Bakken, Katrina K.

Abstract Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1–3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2–M arrest in vitro. In vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp−/− versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b−/− mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood–brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735–46. ©2017 AACR.

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.

Abstract Microtubule-targeting agents (MTA) have been employed in the treatment of many cancers for decades. BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizing agent that induces tumor cell death by activating the ‘spindle assembly checkpoint’. Given intravenously or orally, the drug penetrates the brain and has anti-cancer activity in diverse tumor models refractory to standard MTA or radiotherapy (RT). In this study, BAL101553 was evaluated in orthotopic xenografts from 16 GBM PDX models; 7 of 16 lines demonstrated significant (p<0.01) increases in median survival with BAL101553 versus placebo (range in median survival extension 24-87%). The combination of BAL101553 with conventional therapies for GBM (RT and temozolomide (TMZ) was then evaluated in select lines. In the MGMT methylated GBM12 line, combination of RT with TMZ increased survival compared to placebo (median survival 80 days vs. 23 days, respectively; p<0.001). Extended BAL101553 monotherapy provided a short but significant extension in survival (median survival 31 days, p<0.001), while extended BAL101553 dosing during and after RT/TMZ (median survival 85 days) did not extend survival relative to RT/TMZ alone (p = 0.56). In contrast, in the MGMT unmethylated GBM6 line, combination of RT and extended BAL101553 increased survival (median 90 days, p<0.001) relative to either treatment alone (median survival BAL101553 63 days; RT 69 days) or placebo (46 days). Additionally, the combination of BAL101553 with TMZ (median survival 70 days) was more effective than TMZ alone (median survival 60 days; p = 0.009). Consistent with the unmethylated MGMT status, the TMZ/RT combination (median survival 66 days) was similar to RT alone (p = 0.62), but the combination of extended BAL101553 with RT/TMZ (median survival 101 days; p<0.001 compared to other combination groups) was significantly more effective. To further evaluate whether BAL101553 is a true radiosensitizer, a second GBM6 study was performed. Also here, combination of RT (20Gy, 2wks) with extended BAL101553 dosing (median survival 66 days) significantly extended survival compared to RT alone (median survival 54 days; p = 002). Interestingly, when BAL101553 dosing was limited to 2 weeks with RT, there was no increase in median survival (58 days; p = 0.16). To evaluate effects on tumor repopulation during RT, the efficacy of an extended RT schedule (36 Gy, 6 wks) with or without 6 weeks of BAL101553 was evaluated. In this case, BAL101553 given during the RT schedule (median survival 78 days) extended median survival as compared to RT alone (61 days; p<0.001). Collectively, these data demonstrate that BAL101553 has broad single agent activity across a panel of GBM PDX models and suggests that combination with RT/TMZ therapy may provide additional benefits for survival extension. Citation Format: Ann C. Mladek, Jenny L. Pokorny, Heidi Lane, Felix Bachmann, Mark A. Schroeder, Katrina K. Bakken, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 has anti-cancer activity alone and in combination treatments across a panel of GBM patient-derived xenografts. [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 4781.

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 Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332–43. ©2016 AACR.

Abstract Glioblastoma multiforme (GBM) is an aggressive and fatal disease commonly treated with radiation and temozolomide (TMZ), but emergence of therapy resistance invariably limits treatment efficacy. Inhibition of Poly-ADP ribose polymerases 1 and 2 (PARP1/2) sensitizes GBM cells, and inhibitors of PARP are emerging as combination partners with TMZ. However, limited brain penetration precludes use of several promising PARP inhibitors in GBM. BGB-290 is a relatively new and potent PARP inhibitor that crosses the blood brain barrier with a brain to plasma ratio of ∼0.2. The focus of the present study was to evaluate antitumor effects of BGB-290, as single agent or in combination with TMZ, in patient derived GBM xenografts and glioma cell lines. In a biochemical assay, low doses (30-100 nM) of BGB-290 suppressed PARP activity, while single agent anti-tumor activity of BGB-290 was modest and restricted to concentrations above 3 μM. Combinations of 0.1 μM BGB-290 with 10 μM TMZ significantly diminished primary neurosphere formation in a patient-derived xenograft line, GBM12, decreasing relative neurosphere count to 3.1 ± 1.3% as compared to 9.2 ± 3.1% with TMZ alone, p = 0.03. Consistent with a mechanism involving disruption of DNA repair, treatment of GBM12 cells with TMZ and BGB-290 combination resulted in robust γH2AX foci formation and a synergistic increase in DNA damage signaling as compared to TMZ or BGB-290 alone. TMZ-sensitizing effects of BGB-290 were heterogeneous in TMZ-resistant (GBM12TMZ) sublines that were developed previously by in vivo selection of parental GBM12 with cyclical TMZ therapy. The average neurosphere formation across 4 resistant sublines lacking MGMT expression after 100 μM TMZ only was 75.3 ± 9.0% versus 14.9 ± 7.4% with TMZ and 0.1 μM BGB-290, p<0.01. In contrast, 2 MGMT overexpressing sublines were much less sensitive to the combination with relative neurospheres 91.9 ± 5.2% versus 73.2 ± 9.2% respectively, p< 0.01. Next Gen Sequencing revealed discrete non-synonymous somatic mutations in the mismatch repair pathway of GBM12TMZ sublines lacking MGMT, which led us to examine the relationship between mismatch repair and sensitizing effects of PARP inhibition. Silencing Msh2 resulted in marked TMZ resistance in U251 cells (94.7± 5.4% relative growth with 100 μM TMZ), while co-treatment with 0.1 μM BGB-290 and 100 μM TMZ decreased growth to 23.5 ± 9.6% (p<0.01), which is similar sensitivity to TMZ alone in U251 parental cells. In an initial in vivo study with orthotopic GBM12 xenografts, administration of BGB-290 at 3 mg/kg twice per day combined with 25 mg/kg TMZ extended survival of animals beyond 300 days (80% survival at day 320), while in the group treated with TMZ alone 80% animals were moribund before day 150. In conclusion, BGB-290 is a promising PARP inhibitor with high potency, cytotoxicity and favorable in vivo efficacy in TMZ sensitive and possibly a subset of recurrent TMZ resistant GBM. Citation Format: Shiv K. Gupta, Brett L. Carlson, Mark A. Schroeder, Katrina K. Bakken, Ann C. Tuma, Jann N. Sarkaria. Inhibition of PARP activity by BGB-290 potentiates efficacy of temozolomide in patient derived xenografts of glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3505. doi:10.1158/1538-7445.AM2015-3505

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 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 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