Schroeder, Mark A.

Abstract 3D biomaterial models have potential to explore the influence of the tumor microenvironment on aberrant signaling pathways and compensatory signals using patient‐derived cells. Glioblastoma (GBM) tumors are highly heterogeneous, with both cell composition and extracellular matrix biophysical factors seen as key regulators of malignant phenotype and treatment outcomes. Amplification, overexpression, and mutation of the epidermal growth factor receptor (EGFR) tyrosine kinase have been identified in 50% of GBM patients. Here, hyaluronic acid (HA) decorated methacrylamide‐functionalized gelatin (GelMA) hydrogels are used to examine the synergies between microenvironmental factors and a model EGFR tyrosine kinase inhibitor (TKI) using patient‐derived xenograft cells. The in vitro behavior of 3 patient‐derived xenografts that reflect a clinically relevant range of EGFR variants is characterized: GBM10 (EGFR, wild type), GBM12 (EGFR+), and GBM6 (EGFRvIII). GelMA hydrogels support xenograft culture; cells remain viable, active, respond to matrix‐immobilized HA, and upregulate genes associated with matrix remodeling and tumor growth. Interestingly, matrix‐immobilized HA alters the response of GBM cells to a model tyrosine kinase inhibitor, erlotinib. While constitutively activated EGFRvIII cells are sensitive to TKI in gelatin hydrogels, hyaluronic acid mediated adhesive signaling interacts with EGFRvIII signaling to increase cell metabolic activity, increase soluble hyaluronic acid synthesis, and modify response to erlotinib exposure.

Abstract Mismatch repair (MMR) is essential for temozolomide (TMZ) sensitivity, and MMR deficiency causes TMZ resistance and disease recurrence. However, the role MMR in TMZ response has been linked with Msh2/Msh6 (MutSα), while role of Msh3, a component of MutSβ remains unclear. To delineate role of MutSα and MutSβ in TMZ response, we used siRNA to disrupt MSH6 and MSH3 expression. As expected, silencing of MSH6 led to resistance in a TMZ-sensitive U251 cell line. In contrast, silencing of MSH3 enhanced TMZ sensitivity as compared to the control cells. These results support established role of MutSα in TMZ response via replication stress and DNA double strand breaks (DSB), and reveal that MutSβ may potentially influence TMZ response by modulating DSB repair. Consistently, host cell reactivation (HCR) assays in U251 expressing DRGFP reporter showed decreased homologous recombination (HR) after MSH3 knockdown (KD), while Msh6 KD had no effect on HR efficiency. Supporting the concept of synthetic lethality of HR with PARP inhibition, clinically relevant dose of veliparib (1 µM) showed a modest but consistent increase in TMZ sensitization measured in terms of potentiation factor (PF, ratio of relative growth with TMZ alone/ TMZ and veliparib) in MSH3 KD (PF 1.6±0.2) as compared to control cells (PF 1.1±0.2). Sensitizing effect of 1 µM veliparib in MSH6 KD (PF 1.7±0.5) was comparable to that in MSH3 KD cells. Interestingly, supra-therapeutic dose of veliparib (10 µM) induced more pronounced sensitization in MSH6 KD cells (PF 2.6±0.3) as compared to MSH3 KD (PF 2.4±0.3) or control cells (PF 1.6±0.2). Consistently, veliparib (10 µM) alone had no impact on DNA damage signaling, while TMZ induced prominent increase in phosphorylation of KAP1, Chk1 and Chk2 in control and MSH3 KD cells, but not in MSH6 KD cells; veliparib/TMZ co-treatment led a robust signaling in all 3 types of cells. Increased sensitization by veliparib after MSH3 KD was also examined in a TMZ-resistant (GBM22TMZ) PDX model carrying resistance due to a homozygous mutation in MSH6, and a 4-fold increase in HR efficiency. In clonogenic assays silencing of MSH3 or BRCA1 had no effect on TMZ sensitivity in GBM22TMZ cells. However, veliparib alone (10 µM) decreased colony formation with relative colony formation 0.54±0.03 and 0.32±0.06 in MSH3 and BRCA1 KD cells, respectively as compared to 0.82±0.04 in controls. TMZ/veliparib co-treatment in GBM22TMZ led to further decrease in colony formation in MSH3 KD and BRCA1 KD cells with relative colony formation of 0.30±0.04 and 0.19±0.02, respectively versus 0.46±0.03 in control. Taken together, our results suggest that MutSα and MutSβ differentially regulate cytotoxic effects of TMZ. Decreased HR efficiency and enhanced sensitizing effects of veliparib in MSH3 KD cells partially define synthetic lethality of MMR deficiency with PARP, and may help development of PARP inhibitors as sensitizer of TMZ therapy in GBM. Citation Format: Shiv K. Gupta, Emily J. Smith, Ann M. Tuma, Brett L. Carlson, Mark A. Schroeder, Gasper J. Kitange, Jann N. Sarkaria. MutSα and MutSβ mismatch repair complexes differentially regulate temozolomide efficacy & sensitizing effects of veliparib in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1109. doi:10.1158/1538-7445.AM2017-1109

