Citations

Abstract The clinical efficacies of molecularly targeted glioblastomas therapies have been vastly disappointing. The multitudes of resistance mechanisms suggest that glioblastomas possess highly dynamic molecular circuits grounded in functional redundancy. Emerging data suggests that the expression of functionally redundant oncogenes induced similar forms of cellular stress, requiring hyper-activation of common compensatory pathways to ensure cell viability. Targeting these pathways, therefore, afford potential opportunities for tumor ablation while by-passing the redundancy of oncogenic circuitry. To explore this paradigm, we carried out a siRNA screen to identify synthetic lethal partners of the oncogenic Epidermal Growth Factor Receptor variant III (EGFRvIII) in glioblastoma cells and identified Polo-Like Kinase 1 (PLK1). Treatment using a PLK1 inhibitor, BI2536, or siRNAs induced preferential toxicity to the EGFRvIII expressing glioblastoma cells in multiple in vitro (serum and neurosphere lines) and in vivo models (heterotopic and orthotopic xenograft models). Consistent with the heightened PLK1 requirement, EGFRvIII expressing glioblastomas harbored increased levels of the p-Thr210 PLK1 (an activated form of PLK1). Inhibition of PLK1 by BI2536 treatment induced an increase in the proportion of cells that co-stained for p-Histone H3 and γH2AX foci, suggesting accumulation of mitotic DNA damage. This effect was exacerbated by EGFRvIII expression, implicating induction of mitotic DNA damage as a major contributor to the observed synthetic lethality. Consistent with this observation, EGFRvIII expression induced the formation of aberrant mitosis as well as prolonged mitotic progression. Further supporting an essential role for PLK1 in suppressing DNA damage accumulation, BI2536 treatment significantly enhanced the tumoricidal effect of the DNA damaging chemotherapy, temozolomide. Mechanistically, inhibition of PLK1 suppressed the expression of Rad51, the accumulation of pS14 Rad51 (an active form of Rad51), as well as overall homologous recombination efficiency in vitro. We validated the clinical pertinence of these results using three clinically annotated glioblastoma databases (TCGA, REMEBMRANDT, CGGA). In all three datasets, increased expression of a PLK1 signature consistently associated with increased expression of HR genes and lowered gene expression signature associated with DNA damage accumulation. Supporting our proposed paradigm, the tumoricidal effect of BI2536 was universally observed in a panel of eight murine ink4a/arf (-/-) EGFRvIII expressing glioblastoma clones that developed resistance to EGFR inhibitors by distinct and independent mechanisms. In aggregate, our results support the essential role of PLK1 in suppressing mitotic DNA damage and provide a novel framework for glioblastoma therapy. Citation Format: Ying Shen, Masayuki Nitta, Jie Li, Diahnn Futalan, Tyler Steed, Zack Taich, Jeffrey M. Treiber, Deanna Stevens, Mark A. Schroeder, Jann N. Sarkaria, Hong-Zhuan Chen, Tao Jiang, Bob S. Carter, Fumiko Esashi, Jill Wakosky, Frank Furnari, Webster K. Cavenee, Arshad Desai, Clark C. Chen. Modulation of mitotic DNA damage as a paradigm for glioblastoma therapy. [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 2737. doi:10.1158/1538-7445.AM2014-2737

Abstract GNE-317 is a dual PI3K/mTOR inhibitor with excellent blood-brain barrier penetration. In this study, the efficacy of GNE-317 was evaluated in the Mayo panel of primary glioblastoma multiforme (GBM) xenografts. Based on the concept of oncogene addiction, we hypothesized that GNE-317 would be most efficacious in EGFR-hyper-activated and/or PTEN-deficient lines. In a CyQUANT cell proliferation assay, the inhibitory concentration of 50% cell number (IC50) for GBM6, 10, 22, and 84 were 0.59±0.50, 0.72±0.40, 0.26±0.14, and 3.49±1.64 microM, respectively. In these selected lines, there was a poor correlation between EGFR or PTEN status and in vitro response. The efficacy of GNE-317 also was evaluated in 10 lines in an orthotopic xenograft model. Mice with established tumors were randomized to daily dosing with GNE-317 (30 mg/kg) or placebo until moribund. Similar to in vitro data, there was a spectrum of response to GNE-317 (Table 1). The survival