Pokorny, Jenny L.

Abstract Purpose: Glioblastoma (GBM), the most common primary brain tumor in adults, presents a high frequency of alteration in the PI3K pathway. Our objectives were to identify a dual PI3K/mTOR inhibitor optimized to cross the blood–brain barrier (BBB) and characterize its brain penetration, pathway modulation in the brain and efficacy in orthotopic xenograft models of GBM. Experimental Design: Physicochemical properties of PI3K inhibitors were optimized using in silico tools, leading to the identification of GNE-317. This compound was tested in cells overexpressing P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP). Following administration to mice, GNE-317 plasma and brain concentrations were determined, and phosphorylated biomarkers (pAkt, p4EBP1, and pS6) were measured to assess PI3K pathway suppression in the brain. GNE-317 efficacy was evaluated in the U87, GS2, and GBM10 orthotopic models of GBM. Results: GNE-317 was identified as having physicochemical properties predictive of low efflux by P-gp and BCRP. Studies in transfected MDCK cells showed that GNE-317 was not a substrate of either transporter. GNE-317 markedly inhibited the PI3K pathway in mouse brain, causing 40% to 90% suppression of the pAkt and pS6 signals up to 6-hour postdose. GNE-317 was efficacious in the U87, GS2, and GBM10 orthotopic models, achieving tumor growth inhibition of 90% and 50%, and survival benefit, respectively. Conclusions: These results indicated that specific optimization of PI3K inhibitors to cross the BBB led to potent suppression of the PI3K pathway in healthy brain. The efficacy of GNE-317 in 3 intracranial models of GBM suggested that this compound could be effective in the treatment of GBM. Clin Cancer Res; 18(22); 6239–48. ©2012 AACR.

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.

Abstract INTRODUCTION: The L-Type amino acid transporter 1 (LAT1) transports large neutral amino acids across the plasma membrane in a variety of cells. Positron emission tomography (PET) imaging with the amino acid tracer 6-18F-fluoro-L-dopa (18F-FDOPA) provides useful histological information in human gliomas in addition to providing better localizing information then CT or MRI alone. This study is the first to correlate LAT1 expression with uptake of the 18F-FDOPA analog, 3H-L-DOPA in human glioblastomas. METHODS: Endogenous expression of LAT1 was evaluated in two established glioblastoma multiforme (GBM) cell lines and three primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of FDOPA was assessed in vitro using 3H-L-DOPA as an analog to 18F-FDOPA. Knockdown of LAT1 expression was achieved by lentiviral expression of anti-LAT1 shRNA in GBM lines with high LAT1 expression. The effect of LAT1 knockdown on 3H-L-DOPA uptake was then quantified. RESULTS: Results from Western blotting and qRT-PCR demonstrate that LAT1 is variably expressed in GBM. Levels of 3H-L-DOPA uptake were directly related to LAT1 expression (p<0.001) across the 5 lines. Knockdown of LAT1 was achieved in T98 cells (knockdown efficiency = 82.8%) and xenograft GBM 28 (knockdown efficiency = 73.7%) as confirmed by qRT-PCR and Western blotting. Knockdown of LAT1 reduced 3H-L-DOPA uptake by 57.2% (p<0.0001) in T98 and 52.1% in GBM 28 (p<0.001). CONCLUSIONS: This is the first report of 3H-L-DOPA uptake being driven by LAT1 in human GBM. Our findings suggest LAT1 as a transporter of 18F-FDOPA in GBM. Additional studies are underway to correlate LAT1 expression with PET 18F-FDOPA uptake in patients with GBM. 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 4543. doi:1538-7445.AM2012-4543

Abstract Purpose: The therapeutic benefit of temozolomide in glioblastoma multiforme (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of temozolomide resistance in GBM. Experimental Design: We developed an in vivo GBM model of temozolomide resistance and used paired parental and temozolomide-resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition. Results: Analysis of paired parental and resistant lines showed upregulation of O6-methylguanine-DNA methyltransferase (MGMT) expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative-resistant samples, chromatin immunoprecipitation showed an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased dimethylation (H3K9-me2) in GBM12 and GBM14. In contrast, temozolomide resistance development in GBM22 was not linked to MGMT expression, and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12TMZ-resistant line, MGMT reexpression was accompanied by increased recruitment of SP1, C-JUN, NF-κB, and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with temozolomide and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of temozolomide resistance by MGMT overexpression as compared with treatment with temozolomide alone. Conclusion: This study shows, for the first time, a unique mechanism of temozolomide resistance development driven by chromatin-mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM. Clin Cancer Res; 18(15); 4070–9. ©2012 AACR.

Abstract Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro‐oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short‐term explant cultures or intracranial xenografts. This unit describes detailed procedures for establishment, maintenance, and utilization of a primary GBM xenograft panel for the purpose of using them as tumor models for basic or translational studies. Curr. Protoc. Pharmacol . 52:14.16.1‐14.16.23. © 2011 by John Wiley & Sons, Inc.