Elmquist, William F.

Abstract Clinical data indicate that certain regions within glioblastoma (GBM) have a relatively intact blood-brain barrier (BBB), but controversy surrounds whether associated heterogeneous delivery may limit efficacy for otherwise highly active drugs with poor brain distribution. In this study, the efficacy of molecularly targeted therapies was compared in relevant GBM patient-derived xenograft (PDX) models grown either as heterotopic or orthotopic tumors. The impact of further disrupting BBB integrity in non-responsive orthotopic tumors was evaluated by over-expressing vascular endothelial growth factor (VEGF) and measuring drug distribution by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). The brain:plasma ratio for five targeted agents was determined by LC-MSMS and treatment efficacy was evaluated in target-relevant GBM PDX lines: erlotinib (brain:plasma = 0.02 ± 0.02; efficacy tested in GBM6), palbociclib (brain:plasma = 0.06; efficacy tested in GBM22), AZD1775 (brain:plasma = 0.05; efficacy tested in GBM22), SAR405838 (brain:plasma = 0.01 ± 0.003; efficacy tested in GBM108), and AZD4547 (brain:plasma = 0.045 ± 0.02; efficacy tested in GBM150). Each of these drugs was effective in relevant GBM PDX flank tumor models with a 27% to 218% prolongation in the median time to exceed their pre-specified endpoint compared to placebo treatment (p≤0.02 for each drug). In contrast, none of these drugs were effective in prolonging survival in the same target-relevant orthotopic tumor models (-6% to 8% extension in median survival; p>0.05 for each drug). Consistent with partial and heterogeneous disruption of the BBB, MALDI-MSI of erlotinib, AZD1775 or SAR405838 distribution within orthotopic tumors demonstrated highly heterogeneous drug levels with large regions within each tumor approaching the low drug levels observed within surrounding normal brain. To further evaluate whether heterogenous drug distribution in orthotopic tumors could account for poor treatment efficacy, GBM108 was transduced with lentiviral constructs encoding for VEGF or empty vector (EV). While both GBM108-sublines were readily detectable with gadolinium-enhanced magnetic resonance imaging (MRI), the GBM108-VEGF tumors had a significantly more disrupted BBB, as evidenced by a more uniform and intense distribution of a brain-impenetrant TexasRed-dextran vascular marker. Similarly, the distribution of SAR405838, measured by MALDI-MSI, was markedly higher and more uniform in the GBM108-VEGF tumors when compared to the GBM108-EV tumors. Enhanced delivery of SAR405838 into orthotopic GBM108-VEGF models translated into a marked enhancement in treatment efficacy in comparison to GBM108-EV with a median survival prolongation of 37 vs. 4 days, respectively (p=0.0055). Collectively, these data highlight the importance of testing novel therapeutic agents in orthotopic tumor models and suggest that limited brain penetration for many molecules may significantly limit their efficacy in brain tumors that have a partially intact BBB. In the face of continued failure to develop effective targeted agents for GBM, these in vivo results highlight the importance of re-evaluating the dogma in neuro-oncology that the BBB is fully disrupted in GBM and, therefore, drug delivery across the BBB is not a major factor limiting treatment efficacy. Citation Format: Jann N. Sarkaria, David Calligaris, Daniel Ma, Karen Parrish, Ann C. Mladek, Janice Laramy, Minjee Kim, Shuangling Zhang, Mark Schroeder, Brett L. Carlson, Katrina Bakken, Aaron Johnson, Nathalie Agar, William Elmquist. The critical importance of the blood-brain barrier in modulating the response to otherwise highly effective targeted therapies in patient-derived orthotopic glioblastoma xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B25.

