) to obtain the concentration of test compound that inhibited uridine uptake by 50% relative to Cytotoxicity assays that of untreated cells (IC50 values). Each IC50 value Dojindo CCK-8 was used to quantify drug-induced determination was conducted with nine concentrations and six cytotoxicity. A549, AsPC-1, or Caki-1 cells were seeded in

www.aacrjournals.org Mol Cancer Ther; 14(1) January 2015 237

Damaraju et al.

96-well plates and allowed to attach for 24 hours. Cells were Axitinib then exposed to graded concentrations of sunitinib for 72 H hours. Effects of NBMPR on sunitinib toxicity were tested in N N A549 cells by exposing cells to sunitinib in the absence or O presence of 1 mmol/L NBMPR for 72 hours after which they were treated with CCK-8 reagent for cytotoxicity assessment. HN S For evaluation of in vitro synergy of combinations of gemcitabine with the TKIs, experiments were based on the N individual drug's IC50 value. For sequential treatments, cells were incubated with either drug for 24 hours, followed by incubation in drug-free media for 24 hours and subsequent Pazopanib incubation for 72 hours with the other drug; simultaneous or individual treatments were performed for 72 hours. Drug NH2 synergy was determined by the isobologram and combination OOS index (CI) methods, derived from the median effect principle of Chou and Talalay (37) using the CalcuSyn software (Biosoft). Using data from growth inhibitory experiments and N computerized software, CI values were generated over a range of N fraction affected (Fa) levels from 0.05 to 0.90 (5%–90% growth N N N N inhibition). A CI value of 1 indicates an additive effect between H two agents, whereas a CI value <1or>1 indicates synergism or antagonism, respectively. Sunitinib

Statistical analysis O One-way ANOVA was used for statistical analysis using N GraphPad Prism software. N F H Results NH Inhibition of uridine uptake mediated by recombinant hNTs O produced in Saccharomyces cerevisiae N Axitinib, pazopanib, and sunitinib (chemical structures shown H in Fig. 1) were assessed for their relative abilities to inhibit 3 NBMPR [ H]uridine uptake by each of the five hNTs in concentration- dependent inhibition experiments to determine IC50 (inhibitor N N concentration that produced 50% inhibition of transport) values. HO A representative concentration–effect curve for sunitinib O S inhibition of hENT1-mediated uridine transport in yeast is N − shown in Fig. 2A. IC50 values obtained from such experiments HO N O with axitinib, pazopanib, and sunitinib in yeast producing each of N+ the five recombinant NTs are presented in Table 1. For hENT1, OH O pazopanib, sunitinib, and axitinib IC50 values ( S.E.) were 4 0.3, 31 5, and 46 4 mmol/L, respectively. Inhibition of hENT2, Figure 1. hCNT1, hCNT2, and hCNT3 was seen with IC50 values ranging Structures of axitinib, pazopanib, sunitinib, and NBMPR. from 80 to 210 mmol/L with the exception of axitinib (hCNT1 and hCNT3) and pazopanib (hENT2) where IC50 values were > m 300 mol/L and thus could not be determined. We examined accumulation of [3H]thymidine in all 3 cell lines by following long-term uptake (1 hour) of 1 mmol/L Inhibition of hENT1 mediated [3H]uridine and [3H]thymidine [3H]thymidine in the absence or presence of 5 or 25 mmol/L uptake by sunitinib in human cancer cell lines of sunitinib or 100 mmol/L dilazep. Both sunitinib concentrations A549 cells possess predominantly hENT1 with a minor (5 and 25 mmol/L) inhibited thymidine uptake (Fig. 2C) in all cell component of hENT2 activities (32). Sunitinib, which inhibited lines tested, although neither concentration achieved complete hENT1 in yeast radiotracer experiments, was tested in A549 cells to inhibition as was seen with 100 mmol/L dilazep. evaluate its effects on native hENT1-mediated uptake in human cells. Figure 2B shows inhibition of [3H]uridine uptake in A549 Effect of TKIs on kinetics of uridine uptake in hENT1-producing cells by graded concentrations of sunitinib. Similar concentration– yeast effect studies were conducted with axitinib, pazopanib, and The nature of interaction of TKIs with hENT1 were further sunitinib in A549, AsPC-1, and Caki-1 cells and resulting IC50 examined in hENT1 producing yeast cells by studying effects of values (mean SE) are presented in Table 1, and in all situations, fixed axitinib (Fig. 3A), pazopanib (Fig. 3B), or sunitinib (Fig. 3C) 3 – significant hENT1 inhibition was observed with IC50 values ranging concentrations on kinetics of [ H]uridine uptake (Fig. 3A C). from 1 to 29 mmol/L. Although analysis of results using a Lineweaver–Burk plot showed

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

Human Nucleoside Transporter Inhibition by Kinase Inhibitors

A an apparent competitive nature of inhibition of uridine uptake by axitinib, pazopanib, and sunitinib, suggesting interaction 100 Yeast of the TKIs with the nucleoside-permeant binding site, such inhibition can also be achieved by binding to a separate 75 allosteric site that prevents binding of substrate (i.e., permeant) to its site (38).

