Dinaciclib, a cyclin-dependent kinase inhibitor, is a substrate of human ABCB1 and ABCG2 and an inhibitor of human ABCC1 in vitro
Daniela Cihalovaa, Martina Ceckovaa, Radim Kucerab, Jiri Klimesb, Frantisek Stauda,*
a Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
b Department of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic

A R T I C L E I N F O

Article history:
Received 5 July 2015
Accepted 17 August 2015 Available online xxx

Chemical compounds studied in this article:
Dinaciclib (PubChem CID: 46926350)

Keywords: Dinaciclib ABCB1 ABCC1 ABCG2
Cytotoxicity Multidrug resistance

A B S T R A C T

Dinaciclib is a novel cyclin-dependent kinase inhibitor (CDKI) with significant activity against various cancers in vitro and in vivo. ABC efflux transporters play an important role in drug disposition and are responsible for multidrug resistance in cancer cells. Inhibitors and substrates of these transporters may participate in pharmacokinetic drug–drug interactions (DDIs) that alter drug disposition during pharmacotherapy. To assess such risks associated with dinaciclib we evaluated its possible effects on efflux activities of ABCB1, ABCC1 and ABCG2 transporters in vitro. Monolayer transport, XTT cell proliferation, ATPase and intracellular accumulation assays were employed. Here, we show that the transport ratio of dinaciclib was far higher across monolayers of MDCKII-ABCB1 and MDCKII-ABCG2 cells than across MDCKII parental cell layers, demonstrating that dinaciclib is a substrate of ABCB1 and ABCG2. In addition, overexpression of ABCB1, ABCG2 and ABCC1 conferred resistance to dinaciclib in MDCKII cells. In ATPase assays, dinaciclib decreased stimulated ATPase activity of ABCB1, ABCG2 and ABCC1, confirming it has interactive potential toward all three transporters. Moreover, dinaciclib significantly
inhibited ABCC1-mediated efflux of daunorubicin (EC50 = 18 mM). The inhibition of ABCC1 further led to a
synergistic effect of dinaciclib in both MDCKII-ABCC1 and human cancer T47D cells, when applied in combination with anticancer drugs. Taken together, our results suggest that ABC transporters can substantially affect dinaciclib transport across cellular membranes, leading to DDIs. The DDIs of dinaciclib with ABCC1 substrate chemotherapeutics might be exploited in novel cancer therapies.
ã 2015 Elsevier Inc. All rights reserved.

1. Introduction

Abbreviations: AB, apical-to-basolateral; ABC, ATP-binding cassette; ABCB1, P- glycoprotein; ABCC1, multidrug resistance-associated protein 1; ABCG2, breast cancer resistance protein; ADME, absorption, distribution, metabolism and excretion; BA, basolateral-to-apical; CDK, cyclin-dependent kinase; CDKI, cyclin- dependent kinase inhibitor; CI, combination index; DDI, drug–drug interactions; DMEM, Dulbecco’s modified Eagle’s medium; DNR, daunorubicin; EC50, half maximal effective concentration(s); EMA, the European Medicines Agency; FBS, fetal bovine serum; FDA, the Food and Drug Administration; IC50, half maximal inhibitory concentration(s); IS, internal standard; ITC, International Transporter Consortium; MDCKII, Madin–Darby canine kidney; MDR, multidrug resistance; MFI, median fluorescence intensity; MIT, mitoxantrone; NEM-SG, N-ethylmaleimide- glutathione; PBS, phosphate-buffered saline; PMS, phenazine methosulfate; r, transport ratio; RF, resistance factor; SD, standard deviation; TKI, tyrosine kinase inhibitor; TOP, topotecan; XTT, sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)- 5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt.
* Corresponding author. Fax: +420 495067170.
E-mail addresses: [email protected] (D. Cihalova), [email protected] (M. Ceckova), [email protected] (R. Kucera), [email protected] (J. Klimes), [email protected] (F. Staud).

http://dx.doi.org/10.1016/j.bcp.2015.08.099

0006-2952/ ã 2015 Elsevier Inc. All rights reserved.

Cyclin-dependent kinases (CDKs) are critical regulators of cell cycle progression, and deregulation of their function has been detected in multiple human cancers. Thus, they are attractive targets for cancer treatment and new CDK inhibitors (CDKIs) with favorable pharmacological profiles and minimum adverse effects are being intensively sought. Several small-molecule CDKIs have entered clinical trials [1,2] and a CDK4/6 inhibitor, palbociclib, has been recently approved by the US Food and Drug Administration (FDA) for use in initial endocrine-based therapy for postmeno- pausal women with estrogen-positive, human epidermal growth factor receptor 2-negative advanced breast cancer [3].
Dinaciclib (MK-7965, SCH727965) is an orally administered small-molecule CDKI that selectively inhibits important members of the CDK family (CDK1, CDK2, CDK5 and CDK9) at nanomolar concentrations [4]. In preclinical studies dinaciclib has shown excellent anticancer efficacy, surpassing that of older CDKIs (e.g., flavopiridol and roscovitine), inhibiting the growth of a broad