AbstractBackgroundGlioblastoma (GBM) exhibits profound intratumoral genetic heterogeneity. Each tumor comprises multiple genetically distinct clonal populations with different therapeutic sensitivities. This has implications for targeted therapy and genetically informed paradigms. Contrast-enhanced (CE)-MRI and conventional sampling techniques have failed to resolve this heterogeneity, particularly for nonenhancing tumor populations. This study explores the feasibility of using multiparametric MRI and texture analysis to characterize regional genetic heterogeneity throughout MRI-enhancing and nonenhancing tumor segments.MethodsWe collected multiple image-guided biopsies from primary GBM patients throughout regions of enhancement (ENH) and nonenhancing parenchyma (so called brain-around-tumor, [BAT]). For each biopsy, we analyzed DNA copy number variants for core GBM driver genes reported by The Cancer Genome Atlas. We co-registered biopsy locations with MRI and texture maps to correlate regional genetic status with spatially matched imaging measurements. We also built multivariate predictive decision-tree models for each GBM driver gene and validated accuracies using leave-one-out-cross-validation (LOOCV).ResultsWe collected 48 biopsies (13 tumors) and identified significant imaging correlations (univariate analysis) for 6 driver genes: EGFR, PDGFRA, PTEN, CDKN2A, RB1, and TP53. Predictive model accuracies (on LOOCV) varied by driver gene of interest. Highest accuracies were observed for PDGFRA (77.1%), EGFR (75%), CDKN2A (87.5%), and RB1 (87.5%), while lowest accuracy was observed in TP53 (37.5%). Models for 4 driver genes (EGFR, RB1, CDKN2A, and PTEN) showed higher accuracy in BAT samples (n = 16) compared with those from ENH segments (n = 32).ConclusionMRI and texture analysis can help characterize regional genetic heterogeneity, which offers potential diagnostic value under the paradigm of individualized oncology.

Abstract BACKGROUND: Talazoparib (TAL) is a potent poly (ADP-ribose) polymerase (PARP) inhibitor that robustly enhances in vitro sensitivity to temozolomide (TMZ) in several tumor models. We assessed the in vitro and in vivo efficacy of TAL combined with TMZ in glioblastoma (GBM) models. METHODS: Established glioma cell lines (T98G, U251) and the GBM12 patient derived xenograft (PDX) line were treated in vitro with TAL (1-10 nM) ± TMZ (2-300 μM). Antitumor efficacy was assessed with CyQUANT, clonogenic and primary neurosphere assays; cell cycle analysis with flow cytometry; DNA damage signaling with phospho-specific western blots for pKap1, pChk1, pChk2 and fluorescent immunocytochemistry for γH2AX and replication protein A foci. Brain and plasma concentrations of TAL in wild-type (WT), Mdr1a/b(-/-), Bcrp(-/-) and Mdr1a/b(-/-)Bcrp(-/-) knockout (KO) mice were assessed with LC-MS/MS. The tolerability of TAL (0.05 - 0.30 mg/kg/day orally, divided twice daily (div. bid)) and TMZ (5-50 mg/kg/day orally) was tested in athymic nude mice. Subsequently, in vivo combination TAL/TMZ efficacy was assessed with survival analyses in intracranial and flank GBM12 PDX models. RESULTS: TAL 1-3 nM sensitizes the TMZ resistant T98G glioma cell line to TMZ at high TMZ concentrations (30-300 μM) and is associated with G2 cell cycle arrest and enhanced DNA damage signaling. TAL 1-3 nM also enhances the cytotoxic efficacy of lower concentrations of TMZ (10-30 μM) in the TMZ sensitive U251 cell line. At least 3 nM TAL is required to enhance the in vitro efficacy of low TMZ concentrations (2-10 μM) in the TMZ sensitive GBM12 PDX line. PK studies in non-tumor bearing WT FVB mice treated with a single dose of TAL 0.15 mg/kg revealed mean brain and plasma concentrations of 0.49 ng/g (1.3 nM) and 25.5 ng/ml (67.1 nM) respectively at 2 hours. The mean brain/plasma ratio at 2 hours was unchanged in Bcrp(-/-) KO mice compared to WT mice (1.2% vs. 2.0%), but was significantly increased in Mdr1a/b(-/-) KO mice (22.8%) and Mdr1a/b(-/-)Bcrp(-/-) KO mice (23.9%). Standard doses of TMZ (50 mg/kg/day, days 1-5) combined with low doses of TAL (0.05 mg/kg/day, div. bid) were toxic in nude mice. Higher doses of TAL (0.30 mg/kg/day, div. bid) required substantial TMZ dose reductions (5 mg/kg/day, days 1-5) for tolerability. The addition of TAL (0.30 mg/kg/day, div. bid) to low-dose TMZ (5 mg/kg/day) prolonged tumor stasis in flank GBM12 xenografts (median time to endpoint 76 vs. 50 days, p = 0.005). However this regimen was ineffective in intracranial GBM12 xenografts (median survival 37 vs. 30 days, p = 0.93). CONCLUSIONS: TAL is a potent PARP inhibitor that enhances the efficacy of TMZ in both in vitro GBM models and flank GBM12 PDX models, but not in the corresponding GBM12 intracranial xenografts. This lack of intracranial efficacy is associated with limited brain distribution due to active efflux mediated by Mdr1 at the blood-brain barrier. Citation Format: Sani H. Kizilbash, Kenneth Chang, Shiv K. Gupta, Ryo Kawashima, Karen Parrish, Ann C. Mladek, Brett L. Carlson, Katrina K. Bakken, Mark A. Schroeder, Gaspar J. Kitange, Paul A. Decker, Yuqiao Shen, William F. Elmquist, Jann N. Sarkaria. Talazoparib enhances low-dose temozolomide efficacy in flank glioblastoma models, but intracranial efficacy is constrained by limited brain distribution. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 340.

