Nienke A. de Vries

P-Glycoprotein and Breast Cancer Resistance Protein: Two Dominant Transporters Working Together in Limiting the Brain Penetration of Topotecan

Purpose: The brain is a pharmacologic sanctuary site, due to the presence of the blood-brain barrier (BBB). Whereas the effect of P-glycoprotein (P-gp) at the BBB is well established, the role of breast cancer resistance protein (BCRP) that is also expressed at the BBB is not. Experimental Design: We have studied the effect of BCRP by administering topotecan to wild-type (WT), single Mdr1a/b(−/−) and Bcrp1(−/−), and compound Mdr1a/b(−/−)Bcrp1(−/−) knockout mice. Drug levels in plasma and tissues were determined by high-performance liquid chromatography. Results: The area under the plasma and tissue concentration-time curve (AUC) of topotecan in brains of Mdr1a/b(−/−) and Bcrp1(−/−) mice was only 1.5-fold higher compared with WT mice, but in Mdr1a/b(−/−)Bcrp1(−/−) mice, where both transporters are absent, the AUC increased by 12-fold. The AUC in plasma was ∼0.75-, 2.4-, and 3.7-fold higher in Mdr1a/b(−/−), Bcrp1(−/−), and Mdr1a/b(−/−)Bcrp1(−/−) mice, respectively, resulting in 2.0-fold (P < 0.01), 0.65-fold (P, not significant), and 3.2-fold (P < 0.01), respectively, higher brain-to-plasma AUC ratios. Results using Mrp4(−/−) mice showed that this transporter had no effect on the brain penetration of topotecan. The P-gp/BCRP inhibitor elacridar fully inhibited P-gp–mediated transport of topotecan, whereas inhibition of Bcrp1-mediated transport by elacridar was minimal. Conclusions: Our results using Mdr1a/b(−/−)Bcrp1(−/−) mice clearly show the effect of Bcrp1 at the BBB and also show how two drug transporters act in concert to limit the brain penetration of topotecan. We expect that this finding will also apply to other drugs that are substrates of both P-gp and BCRP. Consequently, to improve the brain penetration of such compounds for targeting intracranial malignancies in patients, it will be essential to use potent inhibitors of both drug transporters.

Effect of the ATP-binding cassette drug transporters ABCB1, ABCG2, and ABCC2 on erlotinib hydrochloride (Tarceva) disposition in in vitro and in vivo pharmacokinetic studies employing Bcrp1−/−/Mdr1a/1b−/− (triple-knockout) and wild-type mice

We tested whether erlotinib hydrochloride (Tarceva, OSI-774), an orally active epidermal growth factor receptor tyrosine kinase inhibitor, is a substrate for the ATP-binding cassette drug transporters P-glycoprotein (P-gp; MDR1, ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance protein 2 (MRP2; ABCC2) in vitro and whether P-gp and BCRP affect the oral pharmacokinetics of erlotinib hydrochloride in vivo. In vitro cell survival, drug transport, accumulation, and efflux of erlotinib were done using Madin-Darby canine kidney II [MDCKII; wild-type (WT), MDR1, Bcrp1, and MRP2] and LLCPK (WT and MDR1) cells and monolayers as well as the IGROV1 and the derived human BCRP-overexpressing T8 cell lines. In vivo, the pharmacokinetics of erlotinib after p.o. and i.p. administration was studied in Bcrp1/Mdr1a/1b−/− (triple-knockout) and WT mice. In vitro, erlotinib was actively transported by P-gp and BCRP/Bcrp1. No active transport of erlotinib by MRP2 was observed. In vivo, systemic exposure (P = 0.01) as well as bioavailability of erlotinib after oral administration (5 mg/kg) were statistically significantly increased in Bcrp1/Mdr1a/1b−/− knockout mice (60.4%) compared with WT mice (40.0%; P = 0.02). Conclusion: Erlotinib is transported efficiently by P-gp and BCRP/Bcrp1 in vitro. In vivo, absence of P-gp and Bcrp1 significantly affected the oral bioavailability of erlotinib. Possible clinical consequences for drug-drug and drug-herb interactions in patients in the gut between P-gp/BCRP-inhibiting substrates and oral erlotinib need to be addressed. [Mol Cancer Ther 2008;7(8):2280–7]

Blood–brain barrier and chemotherapeutic treatment of brain tumors

The blood–brain barrier (BBB) is of pivotal importance to maintain homeostasis of the CNS, as it closely regulates the composition of the interstitial fluid in the brain. Unfortunately, malignancies that grow within the CNS may evade chemotherapeutic drugs using the same barrier, making this disease refractory to most chemotherapy regimens. This review will outline the impact of the BBB in brain cancer and discuss the efforts that have been made to enhance the drug exposure of brain tumors. Although this review will focus on the role of the BBB in primary brain cancer (malignant glioma), its impact on brain metastases will also be briefly discussed.