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spectrum of human cancer cell lines both in vitro and in in vivo xenograft models [4–8]. In addition, 16 clinical trials have been initiated to evaluate its effects, as a single agent or in combination with other anticancer drugs, in patients with hematologic malignancies or solid tumors (clinicaltrials.gov, accessed July 2015). So far, its safety, tolerability and pharmacokinetics have been assessed in phase 1 clinical studies [9,10] and phase 2 studies have evaluated its efficacy relative to erlotinib and capecitabine in patients with non-small cell lung cancer [11] and advanced breast cancer [12], respectively. Dinaciclib also reportedly has encourag- ing single agent activity in patients with relapsed multiple myeloma [13]. Several other clinical studies, including a phase 3 clinical trial (clinicaltrials.gov, ID: NCT01580228), have evaluated therapeutic effects of dinaciclib in the treatment of refractory chronic lymphocytic leukemia.
ATP-binding cassette (ABC) efflux transporters drive substrates across biological membranes, even against concentration gra- dients, using energy from ATP hydrolysis. They are expressed in the liver, kidneys and small intestine, where they modulate absorp- tion, distribution, metabolism and excretion (ADME) of their substrates. ABC transporters protectively limit the entry of xenobiotics into the testes, placenta and blood-brain barrier, and thus may control drug penetration to particularly sensitive organs [14]. In addition to normal tissues, ABC drug transporters are abundantly expressed in cancer cells where they participate in the development of multidrug resistance (MDR). This poses a major obstacle in cancer chemotherapy, as ABC transporters can actively efflux structurally and functionally diverse anticancer drugs, diminishing their intracellular concentrations. Three members of the ABC transporter family contribute most significantly to MDR: ABCB1 (P-glycoprotein, P-gp), ABCG2 (breast cancer resis- tance protein, BCRP) and ABCC1 (multidrug resistance-associated protein, MRP1) [15,16]. Inhibitors and substrates of ABC trans- porters may participate in pharmacokinetic drug-drug interactions (DDIs) that substantially change drug disposition during pharma- cotherapy and affect both the therapeutic efficacy and the severity of adverse effects. Therefore, the International Transporter Consortium (ITC) emphasizes that such interactions must be considered in order to help determine their pharmacokinetic, safety and efficacy profiles [17]. Following these recommenda- tions, both the FDA and the European Medicines Agency (EMA) have issued new guidelines for drug interaction studies, empha- sizing the requirements for in vitro methods in drug transporter interaction assessment [18,19].
In vitro methods, such as ATPase, cellular uptake and monolayer
transport assays, are currently cornerstones for evaluating molecular-level transporter interactions [17,20]. Employing these methods, it has been shown that several CDKIs can interact with ABC transporters by either inhibiting their efflux activity [21,22] or being transported as substrates [23,24]. In these cases, transporter- mediated pharmacokinetic DDIs can occur when the CDKIs are applied in combination with other therapeutic agents [25].
Understanding dinaciclib interactions with ABC transporters is important for determining potential pharmacokinetic DDIs involv- ing the drug, however, to date, no data on ABC transporter substrate specificity has been reported. Thus, in the study reported here we explored interactions of dinaciclib with ABCB1, ABCG2 and ABCC1 transporters in vitro. We also explored the potential ability of these ABC transporters to confer dinaciclib resistance upon cells.

2. Materials and methods

2.1. Chemicals

Dinaciclib was obtained from Axon Medchem (Groningen, the Netherlands). Daunorubicin (DNR), mitoxantrone (MIT),

topotecan (TOP), sodium 2,3-bis(2-methoxy-4-nitro-5-sulfo- phenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT), phenazine methosulfate (PMS), ABCC1 inhibitor MK-571, dual ABCB1/ABCG2 inhibitor GF120918, fluorescein-isothiocya- nate-labeled dextran, roscovitine and HPLC grade solvents (methanol, acetic acid) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The ABCB1 inhibitor LY335979 was obtained from Toronto Research Chemicals (North York, ON, Canada) and the ABCG2 inhibitor Ko143 from Enzo Life Sciences (Farmingdale, NY, USA). Cell culture reagents (media, sera, L-glutamine, DMSO, buffers, trypsin-EDTA) were supplied by Sigma–Aldrich (St. Louis, MO, USA) and Opti-MEM1 from Gibco BRL Life Technologies (Rockville, MD, USA). ABCB1, ABCG2 and ABCC1 PREDEASYTM
ATPase kits (SB MDR1/P-gp, SB BCRP HAM Sf9 and SB MRP1, respectively) were purchased from Solvo Biotechnology (Szeged, Hungary). Transwell inserts were obtained from Corning Inc. (Corning, NY, USA).