Abstract PARP inhibition can enhance the efficacy of temozolomide and prolong survival in orthotopic glioblastoma (GBM) xenografts. The aim of this study was to evaluate the combination of the PARP inhibitor rucaparib with temozolomide and to correlate pharmacokinetic and pharmacodynamic studies with efficacy in patient-derived GBM xenograft models. The combination of rucaparib with temozolomide was highly effective in vitro in short-term explant cultures derived from GBM12, and, similarly, the combination of rucaparib and temozolomide (dosed for 5 days every 28 days for 3 cycles) significantly prolonged the time to tumor regrowth by 40% in heterotopic xenografts. In contrast, the addition of rucaparib had no impact on the efficacy of temozolomide in GBM12 or GBM39 orthotopic models. Using Madin-Darby canine kidney (MDCK) II cells stably expressing murine BCRP1 or human MDR1, cell accumulation studies demonstrated that rucaparib is transported by both transporters. Consistent with the influence of these efflux pumps on central nervous system drug distribution, Mdr1a/b−/−Bcrp1−/− knockout mice had a significantly higher brain to plasma ratio for rucaparib (1.61 ± 0.25) than wild-type mice (0.11 ± 0.08). A pharmacokinetic and pharmacodynamic evaluation after a single dose confirmed limited accumulation of rucaparib in the brain is associated with substantial residual PARP enzymatic activity. Similarly, matrix-assisted laser desorption/ionization mass spectrometric imaging demonstrated significantly enhanced accumulation of drug in flank tumor compared with normal brain or orthotopic tumors. Collectively, these results suggest that limited drug delivery into brain tumors may significantly limit the efficacy of rucaparib combined with temozolomide in GBM. Mol Cancer Ther; 14(12); 2735–43. ©2015 AACR.

Abstract The BBB can critically limit the delivery of several molecularly-targeted agents into the brain. The discovery of focal BRAF mutations in melanoma has stimulated the development of specific BRAF inhibitors. MEK is downstream of BRAF in the MAPK pathway, and has been targeted for combination therapy with BRAF inhibition. Trametinib is a MEK1/2 inhibitor that has improved survival in combination with dabrafenib in clinical trials for metastatic melanoma. We have recently shown that BRAF inhibitors (vemurafenib and dabrafenib) have limited brain distribution due to active efflux at the blood-brain barrier (BBB). Melanoma metastasizes to the brain and effective treatments require adequate CNS exposure of all drugs used in combination therapy. The study purpose was to investigate mechanisms that influence the CNS distribution of trametinib, particularly focusing on the active efflux transport proteins P-gp and Bcrp in the BBB. Intracellular accumulation and bidirectional flux studies were conducted in polarized MDCKII cells that overexpress human P-gp (MDR1) or murine Bcrp (Bcrp1). The brain and plasma AUCs of trametinib were determined after an intravenous dose of 5 mg/kg in WT, Mdr1a/b (-/-), Bcrp1 (-/-) and Mdr1a/b (-/-) Bcrp1 (-/-) (TKO) mice. The steady-state brain distribution of trametinib was determined using osmotic pumps in WT and Mdr1a/b (-/-) (P-gp KO), Bcrp1 (-/-) (Bcrp KO), and Mdr1a/b (-/-) Bcrp1 (-/-) (TKO) mice. The steady-state brain distribution of a combination of trametinib and dabrafenib were determined in WT and TKO mice. IC50s were determined in patient-derived melanoma cell lines (WT and V600E mutant) using the MTT assay. Intracellular accumulation of trametinib was 45% lower in the MDCKII-MDR1 cells and 81% lower in the MDCKII-Bcrp1 cells as compared to WT. Trametinib permeability was ∼ 2.5 and 3.7-fold greater in the basolateral-to-apical direction as compared to apical-to-basolateral direction in the MDR1 and Bcrp1 cells, respectively. Following a single intravenous dose, the brain-to-plasma AUC ratios were approximately 5-fold greater in P-gp KO and TKO mice as compared to the WT. Interestingly, the brain distribution of trametinib in Bcrp KO mice was no different from WT mice. The steady-state brain to plasma (B/P) ratio of trametinib was 5-fold greater in the TKO mice when compared to the wild-type mice. The B/P ratio of both dabrafenib and trametinib were significantly greater in the TKO mice as compared to WT when dosed simultaneously. The IC50 for trametinib alone was > 10 μM in both the WT (M15) and V600E (M12) mutant cells, while a combination of dabrafenib and trametinib had an IC50 of 2.04 μM in the M12 cells. This study shows that P-gp and BCRP restrict the brain distribution of trametinib with P-gp playing a dominant role in the efflux of trametinib at the blood-brain barrier. These findings are clinically relevant to the efficacy of trametinib in mono or combination therapy for the treatment of melanoma brain metastases. Citation Format: Shruthi Vaidhyanathan, Rajendar K. Mittapalli, Daniel J. Ma, Mark A. Schroeder, Jann N. Sarkaria, William F. Elmquist. Trametinib (Mekinist TM) has limited brain distribution in the mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3714. doi:10.1158/1538-7445.AM2014-3714