benefit ratio (ratio of median survival) did not correlate with EGFR amplification (P=0.78), EGFR mutation (P=0.25), or PTEN (P=0.55) status when analyzed singly. However, GNE-317 did extend survival in 4 of 5 lines in which either EGFR or PTEN were deregulated. To evaluate potentially clinically relevant combinations, the efficacy of GNE-317 combined with bevacizumab (BEV) was tested in the same orthotopic model in GBM8, 10, and 59. In GBM10, GNE-317/BEV provided a significant increase in survival compared to the most effective single agent (P<0.0001). In GBM8, 10, and 59, the ratio of median survival for GNE-317/BEV vs. placebo were 1.47, 1.75, and 1.74, respectively (all P≤0.01). In conclusion, there may be a correlation between deregulation of the EGFR/PTEN signaling network and GNE-317 efficacy, and the efficacy of GNE-317 in some models can be extended when combined with bevacizumab. Table 1:Molecular Characteristics and Efficacy of GNE-317 in Mayo Primary GBM XenograftsEGFR AmplifiedEGFR vIII MutatedPTEN MutatedGNE-317 Responder*Benefit Ratio** of GNE-317GBM6YesYesWTYes1.35GBM8YesNoYes, HDYes1.19GBM10NoNoYes, HDYes1.47GBM22NoNoWTNo0.85GBM59YesYesYes, HDYes1.27GBM84YesNoYes, del Exon 1No1.02GBM115NoNoTBDNo0.86GBM116NoNoTBDNo1.05GBM117NoNoTBDYes1.38GBM122YesNoTBDNo0.99Abbreviations: HD=homozygous deleted; del=deletion in; TBD=to be determined; WT=wild type.* If in vivo survival difference reached P<0.05 by Log-rank Kaplan-Meier method between the GNE-317 treated and placebo groups, the GBM line was classified as a responder to GNE-317.** The benefit ratio equals to median survival of the GNE-317 treated group, divided by that of the placebo group. Note: This abstract was not presented at the meeting. Citation Format: Terence T. Sio, Jenny L. Pokorny, Ann C. Mladek, Brett L. Carlson, Mark A. Schroeder, Dennis O. Iyekegbe, Katrina Bakken, Laurent Salphati, Heidi Phillips, Jann N. Sarkaria. Preclinical efficacy of GNE-317, a dual PI3K/mTOR inhibitor, with or without bevacizumab in EGFR and/or PTEN-mutated glioblastoma multiforme. [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 2617. doi:10.1158/1538-7445.AM2014-2617

Abstract Disruption of the MDM2/p53 pathway is an oncogenic driver alteration observed in the vast majority of GBM. This study evaluated the efficacy of the small molecule, human-specific, MDM2 inhibitor SAR405838 across a panel of GBM xenografts with distinct alterations within the MDM2/p53 pathway. Through screening a panel of 56 GBM xenografts with a combination of array CGH and MDM2-specific PCR amplification, five xenograft models were identified with high-level MDM2 amplification (GBM 5, 102, 108, 122, and 143). MDM2 transcript expression was elevated in all of these lines except GBM102, which had expression levels similar to non-amplified lines. The effects of SAR405838 were studied in 2 of the MDM2 amplified lines (GBM102, GBM108), and non-amplified lines with wild-type p53 (GBM10) or mutant p53 (GBM12). Using a combination of neurosphere and Cyquant assays to assess in vitro sensitivity, 100 nM SAR405838 completely inhibited growth of GBM108 (6% +/- 1% absorbance relative to control), partially inhibited GBM102 neurosphere formation (34 +/- 17% relative to control), and had nominal impact on relative neurosphere formation in GBM10 (51% +/- 4% relative to control) or GBM12 (96% +/- 4% relative to control). Twenty-four hours after treatment with 100 nM SAR405838, transcript expression of p53 target genes p21 and PUMA were elevated by 14 and 12 fold relative to control in GBM108, while more limited changes in expression were observed in GBM102 (4 and 2.5 fold increase, respectively). Similarly, analysis of apoptosis after 72h by Annexin-V staining demonstrated a 5-fold increase in apoptotic fraction from control in GBM108 compared to no increase in GBM102. A more striking difference in efficacy was observed in flank tumor xenograft studies where the time to reach a median tumor volume of 1000 mm3 was marginally extended in GBM102 (36 days for placebo vs. 44 days for SAR405838). In contrast, GBM108 placebo-treated mice grew to a median volume of 1000 mm3 in 53 days while treatment with SAR405838 resulted in complete tumor regression at 70 days. SAR405838 is a known substrate for the P-glycoprotein efflux pump