50 Effects of sequencing of administration of TKIs and nucleosides

(% control) on nucleoside retention in cells

H]Uridine uptake H]Uridine uptake Our results thus far showed that TKIs interfered with uptake 3 25 [ of nucleosides in three model cell lines tested. We examined this further in Caki-1 cells to see whether changes in nucleoside 0 uptake occur when two agents are added separately in −7 −6 −5 −4 sequence or simultaneously together. [3H]Uridine (10 mmol/ Log [sunitinib] (mol/L) L), [3H]gemcitabine (1 mmol/L), or [3H]FLT (1 mmol/L) uptake was measured in Caki-1 cells that were either treated without or with 10 mmol/L of each TKI for 15 minutes before or B after exposure to radiolabeled nucleoside or during exposure for 15 minutes. Results of sequencing of administration on 100 A549 cells nucleoside accumulation are shown Fig. 4A–C. Signiﬁcant inhibition (P < 0.05) of nucleoside/drug accumulation occurred when TKIs were combined with uridine or gemcitabine or 75 FLT during simultaneous exposures or were administered before nucleosides, whereas no effects were observed when TKI exposures 50 were after uridine exposure.

(% control) Cytotoxicity and synergy studies H]Uridine uptake H]Uridine uptake 25 3 [ Our results presented above suggested that a sequential schedule for combination of nucleoside drugs with TKI 0 inhibitors in which administration of a nucleoside drug −7 −6 −5 −4 followed by a TKI inhibitor would result in better synergy than Log [sunitinib] (mol/L) any other sequence. Sunitinib cytotoxicity was tested in A549 cells in absence or presence of 1 mmol/L NBMPR, a potent and speciﬁc inhibitor of hENT1 (Fig. 5A). Results showed that NBMPR had no C effect on sunitinib cytotoxicity thus indicating that sunitinib is not a permeant for hENT1. Gemcitabine was equally cytotoxic to A549 AsPC-1 Caki-1 A549, AsPC-1, and Caki-1 cells (data not shown) with IC50 values 100 (mean SE) of 2.2 0.2, 3.5 0.8, and 4.2 0.1 mmol/L, respectively. In cytotoxicity studies conducted over 72 hours, 75 axitinib, pazopanib, and sunitinib were cytotoxic to A549 cells (Fig. 5B). All three cell lines were sensitive to sunitinib with IC50 values of 5 mmol/L. AsPC-1 was insensitive to axitinib but was 50 sensitive to pazopanib with IC50 value of 35 mmol/L. Caki-1 cells (% control) were sensitive to axitinib and pazopanib with IC50 values of 25

H]Thymidine uptake uptake H]Thymidine 25 and 30 mmol/L. 3 [ We explored cytotoxicity of sequential and simultaneous 0 administration of gemcitabine with axitinib or pazopanib in Caki-1 cells. For in vitro combination studies cells were (i) pretreated with axitinib or pazopanib before gemcitabine, Control Control Control (ii) pretreated with gemcitabine before axitinib or pazopanib, or (iii) treated with both agents together or alone as described

5 µmol/L sunitinib 5 µmol/L sunitinib 5 µmol/L sunitinib 25 µmol/L100 µsunitinibmol/L dilazep 25 µmol/L100 µsunitinibmol/L dilazep 25 µmol/L100 µsunitinibmol/L dilazep Error bars are not shown where SE values are smaller than the size of the symbol. Each experiment was repeated three times. B, effects of increasing Figure 2. sunitinib concentrations on [3H]uridine uptake in A549 cells. C, [3H]thymidine Sunitinib's effects on [3H]uridine uptake in yeast and [3H]uridine and accumulation in A549, AsPC-1, and Caki-1 cells. Incubations with 1 mmol/L [3H]thymidine uptake in A549 cells. Yeast cells were incubated with 1 mmol/L [3H]thymidine for 60 minutes were conducted in the absence (open bars) or [3H]uridine for 10 minutes in the absence or presence of increasing sunitinib presence of 5 (dotted bars) or 25 (hatched bars) mmol/L sunitinib or 100 concentrations (0–300 mmol/L; A). Data presented are an average of three mmol/L dilazep (solid bars) and cell-associated radioactivity was measured. experiments each conducted with six replicates per concentration and data Values plotted are percentage of control values obtained in the absence of are expressed as mean SE values. Uptake values represent percentage of additives and average values from two or more experiments are shown in uridine uptake in the presence of TKIs relative to that in its absence (control). each panel.

www.aacrjournals.org Mol Cancer Ther; 14(1) January 2015 239

Damaraju et al.

Table 1. Summary of IC50 values for uridine transport inhibition in yeast and cell and hCNT2 and pazopanib inhibited hENT1 and all three hCNTs lines equally well. In experiments with cultured human cancer cell Transporter (yeast) Axitinib Sunitinib Pazopanib lines, hENT1-mediated uridine was inhibited by sunitinib, IC (mmol/L SE) 50 pazopanib, and axitinib with pazopanib inhibiting hENT1 at hENT1 46 431 54.0 0.3 hENT2 180 44 210 4 >300 very low concentrations followed by axitinib and sunitinib. hCNT1 >300 80 6 170 13 Uridine uptake in hENT1-producing yeast cells was inhibited hCNT2 130 10 200 20 150 12 by axitinib, pazopanib, and sunitinib in a competitive manner hCNT3 >300 130 7120 3 that suggests an apparent competitive inhibition of uridine

