Carlson, Brett L.

Abstract Background: Bioluminescence imaging (BLI) of firefly luciferase expressing cells is widely used for analysis of growth, metastasis and drug response in tumor xenografts. D-luciferin, a commonly used luciferase substrate, has limited brain distribution that may undermine BLI for intracranial xenografts. The synthetic luciferin cyclic alkylamino-luciferin (CycLuc1) is a purported brain penetrant luciferase substrate not yet tested in intracranial xenografts. We compared BLI with CycLuc1 and D-luciferin in a glioblastoma (GBM) xenograft model. Methods: GBM6 cells were lentivirally transduced to express LUC2=tdTomato fusion protein. In vivo BLI were performed in orthotopic and heterotopic xenografts established in athymic nude mice. Concentrations of D-luciferin and CycLuc1 were measured by LC-MS/MS. Results: In a dose-response analysis, CycLuc1 at 5 mg/kg provided robust and reproducible imaging; however, an increased photon flux was observed with 10 mg/kg, while escalation to 15 or 20 mg/kg provided marginal additional gains in photon flux. In comparison of CycLuc1 (5 or 25 mg/kg) and D-luciferin (30 or 150 mg/kg, respectively) with serial, crossover BLI at both dose levels, CycLuc1 resulted in less variability and significantly higher photon flux as compared to D-luciferin in GBM6 intracranial xenografts (15 days after implantation). On average BLI with 25 mg/kg CycLuc1 resulted in ~8 fold greater photon flux from these early-stage xenografts as compared to 150 mg/kg D-luciferin (2.9±0.6x106 vs. 3.3±2.8x105 p/sec/cm2, p<0.001). Interestingly, bioluminescence emission with CycLuc1 and D-luciferin was comparable in xenografts imaged 28 days after implantation. In samples harvested after last imaging session, there was no significant difference in substrate distribution in intracranial tumors (tumor to plasma ratios for CycLuc1 and D-luciferin were 0.012 ± 0.020 and 0.012 ± 0.015, respectively; p=0.89). These results suggest that intracranial tumors at early stage may comprise relatively intact blood-brain barrier, which is disrupted at later stage. Consistent with notion that suboptimal delivery may interfere with D-luciferin mediated BLI in intracranial xenografts, photon flux in heterotopic tumors with D-luciferin or CycLuc1 was comparable (4.9±4.6x108 vs. 1.3±0.8x108 p/sec/cm2, p=0.10). Conclusions: These findings demonstrate that CycLuc1 may be a superior BLI substrate as compared to D-luciferin for monitoring tumor growth of intracranial GBM xenografts. Citation Format: Shiv K. Gupta, Minjee Kim, Bianca-Maria Marin, Lihong He, Katelyn Swanson, Tuma Tuma, Brett L. Carlson, Mark A. Schroeder, William F. Elmquist, Jann N. Sarkaria. Synthetic luciferin, CycLuc1, improves bioluminescence imaging for intracranial glioblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4112.

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 Purpose: CD90 (Thy-1) is a glycophosphatidylinositol-anchored glycoprotein considered as a surrogate marker for a variety of stem cells, including glioblastoma (GBM) stem cells (GSC). However, the molecular and cellular functions of CD90 remain unclear. Experimental Design: The function of CD90 in GBM was addressed using cellular models from immortalized and primary GBM lines, in vivo orthotopic mouse models, and GBM specimens' transcriptome associated with MRI features from GBM patients. CD90 expression was silenced in U251 and GBM primary cells and complemented in CD90-negative U87 cells. Results: We showed that CD90 is not only expressed on GSCs but also on more differentiated GBM cancer cells. In GBM patients, CD90 expression was associated with an adhesion/migration gene signature and with invasive tumor features. Modulation of CD90 expression in GBM cells dramatically affected their adhesion and migration properties. Moreover, orthotopic xenografts revealed that CD90 expression induced invasive phenotypes in vivo. Indeed, CD90 expression led to enhanced SRC and FAK signaling in our GBM cellular models and GBM patients' specimens. Pharmacologic inhibition of these signaling nodes blunted adhesion and migration in CD90-positive cells. Remarkably, dasatinib blunted CD90-dependent GBM cell invasion in vivo and killed CD90high primary GSC lines. Conclusions: Our data demonstrate that CD90 is an actor of GBM invasiveness through SRC-dependent mechanisms and could be used as a predictive factor for dasatinib response in CD90high GBM patients. Clin Cancer Res; 23(23); 7360–74. ©2017 AACR.

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 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.