that functions in the blood brain barrier to exclude xenobiotics from the central nervous system. Consistent with limited penetration of SAR405838 into the brain, a parallel study in an intracranial xenograft model for GBM108 demonstrated no significant survival benefit with SAR405838 treatment compared to placebo. Thus, although SAR405838 provides marked efficacy in vitro and in a flank tumor model of an MDM2 amplified tumor line, the lack of efficacy in an orthotopic model of this study suggests that more prudent investigation is needed before advancing the development of this specific drug in human tumors growing in the brain. Citation Format: Ann C. Mladek, Isabelle Meaux, Katrina Bakken, Pascal Pannier, Cedric Barriere, James W. Watters, Laurent R.J. Debussche, Jann N. Sarkaria. Targeted murine double minute 2 (MDM2) inhibition results in dramatic tumor regression in an MDM2-amplified glioblastoma multiforme (GBM) xenograft 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 1799. doi:10.1158/1538-7445.AM2014-1799

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 Glioblastoma multiforme (GBM) comprises the largest group of brain tumors which are drug resistant and respond very poorly to the current therapies. In this study, we used sulforaphane (SFN), a multi-targeting agent with cancer preventive and anti-cancer activities and showed that it targets GBM established cell lines, early primary cultures, and CD133+ GBM stem cells as well as in GBM stem-like spheroids. SFN at 5-50 μM triggered significant inhibition of cell survival and induced apoptotic cell death in GBM cells and CD133+ stem cells isolated from four GBM cell lines. SFN induced apoptosis in U87MG cells was associated with caspase-7 activation. Moreover, SFN triggered formation of intracellular reactive oxygen species (ROS) and when the cells were pre-treated with 10 mM N-acetyl cysteine (NAC), ROS production and cell survival in cells treated with 5-10 μM were similar to the control untreated U87MG cells, revealing that SFN-triggered cell death is ROS-dependent. Moreover, SFN-generated ROS in U87MG cells were formed at the Mitochondrial Respiratory Chain (MRC) level. SFN also increased expression of the TRAIL receptor DR5 in GBM cells, U87MG and SF767 cells by 24 h post-exposure. Moreover, as revealed by comet assay, SFN increased single- and double-strand DNA breaks in GBM. Compared to untreated control cells, a significantly higher amount of γ-H2AX foci and as consequence higher number of DNA double-strand breaks (DSBs) breaks were observed in the SFN-treated sample. In vivo studies, using NOD/SCID mice revealed that SFN administration via oral gavage at 100 mg/kg for 3 cycles significantly decreases the growth of ectopic xenografts established from the early passage primary cultures of GBM10. Our results show that SFN robustly inhibits growth of GBM cells in vitro and in vivo and induces cell death in established cell cultures, early passage primary cultures, as well as it is effective in eliminating GBM cancer stem cells, which play a major role in drug resistance and disease recurrence. These results suggest that use of SFN alone or in combination with other agents, may potentially improve survival of brain tumor patients. Citation Format: Khadijeh Bijangi-Vishehsaraei, Mohammad R. Saadatzadeh, Haiyan Wang, Malgorzata M. Kamocka, Wenjing Cai, Aaron A. Cohen-Gadol, Stacey L. Halum, Karen E. Pollok, Jann N. Sarkaria, Ahmad R. Safa. Sulforaphane depresses proliferation and induces cell death in glioblastoma multiforme (GBM) cells, GBM stem cell-like spheroids, and tumor xenografts through modulation of multiple cell signaling pathways. [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 2267. doi:10.1158/1538-7445.AM2014-2267