2.2. Cell culture

MDCKII (Madin–Darby canine kidney) cell lines transduced for stable expression of human transporters ABCB1 (MDCKII-ABCB1), ABCC1 (MDCKII-ABCC1) or ABCG2 (MDCKII-ABCG2) as well as the
MDCKII-parent cell line, were obtained from Prof. Piet Borst and Dr. Alfred Schinkel (The Netherlands Cancer Institute, Amster- dam, the Netherlands). The cell lines were grown in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). These cell lines were used for monolayer transport, XTT antiproliferative and intracellular accumulation assays. For in vitro drug combination studies, we also used human ductal breast carcinoma T47D cells, obtained from the European Collection of Cell Culture (PHE, Salisbury, Wiltshire, UK). This cell line was cultured in DMEM without phenol red, supplemented with 10% FBS and 2 mM L-glutamine. DMSO was applied as a solvent in concentrations not exceeding 0.5% (1% in ATPase assays).

2.3. MDCKII monolayer transport assay

MDCKII-ABCB1, MDCKII-ABCC1, MDCKII-ABCG2 or MDCKII-
parent cells were seeded on microporous polycarbonate mem- brane inserts (3 mm pore size, 24 mm diameter; Costar, Cambridge, MA, USA) at a density of 1 106 per insert 72 h before experiments. The medium was replaced after 24 and 48 h of cultivation. The cells were then washed with 1 phosphate-buffered saline (PBS) on both the apical and basal sides and preincubated for 1 h in Opti- MEM1 with or without inhibitors. The appropriate ABC transport- er inhibitor (1 mM LY335979, 25 mM MK-571 or 1 mM Ko143 for ABCB1, ABCC1 or ABCG2 inhibition, respectively) was present in both compartments during the preincubation and the transport experiment, at a concentration known to efficiently inhibit the corresponding transporter. To inhibit any endogenous transporter activity, appropriate inhibitors were also added (1 mM of the ABCB1 inhibitor LY335979 to MDCKII-ABCG2 cultures, 1 mM of the ABCG2 inhibitor Ko143 to MDCKII-ABCB1 cultures, and 2 mM of the dual ABCB1/ABCG2 inhibitor GF120918 to MDCKII-ABCC1 and parent cell cultures). The experiments were started (time = 0) by replacing the medium with fresh Opti-MEM1 containing dinaci- clib, with or without inhibitor, in the appropriate chambers. Samples were taken from opposite compartments after 2, 4 and 6 h, then the concentration of transported dinaciclib was deter- mined by HPLC–MS/MS analysis. Immediately after each experi- ment, cellular monolayer integrity was examined using fluorescein
isothiocyanate-labeled dextran (MW = 40 kDa). Dextran leakage
up to 1% per hour was accepted. Dinaciclib transport in parental and ABC transporter-expressing MDCKII cells was assayed and

D. Cihalova et al. / Biochemical Pharmacology xxx (2015) xxx–xxx 3

transport ratios (r), defined as the dinaciclib transport rate in the basolateral-to-apical (BA) direction divided by the rate in the apical-to-basolateral (AB) direction, were calculated from the observed concentrations at 6 h. In these assays dinaciclib was applied at 100 nM (the lowest concentration allowing sensitive analysis of samples above the detection limits of the HPLC–MS/MS method and instruments, as described in Section 2.4).

2.4. HPLC–MS/MS analysis

Dinaciclib was quantified by HPLC–MS/MS using an LC 20A Prominence chromatograph (Shimadzu, Kyoto, Japan), equipped with an OPTI-GUARD 1 mm C18 guard column (Sigma–Aldrich, St. Louis, MO, USA) and a Hypersil GOLD C18 column (100 4.6 mm, particle size 3 mm; Pragolab, Prague, Czech Republic), coupled to a LCQ Max advantage mass spectrometer (Thermo Finnigan, San
Jose, CA, USA). The mobile phase flow rate was 0.4 mL/min and the column temperature was maintained at 40 ◦C. The acquired MS
data were processed using Xcalibur 2.0 software (Thermo Finnigan, San Jose, CA, USA).
Following optimization of conditions to separate dinaciclib from Opti-MEM1 components and inhibitors (GF120918, MK-571, LY335979, Ko143) a mobile phase consisting of methanol and 0.1% acetic acid (75:25, v/v) was used in all analyses reported here. Roscovitine was added to samples as an internal standard (IS). Retention times were 4.1 and 4.7 min for dinaciclib and the IS, respectively. ESI probe settings were: source voltage 4.5 kV,
capillary temperature 340 ◦C, sheet and auxiliary gas flows
60 and 15 arbitrary units, respectively. The tandem MS was operated in SRM mode, using the molecular ion [M + H]+ (m/z 397 for dinaciclib and 355 for the IS) as precursor ion and product ions with m/z ratios of 240, 268, 285, 379 (dinaciclib) and 313 (IS) for quantifying dinaciclib after collision dissociation. The collision energies were 38% and 40% for dinaciclib and IS, respectively. The linearity of the signals was evaluated in the range of 2.5–500 nM (r2 = 0.9994); the method precision and accuracy were evaluated at analyte concentrations of 500, 50, 10 and 2.5 nM. Samples were found to be stable for at least 40 h.