Abstract Drug delivery to glioblastoma multiforme (GBM) is influenced by the presence of blood-brain barrier (BBB). PI3K/mTOR/AKT is frequently dysregulated in GBM and some agents targeting this pathway have limited distribution across the BBB due to activity of efflux transporters p-glycoprotein (P-gp) and breast cancer resistance protein1 (BCRP1). GNE-317 and GDC-0980 are structurally related PI3K/mTOR inhibitors with the former molecule optimized for brain delivery. Here the influence of the BBB on tumor distribution and resultant efficacy was evaluated in flank and intracranial tumors. Pharmacokinetic-pharmacodynamic approaches were used in GBM10 (patient-derived) and U87 (cell line) orthotopic xenograft models to evaluate the influence of delivery on efficacy. We investigated the brain distribution of GNE-317 and GDC-0980 in tumor-bearing (GBM10 and U87) athymic nu/nu mice and non-tumor-bearing (FVBn) mice. BBB-integrity in both GBM10 and U87-models was evaluated using Texas-Red(TR)-linked 3kD dextran prior to euthanasia. GNE-317 and GDC-0980 were administered orally at 30mg/kg and 7.5mg/kg, respectively, for all studies. Brain distribution studies were performed in FVB-wild type (WT) and P-gp/Bcrp knockout mice (TKO) and brain and plasma exposures (area-under-curve, AUC) were determined. Comparison of tumor pharmacokinetic-pharmacodynamic endpoints at 1- and 6-hr post single oral dose will be reported. GBM10 showed heterogeneous disruption of BBB-integrity, while U87 tumors exhibited homogenous and uniform disruption of BBB by analysis of TR-dextran accumulation within orthotopic xenografts. The brain-to-plasma AUC0-tlast ratio in FVBn-wild type was ∼10-fold higher in GNE-317(0.75) vs. GDC-0980 (0.07). Survival studies in intracranial tumors with U87 demonstrated no difference in survival between the GNE-317 vs. GDC-0980 treated groups (median survival∼26 days (GNE-317), 34 days(GDC-0980), p=0.07), and both treatments significantly extended survival compared to vehicle control (p<0.0003, ∼12 days). Survival with GNE-317 in GBM10 xenografts (median survival 42 days vs. 35 days for control, p<0.0003) was greater than the GDC-0980 treated group (median survival 32 days vs. 35 days in control, p=0.79); both treatments were not significantly different from each other (p=0.08). To evaluate the relative efficacy in the complete absence of the BBB, the effects on tumor growth are being evaluated in flank tumor models. These studies demonstrate that efficacy of a PI3K/mTOR inhibitor with low brain penetrance (GDC-0980) is marginally inferior to the related brain penetrant GNE-317 in tumor models with a partially intact BBB. Since GBM patients have regions of intact BBB, these data suggest that the brain penetration of targeted agents may be a critical factor that determines treatment efficacy in patients. Citation Format: Rajneet K. Oberoi, William F. Elmquist, Rajendar Mittapalli, Jenny Pokorny, Jann Sarkaria. Influence of the blood-brain barrier on drug distribution and efficacy of PI3K/mTOR inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4479. doi:10.1158/1538-7445.AM2014-4479

Abstract Purpose: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide. Experimental Design: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging. Results: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors. Conclusions: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM. Clin Cancer Res; 21(8); 1916–24. ©2015 AACR.