Abstract Glioblastoma multiforme (GBM) is the most common and most lethal primary malignant brain tumor, affecting 25,000 patients per year. Approximately 87% of GBMs have abnormalities in the p53 pathway that affects G2 checkpoint. In the absence of p53, Wee1 kinase has been shown to regulate G2 checkpoint upon chemotherapeutic insult. Wee1 inhibits CDC2 function, thereby promoting G2 phase cell cycle arrest, thus preventing damaged cells from entering into premature mitosis without repairing the DNA. We hypothesize that in p53-deficient tumor cells, inhibitors of Wee1 would be effective to heighten DNA damage due to genetic and pharmacologic loss of both the G1 and G2 checkpoints, respectively. MK-1775 (Merck) is a potent and selective small molecule inhibitor of the Wee1 kinase that has demonstrated enhanced cytotoxic effect in combination with several chemotherapeutic agents in preclinical studies in vitro and in vivo. Here, we selected an array of short term cultures of glioma patient derived xenograft (PDX) models (GBM12, GBM43, GBM79, GBM84) and long term established glioma cell lines (U87, SF767, T98G) with aberrant and wild type G1 checkpoint pathways and investigated cytotoxic effects of MK-1775 as a single agent and in combination with Temozolomide (TMZ). Our results demonstrated differential sensitivity to MK-1775 and TMZ as single agents for different glioma cell lines in vitro. However, no correlation between MK-1775, TMZ sensitivity and p53 status of the cell lines was observed. Combination therapy with MK-1775 and TMZ showed a synergistic effect for one PDX model, GBM43, with combination index (CI) of 0.4. Combination therapy of all other glioma cell lines either showed an additive effect or antagonistic effect with CI of >0.75 irrespective of their p53 status. Our results demonstrated that synergistic effects of MK-1775 in combination with chemotherapy (TMZ) are not solely dependent on p53 status of glioma cells and require a more comprehensivce understanding of the molecular mechanism that dictates GBM's dependence on Wee1 for escaping DNA damage therapy. Supported by the Ben & Catherine Ivy Foundation Citation Format: Harshil D. Dhruv, Andrew D. Nelson, Brock Armstrong, Julie L. Boerner, Jann N. Sarkaria, Nhan L. Tran, Michael Berens. Vulnerability and resistance in glioblastoma to G2-specific drugs. [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 1319. doi:10.1158/1538-7445.AM2014-1319

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 This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM. Mol Cancer Ther; 13(12); 2919–29. ©2014 AACR.

Abstract There is a significant unmet medical need to develop more effective treatments for cancers of the central and peripheral nervous systems. Glioblastoma multiforme (GBM) is the most common form of brain cancer, and successful long-term treatment has not been realized. Neuroblastomas (NB) arise in the sympathetic nervous system and are the most common extracranial solid tumor of childhood. Survival of children with disseminated, high-risk NB tumors is less than 50%, even with intensive multimodal treatment. To improve treatment outcome, inhibition of murine double minute 2 (MDM2) function in the context of front-line therapy is being explored. MDM2 is a multi-functional protein that plays a critical role in controlling cell growth and migration. MDM2 is a negative regulator of p53 and p73 and, under low stress conditions, sequesters them in the cytoplasm. However, inhibition of MDM2 interactions with p53 and p73 by the MDM2 antagonist nutlin3a can lead to activation of p53/p73-mediated cell-death in tumor cells. The objective of the present study was to evaluate the extent to which blockade of MDM2-p53/p73 interactions by nutlin3a, alone or in combination with front-line therapies, could augment cell death in GBM and NB cells with a variety of molecular profiles. Using the median effect model, we found that temozolomide (TMZ) and nutlin3a were synergistic in increasing cell death in wtp53 U87 and U373 but were predominantly additive in mtp53 U118 cells. Primary patient GBM cells were typically resistant to TMZ relative to established cell lines. However, resistance to TMZ was decreased in the presence of nutlin3a. Further, TMZ and nutlin3a were synergistic in primary GBM10 and GBM43 but not in recurrent primary IU-GBM16, IU-GBM23, IU-GBM27, and IU-GBM32 cells. Moreover, in NB cells with wtp53, nutlin3a and cisplatin were synergistic in MYCN nonamplified SK-N-SH and additive in MYCN amplified IMR5. In mtp53 NB cells, nutlin3a and cisplatin were synergistic in MYCN nonamplified SK-N-FI as well as in MYCN amplified SK-N-DZ. Nutlin3a alone produced dose-related increases in cell death of all GBM and NB cell lines irrespective of p53 status, suggesting that p53-independent mechanisms play