2.5. XTT cell proliferation assays

The XTT assay was used to assess effects of dinaciclib and ABC transporter substrates on cell viability and growth. MDCKII-ABCB1, MDCKII-ABCC1, MDCKII-ABCG2 or MDCKII-parent cells were seeded in 96-well culture plates at a density of 1 104 cells and incubated for 24 h. Test compounds diluted with growth medium
were added to the exponentially growing cells and the resulting mixtures were incubated for 72 h at 37 ◦C, 5% CO2. XTT (0.167 mg/ mL) mixed with 4 mM PMS in Opti-MEM1 was then added to the cultures. After a further 2 h incubation the absorbance of the
soluble formazan released was measured at 470 nm using a microplate reader (Tecan, Männedorf, Switzerland), and the half maximal inhibitory concentrations (IC50) of the drugs were calculated using GraphPad Prism 6.00. To determine the influence of ABC transporters on antiproliferative activity of dinaciclib, resistance factors (RFs) were calculated by dividing the IC50 value for each ABC transporter-overexpressing cell line by the value for the correspondingly-treated parental cell line. Thus, the RFs represent fold-increases in resistance caused by the presence of specific ABC transporters [26]. To indirectly assess whether cellular resistance to dinaciclib can be caused by ABCB1, ABCC1 or ABCG2, the cell proliferation assays were repeated with the addition of model inhibitors of the three transporters (1 mM LY335979, 25 mM
MK-571 and 1 mM Ko143 for ABCB1, ABCC1 and ABCG2,
respectively) to abolish the potential influence of ABC transporters on the resistance.

2.6. ABCB1, ABCC1 and ABCG2 ATPase assays

Preparations of membranes with overexpressed ABC trans- porters show vanadate-sensitive ATPase activity that is modulated by interacting compounds. In the activation assay, increases in this ATPase activity associated with increases in substrate transport are measured, while in the inhibition assay reductions in the activity associated with the presence of a known activator of the transporter are measured [27]. ATPase activity was measured by assessing the amount of phosphate liberated from ATP by the ABCB1, ABCC1 or ABCG2 transporters using the corresponding PREDEASY ATPase kits according to the manufacturer’s instruc- tions. For this purpose, Sf9 cell membranes (4 mg protein per well)
were mixed with dinaciclib at concentrations ranging from 14 nM to 300 mM, then incubated at 37 ◦C for 10 min in the presence or
absence of 1.2 mM sodium orthovanadate. The ATPase reaction was started by adding 10 mM ATP magnesium salt to the reaction mixture, stopped 10 min later, and the absorbance at 590 nm was measured after 30 min incubation using the Tecan microplate reader. The ATPase activity in each sample was determined as the difference in liberated amounts of phosphate measured in the presence and absence of 1.2 mM sodium orthovanadate. Phosphate standards were prepared in each plate, using verapamil, N-ethylmaleimide-glutathione (NEM-SG) and sulfasalazine as positive controls for ABCB1, ABCC1 and ABCG2 stimulation, respectively. The results are expressed as vanadate-sensitive ATPase activities.

2.7. DNR and MIT accumulation assays

Effects of dinaciclib on intracellular DNR accumulation in MDCKII-ABCB1 and MDCKII-ABCC1 cells, and MIT accumulation in MDCKII-ABCG2 cells, were examined using a C6 flow cytometer (Accuri, Ann Arbor, USA). MDCKII-parent cells were analyzed as controls for accumulation of each substrate. Cells were seeded at a density of 1.5 105 on a 12-well plate 24 h before each experiment
and treated with dinaciclib at five concentrations, solvent (0.5% DMSO) or Opti-MEM1 (untreated control) for 30 min at 37 ◦C, 5% CO2. DNR or MIT was then added to a final concentration of 2 mM or 1 mM, respectively, and the cells were incubated under the same conditions for a further 60 min. Accumulation was stopped by cooling the samples on ice and washing twice with ice-cold PBS.
The cells were detached with 10 trypsin-EDTA and resuspended in PBS with 2% FBS. Levels of DNR and MIT in individual cells were measured using 488/585 and 488/670 nm excitation/emission filters, respectively, and recorded as histograms. 1 mM LY335979, 50 mM MK-571 and 1 mM Ko143 were used as positive controls for ABCB1, ABCC1 and ABCG2 inhibition, respectively. All inhibitors were used at concentrations that are known to efficiently inhibit the corresponding transporter. Viable cells were gated based on forward and side scatter plots. The median fluorescence intensity (MFI) of 10 000 measured cells was used to compare the
fluorescence resulting from each of the treatments. The relative values were identified by dividing the MFI of each measurement by that of untreated control cells. Where applicable, the half maximal effective concentration (EC50) for ABC transporter inhibition was calculated using GraphPad Prism 6.00.