Cell lines (hENT1) A549 7.0 1.0 26.0 2.0 2 0.3 A ASPC-1 5.0 0.1 25.0 5.0 1 0.1 6.0 hENT1-yeast Caki-1 7.0 1.0 29.0 9.0 2 0.2 Axitinib NOTE: Inhibition of [3H]uridine uptake by TKIs was assessed in yeast producing each of the ﬁve recombinant hNTs in concentration–effect experiments as 4.5 described in Materials and Methods. Inhibition experiments were also conducted in cell lines all of which have major intrinsic hENT1 activity and negligible or no 3.0 CNT or hENT2 activities. IC50 values (mean SE) are listed above. H]Uridine 3 [ (1/pmol 30 min) in Materials and Methods. Isobologram and CI methods 1.5 developed by Chou and Talalay (37) were used to conﬁrm and quantify the synergism observed with various combinations. Isobolograms were constructed for Fa values 0123 of 0.50, 0.75, and 0.90, representing 50%, 75%, and 90% 1/ [uridine] (µmol/L) growth inhibition, respectively, and are shown in Fig. 5C–F. These results indicated that the sequence, gemcitabine followed by TKIs, was synergistic (CI values at Fa 50% were 0.4 and 0.7 B for axitinib and pazopanib; Fig. 5C and D), whereas the sequence hENT1-yeast TKI followed by gemcitabine (CI values at Fa 50% were 2 and 2.2 7.5 Pazopanib for axitinib and pazopanib, Fig. 5E and F) or simultaneous treatment (CI values at Fa 50% were 3 and 2 for axitinib and pazopanib) was antagonistic, suggesting that inhibition of hNTs 5.0 by TKIs would lead to decreased effectiveness of combinations H]Uridine 3

involving these agents and nucleoside chemotherapy drugs. [

(1/pmol 30 min) 2.5 Discussion Several TKIs (e.g., sunitinib, pazopanib, and axitinib) targeting 0.0 VEGFR have been approved by regulatory agencies for treatment 1 23 of RCC and GIST in ﬁrst-, second- or third-line settings (8, 39–41). 1/ [uridine] (µmol/L) In addition, other therapeutic approaches include treatment with sunitinib in advanced pancreatic neuroendocrine tumors C (42). In a recent randomized phase II study in 113 patients hENT1-yeast with advanced pancreatic adenocarcinoma, the combination 2.0 of gemcitabine with sunitinib did not lead to improved Sunitinib progression-free survival when compared with gemcitabine 1.5 alone (23). In a phase III study of patients with advanced pancreatic adenocarcinoma, addition of axitinib to gemcitabine did not lead to improved overall survival (9, 12, 24). In an earlier 1.0 H]Uridine 3

study, we presented results on interactions of erlotinib, geﬁtinib, [

and vandetanib with the hNTs (32). This interaction was (1/pmol 30 min) 0.5 attributed to similarities of these and other TKIs to the classical hENT1 inhibitor NBMPR with the underlying implication that 0.0 TKIs, when combined with nucleoside chemotherapy, inhibit 0123 hNTs and therefore are likely to interfere with nucleoside chemotherapy cytotoxicity. 1/ [uridine] (µmol/L) In this study, we showed that sunitinib, pazopanib, and Figure 3. axitinib inhibited transport of uridine by recombinant hNTs 3 produced individually in yeast. hENT1 was inhibited at low Effects of axitinib or pazopanib or sunitinib on kinetics of [ H]uridine uptake in hENT1-producing yeast. Effects of 0 (&), 5 (~), 25 (~), 50 (^), or 100 (*) concentrations by sunitinib, pazopanib, and axitinib, whereas mmol/L of axitinib (A), pazopanib (B), and sunitinib (C) on uptake of the other hNTs were inhibited at much higher concentrations. [3H]uridine at various concentrations (0–1,000 mmol/L). Values with mean Sunitinib inhibited hENT1 and hCNT1; axitinib inhibited hENT1 SE are shown in each panel. Each experiment was repeated three times.

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

Human Nucleoside Transporter Inhibition by Kinase Inhibitors

A uptake by axitinib, pazopanib, and sunitinib although such Caki-1 cells + 10 µmol/L inhibition could also be achieved by binding to an allosteric Axitinib Pazopanib Sunitinib 100 site (38). [3H]Uridine and [3H]thymidine accumulation in three cell lines was inhibited by 5 and 25 mmol/L sunitinib, respectively. 75 In addition, we tested effects of changing the sequence of administration of nucleosides with TKIs on [3H]nucleoside 50 accumulation in Caki-1 cells. Cells were treated with 3

(% control) (i) [ H]nucleoside alone for 15 minutes, (ii) a combination of 3 H]Uridine uptake H]Uridine uptake [ H]nucleoside with individual TKIs for 15 minutes, (iii) 3 25 [ individual TKIs for 15 minutes followed by [3H]nucleoside, or 0 (iv) combination of TKIs with nucleosides for 15 minutes. Simultaneous as well as sequential (when a TKI was given Urd Urd Urd TKI Urd TKI Urd TKI Urd before a nucleoside) administration of either pazopanib or axitinib with [3H]nucleoside resulted in a large decrease in Urd + TKI Urd + TKI Urd + TKI TKI TKI TKI nucleoside accumulation. Caki IC values (Table 1) were Urd Urd Urd 50 predictive of results observed wherein pazopanib (IC50 value, 2 mmol/L) had large effects in both dosing schedules followed B by axitinib with modest effects, whereas the magnitude of the m Caki-1 cells + 10 µmol/L inhibition by sunitinib (IC50 value, 29 mol/L) was much Axitinib Pazopanib Sunitinib less consistent with its IC value for inhibition of hENT1 100 50 activity. Although TKIs appeared to be interacting with nucleoside- 75 permeant binding sites of hENT1, they appeared not to be transported by hENT1 based on results of cytotoxicity 50 experiments with sunitinib in the absence or presence of the