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and is associated with a poor prognosis. Progress in developing effective therapies for this disease is significantly limited by the blood-brain barrier (BBB), which limits the delivery of many anti-cancer agents to infiltrative tumor cells. In addition to physical barriers, such as tight junctions, the efflux proteins bcrp and P-gp in the BBB limit the brain distribution of numerous anti-cancer agents. Palbociclib (PD0332991) is a potent Cdk4/6 inhibitor which has shown remarkable efficacy in treating peripheral (non-brain) tumors. The Cdk4 pathway is dysregulated in approximately 75% of GBM; most commonly, the pathway is hyperactivated through the homozygous deletion of p16 (52%), amplification of Cdk4 (18%), or amplification of Cdk6 (1%). The purpose of this study is to define the role of the efflux transporters P-gp and bcrp in the brain distribution of palbociclib and to examine if an intact BBB limits efficacy. Palbociclib brain distribution studies were performed in FVB wild-type, P-gp knockout (PKO; Mdr1a/b(-/-)), bcrp knockout (BKO; Bcrp1(-/-)), and triple knockout (TKO; Mdr1a/b(-/-)Bcrp1(-/-)) mice after an oral dose (10mg/kg). The concentrations of palbociclib from all distribution studies were determined by a sensitive and specific LC-MS/MS assay. Survival studies were conducted in patient-derived primary GBM xenograft models in athymic nu/nu mice. The brain exposure of palbociclib (AUCbrain-to-AUCplasma ratio) was ∼ 33.5, 3.2, and 150-fold higher as compared to WT mice (WT: .044; PKO: 1.34; BKO: 0.13; TKO: 6.24). Further, the steady-state brain-to-plasma ratio (B/P) of palbociclib after a constant intra-peritoneal infusion of 10 µg/hr for 48hrs was ∼120-fold higher in the TKO mice than the WT mice [WT: (0.21 ± 0.07); PKO: (2.48 ± .13); BKO: (0.43 ± 0.12); TKO: (26.5 ± 5.4) p < 0.0001]. Inhibition of P-gp and bcrp with elacridar (10 mg/kg IP) resulted in a marked increase in palbociclib brain distribution [control B/P (0.06 ± 0.02); elacridar treatment (2.0 ± 1.4)]. For survival studies, palbociclib was dosed at 150 mg/kg/day continuously. Consistent with limited brain penetration, palbociclib did not improve the median survival of an orthotopic GBM xenograft model. In contrast, treatment of GBM22 xenografts grown as flank tumors resulted in profound efficacy with a 70 day prolongation in the time for tumor volume to reach 1000mm3. These data suggest the clinical paradigm of a potent anti-cancer agent (for instance, palbociclib) used in the treatment of peripheral disease is less effective in the treatment of brain tumors due to the BBB and active efflux by P-gp and bcrp. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C81. Citation Format: Karen E. Parrish, Jenny L. Pokorny, Rajendar K. Mittapalli, Katrina Bakken, Jann N. Sarkaria, William F. Elmquist. BBB efflux pump activity limits brain penetration of palbociclib (PD0332991) in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C81.

Abstract The importance of the blood–brain barrier in preventing effective pharmacotherapy of glioblastoma has been controversial. The controversy stems from the fact that vascular endothelial cell tight junctions are disrupted in the tumor, allowing some systemic drug delivery. P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) efflux drugs from brain capillary endothelial cells into the blood. We tested the hypothesis that although the tight junctions are “leaky” in the core of glioblastomas, active efflux limits drug delivery to tumor-infiltrated normal brain and consequently, treatment efficacy. Malignant gliomas were induced by oncogene transfer into wild-type (WT) mice or mice deficient for Pgp and BCRP (knockout, KO). Glioma-bearing mice were orally dosed with dasatinib, a kinase inhibitor and dual BCRP/PgP substrate that is being currently tested in clinical trials. KO mice treated with dasatinib survived for twice as long as WT mice. Microdissection of the tumor core, invasive rim, and normal brain revealed 2- to 3-fold enhancement in dasatinib brain concentrations in KO mice relative to WT. Analysis of signaling showed that poor drug delivery correlated with the lack of inhibition of a dasatinib target, especially in normal brain. A majority of human glioma xenograft lines tested expressed BCRP or PgP and were sensitized to dasatinib by a dual BCRP/Pgp inhibitor, illustrating a second barrier to drug delivery intrinsic to the tumor itself. These data show that active efflux is a relevant obstacle to treating glioblastoma and provide a plausible mechanistic basis for the clinical failure of numerous drugs that are BCRP/Pgp substrates. Mol Cancer Ther; 11(10); 2183–92. ©2012 AACR.