Abstract The extracellular matrix (ECM) is increasingly recognized as having a key role in cancer development. Advances in biomaterials design and preparation have led to highly defined sophisticated platforms that can recapitulate key features of the complex tumor microenvironment. We have developed a biomaterial tumor mimic that recapitulates systematically the main characteristics and heterogeneities of glioblastoma ECM. With the use of these 3D in vitro platforms we seek to understand the key features that make this cancer so challenging to treat. We are using this device in three different ways: (1) to further understand how particularities of brain tumor ECM affect cancer cells and assist in the diagnostic of different GBM tumor types, (2) provide new models for drug screening to afford more personalized therapies and (3) study the mechanisms of resistance to current therapies in order to identify more effective combinatorial treatments and new therapeutic targets. Glioblastoma (GBM) tumors are highly heterogeneous, both cell composition and ECM biophysical properties are key factors in cancer cell malignancy and treatment outcomes. Hyaluronic acid (HA), is the main component of the brain extracellular matrix and GBM is associated with aberrant HA secretion and overexpression of receptors associated with HA, such as CD44 and EGFR. We use a microfluidic system to culture GBM patient-derived cells within gelatin based hydrogels that contained gradually increasing concentrations of HA, in order to recreate the heterogeneity of the tumor mass. Cell proliferation and gene expression analyses demonstrate that biomimetic hydrogels support xenograft culture; cells remain viable, active, and upregulate matrix remodeling genes and others related to tumor growth. More importantly, we found that GBM cells respond to changes in the ECM composition presenting different gene expression profiles (VEGF, IL6, MMP2, MMP9, HAS3, CD44) depending on the particularities of every tumor, such as EGFR overexpression or PTEN suppression. These findings pave the way for new clinical tools that allow a more precise cataloging of GBM tumor types and tailoring treatments to individual patients. We have also used these platforms to evaluate potential therapeutic strategies for different GBM xenograft cells and to study the mechanisms of cell resistance. For this purpose, we first focused on the epidermal growth factor receptor (EGFR), which has been identified as a molecular target and associated with worse clinical outcomes. Amplification, overexpression and mutation of the EGFR tyrosine kinase has been identified in 50% of GBM patients. The genetic aberration of EGFR (EGFR+) often comes with the overexpression of the mutant EGFR variant III (EGFRvIII). We characterized the 3D in vitro behavior of 3 patient-derived xenografts that represent these EGFR variants: GBM10 (EGFR, wild type), GBM12 (EGFR+) and GBM6 (EGFRvIII). We studied the relationship between the HA contained in the surrounding matrix and response of GBM cells to a tyrosine kinase inhibitor (TKI), erlotinib. We demonstrate that an interaction exists between CD44 and EGF receptors. Results indicate that while EGFR+ cells are sensitive to TKI in HA hydrogels, HA seems to collaborate with EGFRvIII signaling to stir cell activity. Our results indicate that a combination of both bound and cell secreted soluble HA may influence GBM sensitivity and resistance to erlotinib. Moreover, immunoblots that analyze PI3K pathway support the hypothesis that matrix-bound HA is a key factor in the negative response of vIII tumors to EGFR inhibitors, and that the benefits of EGFR inhibition in GBMs may be found in combination to other inhibitors of ECM signaling. In summary, we demonstrate that engineered tumor avatar biomaterials can be used to reproduce some of the main characteristics of brain tumor microenvironment and become a valuable tool in the mechanistic studies of tumor development and treatment efficacy. Citation Format: Sara Pedron, Amanda M. Pritchard, Jann N. Sarkaria, Mark A. Schroeder, Brendan A. Harley. Engineered biomaterials as essential tools to determine the mechanisms of resistance to tyrosine kinase inhibitors in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B05.