H]Gemcitabine hENT1 transport inhibitor NBMPR, although direct evidence 3

[ 25 uptake (% control) uptake for their lack of transportability would require measurement of uptake of [3H]-labeled TKIs. In combination cytotoxicity studies, 0 Caki-1 cells showed greater sensitivity to drug combinations when they were exposed to gemcitabine for 24 hours followed by either Urd Urd Urd Urd TKI Urd TKI Urd TKI axitinib or pazopanib as predicted from our uptake inhibition studies. Earlier reports indicated interaction of TKIs with ATP- Urd + TKI Urd + TKI TKI TKI Urd + TKI TKI Urd Urd Urd binding domains of ATP-binding cassette (ABC) transporter- mediated multidrug resistance (MDR) proteins in cancer cells (43–45). Inhibition of P-glycoprotein (P-gp) activity (46) C resulted in enhanced cytotoxic effects of multiple anticancer Caki-1 cells + 10 µmol/L Axitinib Pazopanib Sunitinib drugs by increasing accumulation of P-gp and ATP-binding 100 cassette subfamily G member 2 (ABCG2) substrates (47, 48). Earlier studies showed inhibition of hENT1-mediated activity in 75 K562 cells by p38 MAPK inhibitors (29), and our current and previous results (32) indicate that another group of potential

H]FLT 50 3 target proteins are hNTs. [ Effects of axitinib, pazopanib, and sunitinib inhibition of hNTs 25 fi uptake (% control) uptake on nucleoside chemotherapy ef cacy need to be addressed and be made known to medical oncologists. There is pharmacologic 0 evidence that suggests that there have been issues with TKIs and nucleoside chemotherapy (9, 12, 24). Although it is Urd Urd Urd TKI Urd TKI Urd TKI Urd difficult to extrapolate in vitro studies to the clinic, especially with drugs that have such extensive protein binding and Urd + TKI Urd + TKI Urd + TKI TKI TKI TKI Urd Urd Urd accumulation in tumors, TKI inhibitors achieve levels in tissues that could inhibit hENT1. In the phase I study of pazopanib, Figure 4. Hurwitz and colleagues (49) found at a dose of 800 mg daily that Effects of sequencing of administration of nucleosides with TKIs on m 3 3 m pazopanib plasma levels were 103 mol/L, and we found that accumulation of [ H]nucleosides in Caki-1 cells. [ H]Uridine (10 mol/L), m [3H]gemcitabine (1 mmol/L), or [3H]FLT (1 mmol/L) uptake was measured in pazopanib's IC50 value for hENT1 inhibition was 2 mol/L for Caki-1 cells that were either treated without (open bars) or with 10 mmol/L of A549 cells. The FDA-approved product monograph indicates that each of the TKIs for 15 minutes after nucleoside (dotted bars), or before pazopanib plasma concentrations are 132 mmol/L which would the nucleoside (hatched bars) or together (solid bars) for 15 minutes as inhibit hENT1 completely thus blocking accumulation of described in Materials and Methods. Effect of TKIs on uptake of 10 mmol/L 3 3 3 cytotoxic nucleoside drugs. In a study of neoadjuvant breast [ H]uridine or 1 mmol/L [ H]gemcitabine or 1 mmol/L [ H]FLT over 15 minutes fi – cancer patients, mean ge tinib plasma levels at steady state are presented in A C, respectively. Values plotted are percentage of control m m values obtained in the absence of additives and average values from two or were 0.18 g/mL (0.40 mol/L) and mean tumor levels were more experiments are shown in each panel. 7.5 mg/g (17 mmol/L), an approximately 42-fold difference

www.aacrjournals.org Mol Cancer Ther; 14(1) January 2015 241

Damaraju et al.

100 100

75 75

50 50 (% control) (% control) Cell viability 25 Cell viability 25 A549 Cells A549 Cells Figure 5. 0 0 − − − − − − − − − Cytotoxicity studies in A549and Caki-1 cell 7 6 5 4 8 7 6 5 4 lines. A, A549 cells were exposed to graded Log [sunitinib] (mol/L) Log [TKI] (mol/L) concentrations (0–100 mmol/L) of sunitinib in absence (*) or presence of 1 mmol/L NBMPR (*) for 72 hours. Cell viability was measured by CDCCK-8 assay kit as described in Materials and Methods. B, A549 cells were treated with axitinib (*), pazopanib (&), or sunitinib (~) 0.04 0.04 for 72 hours. Plotted are percentage of control Caki-1 cells Caki-1 cells values relative to untreated cells and 0.03 0.03 representative experiments with mean SD are shown in each panel. Each experiment was 0.02 0.02 repeated three times. C and D, isobologram analysis of the sequential combination of gemcitabine, followed by axitinib or pazopanib 0.01 0.01 in Caki-1 cells. Isobologram analysis for the sequence axitinib or pazopanib followed by [Gemcitabine] ( µ mol/L) [Gemcitabine] ( µ mol/L) 0.00 0.00 gemcitabine is shown in E and F. Individual 0 5 0 200 400 600 doses of gemcitabine and TKIs to achieve 90% 10 15 20 (dotted line) growth inhibition (Fa ¼ 0.90), [Pazopanib] (µmol/L) [Axitinib] (mmol/L) 75% (hyphenated line) growth inhibition (Fa ¼ 0.75), and 50% (on the axes, closed triangle) growth inhibition (Fa ¼ 0.50) were EF plotted on x-andy-axes. CI values calculated using CalcuSyn software are represented by 0.3 points above (indicate antagonism between Caki-1 cells 0.04 Caki-1 cells drugs) or below the lines (indicate synergy). *,ED50; *,ED75;and&,ED90, respectively. 0.2 0.03