Tumor microvasculature supports proliferation and expansion of glioma-propagating cells.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The identification of ‘cancer stem cells’ (CSC) has shed new light on the potential mechanism of therapy resistance of these tumors. Because these cells appear to be more resistant to conventional treatments, they are thought to drive tumor regrowth after therapy. Therefore, novel therapeutic approaches that target these cells are needed. Tumor cells interact with their microenvironment. It has been reported that close contact between CSCs and tumor microvascular endothelium in GBM is important for CSCs to preserve their undifferentiated state and self‐renewal ability. However, our understanding of this interaction is still rudimentary. This is in part due to a lack of suitable in vitro models that accurately represent the in vivo situation. Therefore, we set up a co‐culture system consisting of primary brain tumor microvascular endothelial cells (tMVECs) and glioma propagating cells (GPCs) derived from biopsies of GBM patients. We found that tMVECs support the growth of GPCs resulting in higher proliferation rates comparing to GPCs cultured alone. This effect was dependent on direct contact between the 2 cell types. In contrast to GPCs, the FCS‐cultured cell line U87 was stimulated by culturing on tMVEC‐derived ECM alone, suggesting that both cell types interact different with their microenvironment. Together, these results demonstrate the feasibility and utility of our system to model the interaction of GPCs with their microenvironment. Identification of molecules that mediate this interaction could provide novel targets for directed therapy for GBM.

Rapid and Robust Transgenic High-Grade Glioma Mouse Models for Therapy Intervention Studies

Purpose: To develop a transgenic mouse model of glioma that can be conveniently used for testing therapy intervention strategies. High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. Typical for high-grade glioma is that glioma cells infiltrate extensively into surrounding pivotal brain structures, thereby rendering current treatments largely ineffective. Evaluation of novel therapies requires the availability of appropriate glioma mouse models. Experimental Design: High-grade gliomas were induced by stereotactic intracranial injection of lentiviral GFAP-Cre or CMV-Cre vectors into compound LoxP-conditional mice, resulting in K-Rasv12 expression and loss of p16Ink4a/p19Arf with or without concomitant loss of p53 or Pten. Results: Tumors reproduced many of the features that are characteristic for human high-grade gliomas, including invasiveness and blood-brain barrier functionality. Especially, CMV-Cre injection into p53;Ink4a/Arf;K-Rasv12 mice resulted in high-grade glioma with a short tumor latency (2-3 weeks) and full penetrance. Early detection and follow-up was accomplished by noninvasive bioluminescence imaging, and the practical utility for therapy intervention was shown in a study with temozolomide. Conclusion: We have developed a realistic high-grade glioma model that can be used with almost the same convenience as traditional xenograft models, thus allowing its implementation at the forefront of preclinical evaluation of new treatments. Clin Cancer Res; 16(13); 3431–41. ©2010 AACR.

Prolonged Ezh2 Depletion in Glioblastoma Causes a Robust Switch in Cell Fate Resulting in Tumor Progression

EZH2 is frequently overexpressed in glioblastoma (GBM), suggesting an oncogenic function that could be a target for therapeutic intervention. However, reduced EZH2 activity can also promote tumorigenesis, leading to concerns about the use of EZH2 inhibitors. Here, we provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and primary tumor-derived human GBM cell lines. Using doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor, we demonstrate that prolonged Ezh2 depletion causes a robust switch in cell fate, including significantly enhanced proliferation, DNA damage repair, and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression. Short-term Ezh2 depletion significantly improved survival without the tumor progression observed upon prolonged Ezh2 depletion, suggesting that precise dosing regiments are very important. These results could be of high clinical relevance with regard to how glioblastomas should be treated with epigenetic therapies.

TRIM28 Is an Epigenetic Barrier to Induced Pluripotent Stem Cell Reprogramming

Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large‐scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2017;35:147–157

Improved Brain Penetration and Antitumor Efficacy of Temozolomide by Inhibition of ABCB1 and ABCG2

The anticancer drug temozolomide is the only drug with proven activity against high-grade gliomas and has therefore become a part of the standard treatment of these tumors. P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) are transport proteins, which are present at the blood-brain barrier and limit the brain uptake of substrate drugs. We have studied the effect of P-gp and BCRP on the pharmacokinetics and pharmacodynamics of temozolomide, making use of a comprehensive set of in vitro transport experiments and in vivo pharmacokinetic and antitumor efficacy experiments using wild-type, Abcg2−/−, Abcb1a/b−/−, and Abcb1a/b;Abcg2−/− mice. We here show that the combined deletion of Abcb1a/b and Abcg2 increases the brain penetration of temozolomide by 1.5-fold compared to wild-type controls (P < .001) without changing the systemic drug exposure. Moreover, the same increase was achieved when temozolomide was given to wild-type mice in combination with the dual P-gp/BCRP inhibitor elacridar (GF120918). The antitumor efficacy of temozolomide against three different intracranial tumor models was significantly enhanced when Abcb1a/b and Abcg2 were genetically deficient or pharmacologically inhibited in recipient mice. These findings call for further clinical testing of temozolomide in combination with elacridar for the treatment of gliomas, as this offers the perspective of further improving the antitumor efficacy of this already active agent.