2.8. Drug combination assays

Combination indices (CIs, derived from the median-effect equation) were calculated to assess combined effects of dinaciclib and DNR or TOP, commonly used anticancer drugs and ABCC1 transporter substrates. CIs provide quantitatively defined indications of additive, synergistic and antagonistic effects, when CI = 0.9–1.1, <0.9, and >1.1, respectively [28]. The XTT cytotoxicity

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assay was used to measure the viability of the tested cells (MDCKII- parent, MDCKII-ABCC1 and T47D) in the presence of dinaciclib and DNR/TOP both alone and in combination, at constant concentra- tion ratios ranging from 0.1 to 2 multiples of the respective, predetermined IC50 values. The data acquired from these drug combination experiments were analyzed using CompuSyn ver.
3.0.1 software (ComboSyn Inc., Paramus, NJ, USA).

2.9. Statistical analysis

Data are presented as means standard deviations (SDs) obtained from at least three independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t tests and one-way ANOVA implemented in GraphPad Prism 6.00, and differences were considered significant if P < 0.05.
3. Results

3.1. Effects of ABCB1, ABCC1 and ABCG2 on transepithelial transport of dinaciclib in vitro

To evaluate whether dinaciclib is a substrate of the tested transporters, we assayed its transport in vitro across polarized monolayers of MDCKII-ABCB1, MDCKII-ABCC1, MDCKII-ABCG2 and
MDCKII-parent cells. In MDCKII cells, ABCB1 and ABCG2 are localized apically, therefore, in this method, the transport of a substrate across the monolayer is greatly accelerated in the

basolateral-to-apical direction. ABCC1, on the other hand, is localized basolaterally, accelerating transport of its substrates in apical-to-basolateral direction.
Under our test conditions, the BA to AB transport ratio (r) of dinaciclib (applied at 100 nM in all assays of this variable) in MDCKII-ABCB1 cells was 30.8. Presence of the model ABCB1 inhibitor LY335979 strongly reduced the ratio to 1.12 (Fig. 1A), confirming the involvement of ABCB1 in dinaciclib transport. Similarly, in MDCKII-ABCG2 cells, the transport ratios in the absence and presence of the model ABCG2 inhibitor Ko143 were 7.84 and
1.33, respectively (Fig. 1B), indicating that dinaciclib is also transported by ABCG2. The transport ratio in MDCKII-ABCC1 cells was 0.91 (Fig.1C), significantly lower than the ratio, under the same conditions, in MDCKII-parent cells (1.29) (Fig. 1D). Presence of the ABCC1 inhibitor MK-571 significantly altered the transport asym- metry in MDCKII-ABCC1 cells, resulting in a transport ratio similar to that recorded for parental cells (1.20). This suggests that ABCC1 might also contribute to the transport of dinaciclib.

3.2. Effects of dinaciclib on the viability of MDCKII cell lines

To determine effects of ABC transporters on the antiprolifer- ative effects of dinaciclib, the substance was used in XTT assays with ABCB1-, ABCC1- and ABCG2-overexpressing MDCKII and MDCKII-parent cell lines. The respective IC50 values of dinaciclib and calculated RFs are shown in Table 1. MDCKII-ABCB1 cells were significantly more resistant to dinaciclib than the parental MDCKII

Fig. 1. Transport of dinaciclib (100 nM) across monolayers of MDCKII-ABCB1 (A), MDCKII-ABCG2 (B), MDCKII-ABCC1 (C) and MDCKII-parent cells (D). ~, basolateral-to-apical transport without inhibitor; ! apical-to-basolateral transport without inhibitor; , basolateral-to-apical transport with inhibitor; , apical-to-basolateral transport with inhibitor; 1 mM LY335979 (LY), 1 mM Ko143 (Ko) and 25 mM MK-571 (MK) were used as model inhibitors of ABCB1, ABCG2 and ABCC1, respectively, in the corresponding overexpressing cell line. Ratios of dinaciclib transport across cell monolayers (dinaciclib transport in basolateral-to-apical direction divided by transport in apical-to- basolateral direction) with or without inhibitor were calculated using data acquired 6 h after dinaciclib addition. Data are presented as means SDs obtained from at least three independent experiments. Asterisks indicate the statistical significance of differences between r values in the absence and presence of the inhibitors (*P < 0.05,
**P < 0.01, ***P < 0.001).

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Table 1
Antiproliferative IC50 values of dinaciclib in MDCKII cells in the absence and presence of ABC transporter inhibitors.