0.02 0.1 0.01

[Gemcitabine] ( µ mol/L) 0.0 [Gemcitabine] ( µ mol/L) 0.00 0 5 0 200 400 600 10 15 20 25 [Axitinib] (mmol/L) [Pazopanib] (µmol/L)

(50, 51). In patients with lung cancer, geﬁtinib tumor levels were Unlike sunitinib, axitinib levels have not been studied in tumor approximately 40-fold higher than plasma levels (22.7 vs. 0.52 tissues. Reyner and colleagues (54) studied axitinib levels in mmol/L; ref. 51). Gotink and colleagues (52) reported tumor normal tissues in mice and found that the highest tissue to sunitinib levels in mice and in patients with RCC treated with plasma partitioning ratio of axitinib was in the liver, around sunitinib. Mice were treated with sunitinib for 4 weeks at a dose of 3.8-fold. Assuming that axitinib would only concentrate 3.8- 40 mg/kg daily after which sunitinib tumor levels were 9 mmol/L fold in tumors, the concentration average at steady state in and plasma levels were 1 mmol/L. Patients were treated with tumors would be 0.1 mmol/L, whereas assuming that it sunitinib doses ranging from 37.5 mg/day to 50 mg/day for 4 concentrates in tumors like sunitinib, the concentration average weeks before tumor biopsy. In patients with RCC, sunitinib tumor at steady state in tumors would be 1 mmol/L. It is also important to levels were 9 mmol/L, 30-fold higher than plasma levels (0.3 consider how axitinib and gemcitabine were administered in the mmol/L). We found that sunitinib's IC50 for hENT1 inhibition clinical trial. Axitinib was administered 0.5 hours before the start in A549 cells was 26 mmol/L with nearly 30% to 40% inhibition of of the gemcitabine infusion (14), yielding axitinib levels at the hENT1 at 10 mmol/L sunitinib. Patients with advanced RCC start of the infusion of approximately 10 ng/mL and 2 hours after taking axitinib 5 mg twice a day had a maximum observed the end of the gemcitabine (half-life 0.3 hours) infusion of plasma concentration of 27.8 ng/mL and an area under the approximately 20 ng/mL including a peak axitinib level of plasma concentration time curve at 24 hours of 265 ng/mL 28.2 ng/mL. It is therefore possible that NTs were exposed to (53) giving a concentration average at steady state of 11 ng/mL axitinib concentrations sufﬁcient to inhibit hENT1 to some extent.

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

Human Nucleoside Transporter Inhibition by Kinase Inhibitors

As our in vitro studies found an IC50 value for axitinib inhibition of results suggest that a sequence of gemcitabine followed by hENT1 of 5 to 7 mmol/L in three cell lines, there could be TKIs would result in synergistic cytotoxic effects. Implications significant (approximately 20%) hENT1 inhibition in tumors of this study in the clinic are that when nucleoside at concentrations less than 2 mmol/L. We cannot rule out chemotherapy drugs are administered concurrently with effects of TKIs on reduction of cell surface expression of hENT1 multitargetedTKIs,attentionshouldbepaidtosequenceof as was shown earlier using a fluorescent probe for evaluation of administration of these agents to achieve better response cell surface hENT1 sites (32). Thus, plasma levels from phase I profiles in patients. In addition, as FLT uptake is also studies of axitinib, pazopanib, and sunitinib taken together with inhibited by these TKIs, early assessment of tumor response studies of tissue and plasma levels of gefitinib suggest that IC50 by PET imaging should be monitored carefully with concerns values for hENT1 inhibition of axitinib, pazopanib, and sunitinib about 18F-FLT's utility as a noninvasive measure of decreased are relevant to the clinic. proliferation of tumors in patients treated with TKI-targeted Preclinical and clinical pharmacologic evidence indicates drug therapies. We are extending these studies to in vivo tumor- interactions between TKIs and nucleoside chemotherapy drugs bearing mice with [18F]FLT PET imaging studies to validate that are consistent with inhibition of hNTs by TKIs providing an our in vitro observations. explanation for the failure of combination therapies with multitargeted TKIs and nucleoside chemotherapy. fl Our study raises doubts about 18F-FLT's utility as a noninvasive Disclosure of Potential Con icts of Interest measure of decreased proliferation caused by targeted therapies V.L. Damaraju has ownership interest in a patent. M.B. Sawyer received speakers' bureau honoraria as a travel grant from Pfizer, has ownership interest such as TKIs. Earlier studies by Paproski and colleagues (55) in a patent regarding methods of combining tyrosine kinase inhibitors and have shown that hENT1 is necessary for uptake and retention methods for treating their side effects, and is a consultant/advisory board of 18F-FLT into cancer cells. Barthel and colleagues (56) showed in member for Pfizer. No potential conflicts of interest were disclosed by the in vivo experiments of 5-fluorouracil in radiation-induced other authors. fibrosarcoma-1 xenograft mouse that 18F-FLT was an earlier and more pronounced marker of decreased tumor proliferation Authors' Contributions 18 18 than F-fluro-deoxy-glucose. Liu and colleagues, (57) used F- Conception and design: V.L. Damaraju, C.E. Cass, M.B. Sawyer FLT to study sunitinib effects on solid tumors. Although they Development of methodology: V.L. Damaraju, C.E. Cass noted significant decreases in 18F-FLT uptake in patients treated Acquisition of data (provided animals, acquired and managed patients, with sunitinib, decreases in 18F-FLT uptake did not correlate with provided facilities, etc.): M. Kuzma, D. Mowles tumor response. We suspect that decreases in 18F-FLT uptake Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V.L. Damaraju, M.B. Sawyer observed by Liu and colleagues were mostly due to inhibition Writing, review, and/or revision of the manuscript: V.L. Damaraju, C.E. Cass, 18 of F-FLT uptake by sunitinib inhibition of hENT1 rather than to M.B. Sawyer decreased proliferation. In another study by Zhao and colleagues Administrative, technical, or material support (i.e., reporting or organizing (58), significant changes in FLT uptake were noticed before any data, constructing databases): M. Kuzma, D. Mowles change in tumor size. Our studies suggest that 18F-FLT might not Study supervision: V.L. Damaraju, M.B. Sawyer be a reliable marker of tumor proliferation when used with a TKI especially as our earlier study (32) showed that TKIs not only Grant Support compete with nucleoside binding on hENT1, but also decrease This work was supported by the Alberta Cancer Foundation (to M.B. Sawyer, cell surface expression of hENT1. C.E. Cass), the Canadian Cancer Society Research Institute (to C.E. Cass), and In summary, we have demonstrated that three multitargeted AstraZeneca (M.B. Sawyer). The costs of publication of this article were defrayed in part by the payment of TKIs, axitinib, pazopanib, and sunitinib, inhibit hENT1, a page charges. This article must therefore be hereby marked advertisement in ubiquitous hNT that is necessary for activity of many accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nucleoside chemotherapy drugs. We have also shown that these agents decrease accumulation of nucleoside drugs Received April 17, 2014; revised October 20, 2014; accepted October 21, when combined together or when TKIs are given first. Our 2014; published OnlineFirst December 17, 2014.