a role in inhibition of cell growth produced by blockade of MDM2-mediated signaling. Consistent with this hypothesis, nutlin3a increased p73 protein levels in mtp53 GBM43 cells that were exposed to TMZ. Inhibition of MDM2 function also inhibited repair of TMZ-mediated DNA damage in GBM. In comet assays, nutlin3a+TMZ decreased DNA repair relative to TMZ alone in wtp53 U87 and GBM10 and in mtp53 GBM43 cells. The present results demonstrate that MDM2 is a valid therapeutic target in GBM and NB cells with varying genetic profiles. In conclusion, inhibition of MDM2 signaling by nutlin3a is generally synergistic to additive in combination with front-line chemotherapeutics used to treat cancers of the central and peripheral nervous systems. Citation Format: Harlan E. Shannon, Haiyan Wang, Shanbao Cai, Wei Michael Liu, Lawrence M. Gelbert, Aaron A. Cohen-Gadol, Jann N. Sarkaria, Lindsey D. Mayo, Karen E. Pollok. MDM2 as a therapeutic target: improving upon front-line chemotherapy for nervous system tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3435. doi:10.1158/1538-7445.AM2013-3435

Abstract Wee1 is a key regulator of both G2/M transition and DNA replication fork stability. In this study, the efficacy of the Wee1 inhibitor MK1775 was evaluated in the Mayo primary glioblastoma multiforme (GBM) xenograft model. Based on western blot expression of Wee1, GBM22, GBM12 and GBM6 were selected for study based on low, intermediate and high-level expression, respectively. Wee1-mediated phosphorylation of Y15-Cdc2 was robustly suppressed in vitro in both GBM22 and GBM6 at 300 nM MK1775. At this concentration, MK1775 had marked anti-proliferative effects on GBM22 with 80±2.5% suppression of absorbance in a CyQUANT cell proliferation assay, while similar treatment of GBM6 had minimal effect on growth (26±21% suppression of absorbance). In a similar primary neurosphere formation assay, 300 nM completely inhibited neurosphere formation in GBM22 and GBM12, while having no effect on GBM6 neurosphere formation. Consistent with the cytotoxicity data, single agent treatment of GBM22 with MK1775 resulted in a marked S-phase arrest 24 hours after treatment (60% vs. 40% cells in S with MK1775 vs. control, respectively) and subsequent accumulation of cells in G2/M 48 hours after treatment (95% vs. 9.5%, respectively). In contrast, relatively minimal cell cycle perturbations were observed with MK1775 in GBM6 (24 hour S-phase accumulation 27% vs. 21%, respectively). Combination studies with MK1775 and temozolomide (TMZ) in a neurosphere formation assay demonstrated enhancement of treatment benefit only in GBM22 and GBM12 but not GBM6 (relative neurosphere numbers for TMZ, MK1775, or combined treatment compared to untreated were 34%, 16% and 7%, respectively for GBM22, 41%, 95% and 15%, respectively, for GBM12; while the same combination had no effect on GBM6 neurosphere formation). In anticipation of evaluating treatment efficacy in animal models, pharmacokinetic modeling of MK1775 brain delivery was performed in nude mice. Following a single MK1775 dose of 50 mg/kg, time to maximum blood concentration was 1.0 hours, AUC was 29.8 microM·h, and Cmax was 7.8 microM in whole blood and 0.24 microM in brain parenchyma. To evaluate whether limitations in brain delivery affected efficacy, parallel in vivo GBM12 heterotopic and orthotopic xenograft efficacy studies were performed with a 5 day dosing regimen of TMZ and MK1775 repeated every 28 days for 3 cycles. In the orthotopic model, MK1775 provided no survival benefit with median survival being 16, 15, 88 and 92 days for placebo, MK1775 alone, TMZ alone, or MK1775 + TMZ treatment, respectively. In contrast, there is some suggestion of MK1775 efficacy in the flank tumor model with median time to reach 1000 mm3 being 45, 56, 101 and 112 days, respectively. Collectively, these data suggest that despite promising in vitro activity for MK1775, limited drug delivery into the brain may reduce the efficacy of MK1775 therapy in an orthotopic GBM xenograft model. Citation Format: Jenny Pokorny, Vincenzo Pucci, Stuart Shumway, Bennett Ma, Katrina Bakken, Mark Schroeder, Brett Carlson, Jann N. Sarkaria. Pre-clinical investigation of the Wee1 inhibitor, MK1775, in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2013-3240