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 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 BACKGROUND/PURPOSE The Mayo Clinic has developed a large panel of patient-derived xenografts (PDX) from patients with glioblastoma multiforme (GBM). Here we report initial molecular profiling of the Mayo PDX panel and a comparison of patient and PDX molecular profile and response to therapy. METHODS & MATERIALS Clinical data was retrieved by retrospective chart review. Animal data from all PDX investigations conducted between 2004-2014 were retrieved from experimental logs and subsequently consolidated into a database for analysis. RESULTS From 1999 to 2014, 182 patient tumor samples of varying histological grades were attempted for xenograft with 73 resulting viable PDX lines. Viable xenografts were only produced from WHO grade IV tumor specimens, yielding an overall success rate of 49% for these tumors. GBM patients that produced viable xenografts compared to those that did not exhibited a trend for decreased overall survival (P = 0.18). There was no significant association between successful xenografting and whether tissue was from newly diagnosed (45/93%) or recurrent (20/40; 50%) tumors. Patient age > 45 at diagnosis was correlated with increased PDX viability in GBMs (p = 0.05). Of the viable PDX models analyzed, EGFR mutation was identified in 17 lines, TERT mutation was found in 13 lines, IDH mutation in 1 line, and MGMT hypermethylation in 25 lines. RNAseq was performed on orthotopic tumor samples from 53 PDX models. After excluding contaminating murine sequence reads, expression analysis demonstrated 32 models with a mesenchymal phenotype. Array comparative genomic hybridization was performed on 9 patient samples and derivative early, mid and late passage PDX tumors. Using unbiased hierarchical clustering, there was a high concordance between patient and xenograft models. Within the PDX panel, fractionated radiation (RT) alone and RT combined with temozolomide (TMZ) was tested in orthotopic tumors in 38 lines. The overall median survival benefit (ratio of median survival for treated vs. placebo) in PDX lines treated with RT only was 1.6 (range: 0.9-2.5) and with RT/TMZ was 2.5 (range: 1.1 - 8.9). There was a positive association between observed patient survival and the corresponding survival benefit in the PDX for subjects treated with RT/TMZ (r = 0.2; n = 17). Response to adjuvant TMZ was evaluated in 42 tumor lines, and response to bevacizumab was tested in 33 tumor lines, and correlations with clinical treatment response are being evaluated. CONCLUSIONS: The Mayo GBM PDX panel is widely used in the neuro-oncology community. The initial molecular analysis suggests a good correlation between patient and PDX at the level of genotypic characteristics and therapeutic sensitivity. Citation Format: Dioval A. Remonde, Brett L. Carlson, Mark A. Schroeder, Brock Armstrong, Sen Peng, Lisa Evers, Paul A. Decker, Jeanette Eckel Passow, Michael E. Berens, Nhan L. Tran, Robert B. Jenkins, Jann N. Sarkaria. Characterization of patient-derived xenograft (PDX) models to evaluate clinical and therapeutic responses 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 1468. doi:10.1158/1538-7445.AM2015-1468

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