References 1. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 6. King JW, Lee SM. Axitinib for the treatment of advanced non-small-cell lung 1971;285:1182–6. cancer. Expert Opin Investig Drugs 2013;22:765–73. 2. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angio- 7. Nishida T, Doi T, Naito Y. Tyrosine kinase inhibitors in treatment of genic switch during tumorigenesis. Cell 1996;86:353–64. unresectable or metastatic gastrointestinal stromal tumors. Expert Opin 3. Appelmann I, Liersch R, Kessler T, Mesters RM, Berdel WE. Angiogenesis Pharmacother 2014;22:1–11. inhibition in cancer therapy: platelet-derived growth factor (PDGF) and 8. Majem M, Pallares C. An update on molecularly targeted therapies in vascular endothelial growth factor (VEGF) and their receptors: biological second- and third-line treatment in non-small cell lung cancer: focus on functions and role in malignancy. Recent Results Cancer Res 2010;180: EGFR inhibitors and anti-angiogenic agents. Clin Transl Oncol 2013;15: 51–81. 343–57. 4. Hellberg C, Ostman A, Heldin CH. PDGF and vessel maturation. Recent 9. Kindler HL, Ioka T, Richel DJ, Bennouna J, Letourneau R, Okusaka T, et al. Results Cancer Res 2010;180:103–14. Axitinib plus gemcitabine versus placebo plus gemcitabine in patients with 5. Escudier B, Porta C, Bono P, Powles T, Eisen T, Sternberg CN, et al. advanced pancreatic adenocarcinoma: a double-blind randomised phase 3 Randomized, controlled, double-blind, cross-over trial assessing treatment study. Lancet Oncol 2011;12:256–62. preference for pazopanib versus sunitinib in patients with metastatic renal 10. Motzer RJ, Escudier B, Tomczak P, Hutson TE, Michaelson MD, Negrier S, cell carcinoma: PISCES Study. J Clin Oncol 2014;32:1412–8. et al. Axitinib versus sorafenib as second-line treatment for advanced renal

www.aacrjournals.org Mol Cancer Ther; 14(1) January 2015 243

Damaraju et al.