Druga
MDCKII MDCKII-ABCB1 MDCKII-ABCC1 MDCKII-ABCG2
IC50 (mM) IC50 (mM) RFb
IC50 (mM) RF IC50 (mM) RF
DIN 0.024 0.0049 0.16 0.054* 6.7 0.088 0.014** 3.7 0.070 0.086** 2.9
+LY 0.016 0.00039 0.017 0.0018 1.1 – –
+MK 0.023 0.0023 – – 0.031 0.0042 1.3 –
– –
–
+Ko 0.029 0.0022 – – – – 0.036 0.0072 1.2
–: not applicable.
a DIN, dinaciclib; LY, LY335979 (1 mM); MK, MK-571 (25 mM); Ko, Ko143 (1 mM).
b RF values were calculated by dividing the IC50 values for ABC transporter-overexpressing cells by those for parental cells. Asterisks indicate the significance of differences between ABC transporter-overexpressing cells and parental cells, as determined by unpaired t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

cell line (RF = 6.7), indicating that ABCB1 mediates dinaciclib efflux. Similarly, both ABCC1 and ABCG2 conferred resistance to dinaciclib in MDCKII-ABCC1 and MDCKII-ABCG2 cell lines, with RF values of
3.7 and 2.9, respectively. To confirm the effect of ABC transporters on sensitivity to dinaciclib, the cell proliferation assays were repeated with combinations of dinaciclib with model inhibitors of the overexpressed transporters. In each case the inhibitors significantly reduced the cells’ resistance to dinaciclib: 1 mM LY335979, 25 mM MK-571 and 1 mM Ko143 reducing the RFs of MDCKII-ABCB1, MDCKII-ABCC1 and MDCKII-ABCG2 cells to 1.1,
1.3 and 1.2, respectively, which further indicates the role of ABCB1, ABCG2 and ABCC1 in the resistance to dinaciclib.

3.3. ATPase assays

To further characterize whether dinaciclib interacts with ABC transporter-associated ATPase activity, we tested its modu- latory effects on vanadate-sensitive ATPase in isolated Sf9 cell membranes overexpressing human ABCB1, ABCC1 and ABCG2. In the ATPase activation assay, dinaciclib did not stimulate increases in vanadate-sensitive ATPase activity of the tested transporters. However, in the inhibition study, dinaciclib significantly lowered the stimulated vanadate-sensitive ATPase activity of all three transporters at concentrations 100 mM, strongly confirming that dinaciclib interacts with ABCB1, ABCC1 and ABCG2 (Fig. 2).

3.4. Effects of dinaciclib on ABC transporter-mediated efflux of
fluorescent substrates

To determine whether dinaciclib can inhibit ABC transporter- mediated efflux we tested its effect on the accumulation of known substrates in MDCKII cells. DNR, a fluorescent substrate of ABCB1 and ABCC1, was used to determine the effect of dinaciclib on ABCB1- and ABCC1-mediated effluxes. In MDCKII-ABCB1 cells,

we observed no significant effect of dinaciclib on intracellular accumulation of DNR (Fig. 3A). However, it significantly and dose- dependently enhanced DNR accumulation in MDCKII-ABCC1 cells (1.5-, 1.9- and 2.4-fold at 10, 30 and 50 mM, respectively) with an EC50 value of 18 5.9 mM (Fig. 3B). Indeed, at the highest tested concentration (50 mM) its inhibitory effect was similar to that of a model ABCC1 inhibitor, MK-571, applied at the same concentration (a 2.5-fold increase in DNR accumulation). In addition, dinaciclib significantly increased the intracellular accumulation of MIT, a fluorescent substrate, in MDCKII-ABCG2 cells, but only at the highest tested concentration (50 mM). At this concentration it induced a 2.5-fold increase in MIT accumulation (Fig. 3C), substantially weaker than the response to the ABCG2 model inhibitor, Ko143, at just 1 mM (a 6.5-fold increase). No effect of dinaciclib on DNR or MIT accumulation was observed in the control MDCKII-parent cells. Our results suggest that dinaciclib can inhibit ABCC1-mediated efflux and may be able to reverse ABCC1-mediated MDR.

3.5. Drug combination assays

Since dinaciclib potently inhibited ABCC1-mediated efflux of DNR, combination studies were performed to assess its ability to sensitize ABCC1-expressing cells to selected cytotoxic ABCC1 transporter substrates through ABCC1 inhibition. Employ- ing the XTT assay, the antiproliferative effect of dinaciclib alone was assessed and compared to the effect of concomitant treat- ments of dinaciclib with DNR and TOP. The combination indices (CIs) obtained for combinations of dinaciclib with DNR or TOP in MDCKII-ABCC1 cells were lower than 0.9 across the entire range of drug effect levels, indicating that these drugs have synergistic antiproliferative effects (Table 2). In contrast, the combinations of dinaciclib with TOP and DNR had significantly weaker and no synergistic effects, respectively, on the control (MDCKII-parent) cells (Fig. 4A and B).