Large variety in a panel of human colon cancer organoids in response to EZH2 inhibition

EZH2 inhibitors have gained great interest for their use as anti-cancer therapeutics. However, most research has focused on EZH2 mutant cancers and recently adverse effects of EZH2 inactivation have come to light. To determine whether colorectal cancer cells respond to EZH2 inhibition and to explore which factors influence the degree of response, we treated a panel of 20 organoid lines derived from human colon tumors with different concentrations of the EZH2 inhibitor GSK126. The resulting responses were associated with mutation status, gene expression and responses to other drugs. We found that the response to GSK126 treatment greatly varied between organoid lines. Response associated with the mutation status of ATRX and PAX2, and correlated with BIK expression. It also correlated well with response to Nutlin-3a which inhibits MDM2-p53 interaction thereby activating p53 signaling. Sensitivity to EZH2 ablation depended on the presence of wild type p53, as tumor organoids became resistant when p53 was mutated or knocked down. Our exploratory study provides insight into which genetic factors predict sensitivity to EZH2 inhibition. In addition, we show that the response to EZH2 inhibition requires wild type p53. We conclude that a subset of colorectal cancer patients may benefit from EZH2-targeting therapies.

Abstract 5718: Implementation of μ-Image Guided Radio-Therapy in the treatment of experimental glioma mouse models: Assessment of the potential of agents that interfere with DNA repair

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. In order to evaluate the potential of novel therapies, we have developed a set of glioma mouse models. These include LoxP conditional mouse models that form spontaneous high-grade gliomas following intracranial injection of lentiviral CMV-Cre vectors and high-grade glioma models that develop following intracranial injection of cell lines obtained from these spontaneous models and maintained as neurosphere cultures. Standard treatment of newly diagnosed high-grade glioma after surgical resection involves radiotherapy (RT) and chemotherapy (CT) with temozolomide. Both modalities are based on inflicting damage to the DNA of tumor cells but have only a limited effect on overall survival. A potential strategy to improve the efficacy of DNA damaging therapies is to combine these with agents that interfere with DNA damage repair. In this study we have concentrated on the inhibition of PARP by ABT-888 and the inhibition of Wee1 kinase by PD0166285 and MK-1775. Importantly, we have mimicked the therapy of patients as closely as possible by implementing μ-Image Guided Radiotherapy (μ-IGRT) using the X-Rad 225Cx (Precision X-Ray Inc). Through cone beam CT guidance this system offers precise delivery of high energy beams (225 KVp) of small field sizes (1 - 5 mm), minimizing the exposure of normal tissues and allowing the delivery of RT doses that can not be given by conventional whole body RT. Here RT was delivered using a fractionated schedule (5 Gy per day x 4) in combination with oral temozolomide (100 mg/kg/day x 4) alone or with ABT-888 (10 mg/kg/bid x 4), PD0166285 (0.25 mg/kg/bid x 4 or MK1775 (20 mg/kg/bid x 4). Treatment of orthotopically injected Ink4a/Arf;P53;K-Rasv12 neurosphere-cultured cells (GBM652457) by RT + CT was much more efficacious than by CT alone. In line with the expectations, the PARP inhibitor ABT-888 significantly improved the response (assessed by bioluminescence monitoring) nd survival. However, the same combination was not more efficacious against spontaneous Ink4a/Arf;P53;K-Rasv12 tumors relative to RT + CT alone. Addition of the Wee1 kinase inhibitors PD0166285 or MK1775 did not improve the efficacy of RT+CT against intracranially injected GBM652457 cells. We are currently investigating the underlying reason of these results. ABT-888, PD0166285 and MK1775 are all substrates of ABCB1 and ABCG2 and especially MK1775 has a very poor BBB penetration. In conclusion, μ-IGRT is an exciting new technique to mimic treatment of glioma patients in mouse models more closely. Since all novel therapies for treatment of high-grade glioma will be given in conjunction to the current chemoradiation therapy, this technique will allow more accurate preclinical evaluation of novel therapies in a clinically relevant setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5718. doi:1538-7445.AM2012-5718