cell carcinoma: overall survival analysis and updated results from a 31. Woodahl EL, Wang J, Heimfeld S, Ren AG, McCune JS. Imatinib inhibition randomised phase 3 trial. Lancet Oncol 2013;14:552–62. of fludarabine uptake in T-lymphocytes. Cancer Chemother Pharmacol 11. Motzer RJ, Hutson TE, Cella D, Reeves J, Hawkins R, Guo J, et al. Pazopanib 2008;62:735–9. versus sunitinib in metastatic renal-cell carcinoma. N Engl J Med 32. Damaraju VL, Scriver T, Mowles D, Kuzma M, Ryan AJ, Cass CE, et al. 2013;369:722–31. Erlotinib, gefitinib, and vandetanib inhibit human nucleoside transporters 12. Plummer R, Madi A, Jeffels M, Richly H, Nokay B, Rubin S, et al. A Phase I and protect cancer cells from gemcitabine cytotoxicity. Clin Cancer Res study of pazopanib in combination with gemcitabine in patients with 2014;20:176–86. advanced solid tumors. Cancer Chemother Pharmacol 2013;71:93–101. 33. Vickers MF, Kumar R, Visser F, Zhang J, Charania J, Raborn RT, et al. 13. Rini BI, Escudier B, Tomczak P, Kaprin A, Szczylik C, Hutson TE, et al. Comparison of the interaction of uridine, cytidine, and other pyrimidine Comparative effectiveness of axitinib versus sorafenib in advanced renal nucleoside analogues with recombinant human equilibrative nucleoside cell carcinoma (AXIS): a randomised phase 3 trial. Lancet 2011;378: transporter 2 (hENT2) produced in Saccharomyces cerevisiae. Biochem 1931–9. Cell Biol 2002;80:639–44. 14. Spano JP, Moore MJ, Pithavala YK, Ricart AD, Kim S, Rixe O. Phase I study 34. Zhang J, Visser F, Vickers MF, Lang T, Robins MJ, Nielsen LP, et al. of axitinib (AG-013736) in combination with gemcitabine in patients with Uridine binding motifs of human concentrative nucleoside transporters advanced pancreatic cancer. Invest New Drugs 2012;30:1531–9. 1 and 3 produced in Saccharomyces cerevisiae. Mol Pharmacol 15. Sternberg CN, Davis ID, Mardiak J, Szczylik C, Lee E, Wagstaff J, et al. 2003;64:1512–20. Pazopanib in locally advanced or metastatic renal cell carcinoma: results of 35. Vickers MF, Zhang J, Visser F, Tackaberry T, Robins MJ, Nielsen LP, et al. a randomized phase III trial. J Clin Oncol 2010;28:1061–8. Uridine recognition motifs of human equilibrative nucleoside transporters 16. van der Graaf WT, Blay JY, Chawla SP, Kim DW, Bui-Nguyen B, Casali PG, 1 and 2 produced in Saccharomyces cerevisiae. Nucleosides Nucleotides et al. Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a rando- Nucleic Acids 2004;23:361–73. mised, double-blind, placebo-controlled phase 3 trial. Lancet 2012;379: 36. Visser F, Vickers MF, Ng AM, Baldwin SA, Young JD, Cass CE. Mutation of 1879–86. residue 33 of human equilibrative nucleoside transporters 1 and 2 alters 17. Blackhall FH, O'Brien M, Schmid P, Nicolson M, Taylor P, Milenkova T, sensitivity to inhibition of transport by dilazep and dipyridamole. J Biol et al. A phase I study of Vandetanib in combination with vinorelbine/ Chem 2002;277:395–401. cisplatin or gemcitabine/cisplatin as first-line treatment for advanced non- 37. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the small cell lung cancer. J Thorac Oncol 2010;5:1285–8. combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme 18. Fountzilas G, Bobos M, Kalogera-Fountzila A, Xiros N, Murray S, Linardou Regul 1984;22:27–55. H, et al. Gemcitabine combined with gefitinib in patients with inoperable 38. Segel IH. Enzyme Kinetics. New York, NY: John Wiley and Sons; 1975. or metastatic pancreatic cancer: a phase II Study of the Hellenic Cooperative 39. Ayllon J, Beuselinck B, Morel A, Barrascout E, Medioni J, Scotte F, et al. Oncology Group with biomarker evaluation. Cancer Invest 2008;26 Long-term response and postsurgical complete remissions after treatment :784–93. with sunitinib malate, an oral multitargeted receptor tyrosine kinase 19. Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, et al. inhibitor, in patients with metastatic renal cell carcinoma. Cancer Invest Gefitinib in combination with gemcitabine and cisplatin in advanced non- 2011;29:282–5. small-cell lung cancer: a phase III trial–INTACT 1. J Clin Oncol 2004;22: 40. Dranitsaris G, Schmitz S, Broom RJ. Small molecule targeted therapies for 777–84. the second-line treatment for metastatic renal cell carcinoma: a systematic 20. Philips GK, Halabi S, Sanford BL, Bajorin D, Small EJ. A phase II trial of review and indirect comparison of safety and efficacy. J Cancer Res Clin cisplatin, fixed dose-rate gemcitabine and gefitinib for advanced urothelial Oncol 2013;139:1917–26. tract carcinoma: results of the Cancer and Leukaemia Group B 90102. BJU 41. Kruck S, Merseburger AS, Gakis G, Kramer MW, Stenzl A, Kuczyk MA. An Int 2008;101:20–5. update on the medical therapy of advanced metastatic renal cell carcinoma. 21. Philips GK, Halabi S, Sanford BL, Bajorin D, Small EJ. A phase II trial of Scand J Urol Nephrol 2008;42:501–6. cisplatin (C), gemcitabine (G) and gefitinib for advanced urothelial tract 42. Mankal P, O'Reilly E. Sunitinib malate for the treatment of pancreas carcinoma: results of Cancer and Leukemia Group B (CALGB) 90102. Ann malignancies–where does it fit? Expert Opin Pharmacother 2013;14: Oncol 2009;20:1074–9. 783–92. 22. Awasthi N, Schwarz MA, Schwarz RE. Antitumour activity of sunitinib in 43. Burger H, van Tol H, Boersma AW, Brok M, Wiemer EA, Stoter G, combination with gemcitabine in experimental pancreatic cancer. HPB et al. Imatinib mesylate (STI571) is a substrate for the breast cancer 2011;13:597–604. resistance protein (BCRP)/ABCG2 drug pump. Blood 2004;104: 23. Richly H, Maute L, Heil G, Russel J, Jager E. Prospective randomized phase II 2940–2. trial with gemictabine versus gemcitabine plus sunitinib in advanced 44. Elkind NB, Szentpetery Z, Apati A, Ozvegy-Laczka C, Varady G, Ujhelly O, pancreatic cancer. J Clin Oncol 31, 2013 (suppl; abstr 4035). et al. Multidrug transporter ABCG2 prevents tumor cell death induced by 24. Spano JP, Chodkiewicz C, Maurel J, Wong R, Wasan H, Barone C, et al. the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib). Efficacy of gemcitabine plus axitinib compared with gemcitabine alone in Cancer Res 2005;65:1770–7. patients with advanced pancreatic cancer: an open-label randomised phase 45. Erlichman C, Boerner SA, Hallgren CG, Spieker R, Wang XY, James CD, II study. Lancet 2008;371:2101–8. et al. The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 25. Young JD, Yao SY, Baldwin JM, Cass CE, Baldwin SA. The human concen- 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast trative and equilibrative nucleoside transporter families, SLC28 and cancer resistance protein-mediated drug efflux. Cancer Res 2001;61: SLC29. Mol Aspects Med 2013;34:529–47. 739–48. 26. Damaraju VL, Sawyer MB, Mackey JR, Young JD, Cass CE. Human nucle- 46. Mi Y, Lou L. ZD6474 reverses multidrug resistance by directly inhibiting the oside transporters: biomarkers for response to nucleoside drugs. Nucleo- function of P-glycoprotein. Br J Cancer 2007;97:934–40. sides Nucleotides Nucleic Acids 2009;28:450–63. 47. Dai CL, Tiwari AK, Wu CP, Su XD, Wang SR, Liu DG, et al. Lapatinib 27. Zhang J, Visser F, King KM, Baldwin SA, Young JD, Cass CE. The role of (Tykerb, GW572016) reverses multidrug resistance in cancer cells by nucleoside transporters in cancer chemotherapy with nucleoside drugs. inhibiting the activity of ATP-binding cassette subfamily B member 1 and Cancer Metastasis Rev 2007;26:85–110. G member 2. Cancer Res 2008;68:7905–14. 28. Huang M, Wang Y, Cogut SB, Mitchell BS, Graves LM. Inhibition of 48. Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, et al. Erlotinib (Tarceva, nucleoside transport by protein kinase inhibitors. J Pharmacol Exp Ther OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and 2003;304:753–60. ATP-binding cassette subfamily G member 2-mediated drug resistance. 29. Huang M, Wang Y, Collins M, Gu JJ, Mitchell BS, Graves LM. Inhibition of Cancer Res 2007;67:11012–20. nucleoside transport by p38 MAPK inhibitors. J Biol Chem 2002;277: 49. Hurwitz HI, Dowlati A, Saini S, Savage S, Suttle AB, Gibson DM, et al. Phase 28364–7. I trial of pazopanib in patients with advanced cancer. Clin Cancer Res 30. Leisewitz AV, Zimmerman EI, Jones SZ, Yang J, Graves LM. Imatinib- 2009;15:4220–7. resistant CML cells have low ENT activity but maintain sensitivity to 50. McKillop D, Partridge EA, Kemp JV, Spence MP, Kendrew J, Barnett S, gemcitabine. Nucleosides Nucleotides Nucleic Acids 2008;27:779–86. et al. Tumor penetration of gefitinib (Iressa), an epidermal growth