Fig. 2. Effects of dinaciclib on the ATPase activity of ABCB1-Sf9 (A), ABCC1-Sf9 (B) and ABCG2-Sf9 (C) membrane preparations. Vanadate-sensitive activity in the presence of dinaciclib in activation (*) and inhibition (&) experiments. Lower dotted line represents baseline vanadate-sensitive ATPase and upper dotted line represents activated ATPase triggered by a reference substrate in all graphs. In the inhibition and activation assays, reductions in the stimulated ATPase activity (indicating interaction of the drug with transporter’s ATPase) and increases in baseline ATPase activity (indicating the drug as a transporter substrate) were measured, respectively. Data are presented as means SDs obtained from three independent experiments. Statistically significant differences between stimulated control and dinaciclib-treated samples in inhibition assays (*P < 0.05) were determined using unpaired t tests.

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Fig. 3. Effects of dinaciclib on the intracellular accumulation of DNR in MDCKII-ABCB1 cells (A) or MDCKII-ABCC1 cells (B) and MIT in MDCKII-ABCG2 cells (C). The results represent fold changes in median fluorescence intensity compared to signals obtained with untreated controls. LY335979 (1 mM), MK-571 (50 mM) and Ko143 (1 mM) were used as model inhibitors for ABCB1, ABCC1 and ABCG2 inhibition, respectively. Data represent means SDs obtained from three independent experiments. Asterisks indicate the significance of differences, relative to controls, determined by unpaired t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

Table 2
Combination indices (CI) for drug combinations assessed after 72 h of simultaneous treatment in MDCKII-parent, MDCKII-ABCC1 and T47D cells.

0.81 0.10

0.44 0.019

a DIN, dinaciclib; DNR, daunorubicin; TOP, topotecan.

0.88 0.30

In the ABCC1-expressing T47D human ductal breast carcinoma cells, the dinaciclib with DNR and TOP combinations showed synergistic cytotoxicity at drug effect levels of >80% and >90%, respectively (Fig. 4C and D).

4. Discussion

Evaluation of new molecules that interact with membrane- bound drug transporters is an integral part of drug development and regulatory review [29]. In vitro studies using ABC transporter- overexpressing polarized epithelial cell lines are considered a critical first step in the assessment of drug interactions with these transporters [18–20]. Dinaciclib, currently in phase 3 clinical trials, is an attractive potential cancer therapeutic because it inhibits several key CDKs. In the study reported here, we used MDCKII cells transduced with human ABC transporters to explore interactions of dinaciclib with ABCB1, ABCC1 and ABCG2 transporters in vitro. According to the ITC white paper [17], a compound is considered a potential substrate of apically localized transporters if its transport ratio is 2 (i.e., transport of the drug is at least twice as high in the transporter-driven direction than in the opposite direction), provided that the epithelial cell system used expresses the studied transporter and a model inhibitor reduces its transport by 50%. Dinaciclib exhibited high transport ratios when applied to ABCB1- and ABCG2-expressing MDCKII cell monolayers (30.8 and 7.84, respectively), and addition of model inhibitors significantly decreased these ratios, by 96% and 83%, respectively. Based on these findings, we conclude that dinaciclib can be a substrate of both ABCB1 and ABCG2 transporters. As ABCC1 is localized basolaterally in MDCKII-ABCC1 cells, the transfer of ABCC1 substrates should be greatly accelerated in apical-to- basolateral direction rather than in the opposite (basolateral-to- apical) direction. We observed a significantly lower transport ratio

of dinaciclib in MDCKII-ABCC1 cells (0.91) than in MDCKII-parent cells (1.29), with significantly accentuated transport in apical-to- basolateral direction, and significant (26%) inhibition of dinaciclib transport in this direction by an ABCC1 inhibitor (MK-571). These findings indicate that ABCC1 contributes to the transport of dinaciclib. However, as the ITC does not provide substrate criteria for basolaterally localized transporters, we cannot conclusively consider dinaciclib an ABCC1 substrate.
We have also shown that overexpression of all three studied transporters (ABCB1, ABCC1 and ABCG2) confers resistance to dinaciclib. In all cases the transduced cells were significantly (2.9- to 6.7-fold) more resistant to dinaciclib than the control parental cell line. Furthermore, co-administration with selective inhibitors resulted in complete reversal of the resistance in the correspond- ing cell lines. These results suggest that efflux of dinaciclib mediated by ABC transporters should reduce its antiproliferative effects. Moreover, application of dinaciclib in a broad range of concentrations significantly decreased activated ATPase activities in the ATPase inhibition assays, which is a typical feature of transporter inhibitors and slowly transported substrates [30].
Since ABC transporters, particularly ABCB1 and ABCG2, consti- tute an effective pharmacological barrier by restricting the passage of drugs through membranes [14], dinaciclib will probably have low permeability to sensitive tissues and organs, e.g., brain and fetal tissues. Its interactions with ABC transporters will also presumably affect its disposition in the body, and the transporters may present important sites for pharmacokinetic DDI if it is used in combination with other therapeutics.
Moreover, as mechanisms of resistance to dinaciclib have not been previously addressed in detail, it should be noted that ABC transporters could diminish the accumulation of dinaciclib in cancer cells, leading to the development of drug resistance and subsequent failure of anticancer therapy. Recent data have