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

Human Nucleoside Transporter Inhibition by Kinase Inhibitors

factor receptor tyrosine kinase inhibitor. Mol Cancer Ther 2005;4: 55. Paproski RJ, Ng AM, Yao SY, Graham K, Young JD, Cass CE. The role of 641–9. human nucleoside transporters in uptake of 30-deoxy-30-fluorothymidine. 51. Haura EB, Sommers E, Song L, Chiappori A, Becker A. A pilot study of Mol Pharmacol 2008;74:1372–80. preoperative gefitinib for early-stage lung cancer to assess intratumor drug 56. Barthel H, Cleij MC, Collingridge DR, Hutchinson OC, Osman S, He Q, concentration and pathways mediating primary resistance. J Thorac Oncol et al. 30-deoxy-30-[18F]fluorothymidine as a new marker for monitoring 2010;5:1806–14. tumor response to antiproliferative therapy in vivo with positron emission 52. Gotink KJ, Broxterman HJ, Labots M, de Haas RR, Dekker H, Honeywell RJ, tomography. Cancer Res 2003;63:3791–8. et al. Lysosomal sequestration of sunitinib: a novel mechanism of drug 57. Liu G, Jeraj R, Vanderhoek M, Perlman S, Kolesar J, Harrison M, et al. resistance. Clin Cancer Res 2011;17:7337–46. Pharmacodynamic study using FLT PET/CT in patients with renal cell 53. Chen Y, Tortorici MA, Garrett M, Hee B, Klamerus KJ, Pithavala YK. Clinical cancer and other solid malignancies treated with sunitinib malate. Clin pharmacology of axitinib. Clin Pharmacokinet 2013;52:713–25. Cancer Res 2012;17:7634–44. 54. Reyner EL, Sevidal S, West MA, Clouser-Roche A, Freiwald S, Fenner K, et al. 58. Zhao S, Kuge Y, Zhao Y, Takeuchi S, Hirata K, Takei T, et al. Assessment of In vitro characterization of axitinib interactions with human efflux and early changes in 3H-fluorothymidine uptake after treatment with gefitinib hepatic uptake transporters: implications for disposition and drug inter- in human tumor xenograft in comparison with Ki-67 and phospho-EGFR actions. Drug Metab Dispos 2013;41:1575–83. expression. BMC Cancer 2013;13:525.

www.aacrjournals.org Mol Cancer Ther; 14(1) January 2015 245

Interactions of Multitargeted Kinase Inhibitors and Nucleoside Drugs: Achilles Heel of Combination Therapy?

Vijaya L. Damaraju, Michelle Kuzma, Delores Mowles, et al.

Mol Cancer Ther 2015;14:236-245. Published OnlineFirst December 17, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-14-0337

Cited articles This article cites 56 articles, 19 of which you can access for free at: http://mct.aacrjournals.org/content/14/1/236.full#ref-list-1

Citing articles This article has been cited by 1 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/14/1/236.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/14/1/236. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.