D. Cihalova et al. / Biochemical Pharmacology xxx (2015) xxx–xxx 7

Fig. 4. Combination indices for applications of dinaciclib with DNR (A and C) and TOP (B and D) in MDCKII-parent (Ⓧ), MDCKII-ABCC1 (&) and T47D (^) cells. Lines represent computer-simulated CI plots in MDCKII-parent (dashed line), MDCKII-ABCC1 (dotted line) and T47D (full line) cells, while symbols represent experimental data points. The concentration ratio was based on the IC50 ratio of individual drugs. CI values < 0.9 indicate synergism, values = 0.9–1.1 indicate additivity, and values > 1.1 indicate antagonism. Drug effect levels were calculated from the cell viability values and correspond to the proportions of cells affected by the drug combination: 0 and 100% indicate no and
absolute antiproliferative effects, respectively. Data are presented as means SDs obtained from three independent experiments.

indicated that cancer cells can develop resistance toward dinaciclib, but the evaluated mechanisms did not include ABC transporter expression [31]. Furthermore, other known resistance mechanisms, like upregulation of antiapoptotic Mcl-1, are unlikely as dinaciclib has been shown to inhibit Mcl-1 transcription, leading to significant apoptotic cell death [32–35]. Thus, the findings presented here appear to be the first indications that ABCB1-, ABCG2- and ABCC1-mediated transport is the causative mecha- nism of cellular resistance to dinaciclib.
Several studies have convincingly shown that tyrosine kinase inhibitors (TKIs) can inhibit ABC transporters and modulate MDR, both in vitro and in vivo [36–39]. Similarly, we have recently demonstrated that several novel small molecule protein kinase inhibitors can modulate MDR [21,40,41]. In the present study, we demonstrated that dinaciclib can effectively inhibit ABCC1- mediated efflux of DNR (EC50 = 18 mM), indicating that this drug may be able to reverse ABCC1-mediated MDR. Interestingly, we observed weak and no inhibitory effects of dinaciclib on ABCG2- and ABCB1-mediated effluxes, respectively.
The new novel kinase inhibitors are believed to have high anti- cancer potential, especially in combination with conventional chemotherapeutics, because they can circumvent compensatory mechanisms that are generally activated when CDKIs interrupt cell functions [42,43]. Dinaciclib has already been found to potently synergize with cisplatin in preclinical models of ovarian cancer, corroborating potential benefits of combinational therapy [44]. Several other combinations of dinaciclib and other drugs, e.g., rituximab or epirubicine, are being evaluated in ongoing trials (clinicaltrials.gov, ID: NCT01650727 and NCT01624441, respective- ly). Therefore, we investigated whether dinaciclib could potentiate the cytotoxic effects of other anticancer drugs through

ABCC1 transporter inhibition in vitro. We hypothesized that by inhibiting ABCC1-mediated effluxes of DNR and TOP, dinaciclib could increase intracellular accumulation of these drugs and enhance their cytotoxic effects. Accordingly, synergistic effects were observed when combinations of dinaciclib with DNR and TOP were applied to ABCC1-transduced and parental MDCKII cells as well as human tumor-derived cells expressing ABCC1. Further- more, combinations of dinaciclib with DNR or TOP had significantly more pronounced synergistic effects in MDCKII-ABCC1 than in MDCKII-parent cells, suggesting the effects are directly related to ABCC1 expression. We believe that inhibition of ABCC1 was also responsible for the synergistic effects of these combinations in human ductal breast carcinoma T47D cells, as they abundantly express ABCC1 [41,45]. Exploiting the synergistic activity of dinaciclib and ABCC1 transporter substrates could therefore offer a promising clinical strategy, particularly for treating resistant tumors.
In conclusion, we have provided the first demonstration that dinaciclib is a substrate of ABCB1 and ABCG2 transporters, and an inhibitor of ABCC1 transporter. These pharmacokinetic features should be considered before bringing dinaciclib into clinical practice as they may result in DDI in normal tissues and/or multidrug resistance in cancer cells. On the other hand, we also show that DDI associated with dinaciclib could be beneficially exploited in cancer treatment if combined with other cytostatic substrates of ABCC1.

Acknowledgement

This work was supported by Charles University in Prague (grant no. SVV/2015/260-185).

8 D. Cihalova et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

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